45 book reviews la revolución del olivar. el cultivo en seto. rius xavier, and josé m. lacarte. agromillora iberia, subirata, barcelona, spain, 2010, pp. 340. isbn 9780-646-53737-5. the need to lower costs to manage olive orchards in order to make the production of extra virgin olive oil more profitable has been for some time an important objective for olive growers worldwide. in this regards, mechanization has helped: today it is at its best when it comes to pruning and harvesting. in this context, the volume by xavier rius and josé m. lacarte is timely and offers a wealth of information. the title itself suggests the quality of the content: research and experimentation that have radically transformed an obsolete management system into new hope for olive growing in the third millennium. the text is divided into four chapters: 1. el aceite de oliva en el mundo (olive oil in the world); 2. la oportunidad de negocio en las plantaciones de olivar superintensivo (the economic opportunity in the superintensive planting system olive groves). 3. el cultivo superintensivo del olivar: respuesta a las necesidades del sector (the superintensive planting system in olive groves: response to sector needs). 4. el cultivo superintensivo en los principales países oleícolas (españa, italia, grecia, portugal, túnez, marruecos, estados unidos, chile, australia, argentina, resto del mundo) (the superintensive planting system in the major olive-producing countries: spain, italy, greece, portugal, tunisia, morocco, usa, chile, australia, argentina, rest of the world). a wealth of tables, figures and color image aid consultation of the text and add value to its presentation. considering the global interest of the topic, translation of the text into english is desiderable and perhaps predictable in the near future. enrico rinaldelli il paesaggio mantovano. nelle tracce materiali, nelle lettere e nelle arti. iv. il paesaggio mantovano dall’età delle riforme all’unità (1700-1865). atti del convegno di studi (mantova, 19-20 maggio 2005)(the mantuan landscape. from material, literary and artistic traces. the mantuan landscape from the period of reform to unity (1700-1865). prooceedings from the study convenction in mantua, 1920 may 2005). camerlenghi e., v. rebonato, and s. tammaccaro (eds.). leo s. olschki, florence (italy), 2010. pp. x + 454 + 7 figures and 70 plates, 63 of which in color. isbn 978-88-222-5845-8. € 55.00. the proceedings, edited by e. camerlenghi, v. rebonato, and s. tammaccaro, of the study convenction held in mantua, 19-20 may 2005 have recently been published. they are the fourth volume of the work on the mantuan lanscape presented by accademia nazionale virgiliana di scienze, lettere, e arti and edit by leo s. olschki. the various contributions which make up the volume, and include images from the period, have been prepared by outstanding experts in the field of historical italian landscapes and offer stimulation for debate about the causes behind the development of the mantua area during the subject period. ample space is dedicated to hydraulic conditions, which were tied to the control of power of padanian principalities, agronomic aspects and the evolution of crops, as well as to literary sources, from folengo to renaissance novelists. all this, without overlooking the urban landscape, which in this historic period in mantua offered moments of great splendor. some of the chapters clearly highlight the changes which occurred in the landscape, above all with regard to the management of water resources, while others focus on the parks, gardens and urban green spaces of mantua during the period. the text, which includes a generous list of bibliographic references, also contains illustrations (both color and black and white) and color photos. this volume is a valuable addition, under a scientific and technical profile as well as from a historical and cultural point of view, to the vast literature that exists on the evolution of historical-landscape areas. the work thus offers the reader insight and knowledge about numerous and important aspects of the landscape which make up our image of the mantuan territory. francesco ferrini 46 l’architetto sapiente. giardino, teatro, città come schemi mnemonici tra il xvi e il xvii secolo. koji kuwakino. giardini e paesaggio, vol. 28. leo s. olschki, florence (italy), 2011. pp. xxiv-326 + 70 figures and 7 plates in colour. isbn 978-88-222-6046-8. € 29.00. in this work, by the japanese scholar koji kuwakino and published by leo s. olschki of florence, the little-known relationship between architecture, the art of memory and encyclopaedism of the first modern age is examined through examples of the fruitful interaction between words, images and space. the subtitle of the book underlines the investigated subject: garden, theater, and city as mnemonic schemes in the 16th and 17th centuries. this architectonic typology offers the schemes of a complex conceptual construction able to collect, order and internalize the enormous quantity of knowledge acquired and accumulated over the centuries, from the ancient world up to the contemporary age. the author analyzes the ideal gardens of agostino del riccio and giovan battista ferrari, the universal theater of samuel von quiccheberg and the mnemonic city of cosma rosselli. as the author points out in the introduction, it is “an ideal journey” aimed at analyzing “three architectonic typologies often utilized as mental spaces: the garden, the theater, and the city”. these spaces “are not so much concretely real, but rather are ideal plans created with words, and the authors are not professional architects but gifted men and virtuosi who understand architecture”. dr. kuwakino goes on to explain that these examples of spaces “set outside the specialized field of ‘ars aedificatoria’, present only in text and for the most part unknown to historians, reveal, as in filigree, aspects that were previously hidden or scarcely examined despite being of considerable interest and rich in suggestion”. the analysis presented in the book aims to offer a complementary reading and enrichment of the history of 16th century architecture. through this work the author succeeds in shining light on fundamental cognitive aspects of the architectonic culture of the first modernity which previously had not been sufficiently emphasized. “l’architetto sapiente” demonstrates clearly how much architecture is under the influence of the spirit of the times architectonic space become, for example, a metaphor for knowledge and how much architecture has contributed to the building of new structures of modern thought. after the author’s introduction, the text is dived into six chapters: “l’arte della memoria architettonica” (the art of architectonic memory); “il giardino del tardo cinquecento come luogo del pensiero” (the garden in the late 16th century as a place for thought); “la rappresentazione emblematica del sapere enciclopedico nel giardino mnemonico di del riccio” (emblematic representation of encyclopaedic knowledge in the mnemonic garden of del riccio); “il ‘paradiso celeste’ nel giardino italiano” (the ‘paradiso celeste’ in the italian garden); “il grande teatro del pensiero creativo” (the great theater of creative thought); and “città celeste e ‘loca communia’”, (the celestial city and the ‘loca communia’), followed by the conclusions. the text includes a ample list of bibliographic references and is enriched by various illustrations and photos (black and white and color) which highlight the presented material. this work is clearly a precious addition to the existing literature on landscape studies, from both scientific and historical-cultural points of view, and offers, page after page, new and significant knowledge to the reader. cinzia silori territori delle acque. esperienze e teorie in italia e in inghilterra nell’ottocento (territories of water: experiences and theories in italy and england in the 19th century). aquae studi e testi sulle terme, 4. gabriele corsani (ed.). leo s. olschki, florence (italy), 2010. pp. 164. isbn 978-88-222-5988-2. € 20.00. water, promethean and domestic, represents the magnificent destiny of progress as the most radical adherence to the spirit and laws of nature. certainly, it is not easy to reduce down to a single text the myriad aspects of water and its interaction between the earth and the living things which occupy our planet. for example, water as power and energy but also as nourishment for life and the human spirit. in this context, gabriele corsani has deftly succeeded in telling the story of water in italy and england over the course of the 19th century. the volume, which contains a wealth of bibliographic information and illustrations from the period, is a complete work from different points of view: an interesting historical analysis and a precious bibliographic source for historians and researchers. 47 the text, compiled by notable authors, includes the following ten contributions: gabriele corsani, territori delle acque: esperienze e teorie in italia e in inghilterra nell’ottocento (territories of water: experiences and theories in italy and england in the 19th century). antonello boatti, storici e scrittori lombardi dell’ottocento di fronte al paesaggio. corsi d’acqua naturali e artificiali tra agricoltura e industria negli scenari disegnati da carlo cattaneo e cesare cantù (lombard historians and writers in the 19th century in a landscape context. natural and artificial waterways in agriculture and industry in scenes drawn by carlo cattaneo and cesare cantù. carlo cattaneo, prospetto della navigazione interna delle province lombarde con alcune notizie sulla loro irrigazione (“il politecnico”, 1841) (prospectives of navigation within the province of lombardy and notes on irrigation) (“il politecnico”, 1841). cesare cantù, storia di milano, diocesi e provincia di milano, cap. v. acque (history of milan, diocese and province of milan, chapter v. water). marco geddes da filicaia, corrado tommasi-crudeli e il tema dell’acqua ai tempi del colera (corrado tommasi-crudeli and the topic of water during times of cholera). corrado tommasi-crudeli, la canalizzazione delle città (canalization of the city). katia caldari, alfred marshall e l’importanza delle risorse naturali (alfred marshall and the importance of natural resources). alfred marshall, l’acqua come un elemento della ricchezza nazionale (water as an element of national wealth). gabriele corsani, la appendix howardiana: tecnica e retorica comunitaria per le acque ludiche di città giardino (the howardian appendix: technique and community rhetoric for water in the city garden). ebenezer howard, appendice. l’approvvigionamento idrico (appendix. the provisioning of water). enrico rinaldelli filippo de pisis botanico flâneur, un giovane tra erbe, ville, poesia. ricostruita la collezione giovanile di erbe secche. roncarati paola and rossella marcucci. giardini e paesaggio, vol. 30. leo s. olschki, florence (italy), 2011. pp. 208. isbn 978-88-222-6139-7. € 28.00. this attractive volume by paola roncarati and rossella marcucci presents ample and detailed documentation about an interesting but rarely mentioned, multifaceted man: filippo de pisis. de pisis is identified principally as a notable painter of the early 20th century but he was also, from a young age, a talented writer and poet as well as a passionate entomologist and botanist. indeed, it is on these latter aspects of his career that the two authors, through a combination of their areas of expertise, have focused the current work, offering an important contribution to the complex and heterogeneous biography of the painter. the volume begins with an introduction by lucia tongiorni tomasi and luigi zangheri entitled “l’erbario essiccato, un inedito tributo alla personalità di filippo de pisis”, (the dried herbarium, a tribute to filippo de pisis), which is then followed by a carefully written preface by gianni venturi, who admirably comments on the meaning and significance of the work: “it is clear that this book is difficult to define as a study of an herbalist, just as every work of poetry includes a multitude of meanings which render it precious, not only for a specific interest, but above all as an important step toward understanding the life and works of the artist in greater depth.” the text is divided into two parts. the first is dedicated to “le implicazioni culturali di una passione botanica” (the cultural implications of a passion for botany) and the second to “luoghi di erborizzazione, escursioni della mente, passeggiate dello sguardo” (places for herbalism, excursions of the mind, and a stroll for the eyes). finally, there is an analytical comment by the authors on the estrosità nell’erbario: lo sguardo “asistematico” di un botanico originale”. (creativity in the herbarium: an asystematic outlook by an original botanist). the work also includes a series of documents, including an article by giuseppe viviani “a passeggio con de pisis” (a walk with de pisis) and a rich bibliography,. enrico rinaldelli anthocyanins. biosynthesis, functions, and applications. gould k., k. davies, and c. winefield (eds.) springer science+business media, llc, new york, ny, usa, 2009. pp. i-xviii + 329. isbn 978-0-387-77334-6. usd 149.00. gbp 79.00. € 99.95. plant colours are attracting not only because they provide spectacular views of nature, but also for their functional roles. it is well known that nothing in nature is made by chance. anthocyanins, molecules of plant secondary metabolism, are those natural pigments giving predominant contributions to the painted word. their appearance on external portions of plant organs seems to be a kind of language to communicate with the animal kingdom, with the aim of attracting or repelling in order to facilitate plant reproduction and diffusion or plant defence, respectively. learning about the beneficial effects of anthocyanins in plants, humans have discovered how beneficial anthocyanins can be to their health as well. considerable literature in plant sciences is devoted to understanding anthocyanin-involved mechanisms, however much remains as yet undisclosed. the book anthocyanins. biosynthesis, functions, and applications edited by kevin gould, kevin davies and chris winefield is certainly a useful reference for all researchers involved in the many multidisciplinary studies of these natural pigments and represents a valuable collection of research results and needed future work for this rapidly expanding field. the different aspects of anthocyanin properties covered by this book include antioxidant activity and photoprotection, the role in plant defence mechanisms, the function in fruits and flowers. extensive chapters are dedicated to plant cell cultures for the biosynthesis of anthocyanins and their biotransformation by microorganisms. in addition, the biochemical pathways of reactions occurring in vivo for the stabilization of anthocyanins and the characterization of new anthocyanin-derived compounds as food colorants are also considered. finally, the last chapter deals with the phytochemical role of anthocyanins to promote human health. being an outsider to the plant science academic world, but working in spectroscopic monitoring of vegetation for about 20 years, i thoroughly enjoyed reading this volume and warmly recommend finding a place for this book on everyone’s desk. giovanni agati la civiltà delle acque tra medioevo e rinascimento. the culture of water between the middle ages and the renaissance. calzona a. and d. lamberin (eds.). collana ingenium, vol. 14. edizioni leo s. olschki, firenze, 2010. volume i and ii pp. xviii + 718, 11 figures, and 83 plates, 22 of which in colour. isbn 978 88 222 5969 1. € 78.00. these two volumes contain the proceedings of the international meeting of the same name held in mantua 1-4 october 2008 and include presentations by 37 authors of national and international level. the works address the literary, philosophic, historic, political-economic and social, historical-artistic and architectural, and scientific and technical-engineering issues relative to the management and significance of freshwater in italy from the middle ages to the renaissance. the works are grouped into five sections: literary and imaginary; political and economic management; art, architecture, landscape and territory; engineering, infrastructures, science and criticism; gardens, parties and spectacles. a final section deals with the magical, thermal, and nutritive aspects of the waters. the overall complex provides a true treatise on the subject matter, making these volumes valuable not only as a guide for those directly involved in the sector but they can also be a source for consultation for others who have personal interest in the topic. various illustrations and tables enrich each paper and there are numerous bibliographic citations, offering the reader the possibility to go into greater depth. this subject is of considerable historical-cultural importance in italy and both the meticulous work and the efforts by the various contributors – some of whom present their life work – attest to just how much there is to know about fresh waters in our country. these two volumes are therefore of great interest and usefulness and are an important contribution to the literature available on the topic and will surely be helpful for not only technical workers and students but also an increasing number of enthusiasts. francesco ferrini 69 book reviews 70 san rossore nella storia: un paesaggio naturale e costruito. san rossore in history: both a natural and built landscape. panattoni r. collana giardini e paesaggio, vol. 27. edizioni leo s. olschki, firenze, 2010. pp. xxxii + 230. 2 figures and 32 plates in colours. isbn 978-88-222-6023-9. € 27.00. this work by rita panattoni was awarded the premio verbania, editoria e giardini in 2009. the title of the volume is, in itself, significant: the subject is not the history but rather the park of san rossore. this difference may not seem important but the title of any publication should define the whole, which in this case is the role played by this park in the history of the region. the book opens with an essay on the evolution of the botanical context by fabio garbari who underlines the extraordinary environmental value of this area which lies somewhere between being a garden and a landscape, and contains a total of 335 pages divided into five parts: the ancient san rossore; san rossore of the medici family; san rossore of the lorena family; san rossore of the house of savoy; the post-world war ii period, the 1950s. overall the work effectively illustrates the story of the transformation of the original selva dei tomboli pisani into the current san rossore estate. the earliest information about the san rossore area – covering an area of 4800 hectares between the serchio and arno rivers, composed of woodlands and pine forests, beaches, dunes, fields and marshlands and approximately 12 km of coast – dates back to about 1000 ad. up until the beginning of the 16th century this vast area was utilized for hunting and fishing or for the cultivation and harvesting of wood. it was the medici family who began the work of transforming the estate, first as renters (in the 16th century) and later as owners. subsequently ownership passed to the lorena family, who used the estate frequently and added improvements. in the mid-1800s, when tuscany became a part of the new kingdom of italy, the house of savoy came to san rossore and they occupied the estate for long periods of the year. after the war and the fall of the monarchy, san rossore became the property of the president of the republic. the volume, which contains an ample list of bibliographic references and numerous illustrations in black and white and in color, is an important addition to the vast literature on the evolution of historical landscapes not only from a scientific and technical profile but also from an historical and cultural point of view. this approach thus offers the reader meaning and knowledge about the important and multiple values of the park of san rossore which has a role in creating an image of tuscany. francesco ferrini paesaggio rurale: strumenti per la pianificazione. strategica. the rural landscape: tools for strategic planning. agnoletti m. edagricole, bologna, 2010. figures 220. pp. xvi + 348. isbn 978-88-506-5226-6. € 39.00. almost always legislation regarding a particular topic is distributed among various laws and decrees, which generally are based on or are handed down from previous periods and are more or less modified as needed. as a consequence, consultation of the regulations pertaining to a subject of interest can be often difficult and laborious and sometimes one has the feeling of not having investigated fully or having interpreted incorrectly, especially when the regulations are apparently contradictory (a not uncommon occurrence). fortunately, prof. agnoletti has prepared this volume which represents a clear and precise compendium of the piano nazionale di sviluppo rurale 2007-2013 (psn). the text is divided into six sections: 1) the evolution of the rural landscape from unification until today; 2) the evolution of forest landscapes from unification until today; 3) spatial characteristics and structural dynamics: the system of tuscan monitoring; 4) the current structure of the italian landscape; 5) the rural landscape in territorial politics; and 6) strategies and actions in order to add value. the appendix contains a section dedicated to analysis methods and management processes. the piano nazionale di sviluppo rurale 2007-2013 (psn) has included landscape amongst its strategic objectives, underlining its role in the new agricultural policy and the transformations which have occurred in terms of understanding the importance of this resource as well as that of the grower and the rural territory. this initiative is in line with the activities set out by the european landscape convention and with landscape planning promoted by the new codice dei beni culturali involving workers in the sector, public decision-makers and universities. the meaning of landscape and the perception of it has, in fact, changed over time. it is the essential element in defining a development model that is particularly suitable to the rural context, representing not only the cultural identity of our country but also economic and environmental values. however it is necessary to elaborate a cultural and scientific proposal that is appropriate for the rural setting, which takes into account the fact that landscape is the result of a combinationof social, economic and environmental factors in space and time where man plays a central, not marginal role. the current volume proposes and adds to the materials utilized to develop a picture and definition of the strategies for landscape by the ministry of agricultural, alimentary and forestry policy, illustrating approaches to analysis and providing additional material for integrated planning. the text includes illustrations, tables, graphs and an ample list of bibliographic references which offer the reader a key to acquiring greater depth of knowledge in the particular aspects of the subject matter. this work is of considerable scientific importance and the book can be of great interest for researchers and students in the field of environmental studies, as well as anyone who deals with landscape on a number of different levels. francesco ferrini 71 handbook of plant nutrition. barker a.v. and d.j. pilbeam (eds.) taylor and francis group. crc press, new york, ny, usa, 2007. pp. xvi + 614 + cd-rom. isbn 0-8247-5904-4. us$ 157.95. the increasing demand on the world food supply, coupled with concern over the use of chemical fertilizers, has determined an interest towards the practice of precision agriculture, has led to a better control and monitoring of plant nutrition to maximize the rate of growth, the yield of crops as well as their nutritional value. this handbook covers principles of plant nutrition from a historical standpoint to current knowledge of the requirements of crops for certain elements and the beneficial effects of others. the book consists of twenty chapters, each one dedicated to an essential macro or micronutrient or beneficial element. more in details, each chapter, written by eminent researchers from across the world, gives historical information on the specific nutrient, explaining why it is either essential or beneficial for the plants; moreover an explanation of how appearance and composition of plants can be used to assess nutritional status is given, as well as recommendations on fertilizers that can be applied to remedy nutritional deficiencies. this handbook, including a cd-rom containing more than 40 illustrations in full colour, can be considered of great value, and for this reason recommended, to growers, agricultural consultants, agronomist and plant scientist, providing a practical easy-to-use reference for determining, monitoring, improving the nutritional needs of plants. the graphical presentations of plant interactions with nutrients and beneficial elements, and the straight-forward explanations of how nutrient deficiencies arise are especially useful to those seeking knowledge of plant nutrition. francesco paolo nicese orticoltura mediterranea sostenibile. tesi r. pàtron editore, bologna, 2010. pp. 504. isbn 978-88-555-3062-0. € 42.00. the original work of romano tesi is inspired and basically derives from the interesting connection among some important facts. first of all, the mediterranean basin is the area of origin and varietal differentiation of many important vegetables, but it also allowed the selection of many others imported from asia and america during the colonization era. to this is added the fact that the countries bordering the mediterranean basin are the most important producers and consumers of those products. in the context of this mixture of cultures and traditions the well-known “mediterranean diet” has developed, which is considered worldwide as a bright example of the highest dietary level. “orticoltura mediterranea” is a new and updated book which gives particular attention to the changing market needs. thus, in addition to general and specific aspects of the traditional horticulture, the book focuses into the composition and nutritional properties of vegetables, together with a outlook on the principal aspects of integrated production and organic farming. the book is divided into two parts. the first part covers the horticulture in the mediterranean basin, which is deeply described in the following eight chapters: 1) l’orticoltura mediterranea (mediterranean horticulture); 2) classificazione degli ortaggi (classification of vegetables); 3) tipi di orticoltura e sostenibilità (sustainability of the different kind of horticulture); 4) i sistemi colturali (cultivation systems); 5) qualità dei prodotti orticoli (quality of horticultural products); 6) mezzi di protezione (protection devices); 7) sementi e vivaismo orticolo (seeds and horticultural nursery); 8) gestione dell’azoto e dell’acqua di irrigazione nel pieno campo (nitrogen and irrigation water management in the field horticulture). the second parts is dedicated to the monographic description of more than one hundred horticultural crops, with broad and updated insights. due to its modern setting, the rich collection of tables, the presence of numerous coloured or black and white images in which the scientific knowledge of the author can be easily recognized, the book may serve to a wide group of readers, from the student to the teacher, from the expert to the manager. enrico rinaldelli impaginato 247 adv. hort. sci., 2022 36(3): 247­251 doi: 10.36253/ahsc­12347 evaluation of hemerocallis germplasm using single nucleotide polymorphisms of nrits and chloroplast interspacer region s.y. park 1, y.h. joung 1, j.k. suh 2, m.s. roh 2, 3 (*) 1 school of biological sciences and technology, chonnam national university, 500‐757 gwangju, korea. 2 department of environmental horticulture, college of bioresource science, dankook university, cheonan, 330‐714 chungnam, korea. 3 current address: the institute of natural resource development, mokpo national university, muan, 58554 jeonnam, korea. key words: daylily, haplotype, nocturnal flowering, polymerase chain reaction, sequence analysis. abstract: this study was initiated to distinguish nocturnal (night) flowering hemerocallis species from day flowering species based on the single nucleotide polymorphisms (snps) of nuclear internal transcribed spacers 1, 2 in a riboso­ mal rna gene (nrits) and a chloroplast interspacer region (cpis). four noctur­ nal flowering species, h. citrina, h. thunbergii, h. minor, and h. lilioasphodelus were collected including korea, and compared with day flowering species that included h. vespertina and h. hongdoensis. based on the haplotypes of nrits and cpis, nocturnal species cannot be distinguished from day flowering species. discrepancies in flowering time and haplotypes among h. minor accessions sug­ gest that more germplasm with diverse geographic origins should be evaluated and identification of other genes is required to effectively distinguish nocturnal species from day flowering species. 1. introduction there are about 15­26 species/varieties in the genus hemerocallis (usda, ars, national genetic resources program, 2015). using amplified fragment length polymorphisms (aflp) markers, h. fulva l. were grouped separately from the nocturnal species h. thunbergii baker and h. lilioas‐ phodelus l., while nocturnal h. minor mill. and h. citrina baroni, were grouped together in a different sub­cluster (tomkins et al., 2001). the flowers of nocturnal hemerocallis open late in the afternoon and wither the next morning (fig. 1) (chen and noguchi, 2000). however, gulia et al. (2009) classified h. minor as a diurnal species rather than a nocturnal species, confirming the study by krestova and nesterova (2003) that h. minor flowered in the morning under sunny weather at >16°c and with­ (*) corresponding author: marksroh@gmail.com citation: park s.y., joung y.h., suh j.k., roh m.s., 2022 ­ evaluation of hemerocallis germpla‐ sm using single nucleotide polymorphisms of nrits and chloroplast interspacer region. ­ adv. hort. sci., 36(3): 247­251 copyright: © 2022 park s.y., joung y.h., suh j.k., roh m.s. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 24 november 2021 accepted for publication 31 august 2022 ahs advances in horticultural science short note https://doi.org/10.36253/ahsc-12347 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2022 36(3): 247­251 248 ered in the afternoon. molecular markers have not previously been test­ e d t o d e t e r m i n e ti m i n g o f fl o w e r i n g a m o n g hemerocallis species. therefore, this study was con­ ducted to investigate the use of snps of nuclear internal transcribed spacers 1, 2 in a ribosomal rna gene (nrits) and a chloroplast interspacer region (cpis) to evaluate the genetic relationships between nocturnal and day flowering species of hemerocallis. 2. materials and methods the nocturnal flowering species hemerocallis thunbergii, h. minor, h. lilioasphodelus, and h. citri‐ na, and the day flowering species h. hongdoensis m.g. chung & s.s. kang, h. vespertina h. hara, h. dumortierii c. morren and hybrid ‘stella de oro’ were collected from korea (k), china (c), united kingdom (uk), united states of america (ua) and germany (ge) (table 1). samples were designated as, for example, k1 (mother plant)/1­3 (seedling 1, 2, 3 from mother plant k1). genomic dna was isolated from young leaves using dneasy plant mini kit (qiagen inc., valencia, ca). polymerase chain reaction (pcr) for nrits was performed with a 18s rrna gene specific forward primer its1 (5’­tag agg aag gag aag tcg taa caa gg­3’) and primer its2 (5’­ gattttcagtcctctgctc­ tac­3’). the reaction mix consisted of 12.5 μl of 2x f­ star taq smartmix (solgent co., daejeon, korea), 2 μl of each primer (0.4 μm final concentration), and 2 μl of genomic dna. for cpis, forward primer (5’ ­tcgt­ gagggttcaagtccctct­3’) and reverse primer (5’­ gattttcagtcctctgctctac­3’) were used. the reaction mix consisted of 12.5 μl of 2x f­star taq smartmix (solgent co., korea), 2 μl of each primer (0.4 μm final concentration), and 2 μl of genomic dna (50 ng) and 5 μl of 5× band doctor buffer (solgent), and made up to the 25 μl final vol­ ume with pcr ultrapure water. the pcr conditions were: 2 min at 95°c, followed by 35 cycles of 20 sec at 95°c, 40 sec at 65°c, and 1 min at 72°c, followed by 5 min of 72°c using an abi veriti dx thermal cycler (life technologies, grand island, ny, usa). pcr products were direct sequenced as described by park et al. (2014). sequences were registered in the national center for biotechnology information genbank (ncbi, http://www.ncbi.nlm.nih.gov/). 3. results and discussion based on the sequences of nrits, three haplo­ types were identified: t i­1, t i­2, and t ii (table 2) and based on the sequences of cpis, 7 haplotypes were identified: t i, ti­1, tii, tiii, tiv, tv, and tvi (table 3). sequences of cpis were more informative to separate accessions than those of nrits, suggest­ ing that the its region in hemerocallis may not be useful as a potential source for species identification, although accessions of cultivated origin kolkwitzia amabilis (graebn.) christenh were derived from the accession of known wild origin (aa816­84a) (park et al., 2014). nocturnal flowering h. citrina, h. thunbergii and h. lilioasphodelus flower in the afternoon and wither the following morning (table 1, fig. 1). the difference between two types of floral morphology of h. thun‐ bergii, based on the presence or absence of bracts subtending the flower buds; k7 lacked bracts while k8­9 had bracts was detected in the haplotype of cpis, with k7 belonging to type i­1 and k8­9 belong­ ing to type i that may result from that the sequence of the its 2 region is more variable than that of the its 1 region (ma et al., 2014). nocturnal flowering h. thunbergii accessions collected from the same loca­ tion in korea (k8­9, and k 8­13) should be evaluated fig. 1 ­ nocturnal h. thunbergii accession 7 collected from korea (k7) showing flower opening at 4, 5, and 6 pm and closed by 10 am following day. http://www.ncbi.nlm.nih.gov/ park et al. ‐ hemerocallis germplasm evaluation 249 further since these accessions were grouped togeth­ er with other h. thunbergii accessions (k1­3). there is no correlation between these nrits hap­ lotypes and timing of flowering observed in h. citrina; collected from china (c2) and the united kingdom (uk5), both belonging to type i, from germany (ge4), belonging to type ii, and from the united kingdom (uk1), belonging to type iii. this requires further examination collecting more accessions from differ­ ent collection sites. further, in cpis, day flowering h. hongdoensis and h. vespertina belonged to type i and ii, respectively. when t was assigned for the ambiguous code c or t at the positions 78, 86, 99, 113, and 120 of nrits sequence for type i­1, and a was assigned for a or g at 231, types i­1 and i­2 can be combined and all accessions can be assigned as type i, separated from h. minor (uk6 and ge3) as type ii (tables 1 and 2). they may be derived from different geographic ori­ gins of k14 or ua5 which were collected from korea, belonging to type i­2, and exhibit some degree of genetic variation revealed in this study using univer­ sal primers for nrits. however, different strains or populations of h. minor may exist since h. minor table 1 ­ accession information on hemerocallis taxa (mother plants and their seedlings) with flowering characteristics. haplotype based on the sequence analysis of nrits and cpis are indicated scientific name mother plant (leaf) seedlings a flowering time b source, country haplotype (t) c nrits cpis h. thunbergii k1 d k1/1­3 n jungseon­gun, korea i­2 i h. thunbergii k2 k2/1­3 n i­2 i h. thunbergii k3 k3/1­3 n i­2 i h. thunbergii k7 n j.w. chang, gomyeong­dong, jecheon­si, chungcheongbuk­do, korea i­2 i­1 h. thunbergii k8,9 e n e.j. kim, gomsi­gil, ungdam­ri, paju­gun, kyunggi­do, korea i­2 i ka8­13 d n e.j. kim, m.s. roh, gomsi­gil, ungdam­ri, paju­gun, kyunggi­do, korea i­2 iv h. thunbergii k11 n hantaek botanical garden, korea i­2 i h. thunbergii uk3, 4 n royal botanical garden edinburgh, uk (19300128a f) i­2 i h. thunbergii ua7 ­ n united states national arboretum, washington, dc, usa (usna; na54757.3 collected from korea) i­2 i h. minor ­ k14/1­3 d hantaek botanical garden, korea i­2 i­1 h. minor uk6 uk6/1­3 n? royal botanical garden edinburgh, uk ii v h. minor ge3/1­3 n? botanischer garten leipzig (xx­o­lz­ad439/2006) ii v h. minor ua5 ­ d usna; na31800.1 i­2 i h. lilioasphodelus ­ c1/1 n x.w. wu, china i­2 i c1/2­3 i­1 i h. liliasphodelus ua6 ­ n usna; na54879.3 i­2 i h. citrina ­ c2/1­3 n x.w. wu, china i­2 i h. citrina uk1 n royal botanical garden edinburgh, uk (19685548a u) i­2 iii h. citrina uk5 n royal botanical garden edinburgh, uk i­2 i h. citrina ge4/1­3 n botanischer garten leipzig (xx­o­lz­aw78/1998, 2000) i­2 ii h. citrina ‘april flower’ ­ c3/1­3 x.w. wu, china i­2 i h. vespertina k12 k12/1­3 d hantaek botanical garden, korea i­2 ii h. dumortierii c. morren k13 d hantaek botanical garden, korea i­2 vi h. hongdoensis ­ k15/1­3 d hantaek botanical garden, korea i­2 i ‘stella de oro’ ua2 ua2/1­3 d m.s. roh, ann arbor, mi., usa i­1 i a seedlings 1, 2, 3 from mother plant k1. designations of accession of mother plant and three seedlings are indicated as k1 and k1/1­3, respectively. b flower opens in the morning and withers in the late afternoon (day, d) or opens in the late afternoon and withers early on the next morning (night, n). flowering characteristics were not evaluated in this study for h. minor collected from royal botanical garden edinburgh, uk and botanischer garten leipzig (xx­o­lz­ad439/2006). c refer to table 2 for nrits and table 3 for cpis haplotypes and single nucleotide polymorphisms. d collected or received from korea (k, ka), united states of america (ua), united kingdom (uk), germany (ge), and china (c). e samples of k8 and 9 and of ka 8­13 were collected from the same location in 2011 and 2014, respectively. f samples were of garden origin from dendrologische gartenerei in pruhonce, czech republic via peter brownless. adv. hort. sci., 2022 36(3): 247­251 250 flowers in the morning under sunny weather at >16oc and withers in the afternoon, as reported at the botanical garden institute, far east branch, r u s s i a n a c a d e m y o f s c i e n c e s ( k r e s t o v a a n d nesterova, 2003). the h. minor, received from the us national arboretum (usna; na31800.1) original­ ly collected from korea, flowers in the morning in ann arbor, mi, usa. therefore, further investigation of hemerocallis minor is needed to determine whether accessions collected from china, korea and the far eastern part of russia are of two different flowering types. hemerocallis hybrid ‘stella de oro’ (ua2), day flowering landscape plant of unknown parentage, was grouped with h. minor collected from korea (k14), but not with h. minor (uk6 and ge3) acces­ sions and their seedlings (k14/1­3) and uk6/1­3, based on the haplotypes of snps with a nrits region (table 2) and with cpis (table 3). grouping of h. dumortieri (k13) with h. minor (uk6 and ge3) with nrits region was also different from that with cpis (table 3). mcgarty (2006) used aflp markers from hemerocallis species, and placed h. lilioasphodelus with h. thunbergii in one sub­group and h. citrina, h. minor, and h. dumortieri in another. this suggests that day and night flowering species cannot be sepa­ rated either by aflp markers, or by snps from a nrits region or cpis, as attempted in this study. difficulties with identifying species of hemerocallis native to korea may not be easy to resolve and flowering time in f1 hybrids between h. fulva (day flowering) and h. citrina (nocturnal flowering) showed discontinuous bimodal distribution (hasegawa et al., 2006). beyond the identification issue for the accessions h. thunbergii ka8­13, grouping of species investigat­ ed in this study differs significantly between nrits and cpis (tables 2 and 3). difficulties with identifying species of hemerocallis native to korea may not be easy to resolve and flowering time in f1 hybrids between h. fulva (day flowering) and h. citrina (night flowering) showed discontinuous bimodal distribu­ tion (hasegawa et al., 2006). distinguishing nocturnal flowering forms of h. minor from day flowering forms is not possible due to the existence of two different genotypes collected from china, korea and far eastern russia and by testing the universal primers by snps of nrits region and cpis. however, these primers were used suc­ cessfully to identify mother plants and seedlings of l i g u s t r u m q u i h o u i c a r r i è r e ( m a e t a l . , 2 0 1 4 ) . table 2 ­ haplotype based on single nucleotide polymorphisms (snps) and insertion and deletion (in/del) of a nuclear internal tran­ scribed spacer 1, 2 in a ribosomal rna gene of hemerocallis accessions haplotype ncbi registration positions and codes of nucleotide single nucleotide polymorphisms in/del 78 86 99 113 120 170 206 210 231 254 432 568 597 435­441 i­1 kt189161 c/t c/t c c/t c/t a g a a/g a a g g c7 i­2 kt189162 c c c c c a g a a a a g g c6­7 ii kt189163 c c t c c g c c c c g a a c7 table 3 ­ haplotypes based on single nucleotide polymorphisms (snps) and insertion and deletion (in/del) of a chloroplast interspacer region of hemerocallis accessions haplotype ncbi registration positions and codes of nucleotide single nucleotide polymorphisms in/del 26 216 243 246 291 315 37 302 i kt189164 t c c c a a t9 t7 i­1 kt189165 t c c c a a t9 t8 ii kt189166 t c c c c a t9 t8 iii kt189167 t c c a a a t9 t8 iv kt189168 c c a a a a t8 t8 v kt189169 c c c a a a t8 t8 vi kt189170 c a c a a a t10 t8 park et al. ‐ hemerocallis germplasm evaluation 251 seedlings grouped in the haplotype with their moth­ er plants, except the mother plant (c1) and seedlings 2­3 of h. liloasphodelus (c1/2­3), which belonged to type i, type i­2 and i­1, respectively, in nr­its region (tables 1 and 2). the current primers for nrits region and cpis cannot be used to differentiate noc­ turnal flowering species from day flowering species. lee and maki (2015) reported that cpdna in the majority of cultivars were inherited from h. albo‐ marginata, although the leaf morphology was simi­ lar to h. sieboldiana, indicating that nrits should fur­ ther be investigated. 4. conclusions the sequence variations of nrits region and cpis cannot be used to distinguish nocturnal flowering species from day flowering hemerocallis species. markers other than those evaluated in this study should be evaluated. genetic variations among s e e d l i n g s o r b e t w e e n m o t h e r p l a n t a n d t h e i r seedlings were observed in h. lilioasphodelus (c1/1 vs. c1/2­3), requiring seedlings of h. lilioasphodelus to investigate to confirm the results of this study. discrepancies in flowering time among h. minor accessions also suggest that more germplasm with diverse geographic origins should be evaluated. acknowledgements critical review and english editing of the manu­ script by dr. c.j. catanzaro, virginia state university, va, usa, is greatly appreciated. references chen x., noguchi j., 2000 ­ 31. hemerocallis linnaeus, sp. p1. 1:324. 1753. ­ flora of china, 24: 161­165. gulia s.k., singh b.p., cartter j., griesbach r.j., 2009 ­ daylily: botany, propagation, breeding. ­ hort. rev., 35: 193­220. hasegawa m., yahara t., yasumoto a., hotta m., 2006 ­ bimodal distribution of flowering time in a nat‐ ural hybrid population of daylily (hemerocallis fulva) and night lily (hemerocallis citrina). ­ j. plant res., 119: 63­68. krestova i.n., nesterova s.v., 2003 ­ flowering and pol‐ lination of the native daylily species (hemerocallis) in the botanical garden institute, far east branch, russian academy of sciences. ­ contemp. problems ecol., 6: 48­ 454. lee s., maki m., 2015 ­ origins of hosta cultivars based on sequence variations in chloroplast dna. ­ hort. j., 84: 350­354. ma s.h., burchi g., suh j.k., roh m.s., joung y.h., 2014 ­ i d e n ti fi c a ti o n o f l i g u s t r u m s e e d l i n g s b a s e d o n sequence analysis of an internal transcribed spacer. ­ hort. environ. biotechnol., 55: 423­427. mcgarty t.p., 2006 ­ phylogenetics, dna, classification and the genus hemerocallis. ­ http://www.telmarcgar­ dens.com. park s.e., burchi g., roh m.s., joung y.h., 2014 ­ characterization of kolkwitzia amabilis accessions based on flowering and molecular markers. ­ scientia hort., 165: 190­195. tomkins j.p., wood t.c., barnes l.s., westman a., wing r.a., 2001 ­ evaluation of genetic variation in the daylily (hemerocallis spp.) using aflp markers. ­ theor. appl. genet., 102: 489­496. usda, ars, national genetic resources program, 2015 ­ germplasm resources information network ‐ ( g r i n ) [ o n l i n e d a t a b a s e ] . n a ti o n a l g e r m p l a s m resources laboratory, beltsville, maryland. 21 1. introduction tecomella undulata (roxb.) seem., known as rohida tree, is an ornamental and medicinal shrub species of the bignoniaceae family widespread in tropical regions such as iran, india, and pakistan. this endangered plant species currently grows in arid and semi-arid regions of southern parts of iran. its bark contains lapachol, a naphthoquinone with anticancer, antibacterial, antifungal, antivirus (consolacao et al., 1975; guiraud et al., 1994; hussain et al., 2007) and analgesic (ahmad et al., 1994) activities. it may have a pivotal role in environmental conservation in the arid parts of iran; furthermore it is an accepted tree species in agroforestry systems (tewari and singh, 2009). due to its beautiful flowers and semi-deciduous habit, it can be used in landscaping with some limitations due to its sterility and low potential germination of the few seeds produced in iran. karami and salehi (2010) reported that rooting of stem cuttings is limited from spring to autumn in this species and it is included in the list of hard to root woody plant species. therefore, micropropagation is necessary to protect this endangered species. plant tissue culture is one of the most important steps in genetic transformation and plant biotechnology studies to produce plantlets from stock plants as a rapid and efficient procedure throughout the year (giri et al., 2004; sarmast et al., 2009). there are few reports about micropropagation of t. undulata; in vitro seasonal effect on shoot proliferation was reported (rathore et al., 1991). attempts have been made to multiply t. undulata plants through micropropagation. rathore et al. (1991) reported that about 8-10 shoots were obtained over two to three weeks at 31ºc in ms medium supplemented with bap (2 mg l-1) and iaa (0.05 mg l-1). robinson et al. (2005) claimed that ms basal medium supplemented with 1.5 mg l-1 bap and 0.02 mg l-1 iaa was the most effective medium for maximum (95%) regeneration of nodal explants. they reported that 29 shoots per nodal segment were observed on ms medium supplemented with 0.75 mg l-1 bap and 0.01 mg l-1 iaa within 3 weeks. furthermore, they observed about 27% mortality after transfer of plantlets to soil mixture. aslam et al. (2009) developed a transformation protocol for osmotin gene in t. undulata. the effects of silver nanoparticles (snps) on decontamination in tissue culture systems have been reported (abdi et al., 2008; sarmast et al., 2011). however, the effect of silver-based material on ethylene mode of action is not well acknowledged in plants (taiz and zeiger, 2006). explants grown in media supplemented with silver ions (eapen and george, 1997; zhang et al., 2001) and snps (sarmast et al., 2011) were relatively healthier than control. the most important step of any in vitro propagation system is mass multiplication of plantlets that are genetically homogenous and phenotypically uniform (sarmast et al., 2012). therefore, micropropagation is restricted to direct regeneration. effects of silver nanoparticles on tecomella undulata (roxb.) seem. micropropagation m. aghdaei, h. salehi(1), m.k. sarmast department of horticultural science, college of agriculture, shiraz university, shiraz, iran. key words: multiplication, rohida tree, snps, tissue culture. abstract: plant tissue culture is a reliable tool for conservation and multiplication of many plants, including medicinal plants. tecomella undulata (roxb.) seem. is a plant native to tropical regions such as iran, india and pakistan; this precious plant which contains lapachol (a strong antiseptic used against jaundice) is an endangered species, therefore its conservation is of prime importance. the aim of this experiment was to evaluate the effects of silver nanoparticles (snps) at concentrations ranging from 5 to 80 mg l-1 alone or combined with 6-benzyl-amino-purine (bap) and indoleacetic acid (iaa) on growth properties of t. undulata in aseptic condition. thidiazuron (tdz) at concentrations from 0.001 to 20 mg l-1 was used in proliferation medium of t. undulata single nodes; combinations of bap (from 0.3 to 1.2 mg l-1), and 2,4-dichloro-phenoxy-acetic acid (2,4-d, 0.2 and 0.4 mg l-1) were also used in callus production and in indirect bud regeneration media. explants were surface sterilized using 10% clorox for 7-8 minutes. results indicated that adding of snps in ms medium increased the mean number of fresh shoots per explants (mnfs/e), the percentage of explants producing shoots (peps) and also plant survival, due to its action on ethylene blockage. tdz at the concentration of 0.1 mg l-1 increased bud proliferation up to two buds per explants, however higher concentration inhibited growth and in some cases caused death of the explants. adv. hort. sci., 2012 26(1): 21-24 (1) corresponding author: hsalei@shirazu.ac.ir received for publication 17 october 2011 accepted for publication 31 january 2012 22 the aim of the present work was to assess snps as ethylene inhibitor for the improvement of micropropagation in t. undulata. a secondary objective was to examine tdz and bap effects on direct and indirect regeneration in t. undulata through single node and callus obtained from the proximal part of the explants. 2. materials and methods more than 15-year-old iranian tecomella undulata plants were chosen for the present study. stem pieces (4 cm long) were cut and prewashed in tap water for 15 min. explants were then treated with 10% clorox (containing 5.25% sodium hypochlorite) plus 0.2% household detergent for 7 min for surface sterilization and then rinsed six times with sterilized distilled water. the stem pieces were finally cut into nearly 10 mm long segments (including a single node) and placed with their proximal ends on ms (murashige and skoog, 1962) basal medium with 3.0% sucrose and 0.8% agar. mean number of fresh shoots per explant (mnfs/e), mean length of shoots per explant (mls/e), mean diameter of callus per explant (mdc/e) and percentage of explants producing shoots (peps) on ms medium supplemented with snps (5 to 80 mg l-1) and combination of bap (2.5 mg l-1) and iaa (0.1 mg l-1) (aslam et al., 2009) along with snps (5 to 80 mg l-1) were evaluated. thidiazuron (tdz) was used at concentrations ranging from 0.001 to 10 mg l-1 for assessment mean number and length of shoots per explant, after two weeks of implementation. the average size of nanoparticles used in this study was 18.5 nm and they were synthesized by nanotechnologies, inc. (nanocid company, tehran, iran) (fig. 1). callus derived from tdz primary treatments was cultured for one week on ms hormone-free medium and then transferred to ms medium supplemented with bap (0.3 to 1.2 mg l-1) in combination with 2,4-d (0.2 and 0.4 mg l-1) for indirect bud formation. the explants were subcultured every two weeks. the ph of the media was adjusted to 5.8 before autoclaving for 15 min at 121°c and 1.5 kg cm-2 pressure. cultures were kept at 25±2°c under cool white fluorescent light (30 µmol m -2 s -1) with 16/8 h photoperiod. the experiment was conducted as a completely randomized design. means were compared using lsd at p≤0.05 with spss software (spss inc., chicago, usa). fig. 1 transmission electron microscopy (tem) micrograph of ag nanoparticles (scale bar of 100 nm). 3. results and discussion completely healthy and disinfected explants were achieved following treatment with 10% clorox for 7 min. explants cultured on ms basal medium supplemented with different concentrations of snps had a higher mnfs/e, mls/e and peps than control (table 1). in other words, snps had positive effects on single node explants of t. undulata, but mdc/e in the control was higher than in other treatments. for this reason, there were not significant differences between 10 mg l-1 snps compared to 80 mg l-1 on mnfs/e and mls/e and, in order to save on cost, the use of 10 mg l-1 snps in tissue culture of t. undulata was proposed. in another experiment (table 1) there were table 1 comparing snps and combination of snps along with bap (2.5 mg l-1) and iaa (0.1 mg l-1) after two weeks on t. undulata (roxb.) single node explants snp (mg l-1) mean number of shoots per explants mean length of shoots per explants mls/e mean diameter of callus per explants percentage of explants producing shoots snp snp+bap+iaa snp snp+bap+iaa snp snp+bap+iaa snp snp+bap+iaa 0 1.06 cd (z) 1.06 cd 2.03 a-c 2.03 a-c 4.27 a 4.27 a 62 ab 62 ab 5 1.00 c 1.24 b-d 1.81 bc 3.01 ab 0.39 c 5.10 a 50 b 67 ab 10 1.65 ab 0.37 e 3.59 ab 1.07 c 0.75 c 2.10 bc 77 a 27 c 20 1.06 cd 1.16 cd 2.33 a-c 2.92 ab 1.19 c 5.02 a 70 ab 70 ab 40 1.82 a 1.41 a-d 3.74 a 1.94 a-c 2.22 bc 5.17 a 80 a 70 ab 80 1.68 a 1.49 a-c 3.02 ab 2.73 a-c 1.27 c 3.81 ab 85 a 70 ab mean 1.37 a 1.12 b 2.75 a 2.28 a 1.68 a 4.24 b 70 a 61 b (z) in each column, means with the same letters are not significantly different at ≤ 0.05 level of probability using lsd. 23 not significant effects between explants grown in medium supplemented with bap (2.5 mg l-1) and iaa (0.1 mg l-1) compared with medium supplemented with different concentrations of snps. when we compared data of snps with snps in combination with plant growth regulators (bap and iaa), there were significant effects between mnfs/e, mdc/e and peps (table 1). our results demonstrate that iranian tecomella undulata (roxb.) seem. had high potential for producing callus from stem segments even in hormone-free medium, but after a couple of days they turned brown and died. even single node explants, after nearly three weeks, had necrotic leaves. the use of common antioxidants such as ascorbic and citric acid or activated charcoal (data not shown), did not have significant effects on survival of single node explants and callus derived from stem segments. leaves of t. undulata produced callus in ms medium but these, when attached to a single node, directly started callus formation. bap at the concentration of 0.9 mg l-1 allowed regeneration of few buds on callus produced from stem segments (data not shown). results indicate that tdz at the concentration of 0.1 mg l-1 increased mns/e and mls/e up to 2 and 3.33 mm respectively from samples taken in autumn (table 2, fig. 2). however tdz at low concentration had a positive effect on callus production, but concentrations of more than 0.01 mg l-1 decreased the dimensions of mdc/e. table 2 regeneration response of t. undulata (roxb.) single node explants on ms medium supplemented with tdz after two weeks snp (mg l-1) number of shoots per explants mean length of shoots per explants (mm) mean diameter of callus per explants (mm) 0.000 0.85 b-d (z) 2.28 a-c 4.23 a-d 0.001 0.90 bc 1.66 a-d 4.80 ab 0.010 1.13 b 2.83 ab 5.33 a 0.100 2.00 a 3.33 a 3.46 a-d 1.000 0.53 b-d 1.66 a-d 4.40 a-c 10.000 0.10 cd 0.40 d 0.16 c-d mean 0.93 1.98 3.63 (z) in each column, means with the same letters are not significantly different at ≤ 0.05 level of probability using lsd. as desert ecosystems currently cover about 35% of the earth’s land surface and also global warming and water deficiency have become worldwide problems, it is urgent to focus on tolerant plant species in these areas (hellen, 1991). utilization of plants for medicinal purposes in iran has been documented in ancient literature. avicenna (abu ali sina) was a persian physician and philosopher whose medical system was for a long time the standard in europe and in the middle east (berman et al., 2009). to protect ecosystems from drought, an option is propagation of tolerant plants such as wild medicinal plants and a rapid and efficient technique for plant propagation is micropropagation (thorpe, 2007). explants used in the present study were collected in october and we believe their low regeneration potential was due to seasonal effects. tdz is one of the most active cytokinin-like substance used successfully for regeneration of recalcitrant woody plants (huetteman and preece, 1993; sarmast et al., 2012). the present study indicates that bap along with iaa did not have a high potential to induce bud regeneration, as previously reported (rathore et al., 1991; robinson et al., 2005). the data presented in this work demonstrated that, at increasing snps concentrations, the dimensions of mdc/e decreased and that buds derived from callus caused somaclonal variation (larkin and scowcroft, 1981; mondal and chand, 2002; sarmast et al., 2012). hence, we conjecture that snps may be helpful in micropropagation to avoid indirect regeneration and the connected somaclonal variability. sarmast et al. (2011) reported that araucaria excelsa explants, grown in ms medium supplemented with snps, were fresher than in ms medium only and that agno 3 in brassica spp. significantly affected bud formation on callus induction medium (akasaka-kennedy et al., 2005) and had positive effects on shoot regeneration in brassica oleracea var. italica (qin et al., 2007). ethylene, the simplest olefin, exists in gaseous state in environmental conditions; it is biologically active in trace amounts and regulates many aspects of the plant life cycle such as senescence (lin et al., 2009; taiz and zeiger, 2006). in tissue culture vessels, plantlet growth and development can be severely influenced by gaseous effects, especially of ethylene at elevated level that may damage explants by suppressing their growth and causing hyperhydration (ziv, 1995). additionally, snps affected ethylene formation and it can be concluded that increasing of durability of explants in culture vessel is due to ethylene blockage. in tissue culture of woody plants, rooting is a severe problem (giri et al., 2004; sarmast et al., 2012), therefore a separate experiment is now in progress to improve rooting of t. undulata with mediating agrobacterium rhizogenes (strain k559). references abdi g.h., salehi h., khosh-khui m., 2008 nano silver: a novel nanomaterial for removal of bacterial contaminants in valerian (valeriana officinalis l.) tisue culture. acta phisial. plant., 30: 709-714. fig. 2 proliferating single node explants of t. undulata in medium supplemented with tdz (0.1 mg l-1). 24 ahmad f., alam khan r., rasheed s., 1994 preliminary screening of methanolic extracts of celastrus paniculatus and tecomella undulata for analgesic and anti-inflammatory activities. j. ethnopharmacology, 42: 193-198. akasaka-kennedy y., toshida h., takahata y., 2005 efficient plant regeneration from leaves of rape seed (brassica napus l.): the influence of agno 3 and genotype. plant cell rep., 24: 649-654. aslam m., singh r., anandhan s., pande v., ahmed z., 2009 development of a transformation protocol for tecomella undulata (smith) seem. from cotyledonary node explants. sci. hort., 121: 119-121. berman p., bianquis t., bosworth c.e., van donzel e., henrichs w.p. brill., 2009 ibn sina (“avicenna”). in: berman p., t. bianquis, c.e. bosworth, e. van donzel, and henrichs w.p. brill (eds.) encyclopedia of islam. 2nd edition. accessed through brill online: www.encislam.brill.nl consolacao m., linardi f., oliveira m.m, sampaio m.r., 1975 a lapachol derivative active against mouse lymphocyte leukemia. j. medicin. chem., 18: 1159-1161. eapen s., george l., 1997 plant regeneration from peduncle segments of oil seed brassica species: influence of silver nitrate and silver thiosulfate. plant cell tiss. org. cult., 51: 229-232. giri c.c., shyamkmar b., anjaneylnu c., 2004 progresses in tissue culture, genetic transformation and application of biotechnology to trees: an overview. trees., 18: 115-135. guiraud p., steiman r., campos-takaki g.m., 1994 comparison of antibacterial and antifungal activities of lapachol and beta-lapachol. planta medica, 60: 373-374. hellen u., 1991 desertification-time for an assessment? ambio, 20: 372-383. huetteman c.a., preece j.e., 1993 thidiazuron: a potent cytokinin for woody plant tissue culture. plant cell tiss. org. cult., 33: 105-119. hussain h., krohn k., ahmad v.u., miana g.a., gree, i.r., 2007 lapachol: an overview. arkivoc., 2: 145-171. karami a., salehi h., 2000 adventitious root formation in rohida (tecomella undulata (am.) seem) cutting. prop. orn. plant., 10: 163-165. larkin p.j., scowcroft w.r., 1981 somaclonal variation a novel source of variability from cell culture for plant improvement. theor. appl. genet., 60: 197-214. lin z., zhong s., grierson d., 2009 recent advances in ethylene research. j. exp. bot., 60: 3311-3336. mondal t.k., chand p.k., 2002 detection of genetic variation among micropropagated tea camellia sinensis (l). by rapd analysis. in vitro cell. dev. biol. plant, 38: 296-299. murashige t., skoog f., 1962 a revised medium for rapid growth and bioassays with tobacco tissue cultures. physiol. plant., 15: 473-497. qin y., li h.l., guo y.d., 2007 highfrequency embryogenesis, regeneration of broccoli (brassica oleracea var. italica) and analysis of genetic stability by rapd. sci. hort., 111: 203-208. rathore t.s., sigh r.p., shekhavat n.s., 1991 clonal propagation of desert teak (tecomella undulata) through tissue culture. plant sci., 79: 217-222. robinson r., bimlendra k., beniwal s.v., 2005 in vitro shoot multiplication of tecomella undulata (sm.) seem: an endangered tree species. indian j. plant physiol., 10: 372-376. sarmast m.k., salehi h., khosh-khui m., 2011 nano silver treatment is effective in reducing bacterial contaminations of araucaria excelsa r. br. var. glauca explants. acta biol. hung., 62(4): 477-484. sarmast m.k., salehi h., ramzani a., abolimoghadam a., niazi a., khosh-khui m., 2012 rapd fingerprint to appraise the genetic fidelity of in vitro propagated araucaria excelsa r. br. var. glauca plantlets. mol. biotechnol., 50(3): 181-188. sarmast m.k., salehi m., salehi h., 2009 the potential of different parts of sansevieria trifasciata l. leaf for meristemoids production. aust. j. basic appl. sci., 3: 2506-2509. taiz l., zeiger e., 2006 plant physiology. sinauer assoc. inc. 4 ed., pp. 700. tewari v.p., singh b., 2009 site index model for tecomella undulata (sm.) seem. (bignoniaceae) plantations in a hot arid region of india. j. arid environ., 73: 590-593. thorpe t.a., 2007 history of plant tissue culture. mol. biotechnol., 37: 169-180. zhang p., phansiri s., kaerlas j.p., 2001 improvement of cassava shoots organogenesis by the use of silver nitrate in vitro. plant cell tiss. org. cult., 67: 47-54. ziv m., 1995 in vitro acclimatization. automation and environmental control in plant tissue culture. aitkenchristie j., t. kozai, and m.a.l. smith (eds.). klumer academic publisher. netherlands, pp. 577. 260 1. introduction salinity is a widespread problem around the world, especially in arid and semi-arid regions. each year more and more land becomes non-productive due to salt accumulation. at least 25% of currently cultivated land throughout the world suffers from excess salinity (bohnert and jensen, 1996) and all major crop species are intolerant to salt (fairbairn et al., 2000). the most economic and sustained way to overcome the problem of salt-stress is to develop salt-tolerant varieties (frommer et al., 1999). in vitro culture has been widely used for the propagation and conservation of crop genetic resources in both agriculture and horticulture crops (barakat and el-lakany, 1992). mutation breeding programs using gamma irradiation on apricot buds were carried out by several investigators (legave and garcia, 1988; ageeva, 1989; gulcan and aksoy, 1995). legave and garcia (1988) reported that bud sticks of five apricot cultivars were exposed to up to 70 gy gamma rays and scored for bud survival and growth. the effect of gamma irradiation in the range 10-70 gy on variation in the characters of apricot was also investigated (ageeva, 1989). the varieties reacted in different ways to treatment. induced mutations in apricot breeding were also investigated (gulcan and aksoy, 1995). apricot (prunus armeniaca) was treated with 0.3 kr gamma irradiation (source 60co). mutagenesis affected vigour, dry matter and vitamin c content, and the level of carotene in fruit. in vitro cultures of japanese plum (prunus salicina) cv. shiro were also gamma-irradiated by predieri and gatti (2000). in vitro culture may offer potential for quick evaluation of germplasm against salt stress (cano et al., 1998). recently, jain (2001) reported that tissue culture generates a wide range of genetic variation in plant species which can be incorporated into plant breeding programs. the effect of nacl and cacl2 in prunus cerasifera was investigated by lucchesini and vitagliano (1993). the in vitro response of peach cv. redhaven and of the peach/almond hybrid rootstock gf677 to increasing concentrations of nacl in the medium was reported (biricolti and pucci, 1995). the response to increasing rates of nacl or cacl2 and proline on ‘mr.s 2/5’ (prunus cerasifera) peach rootstock cultured in vitro has also been reported (dimassi-theriou, 1998). towards in vitro selection studies for salinity tolerance in canino apricot cultivar. effect of gamma irradiation on in vitro mutation and selection for salt-tolerance a.s. el-sabagh*1, m.n. barakat**, e.a-e. genaidy*** * department of horticulture, faculty of agriculture, damanhour branch, university of alexandria, po box 22516, damanhour, egypt. ** department of crop science, faculty of agriculture, alexandria university, egypt. *** pomology department national research center, dokki, geza, egypt. key words: iba, bap, m3 and ms3 medium, mutation gamma ray, propagation, salt tolerance. abstract: in vitro mutation method was used to obtain salt-tolerant clone in apricot. small propagules of canino apricot cultivar were irradiated with gamma ray at doses of 0, 10, 25, 35, 50, 75 and 100 gy. after 30 days from treatment, both the radio sensitivity and post-irradiation recovery were assessed as the number of proliferated shoots per explants, fresh weight of cultures, shoot length and productivity of irradiated explants. a sudden and sharp decrease in the survival percentage occurred with the dose 75 gy, while the highest dose (100 gy) was lethal for all propagules. a marked decline in the number of regenerated shoots per explant and fresh weight of produced cultures was associated with an increase of irradiation doses. doses in the range of 10-75 gy, which preserved high survival percentage of irradiated explants, seemed to be more suitable for in vitro mutation in canino apricot cultivar. irradiated shoots were exposed to different concentrations of nacl which were added to the multiplication medium at the rates of 25, 50, 75, 100, 125 mm and after 30 days, vigorous shoots were selected from salinity treatments. in conclusion, apricot tissues exposed to different doses of gamma irradiation in the range of 10-75 gy, followed by culturing the plantlets produced in a medium containing additional salts (ranging from 25 to 125 mm) can be considered a good method to identify the most tolerant mutants to salts in apricot cultivars. adv. hort. sci., 2011 25(4): 260-263 1 corresponding author: ahmed_elsabagh67@yahoo.com received for publication 12 january 2011 accepted for publication 12 october 2011 261 recently, increased sodium chloride (nacl) salinity effects on bitter almond (amygdalus communis) (prunus dulcis) growth, cell osmolarity and nutrient acquisition were studied in vitro (shibli et al., 2003) and it was found that elevating salinity from 0.0 (control) to 50, 75, 100 mm nacl resulted in reductions in shoot growth (shoot height, shoot dry weight) and rooting (rooting percentage, root number, root length). the objective of the present work was to obtain salttolerant clone (s) in apricot using an in vitro mutation method. 2. materials and methods the present work was carried out in the biotechnology laboratory, crop science department, faculty of agriculture, alexandria university from 2001 to 2005. small propagules of canino apricot cultivar initiated from in vitro culture by the follwing protocol: two explants shoot tips and single node cutting; of 0.5-1 cm in length was used. shoot tips and single node were soaked in 100 mg/l ascorbic acid +150 mg/l citric acid for 30 min. explants were dried for 30 min. before they were immersed in fungicide ridomil (1 g/l solution) for 30 min. and then washed with distilled water, then soaked in clorox (7% sodium hypochlorite) for 7 min and washed with sterile distilled water three times. the explants were aseptically excised and placed in jar containing 50-60 ml of culture medium. each jar contained one explant, considered as one replication. cultures were incubated at 25±2oc under 16 hour’s illumination (2000 lux, day light fluorescent tubes). in order to check the phenol oxidation and to establish the explants with free from phenol, the explants were cultured on two medium (ms and m3,) supplemented with four pvp concentrations (0.0, 40.0, 80.0, 160.0 mg/l). the effect of five medium protocols was examined: modified woody plant medium (m3) (perez-tornero et al., 2000), ms medium (ms1) and ms medium (murashige and skoog, 1962) modified by reducing kno3 and nh4no3 by 25%, 50% and 75% which were designated ms2 , ms3 and ms4, respectively. all media were supplemented with 3.0% sucrose, 4 mg/l adenine sulfate, 160 mg/l pvp, 0.4 mg/l bap and 0.01 mg/l iba. the ph of the media was adjusted to 5.7 by using 1.0 n hcl or 1.0 n naoh and agar was added after adjusting the ph. the best two media (m3 and ms4) for proliferation were used to test the optimum effect of three types of cytokinins benzyladenine (bap), kiniten and isopentenyladenine (2ip) and their concentration on shoot tip proliferation, four different concentration of cytokinin 0.2, 0.4, 0.6 and 0.8 mg/l for each type using m3. also, four different concentration of cytokinin 0.5, 1.0, 2.0 and 4.0 mg/l for each type using ms4. the proliferation was evaluated six weeks after the beginning of the experiment and the number of shoots, (longer than 5 mm) per explant, their length and productivity (number of shoots x the average shoot length) were recorded. shoots of apricot derived from the shoot tips multiplication were cultured on m3 medium perez-tornero et al. (2000). the medium was supplement with either naa (0.0, 0.5, 1.0, 2.0 mg/l) or iba (0.0, 2.0, 4.0, 6.0 mg/l) were employed. in vitro mutation and selection for salt-tolerance effect of gamma irradiation on in vitro shoot culture. small propagules of canino cultivar initiated from in vitro culture (fig. 1) were irradiated in a gamma cell with a cobalt60 source at the middle-east regional radioisotopes centre for arab countries, el-dokki, giza with 10, 25, 35, 50, 75 and 100 gy doses. the irradiated propagules were removed from the jar and recultured on a fresh proliferation medium. after six weeks incubation, impact of the irradiation was assessed by determinating the number of shoots, the fresh weight of shoot multiplication, shoot length and productivity. 0.2 mg bap 0.4 mg bap 0.8 mg bap 0.6 mg bap fig. 1 in vitro micro propagation of apricot cv. canino on m3 medium derived from shoot tip explants. in vitro selection. small propagules of the cultivar (canino) (fig. 1) were irradiated in a gamma cell with cobalt 60 source gy at a dose of 10, 25, 35, 50 and 75 gy. the propagules were subcultured five times before in vitro selection for salt-tolerance. individual shoots from irradiated cultures were grown on the proliferation 0.6 mg/l bap m3 medium supplemented with different concentrations of nacl (25, 50, 75, 100 and 125 mm). after six weeks of incubation, the vigorous shoots were selected and transferred to fresh medium free from salt. analysis of variance with sas software (sas institute, 1988) was carried out. treatment means were compared using the lsd test at 5% level probability. data were analyzed as a factorial arrangement in randomized complete block design according to steel and torrie (1980). 3. results and discussion effect of gamma irradiation on in vitro apricot culture the basic requirement for effective use of mutation induction in plant breeding programs is the analysis of radio sensitivity of the explant material (walther and sauer, 262 1986). predieri (2001) reported that one of the first steps in mutagenic treatment is the estimation of the most appropriate dose to apply. the aim of the present work was to determine the radio-sensitivity of in vitro apricot culture, as assessed by the number of regenerated shoots, the fresh weight of shoot multiplication, the shoot length and productivity in order to select the suitable dose of gamma irradiation to conduct in vitro mutation for improvement. the collected data, reported in table 1, indicate that a clear decrease in in vitro traits occurred with increasing irradiation dose. complete lethality (100% death) was observed with an irradiation dose higher than 75 gy (fig. 2). several other studies have been conducted on the radio-sensitivity of in vitro cultures of fruits, such as prunus avium (walther and sauer, 1985), kiwifruits (shen et al., 1990), grapevine (lima da silva and doazan, 1995; charbaji and nabulsi, 1999) and prunus salicina (predieri and gatti, 2000). previously, laneri et al. (1990), working with gerbera jamesonii, stated that in a mutation breeding experiment, the dose chosen for the main experiment should result in the highest survival of irradiated explants and that a low inhibition of the rate of production of new shoots gives the highest efficiency in recovering useful mutants. in light of these studies, the results obtained in the present investigation suggest that doses of 10 gy to 75 gy seem to be the most suitable for inducing mutation for apricot improvement. in vitro mutation and selection for salt-tolerance in canino apricot cultivar mutation breeding can be employed as a promising technique that allows diversification of apricot. induced mutations change only one or a few specific traits of an elite cultivar without undesired additional variations (predieri, 2001). in fact predieri concluded that the most suitable method may be mutation treatment and propagation of in vitro axillary shoots without passage through undifferentiated growth, and it can contribute to fruit improvements without upsetting the requirements of the fruit industry nor the consumers. through in vitro selection, mutation with a useful agronomic trait, e.g. salt or drought tolerance or disease resistance, can be isolated in a short time (jain, 2001). the present work was conducted to obtain salt-tolerant clone(s) in apricot cv. canino using in vitro shoot mutation. small propagules were irradiated with 0, 10, 25, 35, 50, or 75 gy and explants were multiplied for five subcultures. the generated irradiated shoots were subjected to a salt (nacl) which was added to the medium with the concentrations 25, 50, 75, 100, or 125 mm. the number of vigorous shoots of cv. canino showed marked differences in their in vitro salinity tolerance (table 2). it is clear that the number of vigorous shoots decreased rapidly with increasing salinity. the highest number of vigorous shoots was obtained in medium supplemented with 25 mm selective agent of salinity when the propagules were exposed to 25 and 50 gy, respectively (table 2). from these results, it can be concluded that apricot cv. canino tissues exposed to different doses of gamma irradiation in the range 10-75 gy, followed by culturing in medium containing a higher concentration of additional salts (ranging from 25 to 100 mm) can be considered a good method to identify mutants in apricot cv. canino which are the most tolerant to salts. fao/iaea (1997) reported that plant biotechnology in combination with mutation induction and conventional breeding might open new frontiers for obtaining salt-tolerance rice varieties. the application of mutation techniques in breeding has increased constantly over the past years. these techniques must be rapid to keep pace with the large quantity of breeding materials generated after mutagenesis. screening under field conditions is difficult due table 1 effect of gamma irradiation six weeks after the treatment on in vitro apricot shoot gamma irradiation (gy) doses weight number of shoots shoot length productivity control 5.85 a 23.40 a 6.15 a 147.36 a 10 3.58 b 14.30 b 2.99 b 42.27 b 25 3.45 b 13.80 b 3.14 b 42.52 b 35 2.40 bc 9.60 bc 3.63 b 41.68 b 50 1.50 cd 6.00 cd 0.77 c 11.05 c 75 0.68 d 2.70 d 0.48 c 4.18 c means within a column or a row followed by the same letter(s) are not significantly different at the 0.05 level of probability. table 2 effect of irradiation doses on the number of vigorous shoots of apricot cv. canino, after six weeks from culturing in media containing different salt concentrations salt concentration mm irradiation dose (gy) 0 10 25 50 75 total 25 0 2 5 4 3 14 50 0 2 3 1 2 8 75 0 0 0 2 2 4 100 0 0 0 1 2 3 125 0 0 0 0 0 0 total 0 4 8 8 9 29 fig. 2 effect of gamma irradiation 100 gy, six weeks after the treatment on in vitro apricot shoot. 263 to stress heterogeneity, presence of salt-related stress and the significant influence of environmental factors such as temperature, relative humidity and solar radiation. genetic modification of crop plants to improve their salt-tolerance is a possible way of increasing production, especially for regions of the world where arable lands must be extended to marginal areas, and sometimes irrigated with saline water (dorion et al., 1999). references ageeva n.g., 1989 effect of gamma irradiation on variation in the characters of apricot. bulletin’ gosudarstvennogo nikitskogo botanicheskogo, 68: 67-70. barakat m.n., el-lakany m.h., 1992 clonal propagation of acacia saligna by shoot tip culture. euphytica, 59: 103-107. biricolti s., pucci s., 1995 effects of increasing nacl rates on ‘redhaven’ peach and ‘gf677’ rootstock cultured in vitro. adv. hort. sci., 9(2): 75-78. bohnert h.j., jensen r.g., 1996 metabolic engineering for increased salt tolerance. the next step. australian journal of plant physiology, 23: 661-667. cano e.a., perez-alfocea f., moreno v., caro m., bolarin m.c., 1998 evaluation of salt tolerance in cultivated and wild tomato species through 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growth and bioassays with tobacco cultures. physiol. plant., 15: 473-497. perez-tornero o., lopez j.m., egea j., burgos l., 2000 effect of basal media and growth regulators on the in vitro propagation of apricot (prunus armenica l.) cv. canino. journal of horticultural science & biotechnology, 75(3): 283-286. predieri s., 2001 mutation induction and tissue culture in improving fruits. plant cell, tissue and organ culture, 64: 185-210. predieri s., gatti e., 2000 effects of gamma radiation on plum (prunus salicina lindl.) ‘shiro’. adv. hort. sci., 14: 215-223. predieri s., gatti e., 2000 effects of gamma radiation on micro cuttings of plum (prunus salicina lindl.) ‘shiro’. istituto di ecofisiologia delle piante arboree da frutto, cnr, 14(1): 7-11. shen x.s., wan j.z., luo w.y., ding x.l., 1990 preliminary results of using in vitro axillary and adventitious buds in mutation breeding of chinese gooseberry. euphytica, 49: 77-82. shibli r.a., shatnawi m.a., swaidat i.q., 2003 growth, osmotic adjustment, and nutrient acquisition of bitter almond under induced sodium chloride salinity in vitro. communications in soil science and plant analysis, 34(13/14): 1969-1979. steel r.g.d. torrie j.h., 1980 principles and procedures of statistics: a biomaterical approach. mcgrow hill book co. inc., new york, usa, pp. 196-201. walther f., sauer a., 1985 analysis of radio-sensitivity a basic requirement for in vitro somatic mutagenesis. i. prunus avium l. acta horticulture, 169: 97-104. walther f., sauer a., 1986 analysis of radio-sensitivity: a basic requirement for in vitro somatic mutagenesis. ii. gerbera jamesonii. proc. int. symp. nuclear techniques and in vitro culture for plant improvement. iaea/fao, vienna, pp. 155-159. 187 1. introduction an increase in fruit quality is a key objective of fruit tree cultivation, while keeping in mind at the same time the relevance of production cost reduction and environmental issues. the citrus industry is increasingly oriented toward upgrading groves, discarding obsolete plantings and introducing new rootstock/scion combinations which are more tailored to local environmental conditions. moreover, increases in production costs, without a proportionate profit increase, and innovation at the technological level have led to many changes in citrus orchard management and the updating of cultural techniques. new plantings have been realized with regular planting distances for a fully mechanised approach to all cultural practices. in this context, technical evolution also includes pruning, essential for healthy and fruitful orchard management, but without the clear push towards mechanisation as in other countries. this cultural technique as well as all other practices, even though respecting plant physiology, has to be evaluated according to the economic impact. furthermore, it is wise to consider the manifold factors that affect the final result, such as rootstock/scion combination, tree age and development, planting distance, soil and climate conditions. citrus groves in their former conception, although still present in many citrus cultivation areas in italy, were highdensity based (more than 800 plants ha-1) with narrow planting distances. citrus growers were forced to carry out frequent pruning on bearing plants, repeated in spring and at the end of summer, as the only available way for both high plant density and light penetration between and within plant canopies to coexist (rebour, 1971). this situation led to the development of trees with high scaffold (frequently more than 1 m high), usually lacking in skirt and with poor yield. the transition to modern citrus production, based on greater planting distance and average densities of 416 plants ha-1, at least for standard grafting combinations, has led to a new concept of pruning, which in turn has meant substantial changes in the management of this technique, with sometimes substantial negative effects on citrus production (intrigliolo, 1998). similarly to other fruit tree cultivations (giacalone et al., 2004; neri and sansavini, 2004; peano and giacalone, 2004; ventura and sansavini, 2005), pruning practices in citriculture are important to support plant health and reduce stress in order to reach an acceptable balance between vegetative and reproductive activities, a key factor in many stages of citrus grove development. citrus bearing trees in semi arid environments have main shoot growth flushes during the year (spring, summer and autumn flush), with growth stasis periods overlapped with periods of higher and lower temperatures. only for lemon (c. limon (l.) bern) does flowering occur during all growth flushes, whereas for other citrus species flowering is mainly bound to spring flush. flowers, solitary or in inflorescence, can be terminal or axillary and are normally produced on one-year modern trends of citrus pruning in italy f. intrigliolo, g. roccuzzo* centro di ricerca per l’agrumicoltura e colture mediterranee, cra-acm, corso savoia, 190, 95024 acireale (ct), italy. key words: fruit quality, mechanical pruning, rootstock, tree habit. abstract: the citrus industry is at present increasingly oriented toward upgrading groves, discarding obsolete plantings and introducing new rootstock/scion combinations which are more tailored to local environmental conditions. a rise in production costs (but without a proportionate increase in profit) together with technological innovation have both led to many changes in citrus orchard management, and consequently there have been changes in cultural techniques. many cultural and edaphic factors influence the choice of pruning frequency and intensity. the main goal of pruning is to reach a useful balance between yield and growth, and to reduce cultivation costs. drastic pruning in young citrus trees with a vigorous vegetative habit causes an excess of shoot growth, thereby extending the juvenile stage and delaying fruiting. adaptation of the technique to local conditions in bearing groves (species, cultivars, planting distances, etc.) is mandatory for pruning optimization. a fully mechanised approach to pruning leads to substantial changes in citrus grove management and sometimes to negative effects on yield. functional integration of mechanical and assisted pruning seems to be the right choice for the italian citrus industry. adv. hort. sci., 2011 25(3): 187-192 * present address: fruit tree research centre (cra-fru), via di fioranello, 52, 00134 rome, italy. received for publication 20 may 2011 accepted for publication 9 september 2011 188 shoots. in young citrus trees shoots are normally vegetative, since the productive stage begins with flower emission mainly on lateral drooping shoots. in citrus species, like in other tree fruit species, bud differentiation occurs in response to chilling temperatures and mainly depends on interactions at physiological and nutritional level (garcia-luis et al., 1995). drastic pruning in young citrus trees with a vigorous vegetative habit causes an excess of shoot growth, thereby extending the juvenile stage and delaying the beginning of fruiting. in adult trees heavy pruning of branches, twigs and leaves means the removal of reserve substances (i.e. carbohydrates, nitrogen), thus leading to serious reductions in plant growth and overall development. furthermore, in this way pruning in adult trees favors excessive sucker production especially in upright-growing species. on the contrary, in senescent or declined trees light or even hard thinning may be useful to promote growth and healthy fruitwood. 2. pruning of young trees it is essential to take care of citrus trees during the juvenile stage to obtain a balanced scaffold with three to four principal branches developing at 30-50 cm from the collar (fig. 1). if citrus trees are correctly managed in the nursery (cut back or headed) they require little pruning once in the field. trees will grow naturally and they will take the growth habit typical of the species or cultivar. in this case trees assume a drooping shape, ranging from spheroid to ellipsoid (fig. 2). at this stage the most common and severe mistakes are the removal of apical or more drooping branches. in such cases the development of both upper and lower part (the first to produce) of the tree are limited. however, pruning during the first period should be limited to removing an occasionally unwanted branch or buds on the rootstock, to regulate the final scaffold and reduce future severe cuts. 3. pruning mature trees for many years after transplanting citrus trees require no relevant pruning. it is not easy to determine a general rule for the beginning and frequency of regular pruning since this practice depends on many factors: species, cultivar, planting distance, soil and climate conditions and, more relevant, growth status (crowding, presence of deadwood in the internal part, upright shoots exhaustion) and its balance with fruitfulness. pruning frequency can be annual or long-standing, with frequency and severity closely linked. longer time intervals imply more drastic pruning, with wider wounds and a subsequent massive influence on plant growth. in a field trial on full-bearing trees of tarocco orange (citrus sinensis osbeck) the effects of hand pruning with annual, biennial and quadrennial frequency were evaluated (calabretta et al., 2008). quadrennial frequency of pruning showed a decline of fruit quality parameters (above all for average fruit weight), even though they were linked to higher yield and shortening of work time. biennial frequency of pruning showed the best balance fig. 1 young ‘tarocco’ orange tree. fig. 2 ‘navelate’ orange tree with drooping canopy and full skirt. 189 as far as work time (costs), yield and fruit quality were concerned. in the case of aged trees, low in vigor or presenting other problems, it is wise to increase the frequency (intrigliolo, 1984; intrigliolo, 1998). for wider planting distances progressive exhaustion of the internal part of the canopy can be balanced by lateral and vertical expansion, without any influence on yield. therefore in these conditions pruning initially plays a minor role and it could be delayed. on the contrary if pruning is carried out at maturity stage of the orchard, in conjunction with the right fulfillment of all other cultural practices, it becomes necessary to sustain growth vigor, high yield and fruit quality standards. it is not easy to choose the right time to start regular pruning. in the case of a delayed start, the citrus orchard could grow old prematurely, while the opposite case could cause an increase in costs and severe yield reduction (intrigliolo, 1998). another important factor is the timing of pruning. intervention is often undertaken without distinction from january to june, and sometimes is repeated at the end of august or beginning of september to eliminate vigorous upright suckers, due to severe spring cuts. in any case, the right pruning time is extremely variable, depending on species, variety, climate conditions and severity of the previous treatment. early varieties are usually pruned before late ones, either because of earlier harvest times or because of an absence of frost risk. frosts are the main deterrent to early pruning (fisher, 1977; phillips, 1980 b; cutuli, 1985; intrigliolo, 1986 b). as a matter of fact, the removal of the outer part of foliage makes trees more subject to frost injuries since it stimulates the emission of new soft shoots that can be easily damaged by low temperatures. severe treatments should be properly scheduled and deferred until after the juvenile stage: moderation is still the key word. light pruning is advisable to provide deadwood removal and to increase light interception in the internal part of the canopy and between rows. this improves yield and fruit quality, especially fruit size, thus making other cultural techniques easier and less expensive (intrigliolo, 1984; calabretta et al., 2008). studies on pruning timing showed the positive effects of early treatments (end of winter to early spring) in comparison with late summer ones (cameron and hogdson, 1943; milella, 1967; turpin, 1973; fisher, 1977; fucik, 1979). if pruning takes place before spring flush, removal of new vegetation is prevented (turpin, 1973). early pruning is thus highly recommended for skeletonisation and as a general rule for trees low in vigor or weakened by biotic or environmental stresses (cameron and hogdson, 1943; bevington and bacon, 1976; phillips, 1980 a, 1980 b). vigorous trees, on the other hand, will react improperly with extra shooting and will lose fruit bearing surface, as reported by bevington and bacon (1976). phillips (1980 a) reported that light pruning in july and august implied useful fruit thinning, with the consequence of increased fruit size, especially in case of top dressing. as far as yield is concerned, fucick (1979) reported higher levels in texas with grapefruit (c. paradisi macfadyen) resulting from december treatment, whereas in australia bevington and bacon (1976) and bevington (1980), working with valencia late oranges, found similar levels comparing summer and autumn treatments. tree phenological stage as well as scion/rootstock combination and species/variety habit are decisive factors in determining pruning effects. ‘moro’ in the bloody group and many accessions in the navel group represent low vigor orange cultivars; low vigor trees are found among a few lemon cultivars, most bergamot (c. bergamia rissi) and citron (c. medica l.) (fig. 3) cultivars. given the vegetative habit of these genotypes, pruning has the main function of increasing air and light penetration in the internal part of the canopy, avoiding backcuts that would thicken the tree and preferring a balanced thinning. in the case of mandarin (c. reticolata blanco) (fig. 4) and its hybrids these treatments are essential, the canopy being extremely dense due to huge branch production. fig. 3 ‘navelina’ orange tree. fig. 4 ‘avana’ mandarin tree. 190 satsumas (c. unshiu marcovitch) and many clementines (c. clementine hort.) (fig. 5) show an intermediate growth habit. expanded growth habit is normally shown by bloody orange ‘sanguinello’ and ‘tarocco’ clones, with a tendency to upright growth habit in nucellar lines (fig. 6) and in triploids, such as ‘tacle’ and ‘alcantara’. with the aim of obtaining virus-free and viruslikefree accessions, nucellar progeny strategy has been largely adopted in breeding projects in italy. these lines are characterized by their large size (although with some exceptions) thus conditioning plant spacing and orchard management. in this situation concerns arise about the extremely reduced density and about pruning practices, that should be reduced in number and intensity. branches shortening cuts should be preferred mainly in the first years after planting, even though this practice induces very vigorous growth reactions and delay of productive stage. these effects are linked to the increase of costs for future pruning, harvest and other cultural practices. in order to increase yield and reduce the costs of cultural practices (especially pruning), recovery strategies based on micrografting technique should be encouraged, as well as the adoption of new rootstocks able to reduce tree vigor (russo et al., 2011). ‘femmiminello’ lemon trees are characterised by upright irregular shoots (fig. 7), with pronounced apex dominance; young trees show long, thin shoots which are weak and easily prone to breakage. for these trees it is necessary to shorten the branches to reduce plant height, favoring their strength and thus their stability. in late summer, pruning treatments play a prominent role in the eradication of phoma tracheiphila (kanc et ghik) infections, making clear the necessity for tree removal in extreme situations. more extensive pruning (i.e. skeletonisation) is essential in cases of old, decadent trees or in case of damage due to environmental or biotic stresses. in these situations removal of deadwood is useful or absolutely necessary for tree rejuvenate, thus leading to normal growth and production conditions. 4. mechanical pruning in citrus orchard management, pruning is increasingly oriented toward greater levels of mechanization with the aim of combining cost reduction with a proper balance between yield and plant growth. research activity in this field started in italy at the end of 1970s with several experiences of mechanical pruning (giuffrida et al., 1979; blandini et al., 1981; raciti et al., 1981; intrigliolo et al., 1986) with the integration of internal thinning of deadwood and upright shoot removal, and aided pruning by means of pneumatic saws and clippers (intrigliolo and barbagallo, 1987; schillaci, 1988). fig. 5 clementine tree. fig. 6 ‘tarocco’ clone nl 57-e-1 orange tree. fig. 7 ‘femminello’ lemon tree. 191 experiments carried out in italy on mechanic pruning in citrus (giuffrida et al., 1979; blandini et al., 1981; raciti et al., 1981; giametta, 1983; spina et al., 1984; intrigliolo, 1986 a; intrigliolo et al., 1986; intrigliolo et al., 1988; intrigliolo and giuffrida, 1990; raciti et al., 1991) gave largely positive results. trials were carried out on several species and cultivars under different environmental and cultivation conditions, utilizing different equipment and operating systems mainly associated with a traction engine. up till now, however, hand pruning seems to be the most widespread approach in italy and in other advanced citrus cultivation areas like spain (agustì, 2003). frequency and intensity of mechanical pruning represent the key choices to attain high yields and delayed tree senescence (zaragoza-adriaensens and alonso cabo, 1981; intrigliolo, 1986 b; raciti and intrigliolo, 1989). results of a two-year trial on ‘tarocco’ orange trees mechanically pruned with the same intensity in april, june and august, showed that full summer treatments were useful to control plant growth, whereas early treatments stimulated the spring flush (intrigliolo and giuffrida, 1990). yield and fruit quality were only slightly influenced by treatment time. the main purposes of fully mechanized pruning are the fulfillment of the tree’s physiologic demand and the massive reduction of production costs. mechanical pruning is not a selective or thinning practice, but it follows rigid patterns by cutting trees back vertically (hedging) (fig. 8) or removing their tops (topping) (fig. 9) and it is adapted to wide planting distances. thus, the grove is sufficiently open for the passage of equipment for spraying and other cultural practices, reducing shady areas and removing dead or decadent wood (intrigliolo, 1986 b). pneumatic tools reduce the physical effort of workers, amplifying their performance both by replacing hand pruning with traditional tools and complementing mechanical pruning. the economic convenience of pneumatic tool utilization increases as the time needed for the intervention increases. assisted pruning loses its economic convenience in comparison to traditional pruning (intrigliolo and barbagallo, 1987). the reduction of working time accounts for up to 30-40% for assisted pruning, 90% for mechanical pruning and an average of 60-70% when integrated with the latter (fig. 10) (intrigliolo, 1986 a, 1998). the functional integration of mechanical and assisted pruning could be the best way to achieve useful results, hopefully in economic and agronomic terms, at least until further profit loss forces growers toward full mechanizaten. even though experimental results with fully mechanized pruning have to date shown limited effects, it seems that in the near future it will spread to largeand medium-sized citrus orchards. in the traditional italian citrus industry, with terrace cultivation and small-sized farms, for many years pruning has been carried out using pneumatic tools permitting workers to use their own judgment in terms of frequency and limiting costs.fig. 8 mechanical hedging and topping done at the same time. fig. 9 mechanical topping. fig. 10 work time in different kinds of pruning. 192 references agustì m., 2003 citricultura. mundi-prensa, madrid, spain, pp. 422. bevington k.b., 1980 response of valencia orange trees in australia to hedging and topping. proc. fla. state hort. soc., 93: 65-66. bevington k.b., bacon p.e., 1976 response of valencia orange trees to hedging. australian citrus news, 52: 2. blandini g., petrone f., sisinna s., 1981 trials of mechanical pruning machines in italian citriculture. proc. int. soc. citric., vol. i: 180-185. calabretta m.l., giuffrida a., intrigliolo f., 2008 la periodicità della potatura, fattore determinante per la qualità delle arance. rivista di frutticoltura e di ortofrutticoltura, 70(1): 2-5. cameron s.h., hodgson r.w., 1943 effect of time of pruning on the rate of top regeneration of valencia orange trees. proc. am. soc. hort. sci., 42: 280-282. cutuli g., 1985 potatura, pp. 423-451. in: spina p. (ed.) trattato di agrumicoltura. edagricole, bologna, italy, pp. 552. fisher i.e., 1977 hedging and topping. the citrus industry, 57: 6-12. fucik i.e., 1979 long term responses of texas grapefruit trees to hedging and topping. the citrus industry, 59: 2527. garcia-luis a., fornes f., guardiola j.l., 1995 leaf carbohydrates and flower formation in citrus. j. amer. soc. hort. sci., 120(2): 222-227. giacalone g., la iacona t., vittone g., 2004 valutazione di parametri produttivi e qualitativi in impianti di melo condotti con differenti metodi di potatura. proc. vii giornate scientifiche soi, pp. 763-765. giametta g., 1983 prove sperimentali di potatura meccanica degli agrumi. macchine e motori agricoli, 7: 7-20. giuffrida a., melita e., romeo m., 1979 prove di potatura meccanica degli agrumi. rivista di frutticoltura, 41: 23-28. intrigliolo f., 1984 la potatura degli agrumi. l’informatore agrario, 40(22): 49-53. intrigliolo f., 1986 a la potatura meccanica e agevolata in agrumicoltura. l’informatore agrario, 42(9): 93-105. intrigliolo f., 1986 b potatura meccanica e tradizionale a seguito di danni da gelo su arancio cv. ‘sanguinello moscato’. proc. ‘il recente contributo della ricerca allo sviluppo dell’agrumicoltura italiana’, cagliari 29 april-3 may, pp. 267-277. intrigliolo f., 1998 differenti frequenze di potatura meccanica su piante di arancio. atti giornate scientifiche soi, 1: 157-158. intrigliolo f., barbagallo a., 1987 confronto in diverse condizioni di campo fra potatura tradizionale ed agevolata in agrumicoltura. l’informatore agrario, 43(25): 69-72. intrigliolo f., giuffrida a., 1990 interventi di potatura meccanica sull’arancio cv. ‘tarocco’ effettuati in diverse epoche. frutticoltura, 52(5): 91-95. intrigliolo f., raciti g., scuderi a., 1988 mechanical and aided pruning combined with tree removal of nucellar ‘tarocco’ orange. proc. sixth int. citrus congress, vol. ii: 947-952. intrigliolo f., raciti g., scuderi a., giuffrida a., 1986 prove quadriennali di potatura meccanica sul mandarino cv. ‘avana’. proc. ‘il recente contributo della ricerca allo sviluppo dell’agrumicoltura italiana’, cagliari 29 april-3 may, pp. 297-307. milella a., 1967 un quadriennio di osservazioni sull’epoca di potatura dell’arancio. studi sassaresi, sez. iii, ann. fac. agraria, 1: 1-15. milella a., 1980 potatura. in: gli agrumi, reda, pp. 165. neri d., sansavini s., 2004 attualità della potatura nella frutticoltura intensiva. rivista di frutticoltura, 1: 14-23. peano c., giacalone g., 2004 potatura del melo: dalla taille longue all’extinction. proc. vii giornate scientifiche soi, pp. 196-198. phillips r.l., 1980 a hedging and topping practices for florida citrus. the citrus industry, 55: 5-10. phillips r.l., 1980 b rejuvenation pruning of citrus. citrus and vegetable magazine, 44: 6-22. raciti g., cutuli g., intrigliolo f., giuffrida a., 1991 indagine sull’influenza delle tecniche colturali sul mal secco degli agrumi. 2a nota: conduzione del terreno e potatura. l’informatore agrario, 47(22): 47-49. raciti g., intrigliolo f., 1989 nuovi orientamenti sulla potatura degli agrumi. rivista di frutticoltura, 51(1): 2530. raciti g., spina p., scuderi a., intrigliolo f., 1981 three years of mechanical pruning of citrus in italy. proc. int. soc. citriculture, vol i: 175-180. rebour h., 1971 gli agrumi. edagricole, bologna, italy, pp. 371. russo g., reforgiato recupero g, recupero s., intrigliolo f., 2011 la scelta dei nuovi portinnesti i risultati delle ricerche. rivista di frutticoltura, 73(1-2): 6264. schillaci g., 1988 la potatura agevolata in agrumicoltura. macchine e motori agricoli, 4: 55-64. spina p., giuffrida a., melita e., 1984 comparative trials of mechanical and aided pruning. proc. int. soc. citriculture, vol. i: 106-109. turpin i.w., 1973 the place of hedging in the management of citrus orchards. australian citrus news, 49: 6-7. ventura m., sansavini s., 2005 tipologia di rami a frutto e qualità delle mele cvs braeburn, golden delicious e jonagold. rivista di frutticoltura, 67(11): 52-60. zaragoza-adriaensens s., alonso-cabo e., 1981 la frecuencia de la poda manual de los agrios. primeros resultados. anales del instituto nacional de investigaciones agrarias. serie: agrìcola, nùm. 15, separata nùm. 11. impaginato 99 adv. hort. sci., 2011 25(2): 99-105 received for publication 6 april 2011. accepted for publication 13 may 2011. growth reduction in root-restricted tomato plants is linked to photosynthetic impairment and starch accumulation in the leaves s. mugnai*, h.s. al-debei** * dipartimento di scienze delle produzioni vegetali, del suolo e dell’ambiente agroforestale, sezione di coltivazioni arboree, università degli studi di firenze, viale delle idee, 30, 50019 sesto fiorentino (fi), italy. ** department of horticulture and crop science, faculty of agriculture, university of jordan, 11942 amman, jordan. key words: chlorophyll, leaf gas exchange, solanum lycopersicum l., sugar content, total water potential. abstract: the mechanisms responsible for reduced shoot growth due to restricted root growth is still not fully understood. therefore, this investigation was planned to determine the morphological and physiological changes induced in response to root restriction conditions and to determine the time frame within which these changes occurred. in particular, this research aims to evaluate the effect of root restriction on growth, leaf gas exchange parameters, carbohydrate production and water relations in tomato (solanum lycopersicum l.). our results show that growth reduction by root restriction is mainly linked to a photosynthetic impairment, caused by a concurrent limited stomatal conductance (probably driven by stomatal factors and hormonal substances) together with a strong accumulation of starch in the tissues, which led to a feedback inhibition of the photosynthetic process. 1. introduction the use of root-restricted cultivation for vegetable production has significantly grown in the last decades (shi et al., 2008), as it appears an effective technique for saving resources, controlling root environment, and regulating early yield and quality (marsh and paul, 1988; shi et al., 2008). root restriction may occur wherever pot size or rooting volume is physically limited (tschaplinski and blake, 1985; ismail and noor, 1996; saito et al., 2008; mugnai et al., 2009), mostly with greenhouse-grown horticultural crops (thomas, 1993). root restriction leads to a denser root mass and a reduced root growth (ismail and noor, 1996). besides limiting the volume of the soil available to the root system for water and nutrient uptake, it also suppresses canopy growth (ismail and noor, 1996; shi et al., 2008) via many plant physiological and biochemical processes. the mechanisms responsible for reduced shoot growth due to restricted root growth is still not fully understood. several hypotheses were investigated including water and nutrient stresses (hameed et al., 1987), decrease in root respiration (shi et al., 2007) and photosynthesis (shi et al., 2008), and production of plant hormones (liu and latimer, 1995), but reports indicated that there are contradictory results as to which of these factors play a significant role in the response of aerial plant parts to restricted root growth and indicated differences between species. leaf photosynthesis strongly depends on environmental conditions such as radiation, co2 concentration and temper-ature. in addition to these environmental conditions, photosynthesis is subjected to internal regulation associated with sink demand for assimilates (marcelis, 1991). the presence of a physical restriction to root growth, a major metabolic sink for photsynthetically fixed carbon at seedling stage (thomas and strain, 1991) resulted in feedback inhibition mechanisms, with lower rates of carbon metabolism and photosynthesis as a result of carbohydrate accumulation (schaffer et al., 1996; shi et al., 2008). therefore, this investigation was planned to determine the morphological and physiological changes induced in response to root restriction conditions and to determine the time frame within which these changes occurred. in particular, this research aims to study the effect of root restriction on growth, leaf gas exchange parameters, carbohydrate production and water relations in tomato (solanum lycopersicum l.). 100 2. materials and methods plant material experiments were carried out at the department of plant biology, university of pisa (italy). seeds of tomato (solanum lycopersicum l.) cv. ‘cal j’ were sown in seedling flats filled with vermiculite and placed in a germinating room at constant temperature (25°c) and light intensity (300 mol m-2 s-1 of par). after germination, seedlings with the first true leaves were selected for uniformity and single plants were transplanted into 7 ml (root restricted, rr) and 230 ml (control) speeding flats filled with vermiculite. flats were placed in a greenhouse and suspended 15 cm above the benches to facilitate air pruning of roots and to induce root restriction treatment through out the experiment period. in each flat 24 seedlings were planted regardless of the original number of cells per flat to minimize the effect of mutual shading, to avoid light competition between plants and to allow for uniform plant density. in order to avoid any water or nutrient stress, a closed fertirrigation system controlled by a timer was established to supply water and nutrients at frequent and regular intervals. the nutrient solution was composed thus: 10 mm no3-, 1 mm h2po4-, 8mm k+, 4 mm ca2+, 1.5 mm mg2+, 1 mm so42-, 0.04mm fe2+ and microelements (ph 6.0, ec=1.2 ms cm1). the nutrient solution was renewed every week. growth measurements five plants per treatment were sampled at weekly intervals. roots were carefully washed, then plants were separated into leaves, stems and roots. leaf area was measured with an area meter (delta t-devices ltd., cambridge, uk), plant height was estimated using a ruler and dry weight for each organ was obtained after oven drying (48 hr at 70°c). leaf gas exchange measurements net co2 assimilation (a), stomatal conductance (g)and transpiration (e) measurements were performed weekly (n=5) on the central sector of the youngest fully-expanded leaf using an open system (cms 400, heinz walz, effeltrich, germany) connected to an assimilation chamber and equipped with a high sensitivity irga (binos, leybold haeraeus, germany) under temperature (24°c) and growing light (400 mmol m-2 s-1 par) conditions provided by a mercury vapour lamp (osram hqi-ts 250 w/ndl). calculation of all the parameters was performed following von cammerer and farquhar (1981) using a specific software (diagas 2.02, walz, effettrich, germany). chlorophyll content five leaf disks (10 mm diameter) were randomly taken from the uppermost fully-expanded leaves at weekly intervals, and extracted in 2 ml of n,ndimethylformamide for 24 hr in the dark. absorbance was then determined for each sample using a spectrophotometer at 647 and 663 nm. chlorophyll a and b contents, and a/b ratio were calculated according to moran (1982). determination of total, osmotic and turgor potentials leaf water potential measurements were taken on the same leaf immediately after measuring gas exchange (n=5). total water potential (ψw) was determined using a pressure chamber (pardossi et al., 1991). osmotic potential (ψs) of the leaf xylem sap was determined using an osmometer (precision system, usa) by determining the freezing point depression of the sample. leaf turgor potential (ψp) was calculated using the following equation (eq. 1): ψp = ψw ψs (eq. 1) measurement of sugar content leaf, stem, and root samples (approx. 50 mg each) were taken at weekly intervals (n=5) and directly freeze-dried in liquid nitrogen. samples were homogenized and extracted with 1 ml hot 80% ethanol, boiled for 5 min, and centrifuged at 12000 rpm for 15 min; the supernatant was then collected. the pellet was extracted again as described above, and the supernatant was collected again. at the end of the procedure, the pellet was evaporated to remove any excess ethanol. particulates including starch were suspended in 1 ml of koh 20 mm, boiled and centrifuged at 8000 rpm for 15 min and the supernatant was collected. the extract from ethanol was used for sucrose, glucose and fructose determinations, and the extract from koh was used for starch determination. for sugar determination, two 200 µl aliquots from the ethanol extract were taken, one incubated for 30 min at 37°c with 100 µl solution containing invertase (1 mg invertase ml-1 na-acetate 50 mm at ph 4.6), the other with 100 µl solution containing na-acetate 50 mm at ph 4.6, then both brought to the final volume (1 ml) with a solution containing 100 mm tris-hcl, ph 7.6, 3 mm mgcl2, 2 mm atp, 0.6mm nadp, 1 unit hexokinase and 1 unit glucose-6-pdehydrogenase (incubated at 37°c for 30 min). absorbance at 340 nm was then measured using a spectrophotometer. the concentration of glucose in each solution was determined from glucose standard curves according to guglielminetti et al. (1995). the solution without invertase was used to calculate the amount of free glucose in the sample and the difference between the two gave the amount of sucrose (as glucose equivalent). for each of them 10 µl of solution containing 15 µl of phosphoglucoisomerase in 150 µl of tris-hcl 300 mm at ph 7.6 were incubated at 37°c for 15 min, then absorbance at 340 nm was determined. the difference between the one without invertase and treated with phosphoglucoisomerase and the other without invertase at the first determination gave the amount of free fructose (as glucose equivalent). for starch determination, 100 µl of extract was incubated at 101 37°c for 1 hr with 100 µl solution of na-acetate 100 mm ph 5.2/10 α-amylase. this solution was incubated with 100 µl of na-acetate 100 mm ph 4.6/10 u amyloglucosidase at 55°c for 1 hr. finally, the solution was boiled and centrifuged to eliminate denaturated protein from α-amylase and amylogluco-sidase. 100 µl from this solution was taken and brought to 300 µl with distilled water, then starch analysis (as glucose equivalent) was carried out as mentioned above for glucose. statistical analysis data were analyzed by one-way anova, and means (n=5) were separated using duncan’s multiple range test (p≤0.05). statistical analysis was performed using graphpad prism 4.0 (graphpad software). 3. results growth parameters were greatly affected by root restriction treatment (rr), with significant reductions in total dry weight, leaf area and plant height (fig. 1a, b and c) starting from an early stage of seedling development. rr plants also showed a significantly higher root:shoot ratio (fig. 1d), due to a higher allocation of biomass in the root system compared to canopy (stem and leaves). during the first month, no significant differences were noticed in leaf gas exchange parameters. from day 29, however, stomatal conductance (g) started to significantly decrease in rr plants (fig. 2a), leading to a significant reduction from day 36 in both net co2 fig. 1 growth parameters measured at weekly intervals from day 22 to the end of the experiment in both control and root-restricted (rr) plants: total dry weight (a), leaf area (b), plant height (c) and root:shoot ratio (d). * indicates significantly different values for p≤0.05 (n=5), when means were separated by duncan’s test. fig. 2 leaf gas exchange parameters measured at weekly intervals from day 22 to the end of the experiment in both control and root-restricted (rr) plants: stomatal conductance (a), net co2 assimilation (b) and transpiration (c). * indicates significantly different values for p≤0.05 (n=5), when means were separated by duncan’s test. assimilation (fig. 2b) and transpiration (fig. 2c) until the end of the experiment. the reduction in net co2assimilation was not related to a decrease in the chloro102 phyll content of rr plants (table 1), as no significant differences were noticed for chlorophyll a, b, and a/b ratio between the two treatments. leaf water status did not affect stomatal closure, as total water potential (fig. 3a) and turgor potential (fig. 3c) did not show any significant difference throughout the entire experiment in both the treatments, even if a slight, but not significant, reduction in total water potential was measured on day 43 in rr plants. this behaviour also confirmed the fact that no water stress symptoms occurred during the experimental period, giving a positive feedback of our experimental system. on the contrary, sugar content determination led to interesting results. while sucrose content trend was not uniform during the experiment, leading to contradictory results (fig. 4a), rr treatment led to a clear increase in glucose content (fig. 4b) and a concurrent decrease in fructose content (fig. 4c) together with a great accumulation of starch (fig. 4d). in particular, starch accumulation in the tissues began early in the developmental process (day 29). starch was mainly compartimentalized in the leaves (fig. 5a) and stems (fig. 5b) of rr plants, whereas no significant differences were noticed in roots between control and rr plants (fig. 5c). 4. discussion and conclusions our growth data are in line with several previous results concerning growth depression induced by root restriction in other horticultural crops (carmi and heuer, 1981; tschaplinski and blake, 1985; thomas and strain, 1991; rieger and marra, 1994; liu and latimer, 1995; van iersel, 1997; kharkina et al., 1999; saito et al., 2008; shi et al., 2008). root restriction generally caused an increase in root:shoot ratio (carmi et al., 1983; mugnai et al., 2000); roots in smaller volume formed a highly branched mat, whereas plants in large volume had long tap roots and showed little branching. the increased root:shoot ratio reported by some researchers for many crop species subjected to table 1 chlorophyll content (a, b and a/b ratio) measured at weekly intervals from day 22 to the end of the experiment in leaves collected from control and root-restricted (rr) plants control plants root-restricted plants (rr) 22 29 36 43 50 chl a (mg cm-2) chl b (mg cm-2) a/b 8.075 7.403 7.414 9.076 10.924 3.221 3.274 3.276 3.686 4.320 0.484 0.703 0.702 0.596 0.624 chl a (mg cm-2) chl b (mg cm-2) a/b 8.423 9.261* 9.949* 9.809 10.873 3.333 3.708 3.500 3.863 4.054 0.483 0.566 0.216* 0.547 0.422* * indicates significantly different values between the two treatments for the same parameters for p≤0.05 (n=5), when means were separated by duncan’s test. fig. 3 leaf water status determined at weekly intervals from day 22 to the end of the experiment in both control and root-restricted (rr) plants: total water potential (a), osmotic potential (b) and turgor (c). * indicates significantly different values for p≤0.05 (n=5), when means were separated by duncan’s test. root restriction might be attributed to an increased substrate temperature in smaller containers in conjunction with a possible temperature dependence of root elongation as suggested by hurley et al. (1998). our results reveal that root restriction significantly reduces stomatal conductance, as previously noted by other authors for different species (carmi et al., 1983; thomas and strain, 1991; ismail and noor, 1996; day 103 fig. 4 sugar content measured at weekly intervals from day 22 to the end of the experiment in both control and root-restricted (rr) plants: total sucrose (a), total glucose (b), total fructose (c) and total starch (d). * indicates significantly different values for p≤0.05 (n=5), when means were separated by duncan’s test. fig. 5 starch content in the different plant organs measured at weekly intervals from day 22 to the end of the experiment in both control and root-restricted (rr) plants: leaf (a), stem (b) and roots (c). * indicates significantly different values for p≤0.05 (n=5), when means were separated by duncan’s test. kharkina et al., 1999), and that stomatal conductance was the primary cause of decrease in co2 assimilationin root-restricted plants suggesting a stomatal factor limiting the photosynthetic rate under root-restriction conditions (shi et al., 2008). the decline in stomatal conductance was not correlated to a concurrent decline in total water potential, as leaf tissues were able to maintain a high level of turgor during the whole experiment. this means that other factors are largely involved in the stomatal closure. it has been suggested that root volume restriction induces a reduction in the stomatal conductance via a decrease in the supply of growth substances from roots to shoots and/or an imbalance in root and shoot hormones. for example, shi et al. (2008) reported that shoot growth suppression may be caused by the influence of aba originating from the restricted roots. carmi (1995) found that the higher level of aba in the leaves of root-restricted plants was not a consequence of an enhanced transport from the restricted roots, concluding that root-zone restriction might promote aba accumulation in the root and the shoot, with a possible influence of such accumulation on other processes in root-restricted plants, such as leaf gas exchange. the decline in net co2 assimilation observed inroot-restricted conditions was also interpreted as a feedback inhibition by carbohydrate accumulation (pezeshki and santos, 1998). plant growth is strongly affected by leaf photosynthetic activity, since photosynthates are essential either as the source of carbon used for the build-up of organic compounds or as the source of energy needed for biochemical reactions involved in growth and maintenance processes. growth rate may regulate photosynthesis either through effects on the supply of growth substances translocated into leaves or through the effect on the translocation rate of photosynthates from leaves to the growing organs (carmi et al., 1983). the accumulation of photosynthates is influenced by the rate of their translocation to the sink organs (sonnewald and willmitzer, 1992), and sink demand for photosynthates has a marked influence on source leaf photosynthesis, which is greatly dependent on sink strength, considered as a product of sink size and sink activity (sonnewald and willmitzer, 1992). however, sink size is determined by different parameters. roots are recognized as 104 a metabolic sink that influences the partitioning of photosynthetically fixed carbon (gifford and evans, 1981; robbins and pharr, 1988). sink limitation caused by root restriction can greatly reduce leaf photosynthetic rate in many crop species (hameed et al., 1987; ismail and noor, 1996; schaffer et al., 1996; whiley et al., 1999; shi et al., 2008), and reduced translocation of assimilates from leaves (robbins and pharr, 1988; kharkina et al., 1999). root volume restriction often promotes an accumulation of nonstructural carbohydrates in the stem and leaves in response to the lack of the active sinks (nishizawa and saito, 1998), meaning that the difference in the growth rate between root-restricted and control treatments was not due to a decrease in assimilates supply to the organs whose growth was restricted (mandre et al., 1995). our results suggest that the role of the leaves and stem as sink organs may increase when root growth is extremely limited by volume restriction and a relatively larger amount of carbohydrates may accumulate in the canopy. a new shoot to root equilibrium may be established for an increased function of leaves and stem, together with a concurrent diminished function of the roots. therefore, it can be concluded that as a result of reduced vegetative growth an excess of assimilates was produced which could not be used for growth, and thus accumulated in the form of starch, as also indicated by carmi and heuer (1981), robbins and pharr (1988) and shi et al. (2008). accumulation of non-structural carbohydrates in the leaves in response to root restriction could provide a feedback mechanism that reduces carbon metabolism (thomas and strain, 1991). starch accumulation may reduce net photosynthetic rate by avoiding intracellular co2 transport (shi et al., 2008). however, contradicto-ry results were obtained by rieger and marra (1994), who suggested that reduced co2 assimilation cannotalways be explained by a feedback inhibition of carbohydrates. the relatively low maximum assimilation (amax) rates for container-grown plants compared tofield-grown plants may be attributed to containers restricting the root sink, thus causing the photoassimilate supply to exceed the capacity of demand (i.e. endproduct inhibition of photosynthesis) as indicated by arp and drake (1991) and whiley et al. (1999). in conclusion, our results show that growth reduction by root restriction is mainly linked to a photosynthetic impairment, caused by a limited stomatal conductance (probably driven by both stomatal factors and hormonal substances) and a strong accumulation of starch in the tissues, which probably leads to a feedback inhibition of the photosynthetic process. acknowledgements the authors are particularly indebted to prof. franco tognoni for scientific support. references arp w.j., drake b.g., 1991 increased photosynthetic capacity of scirpus olneyi after 4 years of exposure to elevated co2. plant cell environ, 14: 1003-1006. carmi a., 1995 growth, water transport and transpiration in root-restricted plants of bean, and their relation to abscisic acid accumulation. plant science, 107: 69-76. carmi a., hesketh j.d., enos w.t., peters d.b., 1983 interrelationships between shoot growth and photosynthesis, as affected by root growth restriction. photosynthetica, 17(2): 240-245. carmi a., heuer b., 1981 the role of roots in control of bean shoot growth. ann. bot., 48: 519527. gifford r.m., evans l.t., 1981 photosynthetic carbon partitioning and growth. ann. rev. plant physiol., 32: 485-509. guglielminetti l., perata p., alpi a., 1995 effect of anoxia on carbohydrate metabolism in rice seedlings. plant physiol., 108: 735-741. hameed m.a., reid j.b., rowe r.n., 1987 root confinement and its effects on the water relations, growth and assimilate partitioning of tomato. ann. bot., 59: 685-692. hurley m.b., rowarth j.s., trought m.c.t., barnes m.f., rowe r.n., 1998 variations in water availability and temperature in the root environment during root volume restriction studies. new zeal. j. crop hort. sci., 26: 127-134. ismail m.r., noor k.m., 1996 growth, water relations and physiological processes of starfruit plants under root growth restriction. scientia hort., 66: 51-58. kharkina t.g., ottosen c.o., rosenqvist e., 1999 effects of root restriction on growth and physiology of cucumber plants. physiol. plant., 105: 434-441. liu a., latimer j.g., 1995 water relations and abscisic acid levels of watermelon as affected by rooting volume restriction. j. environ. bot., 289: 1011-1015. mandre o., rieger m., myers s.c., seversen r., regnard j.l., 1995 interaction of root confinement and fruiting in peach. j. amer. soc. hort. sci., 120(2): 228-234. marcelis l.f.m., 1991 effects of sink demand on photosynthesis in cucumber. j. exp. bot., 42: 1387-1392. marsh d.b., paul k.b., 1988 influence of container type and cell size on cabbage transplant development and field performance. hortscience, 23(2): 310-311. moran r., 1982 formula for determination of chlorophyll pigments extracted with n,n-dimethylformamide. plant physiol., 69: 1376-1381. mugnai s., ferrante a., petrognani l., serra g., vernieri p., 2009 stress-induced variation in leaf gas exchange and chlorophyll a fluorescence in callistemon plants. res. j. biol. sci., 4: 913-921. mugnai s., vernieri p., tognoni f., 2000 container volume effects on morphology and physiology of tomato seedlings. acta horticulturae, 516: 49-56. nishizawa t., saito k., 1998 effects of rooting volume restriction on the growth and carbohydrate concentration in tomato plants. j. amer. soc. hort. sci., 123(4): 581-585. pardossi a., vernieri p., tognoni f., 1991 evaluation of the pressure chamber method for the assessment of water status in chilled plants. plant cell environ., 14: 675-682. pezeshki s.r., santos m.i., 1998 relationships among rhizosphere oxygen deficiency, root restriction, photosynthesis, and growth in baldcypress (taxodium distichum l.) seedlings. photosynthetica, 35: 381-390. rieger m., marra f., 1994 response of young peach trees to root confinement. j. amer. soc. hort. sci., 119: 223-228. robbins n.s., pharr d.m., 1988 effect of restricted root growth on carbohydrate metabolism and whole plant growth of cucumis sativa l. plant physiol., 87: 409-413. 105 saito t., fukuda n., iikubo t., inai s., fujii t., konishi c., ezura h., 2008 effects of root-volume restriction and salinity on the fruit yield and quality of processing tomato. j. japan. soc. hort. sci., 77: 165-172. schaffer b., searle c., whiley a.w., nissen r.j., 1996 effects of atmospheric co2 enrichment and root restriction on leaf gas exchange and growth of banana. physiol. plant., 97: 685-693. shi k., fu l.j., ding x.t., dong d.k., zhou y.h., yu j.q., 2008 root restriction-induced limitation to photosynthesis in tomato leaves scientia hort., 117: 197-202. shi k., hu w.h., dong d.k., zhou y.h., yu j.q., 2007 low o2 supply is involved in the poor growth in root-restricted plants of tomato. env. exp. bot., 61: 181-189. sonnewald u., willmitzer l., 1992 molecular approaches to sink-source interactions. plant physiol., 99: 1267-1270. thomas r.b., strain b.r., 1991 root restriction as a factor in photo-synthetic acclimation of cotton seedlings grown in elevated carbon dioxide. plant physiol., 96: 627-634. thomas t.h., 1993 effects of root restriction and growth regulator treatments on the growth of carrot (daucus carota l.) seedlings. plant growth regul., 13: 95-101. tschaplinski t.j., blake t.j., 1985 effects of root restriction on growth correlations, water relations and senescence of alder seedlings. physiol. plant., 64: 167-176. van iersel m., 1997 root restriction effects on growth and development of salvia (salvia splendens). hortscience, 32(7): 1186-1190. von cammerer s., farquhar g.d., 1981 some relationships between the biochemistry of photosynthesis and the gas exchange of leaves. planta, 153: 376-387. whiley a.w., searle c., schaffer b., wolstenholme b.n., 1999 cool orchard temperatures or growing trees in containers can inhibit leaf gas exchange of avocado and mango. j. amer. soc. hort. sci., 124(1): 46-51. 26 adv. hort. sci., 2011 25(1): 26-31 *corresponding author efallik@volcani.agri.gov.il received for publication 10 february 2011. accepted for publication 18 february 2011. photoselective shade nets reduce postharvest decay development in pepper fruits a. goren, s. alkalia-tuvia, y. perzelan, z. aharon, e. fallik* department of postharvest science of fresh produce, aro, the volcani center, p.o. box 6, 50250 bet dagan, israel. key words: capsicum annuum, postharvest, shelf life, storage. abstract: during two-year studies, we evaluated the influence of photoselective coloured shade nets on the quality of fresh harvested pepper fruits (capsicum annuum) after prolonged storage and shelf life simulation. pepper cultivar ‘romans’ grown in a semi arid region under 35% pearl and yellow shade nets significantly maintained better pepper fruit quality after 16 days at 7°c plus three days at 20°c, mainly by reducing decay incidence during two consecutive years (2008 and 2009), compared to commercial black and red nets. no significant differences were observed in percentage of weight loss, firmness and total soluble solids in fruit harvested under the different coloured shade nets. the skin colour of fruit harvested under pearl net was significantly lighter than that of fruit harvested under red and black shade nets and this fact can be associated with inhibition of fruit ripening during growth. after storability and shelf life simulation however skin colour was red to dark red under all shade nets. pearl and yellow shade nets significantly reduced alternaria spp. population in the field, which was evaluated with alternaria-selective growing medium. the highest alternaria population was found under red shade net. the significant low decay incidence in fruit harvested under pearl and yellow shade nets can be explained by the low inoculum level of alternaria spp. in the field, and inhibition of fungal sporulation, and/or by a slowing of fruit ripening during its growth, reducing fruit susceptibility to fungal infection in the field due to the scattered light, its quality and the ratio between the light spectrum under the two shade nets. 1. introduction sweet bell pepper (capsicum annuum l.) is an important export commodity in israel with more than 120,000 tons per year. fruits may be green (unripe), red, yellow, orange, or brown when ripe. peppers are rich in vitamins, minerals and dietary fibres, and are low in calories (kevers et al., 2007) and they have become popular decorative items (frank et al., 2001). however, to extend the export season with high pepper quality during the summer season, shade netting is necessary to protect agricultural crops from excessive solar radiation and pests (shahak, 2008). in recent years coloured shade netting (photoselective shade nets), designed specifically to manipulate plant development and growth, has become available. these nets can be used outdoors as well as in greenhouses. they can provide physical protection (from birds, hail, insects, excessive radiation), affect environmental modification (humidity, shade, temperature) (perez et al., 2006), and increase the relative proportion of diffuse (scattered) light as well as absorb various spectral bands, thereby affecting light quality (shahak et al., 2004). these effects can influence crops as well as the organisms associated with them. the effect of colour shade nets on plant development in crops, foliage crops, fruit trees and vegetables has been studied in recent years (nissim-levi et al., 2008; shahak et al., 2008). bell peppers are grown commercially in semi-arid regions of israel under black shade nets. netting is frequently used to protect pepper plants from excessive solar radiation (shade-nets), environmental hazards (e.g. hail-nets), or pests (bird, or insect-proof nets) (shahak et al., 2004). shahak (2008) reported that productions of three cultivars of bell pepper were increased by 16 to 32% under pearl and red netting compared with equivalent black shade netting, or with open field production. in addition, elad et al. (2007) reported that blue-silver, green and red nets were associated with lower levels of powdery mildew disease, caused by leveillula taurica in pepper field experiments. other studies have suggested differential effects of photoselective screens and shade nets on pest infestation and vector-borne viral diseases (ben-yakir et al., 2008). in a preliminary research conducted in 2007, 27 fallik et al. (2009) demonstrated the potential use of coloured shade nets to maintain the postharvest quality better of sweet pepper grown under red and yellow nets, during prolonged storage and shelf life. therefore, we undertook the two-year study presented in this work to evaluate, on a commercial growth scale, the influence of different coloured shade nets on the quality of fresh harvested bell pepper after prolonged storage and shelf life in order to understand, in part, the mode-of-action of those shade nets on harvested fresh produce. 2. materials and methods plant materials and coloured net shades red sweet bell pepper (capsicum annuum l.) cv. ‘romans’ was grown at the b’sor experimental station in the south-west of israel (31.271° n; 34.389° e), using commercial cultivation practices (planting seedlings directly into the soil [~ 90% sandy regosol and ~ 10% arid brown soil], automatic drip fertigation), under four different coloured shade-nets, as follows: red, yellow, pearl and black (commercial shade net) with 35% relative shading (in par) (polysack plastics industries, nir-yitzhak, israel under the trade mark chromaticnet), in four random replicates of 18 x 18 m each, all within one large horizontal net-house, 2.5 m high. plants were planted during the third week of may, both in 2008 and 2009, and harvested as described below. light measurements under the nets measurements of light spectra, light scattering and microclimate parameters under the nets were taken as described previously by shahak et al. (2008). spectra of solar radiation outside and under the nets were measured by a spectroradiometer ps-100 (apogee instruments inc. logan ut, usa) which employs a light diffuser of 4 cm diameter above the 300 µm fiber optics. the diffuser was oriented along the sun beams. a round opaque plate of 6 cm diameter was held at 40 cm above the diffuser to block the direct light to measure the scattered light. direct radiation was measured through a 4 x 45 cm tube placed on top of the diffuser. the radiation was measured three times at noon in the middle of august 2009 (fig. 1). quality parameters fruit samples for storability and shelf-life were harvested five times each year, every three weeks, between the beginning of september and the end of november 2008 and 2009. fruits (185±10 g) without defects or diseases were harvested without a calyx, at ~85% light red/red colour (~16% green ‘cheek’), rinsed and brushed in hot water as described by fallik et al. (1999) and packed in four 6.5 kg corrugated cartons. fruit quality parameters were evaluated immediately after each harvest and at the end of 16 days storage at 7°c and relative humidity (rh) of ~ 94%, plus three days at 20°c (rh ~ 70-75%) as follows: weight loss was expressed as percentage of weight loss from the initial weight of ten fruits. fruit firmness was measured on ten fruit as described by ben-yehoshua et al. (1983). each fruit was placed horizontally between two flat surfaces and a 2 kg weight loaded on top of the flat surface. a dial fixed to a graduated plate recorded the deformation of the fruit in millimetres. full deformation was measured 16 s after exerting the force on the fruit, then the weight was removed and the residual deformation was measured 16 s later. fruit was considered very firm with 0-1.5 mm deformation; firm = 1.63 mm deformation; soft = 3.1-4.5 mm deformation; very soft = above 4.6 mm deformation. total soluble solids (tss) were measured on the same ten fruits tested for firmness, by squeezing out juice from fruits onto an atago digital refractometer (atago, tokyo, japan) and taking readings. the development of fruit colour was expressed as colour index (cinx) – on a scale of 1 to 4 with 1 = light red with 10-16% green peel; 2 = light red; 3 = red; and 4 = dark red. the index was calculated as follows: (number of fruits at light red colour x 1 + number of fruits at light red x 2 + number of fruit at red colour x 3 + number of fruits at dark red x 4)/total number of fruits. decay incidence was considered once fungal mycelia appeared on fruit pericarp and/or calyx. decay was expressed as a percentage of the total initial fruit number. evaluation of total microorganism population levels and alternaria spp. levels under coloured shade nets population levels of conidia in the greenhouse envifig. 1 influence of coloured shade nets on the scattered light under the different nets. measurements were taken at noon in august 2009. scattering relate to par (in µmol m-2 s-1). the values represent integrals of the spectra over the following wavelength ranges: par: 400-700 nm; uv: 300-380 nm; blue: 410-470 nm; red: 640-680 nm; far red: 690-750 nm (means of 3 replicates ± se). 28 ronment were evaluated according to elad (1997) by exposing 90 mm petri dishes, under each net, containing pda (potato dextrose agar, difco) supplemented with chloramphenicol (sigma chemicals, 50 µg/ml) at a height of 60 cm above the ground, in five different places (five plates) under each coloured shade net for 60 min. plates were then incubated for four days at ~22°c and colonies were counted. based on preliminary results obtained in 2007, the main decay-causing agent on pepper fruits after harvest was alternaria alternata. population levels of alternaria spp. were evaluated by exposing 90 mm petri dishes containing selective medium for alternaria (soon gyu and pryor, 2004), as described above. colonies were counted after 10 days incubation at ~22°c. the plates were exposed horizontally 60 cm above the ground. each evaluation was repeated three times from the beginning of october to the end of november. statistical analysis all the results were analyzed using a oneor twoway anova statistical analysis at p=0.05 using jmp 6 statistical analysis software program (sas institute inc. cary, nc., usa) (sall et al., 2001). 3. results light quality under different shade nets figure 1 shows the spectra of the scattered light under the different nets. the pearl net significantly reduced uv, while it significantly increased the blue light, thus significantly increasing the ratio between blue/uv compared to all other shade nets. the yellow net significantly increased the scattered red light compared to all other shade nets, while the blue light under this net was significant lower compared to red and pearl nets (fig. 1). the three shade nets significantly increased the far-red scattered light compared to the commercial black net. quality evaluation after 16 days storage at 7°c plus three days at 20°c, the major differences in fruit quality were found between 2008 and 2009 in weight loss and fruit firmness. however, tss was similar in the two years (table 1). in 2008, fruits lost less weight under the red net (2.9%), while under the yellow net weight loss was higher (3.5%). in 2009, percentage of weight loss under the four shade nets was similar (between 3.4 to 3.6%). immediately after harvest, fruits picked from the pearl net treatment were significantly lighter in their red colour index (2.33) than fruit picked from the commercial black or red shades (2.60 and 2.64, respectively) (table 2). no significant differences were observed in fruit colour index between the yellow and pearl treatments. after 16 days at 7°c plus three additional days at 20°c, all fruits turned almost dark red, however fruits picked under the red shade were significantly darker (3.86) than fruits picked under yellow and pearl shades (3.76 and 3.75, respectively) (table 2). decay incidence in both years, decay incidence under yellow and pearl shade nets were significantly lower compared to fruit picked under the commercial black or red net (fig. 2). in 2009, the average decay incidence was higher than in 2008. in 2008, the highest decay incidence was observed on fruit picked under black and red shade nets, which was significantly higher than on fruit picked under the yellow and pearl shade nets. in 2009, black red yellow pearl weight loss (%) z 3.2 abb w 2.9 bb 3.5 aa 3.3 ab firmness (mm) y tss (%) x 3.6 aa 3.4 aa 3.5 aa 3.6 aa 2.1 ab 2.3 ab 2.3 ab 2.0 ab 3.0 aa 3.0 aa 2.7 ba 2.9 aba 6.7 aa 6.4 aa 6.6 aa 6.5 aa 6.6 aa 6.4 aa 6.6 aa 6.4 aa colored shade net 2008 2009 2008 2009 2008 2009 table 1 the influence of coloured shade nets on weight loss, firmness and tss after 16 days at 7°c plus three days at 20°c (means of five experiments each year, with four 6.5 kg boxes per treatment) z percent weight loss from initial. y firmness millimeter deformation (flexibility). x percent total soluble solids. w values followed by the same upper-case letter do not significantly differ between the treatments, while values followed by the same lower-case letter, do not significantly differ between the harvest seasons at p=0.05 according to duncan’s multiple range test. colored shade net immediately after harvest (1-4) z after storage and shelf life (1-4) black red yellow pearl 2.60 a y 2.64 a 2.46 ab 2.33 b 3.80 ab 3.86 a 3.76 b 3.75 b table 2 the influence of coloured shade net on fruit colour index (cinx) development immediately after harvest and after 16 days at 7°c plus three days at 20°c (means of five experiments each year, with four 6.5 kg boxes per treatment) z colour index: 1= light red with 10-16% green peel; 2= light red; 3= red; and 4= dark red. y values within a column followed by the same letter do not significantly differ at p= 0.05 according to duncan’s multiple range test. 29 fig. 2 influence of coloured shade nets on decay incidence in fruit of cv. romans, in 2008 and 2009 after 16 days at 7°c plus three days at 20°c. (means of five experiments with four 6.5 kg boxes per treatment ± se). fig. 3 influence of coloured shade nets on the epiphytic population of fungi detected under the nets (cfu/plate – means of 3 experiments conducted in 2009 ± se). a significantly lower decay incidence was observed on fruit picked under the pearl shade net, whereas the highest one was observed on fruit picked under the red shade net (fig. 2). fig. 4 influence of coloured shade nets on the alternaria spp. population detected under the nets using selective medium (cfu/plate – means of 3 experiments conducted in 2009 ± se). evaluation of total microorganisms population levels and alternaria spp. levels under coloured shade nets the epiphytic population of fungi evaluated on a regular pda medium was lower under the pearl net, compared to the population that was counted under the red or black shade nets. however, no significant difference between treatments was found (fig. 3). using a selective medium for alternaria spp., a significant reduction in its population was observed under pearl and ellow shade nets (fig. 4). the highest alternaria population was found under the commercial black shade net (fig. 4). 4. discussion photoselective coloured shade nets (chromatinets™) have been developed over the last decade to filter selected regions of the spectrum of sunlight, concomitantly with inducing light scattering, and are designed specifically to modify the incident radiation (spectrum, scattering and thermal components) (shahak et al., 2004). depending on the pigmentation of the plastic and the knitting design, the nets provide varying mixtures of natural, unmodified light together with spectrally modified scattered light. they are aimed at optimizing desirable physiological responses, in addition to providing physical protection to the crop, thus improving plant growth, flowering and yield and fruit quality (rajapakse and shahak, 2007; shahak, 2008; shahak et al., 2008). the most prominent effect of the coloured shade nets was found on decay incidence in fruit harvested under pearl and yellow nets, without affecting fruit quality after prolonged storage and shelf life. it is well known that light plays an important role in plant growth. red light induced resistance in broad bean against botrytis cinerea (islam et al., 1998) and alternaria tenuissima (rahman et al., 2003), and in rice against magnaporthe grisea (arase et al., 2000). tabira et al. (1989) reported that continuous irradiation of visible light of 570-680 nm protected apple leaves by inducing insensitivity to alternaria alternata apple pathotype. disease suppression under red light was also observed in glasshouse-grown corynespore cassiicola-inoculated cucumbers, and indicated that delay and suppression of corynespora leaf spot of cucumber were due to induction of resistance in cucumber, and not to differences in environmental conditions or fungal populations between the two greenhouses (rahman et al., 2009). we therefore speculate 30 that under yellow net, significantly more scattered red light penetrates into the plant and fruit (fig. 1), which in turn inhibits fruit ripening as shown by fruit colour index (table 2) and/or, indirectly, induces resistance against alternaria alternata infection after harvest. preliminary results have revealed high amounts of chlorophyll in fruit harvested under pearl and yellow nets and relatively low amounts of carotenoids, which might indicate slow fruit ripening (fallik and goren, unpublished). in parallel, it was found that under the pearl and yellow shade nets the population of alternaria alternata, the main decay-causing agent after harvest, was significantly low compared to commercial black nets. light has profound effects on fungal biology. growth and development of many fungal species are intricately regulated by light (springer, 1993; purschwitz et al., 2006). fungal responsiveness to different wavelengths of light has been well documented, and blue light/uv and red/far-red light are two types of photoresponses primarily observed (mooney and yager, 1990; griffith et al., 1994; purschwitz et al., 2006, 2008; olmedo et al., 2010). blue light or a high ratio of blue to uv radiation was inhibitory to the sporulation of many important phytopathogens (raviv and antignus, 2004; paul et al., 2005). increases in the spectral ratios between transmitted light:blue/near-uv was found to inhibit the sporulation of several isolates of the fungal pathogen botrytis cinerea in tomato (kotzabasis et al., 2008). hence, the ability of the pearl and yellow shade nets to filter uv light and enrich the blue spectrum (shahak et al., 2008) and the blue/uv (fig. 1) may be involved in alternaria alternata inhibition in the field, thus reducing inoculum levels and fruit infection during its growth and thereafter decay development during prolonged storage and shelf life (barkai-golan, 2001). in conclusion, based on our results from two consecutive years of study which have shown a significant reduction in decay development on pepper fruit harvested under pearl and yellow shade nets, it seems that these two coloured shade nets influence both the pathogen in the field and the fruit ripening and its susceptibility to pathological deterioration after harvest. acknowledgements this research was supported by grant no. 430-0184-08/09 from the chief scientist of the ministry of agriculture and rural development, 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apple and japanese pear leaves to am-toxin from alternaria alternata apple pathotype. ann. phytopathol. soc. japan, 55: 567-578. 81 1. introduction due to consumer demand for high-convenience foods, fresh-cut peppers may represent an interesting product to add to the existing fresh-cut products. the quality of a fresh-cut product is generally affected by pre-harvest and post-harvest factors, including processing. genotype, growing conditions, cultural practices, and maturity stage at harvest, from a pre-harvest point of view, may greatly influence initial quality of the product to be processed; on the other hand, postharvest handling and storage of raw materials, and processing conditions, including finished product fate throughout the distribution chain, markedly determine its final quality. maturity stage is an important factor conditioning final quality and processability of fresh-cut products, particularly fruits. less mature fruits, in fact, are more suitable for processing due to their greater firmness, compared to more mature fruits, but this can result in lower sensorial quality as observed in melons (watada and qi, 1999) and mangoes (bender et al., 2000). in peppers, the maturity stage was found to have an impact on flavonoids, carotenoids, and ascorbic acid concentration (marin et al., 2004; fox et al., 2005), with carotenoids and ascorbic acid increasing, and flavonoids decreasing, as maturity proceeds, while deepa et al. (2007) observed an increase in the main antioxidant compounds, including phenols ascorbic acid, capsicin, and carotenoids for 10 different genotypes. moreover, in ‘domino’ bell peppers, an increase in firmness was observed with the increasing of fruit size, occurring with ripening, most probably due to the increase of pericarp thickness (tadesse et al., 2002). the same authors reported an increase in soluble solids, and a decrease in respiration rate and ethylene production and suggested as maturity index at harvest for this cultivar firmness values of 35 n and a minimum of 6°brix. molinari et al. (1999) reported greater titrable acidity on full-ripe peppers ripened on the plant compared to those harvested at the color-break stage and ripened in storage. in addition, agricultural practices, soil, and climate conditions may differently affect quality attributes of fresh produce, including external attributes (kays, 1999), firmness (sams, 1999), and nutritional composition (lee and kader, 2000). in particular, nitrogen fertilization seems to decrease the concentration of vitamin c in many fruits and vegetables (lisiewska and kmiecik, 1996). all these factors can consequently affect quality of fresh-cut produce effect of type of fertilization and maturity on quality of fresh-cut red and yellow peppers (capsicum annuum l. ) f. piazzolla*, m.l. amodio*, r. rinaldi*, f. raimo**, g. colelli* * dipartimento di scienze delle produzioni e dell’innovazione dei sistemi agroalimentari mediterranei, università degli studi di foggia, via napoli, 25, 71121 foggia, italy. ** consiglio per la ricerca e sperimentazione in agricoltura, unità di ricerca per le colture alternative al tabacco, cra-cat, via p. vitiello, 108, 84018 scafati (sa), italy. key words: appearance, organic fertilization, phenolics, vitamin c. abstract: the aim of this work was to evaluate the effect of the type of fertilization (mineral and combined fertilization with compost in pre-transplant plus mineral addition during cultivation) and stage of maturity at harvest (mature-green and full-colored) on post-cutting quality of red and yellow ‘cazzone’ peppers. peppers were cut into strips, and air-stored for 8 days at 5°c. during storage, color, appearance score, firmness, respiration rate, soluble solids, acidity, ph, vitamin c, total phenols, and antioxidant activity were measured. the maturity stage influenced color parameters and soluble solids, acidity and ph for both yellow and red types. full-colored peppers showed a lower respiration rate, and higher ssc than mature-green peppers; for the yellow type, a lower firmness value was observed for full-colored fruits compared to the mature-green ones. a lower antioxidant activity was also observed in the yellow type fertilized with the combined treatment, while phenol content in full-colored peppers was higher than in mature-green ones. fresh-cut yellow peppers showed higher susceptibility to decay compared to red types: after 8 days of storage, the appearance score in mineral fertilized full-colored yellow peppers dramatically decreased below the limit of marketability. the results of this experiment show that the type of fertilization and maturity stage can have varying impact on the quality of yellow and red peppers. adv. hort. sci., 2012 26(2): 81-87 received for publication 26 april 2012. accepted for publication 25 june 2012. 82 and their storability, but very few works have directly investigated the impact of pre-harvest factors on the quality of fresh-cut products, and none of them regard bell peppers. more works are available on the impact of processing, and among them artés-hernández et al. (2010) studied the effect of the cut type and of the modified atmosphere packaging on quality of bell peppers (cv. requena) observing that in general peppers cut in a ‘ring’ suffer greater weight loss than peppers cut in strips or dice, although respiration was not affected by the cutting mode and was not significantly different from the whole product. the objective of the present work was to evaluate the effect of the type of fertilization (mineral and combined fertilization with compost in pre-transplant plus mineral addition during cultivation) and stage of maturity at harvest (mature-green and full-colored) on post-cutting quality of red and yellow ‘cazzone’ peppers. 2. materials and methods plant material the experiment was carried out in scafati (sa, italy, coordinates 40° 44’n, 14° 30’e, 10 m a.s.l.), on soil of sandy loam texture, basic ph and with organic and mineral content as reported in table 1. table 1 soil parameters soil parameters ph in water (1:2.5) 8.4 ec 25°c (1:2) (ds/m) 0.6 c (g/kg) 15.4 organic matter (g/kg) 26.6 n (g/kg) 1.3 c/n 11.6 assimilated p 2 o 5 (mg/kg) 85.0 exchangeable k (ppm) 917.1 exchangeable na (ppm) 251.8 exchangeable ca (ppm) 2090.9 exchangeable mg (ppm) 497 ecotypes of red and yellow ‘cazzone’ pepper (capsicum annuum l.) (density of 3.3 plants m-2) were subjected to two different tecniques of fertilization: mineral and combined fertilization with compost in pre-transplant plus mineral addition during cultivation. mineral fertilization was in compliance with the campania region guidelines: 100 kg ha-1 of nitrogen were applied for 1/3 in pre-transplant and 2/3 during plant growth. for the combined fertilization, 20 t ha-1 of dry organic compost obtained from urban organic waste (characteristics reported in table 2) were applied in pre-transplant, and integrated with 50 kg ha-1 of mineral nitrogen during plant growth. transplanting was carried out on 25 may. pepper fruits were harvested on 15 september at two stages of maturity (mature-green and full-colored), and transported to the laboratory of postharvest technology at the university of foggia (italy). table 2 compost parameters compost parameters humidity (%) 31 ph 6-8 c (% dm) 28 humic and fulvic carbon 8 n (% dm) 2 c/n 14 cu (mg/kg dm) 110 zn (mg/kg dm) 250 salinity (meq/100 g) 21 experimental design and protocol for each ecotype/fertilization/stage of maturity combination, six lots (two replicates x three storage sampling) of 15 strips were individually placed in plastic trays closed in pet macroperforated bags, together with wet paper (to maintain high level of rh), and stored at 5°c for eight days. initially, and after four and eight days of storage at 5°c quality attributes (including color, appearance score, firmness, respiration rate, weight loss, soluble solids, titrable acidity, ph, vitamin c, phenols content, and antioxidant activity) were monitored. respiration rate and weight loss respiration rate (ml co 2 kg−1 hr−1) was measured using the static system, measuring the amount of co 2 accumulated in the headspace of sealed pvc containers (5 l). co 2 concentration, determined by a shimadzu gas chromatograph (model 17a) equipped with a tcd detector, was then referred to the weight of the sample, to the volume of the headspace, and to the elapsed time. samples were individually weighed and the weight loss was calculated as % of the initial fresh weight. physical analysis the colour of the strips was measured in two different points of the mesocarp, randomly selected, using a spectrophotometer (cm 2600d konica minolta, osaka, japan) in the reflectance mode using the cie l*a*b* colour scale. hue angle and saturation were then calculated. appearance score evaluation was subjectively assessed using a scale of 5 to 1 where 5= excellent, no defects; 4= very good, few defects; 3= good, moderate defects, limit of marketability; 2= poor, many defects; and 1= inedible. a score of 3 was considered as the limit of marketability and a score of 2 as the limit of edibility (amodio et al., 2007). firmness was measured on two points of the mesocarp, as resistance of the strips to a 2-mm penetration by a probe 83 of 6-mm diameter, using a digital penetometer (tierre s.r.l., torino). chemical analysis ascorbic acid (aa) and dehydroascorbic acid (dhaa) contents were determined as described by zapata and dufour (1992), with some modifications. samples of 20 μl were analysed with an agilent 1200 series hplc (waldbronn, germany) equipped with a binary pump, an autosampler, and a photodiode array detector. separations of dhaa and aa were achieved on a zorbax eclipse xdb c18 column (150 mm × 4.6 mm; 5 μm of particle size; agilent technologies, santa clara, ca, usa). aa and dha contents were expressed as milligrams of ascorbic or dehydroascorbic acid per kilogram of fresh weight (mg kg−1). total phenols were determined according to the method of singleton and rossi (1965). the absorbance was read at 725 nm against a blank using a uv-1700 shimadzu spectrophotometer (jiangsu, china). the content of total phenols was calculated on the basis of the calibration curve of gallic acid, and was expressed as grams of gallic acid per kilogram of fresh weight (g ga kg−1). antioxidant assay was performed following the procedure described by brand-williams et al. (1995), with minor modifications. trolox was used as a standard and the antioxidant activity was reported in milligrams of trolox equivalents per kilogram of fresh weight (g te kg−1). four grams of fresh juice were used to determined total soluble solids (tss) (measured with a digital refractometer, atago pr32), ph and titratable acidity (ta). titratable acidity was determined with an automatic titrator (titromatic crison 1s), using the juice samples and titrating with 0.1 n naoh up to ph 8.1, and the value was expressed as percentage of citric acid. data analysis for mean data at harvest of peppers of both ecotypes, a two-way anova for stage of maturity and fertilization effects was run, while on the whole data set, a three-way anova was performed with stage of maturity, fertilization treatment and time of storage as factors. when interactions among factors were not significant, main effects were analyzed. mean separation among treatments at each time of storage was performed with the tukey test. (p< 0.05, n=10). 3. results and discussion most of the quality attributes were not affected by the type of fertilization, except respiration rate, a* value and ph, whereas, as expected, most of them were affected by the stage of maturity. the interaction between the type of fertilization and the stage of maturity was statistically significant for respiration rate and ph (table 3). pepper fruits treated with mineral fertilization showed a higher respiration rate (9.6 ml co 2 kg-1 hr-1) than fruits treated with the combined fertilization (8.8 ml co 2 kg-1 hr-1 ), a higher a* value (0.6 vs. -1.1) and ph (5.44 vs. 5.35) although the absolute ph difference was very little (table 4). table 4 illustrates the effect of stage of maturity on quality attributes of fresh-cut yellow peppers at harvest. in particular, the stage of maturity affected the respiration rate, l*, a*, and b* values, hue angle, chroma, soluble solids, and ph. full-colored peppers showed lower respiration rate than mature-green peppers (7.6 and 10.7 ml co 2 kg-1 hr-1 respectively), higher soluble solids (6.1 vs. 4.7°brix ) and lower ph (5.75 vs. 5.04). these findings confirmed that reported by tadesse et al. (2002) for ‘domino’ bell peppers, and mainly that soluble solids, respiration rate and firmness (other than color) evolve during fruit ripening, and may be used as maturity index at harvest. the increase of titratable acidity and decrease of ph during bell pepper growth and ripening has also been observed in several studies (fox et al., 2005), during plant ripening (molinari et al., 1999; serrano et al., 2010) and, in particular serrano et al. (2010) observed an increase of citric acid during ripening. for red ‘cazzone’ peppers, no differences were observed for the measured attributes according to the type of fertilization and interaction between the type of fertilization and the stage of maturity, whereas the stage of matutable 3 effect of type of fertilization (mineral and combined) and stage of maturity (mature-green and full-colored), and their interaction on quality attributes of fresh-cut yellow ‘cazzone’ peppers at harvest quality attributes fertilization (f) stage of maturity (s) fxs firmness (n) ns ns ns respiration rate (ml co 2 kg-1 hr-1) * *** *** l* ns **** ns a* * **** ns b* ns **** ns hue angle (*) ns **** ns chroma ns **** ns soluble solids (°brix) ns *** ns titrable acidity (% citric acid) ns ns ns ph ** **** *** vitamin c (mg/100 g fw) ns ns ns ascorbic acid (mg/100 g fw) ns ns ns l-dehydroascorbic acid (mg/100 g) ns ns ns antioxidant activity (mg trolox/100 g fw) ns ns ns total phenol content (mg gallic acid/100 g fw) ns ns ns * when p≤ 0.05; ** when p≤ 0.01; *** when p≤ 0.001; **** when p≤ 0.0001; ns when not significant; n= 10. 84 rity affected several quality attributes (table 5). the respiration rate of full-colored peppers was about half that of peppers at a mature green stage; soluble solids increased by about 2°brix with ripening. in addition, an increase of acidity from 0.1% to 0.15% and a decrease of ph were also observed (5.93 and 4.93, respectively) (table 6). most of the differences observed at harvest were maintained during storage for yellow peppers. some differences among chemical constituents were found for yellow table 4 main effect of type of fertilization (mineral and combined) and stage of maturity (mature-green and full-colored) on mean values of quality attributes of fresh-cut yellow ‘cazzone’ peppers at harvest quality attributes type of fertilization stage of maturity mineral combined mature green full-colored firmness (n) 19.0 nss 18.3 ns s 19.2 nss 18.1 ns s respiration rate (ml co 2 kg-1 hr-1) 9.6 as s 8.8 b s 10.7 a s 7.6 b s l* 45.2 nss 46.1 ns s 39.3 b s 52.1 a s a* 0.6 a ss -1.1 b s -13.2 b s 12.7 a s b* 37.7 ns s 38.3 ns s 26.4 b s 49.6 a s hue angle (*) 95.0 ns s 97.3 ns s 116.6 a s 75.6 a s chroma 40.3 ns s 40.5 ns s 29.5 b s 51.3 a s soluble solids (°brix) 5.4 nss 5.5 ns s 4.7 b s 61.0 a s titrable acidity (% citric acid) 0.16 ns 0.14 ns 0.14 ns 0.15 ns ph 5.44 a s 5.36 b s 5.75 a s 5.04 b s vitamin c (mg/100 g fw) 59.4 ns s 58.8 ns s 61.4 nss 56.8 ns s ascorbic acid (mg/100 g fw) 58.6 ns s 58.0 ns s 60.4 nss 56.2 ns s l-dehydroascorbic acid (mg/100 g) 0.8 ns s 0.8 ns s 1.0 ns s 0.7 ns s antioxidant activity (mg trolox/100 g fw) 169.8 ns s 155.0 ns s 153.7 ns s 171.1 ns s total phenol content (mg gallic acid/100 g fw) 114.1 ns s 119.5 ns s 108.0 ns s 125.5 ns s for each row, mean values followed by a different letter are significantly different (n=10 and p≤0.05). table 5 effect of type of fertilization (mineral and combined) and stage of maturity (mature-green and full-colored), and their interaction on quality attributes of fresh-cut red ‘cazzone’ peppers at harvest quality attributes fertilization (f) stage of maturity (s) fxs firmness (n) ns ns ns respiration rate (ml co 2 kg-1 hr-1) ns ** ns l* ns **** ns a* ns **** ns b* ns *** ns hue angle (*) ns **** ns chroma ns ** ns soluble solids (°brix) ns *** ns titrable acidity (% citric acid) ns *** ns ph ns *** ns vitamin c (mg/100 g fw) ns ns ns ascorbic acid (mg/100 g fw) ns ns ns l-dehydroascorbic acid (mg/100 g) ns * ns antioxidant activity (mg trolox/100 g fw) ns ns ns total phenol content (mg gallic acid/100 g fw) ns ns ns * when p≤ 0.05; ** when p≤ 0.01; *** when p≤ 0.001; **** when p≤ 0.0001; ns when not significant; n= 10. table 6 main effect of stage of maturity (mature-green and fullcolored) on mean values of quality attributes of fresh-cut red ‘cazzone’ peppers at harvest quality attributes mature green full-colored firmness (n) 18.9 ns 18.9 ns respiration rate (ml co 2 kg-1 hr-1) 9.8 a 4.9 b l* 40.2 b 31.8 b a* -13.1 b 32.4 a b* 27.0 b 16.6 b hue angle (*) 115.9 a 27.2 b chroma 30.0 b 36.4 a soluble solids (°brix) 4.1 b 6.3 a titrable acidity (% citric acid) 0.10 b 0.16 a ph 5.93 a 4.93 b vitamin c (mg/100 g fw) 55.0 ns 52.0 ns ascorbic acid (mg/100 g fw) 53.6 ns 51.4 ns l-dehydroascorbic acid (mg/100 g) 1.4 a 0.6 b antioxidant activity (mg trolox/100 g fw) 149.1 ns 163.9 ns total phenol content (mg gallic acid/100 g fw) 107.2 ns 123.2 ns for each row mean values followed by a different letter are significantly different (n=10; p≤0.05). 85 ‘cazzone’ peppers, depending on the fertilization treatment. for this last ecotype, table 7, the main effect of the fertilization treatment over storage on quality attributes is shown. in particular, a lower antioxidant activity was observed for peppers fertilized with the combined treatment (139 mg trolox/100g fw) compared to mineral fertilization (157 mg trolox/100g fw), whereas phenol content was positively affected by the stage of maturity, showing a value 10% higher for full-colored peppers compared to mature green (table 7). the higher phenol content of fullcoloured peppers compared to mature green ones confirms that phenols and antioxidant compounds increase during ripening, as found by marin et al. (2004) and deepa et al. (2007). in addition, a higher susceptibility to decay of fresh-cut yellow peppers compared to red type was observed. as shown in figure 1, after eight days of storage the appearance score in mineral-fertilized full-colored yellow peppers dramatically decreased below the limit of marketability (score 3) due to the presence of decay and most likely to the high respiration rate, even if it was found not significant among treatments (fig. 1). for red peppers, no differences related to the fertilization treatment were observed during storage; on the other hand, the stage of maturity had effects on many attributes of fresh-cut red peppers after storage (table 8). in particular, the mature-green peppers showed a higher respiration rate (8.6 ml co 2 /kg/h) than full-colored peppers (5.9 ml co 2 /kg/h), probably causing the greater weight loss (0.9% and 0.5%, respectively). full-colored red peppers accumulated 2.4°brix with respect to the mature green fruits, also showing an increase of titrable acidity and lower ph (0.20% citric acid vs. 0.11%) which resulted in a lower ph (4.72 vs. 5.80). table 7 main effect of type of fertilization (mineral and combined) and stage of maturity (mature-green and full-colored) on mean values of quality attributes of fresh-cut yellow ‘cazzone’ peppers during storage quality attributes type of fertilization stage of maturity mineral combined mature green full-colored firmness (n) 16.5 ns 17.0 ns 15.9 ns 17.5 ns respiration rate (ml co 2 kg-1 hr-1) 9.7 ns 7.8 ns 9.2 ns 8.3 ns l* 44.6 ns 45.0 ns 39.0 b 50.6 a a* 0.4 a -0.8 b -12.6 b 12.2 a b* 37.5 ns 37.7 ns 26.4 b 48.8 a hue angle (*) 95.0 ns 96.6 a 115.6 a 76.0 b chroma 39.8 ns 39.8 ns 29.3 b 50.3 a appearence score 4.0 b 4.2 a 4.1 ns 4.0 ns soluble solids (°brix) 5.9 ns 5.8 ns 4.8 b 6.9 a titrable acidity (% citric acid) 0.15 ns 0.14 ns 0.11 b 0.18 a ph 5.26 ns 5.26 ns 5.66 a 4.86 b weight loss (%) 0.8 ns 0.7 ns 0.9 ns 0.6 ns vitamin c (mg/100 g fw) 60.9 ns 61.8 ns 58.5 ns 64.2 ns ascorbic acid (mg/100 g fw) 58.6 ns 59.3 ns 55.9 ns 62.1 ns l-dehydroascorbic acid (mg/100 g) 2.3 ns 2.5 ns 2.6 ns 2.2 ns antioxidant activity (mg trolox/100 g fw) 157.0 a 139.0 b 141.7 ns 154.3 ns total phenol content (mg gallic acid/100 g fw) 111.4 ns 106.8 ns 103.6 b 114.6 a for each row mean values followed by a different letter are significantly different (n=10; p ≤0.05). table 8 main effect of type stage of maturity (mature-green and fullcolored) on mean values of quality attributes of fresh-cut yellow ‘cazzone’ peppers during storage quality attributes mature green full-colored firmness (n) 16.5 ns 17.2 ns respiration rate (ml co 2 kg-1 hr-1) 8.6 a 5.9 b l* 39.7 a 30.9 a a* -12.6 b 31.4 a b* 26.6 a 16.3 b hue angle (*) 115.3 a 27.4 b chroma 29.5 b 35.4 a appearance score 4.2 ns 4.2 ns soluble solids (°brix) 4.4 b 6.8 a titrable acidity (% citric acid) 0.11 b 0.20 a ph 5.80 a 4.72 b weight loss (%) 0.9 a 0.5 b vitamin c (mg/100 g fw) 54.5 ns 58.8 ns ascorbic acid (mg/100 g fw) 51.8 ns 56.3 ns l-dehydroascorbic acid (mg/100 g) 2.7 ns 2.5 ns antioxidant activity (mg trolox/100 g fw) 136.6 ns 147.9 ns total phenol content (mg gallic acid/100 g fw) 96.1 ns 107.2 ns for each row mean values followed by a different letter are significantly different (n=10; p ≤0.05). 86 fig. 1 effect of storage at 5°c on appearance score, respiration rate and firmness of fresh-cut yellow and red ‘cazzone’ peppers (m=mineral fertilization; c=combined fertilization). at each storage evaluation different letters indicate significant differences among treatments (n=10; p≤0.05). 87 all red peppers received a score higher than 3 at the end of storage, without differences among treatments, whereas a lower firmness was observed for peppers fertilized with the combined treatment (fig. 1). particularly, after four days of storage, the mature-green peppers fertilized with the mineral system showed greater firmness (19.80 n) than mature-green peppers fertilized with the combined system (12.09 n), while intermediate results were observed for full-colored peppers. at eight days of storage, full-colored and mature-green peppers treated with mineral fertilization showed greater firmness than peppers fertilized with the combined treatment. for yellow peppers, no significant differences in firmness were observed. in conclusion, the results of this experiment show that the type of fertilization and the maturity stage had a different impact on quality of yellow and red peppers. in particular, for yellow full-colored peppers, the combined fertilization treatment allowed a longer shelf-life than the mineral treatment, and this should be considered when processing fresh-cut peppers. these results may also encourage further study of the feasibility of using ‘environmentally friendly’ fertilization techniques on bell peppers and eventually to extend these trials to other species. acknowledgements we thank consiglio per la ricerca e sperimentazione in agricoltura, unità di ricerca per le colture alternative al tabacco (cra-cat) for supplying the peppers. references amodio m.l., cabezas-serrano a.b., rinaldi r., colelli g., 2007 implementation of rating scales for visual quality evaluation of various vegetable crops. in: kader a.a., and m. cantwell (eds.) produce quality rating scales and color charts. postharvest horticulture series, no. 23. university of california, davis, ca, usa, pp. 151. artés-hernández f., conesa a., artés f., 2010 minimally fresh processed pepper under different kind of cuts. acta horticulturae, 857: 25-30. bender r.j., brecht j.k., baldwin e.a., maludo t.m.m., 2000 aroma volatiles of mature-green and treeripe ‘tommy atkins’ mangoes after controlled atmosphere versus air storage. hortsci., 35(4): 684-686. brand-williams w., cuvelier m.e., berset c., 1995 use of a free radical method to evaluate antioxidant activity. food sci. and technol., 28(1): 25-30. deepa n., charanjit k., binoy g., balraj s., kapoor h.c., 2007 antioxidant constituents in some sweet pepper (capsicum annuum l.) genotypes during maturity. food sci. and technol., 40(1): 121-129. fox a.j, del pozo-insfron d., lee j.h., sargent s.a., talcott s.t., 2005. ripening-induced chemical and antioxidant changes in bell peppers as affected by harvest maturity and postharvest ethylene exposure. hortsci., 40(3): 732-735. kays s.j., 1999 preharvest factors affecting appearance. postharv. biol. and technol., 15: 233-247. lee s.k., kader a.a., 2000 preharvest and postharvest factors influencing vitamin c content of horticultural crops. postharv. biol. and technol., 20: 207-220. lisiewska z., kmiecik w., 1996 effect of level of nitrogen fertilizer, processing conditions and period of storage for frozen broccoli and cauliflower on vitamin c retention. food chem., 57: 267-270. marin a., ferreres f., tomàs-barberan f.a., gil m.i., 2004. characterization and quantification of antioxidant constituents of sweet peppers (capsicum annuum l.). j. agric. food chem., 52: 3861-3869. molinari a.f., de castro l.r., antoniali s., pornchaloempong p., fox a.j., sargent s.a., lamb e.m., 1999 the potential for bell pepper harvest prior to full color development. proc. fla. state hort. soc., 112: 143-146. sams c.e., 1999 preharvest factors affecting postharvest texture. postharv. biol. and technol., 15(3): 249-254. serrano m., zapata p.j., castillo s., guillén f., martínez-romero d., valero d., 2010 antioxidant and nutritive constituents during sweet pepper development and ripening are enhanced by nitrophenolate treatments. food chemistry, 118(3): 497-503. singleton v.l., rossi j.a., 1965. colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. am. j. enol. vitic., 16(3): 144-158. tadesse t., hewett e.w., nichols m.a., fisher k.j., 2002 changes in physicochemical attributes of sweet pepper cv. domino during fruit growth and development. sci. hort., 93(2): 91-103. watada a.e., qi l., 1999 quality of fresh-cut produce. postharv. biol. and technol., 15: 201-205. zapata s., dufour j.f., 1992 ascorbic, dehydroascorbic and isoascorbic acid simultaneous determinations by reverse phase ion interaction hplc. j. food sci., 57(2): 506-511. impaginato 37 adv. hort. sci., 2011 25(1): 37-43 received for publication 4 march 2011. accepted for publication 15 march 2011. foliar application of molybdenum: effects on yield quality of the grapevine sangiovese (vitis vinifera l.) e. masi*, m. boselli** * dipartimento di scienze delle produzioni vegetali, del suolo e dell’ambiente agroforestale, università degli studi di firenze, viale delle idee, 26, 50019 sesto fiorentino (fi), italy. ** dipartimento di biotecnologie, università degli studi di verona, villa lebrecht, via della pieve, 70, 37029 san floriano (vr), italy. key words: grapevine, foliar spray, molybdenum, yanc. abstract: field experiments with sangiovese vines were carried out in the chianti classico region over a period of two years to examine the effect of molybdenum (mo) foliar sprays on nutrient composition of leaves, petioles and berries, leaf gas exchanges, must composition, total yield, bunch size and pruning weight. two mo foliar doses and time sprays (mox1: one application in early flowering; mox3: three applications in early flowering, early fruit set and veraison) were applied. basal sample of petioles, leaves and berries collected at fruit set (except berries), veraison and harvest for mineral analyses showed not relevant interactions between mo and the main macro and micronutrients. leaf gas exchanges monitored after the applications, as well as spad units, showed a higher activity in the mox3-treated vines. increased vigour was also confirmed by the slightly higher total yield, bunch size and pruning weight, as well as the delay in fruit maturation (lower sugar and polyphenol contents at harvest). no relevant discrepancy between mox1 and the control was found, except for higher soluble solid and yeast-assimilable nitrogen contents (yanc) in the treated vines. yanc was positively influenced also in the mox3 vines, however with no significant differences towards the mox1 treatment. the application of mo as a useful tool to stimulate nitrogen metabolism, as well as indications about dose and time of mo application, are discussed. 1. introduction the transition element molybdenum (mo) is a very rare but essential micronutrient for all organisms (bortels, 1930; fortescue, 1992), and understanding of its role and function in plants is progressing rapidly. its importance for plants has been known for a long time (coughlan, 1980), even though mo itself seems to be catalytically inactive in biological systems until it is complexed by a special cofactor, the pterin (mendel and hänsch, 2002) which binds to diverse apoproteins. this latter compound is a unique pterin named molybdopterin or metal-containing pterin. in this form, it occurs in more than 40 enzymes catalysing many redox reactions, four of which have been found in plants (hille, 1996; kappl et al., 2002). one of these is nitrate reductase (nr) that catalyses the first step in nitrate assimilation, a pathway of key importance for plant nutrition. nitrate reductase is the key-enzyme for nitrate assimilation while nitrogenase is found in nitrogen-fixing bacteria inside nodules of symbiotically growing species. the last step of abscisic acid biosynthesis is catalyzed by the molybdenum-enzyme aldehyde oxidase, and sulfite oxidase protects the plant against toxic levels of sulfite (hänsch and mendel, 2009). nitrogen (n) is assimilated into the cell as a fully reduced form, ammonia (nh3). nh3 may be obtainedin free form (e.g. by fertilizers) and from the degradation of amino acids. if these sources are not available, then ammonia must be produced via nitrate (no3)reduction. when nr is a limiting factor, plant growth, development and protein synthesis by plants are reduced (solomonson and barber, 1990). hence a shortage of mo in the soil, even though plants’ requirement for it is very low, or a mutational block of the cellular ability to use mo leads to the loss of essential metabolic functions and can cause the death of the plant. mo deficiency has been reported for many plant species including herbs, crops and trees (gupta, 1997) and could determine poor nr activity (hewitt, 1983), causing an inability to utilize n, with visible symptoms of chlorosis or yellowing of the leaves. 38 in grapevine, n is essential in overall vine establishment and maintenance, fruit quality, and the conversion of grape juice to wine. in grape berries, n is found primarily as ammonium cations and organic compounds such as amino acids (proline, arginine, glutamine, glutamate, etc.), hexose amines, peptides, nucleic acids and proteins. there are two phases of intense n incorporation in the fruit: the first takes place during the two weeks before the “pea-size” stage of the berries; the second starts one month later at veraison and lasts an additional two weeks (löhnertz, 1991). the amount of n in the clusters at harvest is approximately 40-44% of total available n of the entire plant (alexander, 1957; conradie, 1991; weinbaum et al., 1984). furthermore, n compounds are required by yeast for the production of cell biomass and the synthesis of proteins and enzymes necessary for the biochemical process of fermentation. the readily (easily assimilable) fermentable n compounds in juice and must consist primarily of nh3 and n available from the alphaamino acids present at harvest, particularly arginine (bisson, 1991). low levels of yeast-assimilable n content (yanc) in grape juice and must at harvest have been associated with sluggish and stuck fermentation and consequent undesirable levels of residual sugar in wines (kunkee, 1991; jiranek et al., 1995). slow and stuck fermentations are sometimes associated with grapes from vineyards composed of not vigorous plants or with high planting density, clearly characterized by scarce availability of n (masi and boselli, 2007). in some cases, despite plants having a sufficient level of n in their tissues, plant growth and crop yield show a clear n deficiency making it possible to hypothesize that most of the n in the plant is stored in non available forms. however, fertilizations with n are considered to be difficult, especially concerning choice of the right dose for optimum plant and yeast growth: even a low dose of n frequently causes excessive canopy growth and an unacceptable delay in fruit maturation being, at the same time, not sufficient in order to assure an optimal assimilable n concentration of the juice (spayd et al., 1995). assuming the inadequacy to reach optimal levels of available forms of n by chemical fertilization, mo supply could be a possible solution since its key role in the activation of n metabolism could be compared to a n fertilization itself. moreover, mo application has been shown to increase fruit quality, sugar content (rus’-ko, 1979; strakhov, 1988) and qualitative and quantitative composition of free amino acids (veliksar, 1977) in grapevine. mo is important in nitrogen nutrition of vines and has been suggested as a primary cause of millerandage in merlot vines (longbottom et al., 2004; williams et al., 2004). it is thought that mo directly affects the development of reproductive structures. molybdenum is necessary for successful pollen tube growth, ovule penetration, and fertilization (longbottom et al., 2004). williams et al. (2004) showed that mo increased the percent of coloured berries with one or more functional seeds and decreased the proportion of green berries, thus suggesting that mo application affected pollination and/or fertilization, and thereafter berry development. the present study, comprising two different levels and time of mo supply, was undertaken to determine the effect of mo on n metabolism by studying fruit and vegetative development in the grapevine sangiovese (vitis vinifera l.). 2. materials and methods plant material the study was carried out in a six-year-old vineyard of sangiovese vitis vinifera (l.) cultivar, grafted onto berlandieri x riparia 420a, located in the chianti classico docg region (elevation about 380 m asl; weather was characterized by mild winter, hot and dry season during the summer and average yearly rainfall of 700 mm, with the highest levels in november and march). the vines were spaced 0.8 m within the row and 2.4 m between rows and pruned to a single cordon system with four spurs, with an average of eight buds growing off the cordon. soil was rich in clay and “skeletal” material. pre-planting studies showed that the soil chemical fertility was good for most of the elements except for n (table 1). for that reason, every year, soil fertilizations rich in n (400 kg of npk-fertilizer 12+10+20) had been executed since the vineyard was established; although tissue analysis did not show any mineral deficiency, neither for microelements nor macroelements (fig. 1), light symptoms of n-deficiency were present on the vines at the moment the experiment was performed, consisting in low vigour and poor yanc must content in the previous vintages. treatments during two consecutive years (2002-2003), the vineyard was divided into three homogeneous areas, of six rows each, for two mo treatments and a control table 1 soil chemical analysis soil parameter ph (h2o) electrical conductivity total lime (caco3) active lime (caco3) cation exchange capacity organic matter n ca mg k (k2o) p (p2o5) fe mn b 8.2 126.5 µs ·cm-1 7.6% 0.7% 18.5 me·100g-1 0.9% 0.4% 1157 mg·kg-1 134 mg·kg-1 146 mg·kg-1 12.5 mg·kg-1 78.3 mg·kg-1 85.7 mg·kg-1 2.8 mg·kg-1 39 (untreated vines). foliar applications were sprayed using 350 g·ha-1 of a custom-made mo fertilizer (12% w/w) dissolved in 200 l of water. the first treatments provided a single application in early flowering (mox1); the second provided three applications, respectively in early flowering, early fruit set and early veraison (mox3). recommended cultural practices of the vineyard were applied. data collection three basal leaf and petiole samples were collected (at fruit set, veraison and harvest); two berry samples were also collected from veraison to harvest. each sample was the result of 50 to 60 organs harvested from random positions among the six rows of each treatment area. the material was rinsed with distilled water in order to take away any pesticide or fertilizer traces, then oven dried at about 80°c until thoroughly dry. each dried sample was then ground and sent to the laboratory for chemical analysis of macro and microelements; the analyses were performed three times and results averaged. gas exchange measurements were taken after veraison on 25 replications obtained by selecting five leaves on five plants per treatment: the leaves were of the same age, spatial orientation and light exposure. net photosynthetic rate (pn) and transpiration rate (e)were recorded with a portable photosynthesis system (ciras-i, ppsystems, hertfordshire, uk). leaves were enclosed in a ventilated leaf cuvette and exposed to saturating irradiance (par≥1200 µmol·m-2); concentration of co2 used for the measurements was 350mg·l-1. in addition, water user efficiency (wue) was calculated as the rate of net photosynthesis per unit of transpired water. according to the same statistical design, after veraison (and 30 days later) on the same plants and leaves leaf chlorophyll and nitrate content (westerveld et al., 2003) was estimated non-destructively using a portable chlorophyll meter (spad-502, minolta corp., ramsey, n.j., u.s.). at fruit set, veraison and harvest, berry samples were picked randomly from 10 plants per treatment, crushed, pressed and the juice analyzed for the yeast fermentable nitrogen content. the yanc is expressed as mg·l-1 of n as the sum of the assimilable nitrogen from ammonia plus the assimilable nitrogen from alpha amino acids. fig. 1 primary macronutrient (n, p, k) concentrations in basal leaf, petiole and berry at fruit set, veraison and harvest in mo-treated and untreated grapevine plants. error bars reflect the lsd at p ≤ 0.05. mox1= mo treatment in early flowering; mox3= mo treatments in early flowering, early fruit set and early veraison. 40 at harvest, 300 berries were collected, weighed and then squeezed by hand and filtered through a strainer. samples were harvested randomly from 10 plants (30 berries per plant) per treatment. the expressed juice was measured to determine total soluble solids (brix) with a hand refractometer and titratable acidity by titration with 0.1 n naoh to ph 7. titratable acidity was expressed by g·l-1 of tartaric acid. anthocyanin content of the must was performed via glories indices (glories, 1984). indices evaluated were: total anthocyanins a1 (mg·l-1);extractable anthocyanins a3.2 (mg·l-1); extractabilityassay ea (based on the ratio of the above two antho-cyanin values); and extractability of the tannins contained in the seed mp.during the season, five plants per treatment were labelled; they were harvested separately in order to calculate average yield per vine and their pruning materials were weighed as an indicator of the vegetative vigour. the ratio of total yield to total pruning weight per vine, referred to as the ravaz index (champagnol, 1984) was used as an indicator of balance between fruit and vegetative growth. statistical analysis of variance on the obtained data was performed with separation of the means by lsd test at 5%. no year effect was observed, thus data shown are the average of the measurements of two years. 3. results tissue analysis showed in general no deficiency or excess in the levels. the effect of applied mo on the concentration of the nutrients in basal petioles, leaves and berries was small and of little practical importance. nitrogen (n) content of leaf, petiole and berry compared with control treatment was not influenced by mo (fig. 1 a). leaf and petiole phosphorous (p) content tended to decrease in leaf and petiole after mo supply, especially in the mox3 treatment (fig. 1 b), while berry content was not influenced. on the contrary, potassium (k) level was significantly higher in berries of motreated plants (fig. 1 c) at harvest. calcium (ca) content was not affected by any kind of treatment in all analyzed organs (fig. 2 a), while petiolar and leaf manganese (mn) tended to increase (fig. 2 b), especially at harvest time and for mox3 treatment. moreover, for the same treatment, a higher and significant level of mn was found in berries during both measurements (at veraison and harvest time). no significant differences were detected for the level of iron (fe) (fig. 2 c). co2 assimilation rate of mox3-treated vines wassignificantly higher than those of the mox1-treated and untreated vines (fig. 3 top). mox3 showed also relevant difference towards the other treatments in transpifig. 2 secondary macro (ca) and micronutrient (mn, fe) concentrations in basal leaf, petiole and berry at fruit set, veraison and harvest in motreated and untreated grapevine plants. error bars reflect the lsd at p ≤ 0.05. mox1= mo treatment in early flowering; mox3= mo treatments in early flowering, early fruit set and early veraison. 41 ration rate (fig. 3 middle); as a result of the higher transpiration rate, however, the mox3 vines showed more critical water use efficiency values (fig. 3 middle). accordingly, the spad unit measurements were significantly influenced by mo treatments (fig. 3 bottom). mox3 showed the highest values in both sampling data; the values decreased for mox1 treated vines after veraison, although remained higher than those of the control plants. fig. 3 top: assimilation rates measured after veraison in mo-treated and untreated grapevine plants. middle: transpiration rates and water use efficiency measured after veraison in mo-treated and untreated grapevine plants. bottom: spad units measured after veraison and a month later in mo-treated and untreated grapevine plants. error bars reflect the lsd at p ≤ 0.05. different letters correspond to significant differences (p ≤ 0.05). mox1= mo treatment in early flowering; mox3= mo treatments in early flowering, early fruit set and early veraison. the yanc content increased significantly after the applications showing the positive role of mo on nr activity to provide an available form of n for the plant (table 2). furthermore, at veraison, after more than a month from the first (and single, for mox1) application, both mox1 and mox3 berries had higher levels of yanc compared to the control ones. surprisingly, no significant difference was found between the two mo treatments, not showing a linear correlation between mo dose and nr activity. in any case at harvest time, no effect was found for either of the mo treatments: the difference tended to decrease during berry ripening, disappearing at harvest when both the treated and untreated vines had quite the same amount of available n. significant differences in must soluble solids (ss) analysis at harvest were observed between mox1 treatment (higher value) and both mox3 and control treatments (lower values). titratable acidity (ta) and ph levels were similar for treated and untreated vines (table 3). total anthocyanin content was significantly greater in the untreated vines compared to the treated ones while extractable anthocyanins in mox1-treated and control plants were similar and higher than in mox3-treated vines (table 3). mox1-treated vines must extractability assay and mp value suggest a betterpolyphenolic evolution during ripening. mox3 treatment negatively influenced anthocyanin extractability and polyphenol evolution as confirmed by the highest value of mp. table 2 yeast assimilable nitrogen content (yanc) yanc (mg·l-1 n) mox1 mox3 untreated 96 a 92 a 74 b fruit set veraison harvest 66 a 62 a 34 b 68 ns 64 ns 55 ns different letters within the same column correspond to significant differences (p ≤ 0.05). non-significant results are marked ns. mox1= mo treatment in early flowering; mox3= mo treatments in early flowering, early fruit set and early veraison. table 3 berry soluble solids (ss), ph, titratable acidity (ta), berry glories indices (a1: total anthocyanins; a3.2: extractable anthocyanins; ea: extractability assay; mp: extractability of the tannins contained in the seed analysis results) technol. analysis mox1 mox3 untreated 23.9 a 22.1 b 22.6 b ss (brix) ph ta (g·l-1) 3.1 ns 3.0 ns 3.0 ns 7.3 ns 7.5 ns 7.3 ns different letters within the same column correspond to significant differences (p ≤ 0.05). non-significant results are marked ns. mox1= mo treatment in early flowering; mox3= mo treatments in early flowering, early fruit set and early veraison. polyphenol. analysis a1 (mg·l-1) a3.2 (mg·l-1) ea (%) mp (%) mox1 mox3 untreated 1535 b 1437 b 2038 a 1007 a 706 b 1005 a 34.4 b 50.9 a 50.7 a 29.6 b 40.5 a 32.0 b 42 the different ripening development between mox3 and untreated vines probably has to be linked to the influence of mo on the fruit and vegetative activity of the plant, suggested also by the average bunch weights (fig. 4 right) and the total pruning weight, which were higher in the mox3 treatment (fig. 4 left): plants treated with higher dose of mo had greater difficulty completing the ripening process within the same time as the other vines. no significant differences were however revealed with regard to total yield per vine (data not shown) and ravaz index (fig. 4 left). 4. discussion and conclusions the present study supports earlier suggestions that mo affects the n metabolism in grape, whose unquestionable evidence is in the yanc analysis of the berry. the fact that the reaction of the plants to mo applications was measurable, in terms of yanc, as early as a few days after the first treatments performed before fruit set gives interesting indications about its rate of speed. the absence of differences in yanc content between the two mo treatments suggests that when the general plant availability of n is good (like in this case), higher doses of mo result to be useless. this hypothesis, therefore, is not confirmed by the analysis of the bunch weight and total yield per vine nor in the development of the ripening process, when relevant differences were found between the two mo treatments, as well as a clearly positive correlation between the quantity of mo supplied and the appearance of such phenomena, well known to be connected with an increased n-availability for the plant. in fact, mo, especially when supplied at high rate, is able to increase plant activity (see results about gas exchange analysis) as the result of rendering the n of the plant more available for the metabolisms and, consequently, plants tend to be generally more vigorous. finally, important information about fertilization management can be underlined: a high dose treatment (mox3) seems to be inadequate and sometimes contrary to the quality of production. mox1-treated vines, on the other hand, did not show an influence of the treatment on vegetative and fruit balance nor on phenolic evolution and, interestingly, differentiated positively from the control regarding the sugar content of the berry and the yanc, even though its level at harvest was not optimal. assuming the mox1 dose and time optimal for quality, further analysis should be aimed at correlating the role of mo on nr activity towards n availability in order to understand how to increase must quality. references alexander d., 1957 seasonal changes in the nitrogen content of the sultana vine. aust. j. agr. res., 8: 162-178. bisson l.f., 1991 influence of nitrogen on yeast and fermentation of grapes, 128-131. in: rantz j.m. (ed.). proceedings of the international symposium on nitrogen in grapes and wines. am. soc. enol. vitic., davis, ca, usa. bortels h., 1930 molybdan als katalysator bei der biologischen stickstoffbindung. archiv für mikrobiologie, 1: 333342. champagnol f., 1984 élements de physiologie de la vigne et de viticulture générale. saint-gély-du-fesc, france, pp. 351. conradie w.j., 1991 distribution and translocation of nitrogen absorbed during early summer by two-year-old grapevines grown in sand culture. am. j. enol. viticult., 42(3): 180-190. coughlan m.p., 1980 aldehyde oxidase, xanthine oxidase and xanthine dehydrogenase: hydroxylases containing molybdenum, iron-sulfur and flavin, 119-185. in: coughan m.p. (ed.) molybdenum and molybdenum-containing enzymes. pergamon press, oxford, uk. fortescue j.a.c., 1992 landscape geochemistry: retrospect and prospect 1990. appl. geochem., 7: 1-53. glories y., 1984 la couleur des vins rouges. 2e. partie: mesure, origine et interpretation. conn. vigne vin, 18(4): 253-271. gupta u.c., 1997 soil and plant factors affecting molybdenum uptake by plants, 71-91. in: gupta u.c. (ed.) molybdenum in agriculture. cambridge university press, cambridge, uk. hansch r., mendel r.r., 2009 physiological functions of mineral micronutrients (cu, zn, mn, fe, ni, mo, b., cl). curr. fig. 4 bunch weight (left), total pruning weight and ravaz index (right) measured in mo-treated and untreated grapevine plants. right: error bars reflect the lsd at p ≤ 0.05. different letters correspond to significant differences (p ≤ 0.05). non-significant results are marked ns. 43 opin. plant biol., 12(3): 259-266. hewitt e.j., 1983 molybdenum requirements of plants, pp. 277-330. in: robb d.a., and w.f. pierpoint (eds.) metals and micronutrients. uptake and utilisation by plants. pergamon press, oxford, uk. hille r., 1996 the mononuclear molybdenum enzymes. chem. rev., 96: 2757-2816. jiranek v., langridge p., henschke p.a., 1995 amino acid and ammonium utilization by saccharomyces cerevisiae wine yeast from a chemically defined medium. am. j. enol. viticult., 46(1): 75-83. kappl r., hüttermann j., fetzner s., 2002 the molybdenum-containing hydroxylases of quinoline, isoquinoline, and quinaldine, 481-538. in: sigel a., and h. sigel (eds.) molybdenum and tungsten. their roles in biological processes. marcel dekker, new york, usa. kunkee r.e., 1991 relationship between nitrogen content of must and sluggish fermentation, pp. 148-155. in: rantz j.m. (ed.) proceedings of the international symposium on nitrogen in grapes and wines. am. soc. enol. vitic., davis, ca, usa. löhnertz o., 1991 soil nitrogen and the uptake of nitrogen in grapevines. international symposium on nitrogen in grapes and wine, seattle, washington, pp. 1-11. longbottom m., dry p., sedgley m., 2004 foliar application of molybdenum pre-flowering: effects on yield of merlot. aust. grapegrower, 491: 36-39. masi e., boselli m., 2007 molibdeno si, ma con misura. vq, 1: 54-58. mendel r.r., hänsch r., 2002 molybdoenzymes and molybdenum cofactor in plants. j. exp. bot., 53(375): 16891698. rus’-ko e.a., 1979 the effect of soil and foliar nutrition with minor element on grapevine growth and productivity. sadovodstvo vinogradarstvo i vinodelie moldavii, 1: 30-32. solomonson l.p., barber m.j., 1990 assimilatory nitrate reductase: functional properties and regulation. annu. rev. plant physiol. plant mol. biol., 41: 225-253. spayd s.e., nagel c.v., edwards c.g., 1995 yeast growth in riesling juice as affected by vineyard nitrogen fertilization. am. j. enol. viticult., 46(1): 49-55. strakhov v.g., 1988 trace elements fertilizer application in vineyards. vliyanie udobrenii na obmen veshchestv i produktivnost’ rastenii, pp. 153-160. veliksar s.g., 1997 the effect of molybdenum on the changes in the aminoacid composition of grapevine shoots. sadovodostvo, vinogradarstvo i vinodelie moldavi, 5: 25-27. weinbaum s.a., klein i., broadent f.e., micke w.c., muraoka t.t., 1984 effects of time of nitrogen application and soil texture on the availability of isotopically labelled fertilizer nitrogen to reproductive and vegetative tissue of mature almond trees. j. am. soc. hortic. sci., 109: 339-343. westerveld s.m., mckeown a.w., mcdonald m.r., scott-dupree c.d., 2003 chlorophyll and nitrate meters as nitrogen monitoring tools for selected vegetables in southern ontario. acta horticulturae, 627: 259-266. williams c.m.j., maier n.a., bartlett l., 2004 effect of molybdenum foliar sprays on yield, berry size, seed formation, and petiolar nutrient composition of ‘‘merlot’’ grapevines. j. plant nutr., 27: 1891-1916. impaginato 269 adv. hort. sci., 2021 35(3): 269­275 doi: 10.36253/ahsc­10193 effect of vine and fruit pruning on yield attributes of two watermelon (citrullus lanatus) cultivars a. ndereyimana 1 (*), b.w. waweru 1, b. kagiraneza 1, a.n. niyokuri 2, p. rukundo 1, g. hagenimana 1 1 rwanda agriculture and animal resources development board (rab), agriculture innovation and technology transfer department, p.o. box 5016, kigali, rwanda. 2 department of crop sciences, college of agriculture, animal sciences and veterinary medicine, university of rwanda, p.o. box 210, musanze, rwanda. key words: apical shoot pinching, cucurbits, fruit pinching, fruit size, hybrid. abstract: this study was carried out to determine the effect of vine and fruit pruning on watermelon (citrullus lanatus) yield. five pruning methods: p1=no pruning at all, p2=pruning to four vines with two fruits per vine, p3=pruning to four vines with one fruit per vine, p4=pruning to three vines with two fruits per vine and p5=pruning to three vines with one fruit per vine were evaluated on two watermelon cultivars: ‘sugar baby’ and ‘julie f1’ under a factorial random­ ized complete block design with three replications. investigations were carried out in the seasons 2017a (short rains) and 2017b (long rains) at karama and rubona experimental sites belonging to rwanda agriculture and animal resources development board. the obtained results indicated a significant dif­ ference among the different cultivars and pruning methods tested during both seasons and at two sites. generally, all studied parameters recorded higher val­ ues during season 2017b than in season 2017a at rubona site. a similar trend was recorded at karama site except that the fruit yield per plant and per hectare for plants which were pruned to three vines with one fruit reduced dur­ ing season 2017b as compared to season 2017a. the highest number of fruits per plant, fruit weight, fruit yield per plant and per hectare was recorded in ‘julie f1’ compared to ‘sugar baby’ at both sites and during both seasons. higher fruit weight was obtained when both cultivars were pruned to three or four vines with one fruit per vine. higher number of fruits per plant and higher fruit yield per plant was observed under pruning to four vines with two fruits per vine at rubona site; while at karama site, higher fruit yield per plant was recorded under pruning to three vines with one fruit or two fruits per vines and pruning to four vines with two fruits per vine. a similar trend was observed in fruit yield per hectare. based on results of the current study, cultivation of the hybrid ‘julie f1’ and pruning to three vines with one fruit per vine is recom­ mended for optimum watermelon yield with big­sized fruits. 1. introduction watermelon is a crop belonging to the cucurbitaceae family that has gained a great economic importance due to its delicious fruits that are also rich in various nutritional compounds (kong et al., 2017). during the (*) corresponding author: assinapol.ndereyimana@rab.gov.rw citation: ndereyimana a., waweru b.w., kagiraneza b., niyokuri a.n., rukundo p., hagenimana g., 2021 ­ effect of vine and fruit pruning on yield attributes of two watermelon (citrullus lanatus) cultivars. ­ adv. hort. sci., 35(3): 269­275 copyright: © 2021 ndereyimana a., waweru b.w., kagiraneza b., niyokuri a.n., rukundo p., hagenimana g. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 27 december 2020 accepted for publication 16 july 2021 ahs advances in horticultural science https://doi.org/10.36253/ahsc-10193 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2021 35(3): 269­275 270 year 2018, the worldwide watermelon production w a s 1 0 3 , 9 3 1 , 3 3 7 t h a r v e s t e d o v e r a n a r e a o f 3,241,239 ha with the yield of 32.1 t ha­1 (fao, 2020); while in east africa, it was 230,729 t from an area of 12,110 ha with the yield of only 19.1 t ha­1. the yield gap of 13 t ha­1 compared to the world’s yield can be addressed by improving production practices, includ­ ing regulation of number of vines and fruits per plant (oga and umekwe, 2016; dube et al., 2020). as other cucurbits, watermelon is also a crop with vines on which female flowers appear after about every five male flowers (dube et al., 2020). the num­ ber of vines per plant is an important parameter determining the performance of cucurbitaceae crops, including watermelon (gomes et al., 2019). on the other side, the number of fruits per vine is also an important parameter that determines fruit size, mass and yield (lins et al., 2013). therefore, due con­ sideration given to these aspects of cucurbit manage­ ment is of utmost importance. in cucurbits, regula­ tion of number of vines per plant and fruits per vine can be achieved through different methods including vine and fruit pruning (campos et al., 2019). pruning is a special horticultural practice that is carried out by removing some parts of plant to boost flowering and subsequent fruiting. this leads to i m p r o v e d y i e l d w i t h e n h a n c e m e n t i n f e a t u r e s required by consumers, such as fruit size and appear­ ance (oga and umekwe, 2016). the main purpose of pruning is to promote balance between vegetative growth and fruit load (anwar et al., 2019). pruning has been reported to increase yield of cucumber (nayak et al., 2018) and butternut (eve et al., 2016). pruning is also a common practice in watermelon production that can lead to synchronization of har­ vesting period and production of uniform fruits (oga and umekwe, 2016). in east african countries, limited investigations have been carried out on watermelon as compared to legume and cereal crops (dube et al., 2020). besides, east african farmers rarely practice water­ melon pruning due to limited knowledge on appro­ priate pruning method. the study was carried out to determine the effect of vine and fruit pruning on yield attributes of two watermelon cultivars. 2. materials and methods sites and seasons of study field experiment was carried out at karama and rubona experimental sites belonging to rwanda agriculture and animal resources development board (rab). karama site is located in eastern province, bugesera district, on longitude 02°23’15”s, latitude 30o11’27”e, and at an altitude of 1524 m above sea level (ndabamenye et al., 2013; kabirigi et al., 2017). its annual average rainfall is 850­1100 mm and the average temperature is 20­21oc (verdoodt and van ranst, 2003). rubona site is located in southern province, huye district, on longitude 029o46’475”e, latitude 02o29’327”s, and at an altitude of 1727 m above sea level (ndabamenye et al., 2013; kabirigi et al., 2017). the annual average rainfall at rubona is 1100­1400 mm and the average tempera­ ture is 17­20oc (verdoodt and van ranst, 2003). at both sites, the study was conducted in two cropping seasons: season 2017a (short rain season) covering the period from september to december 2016 and season 2017b (long rain season) from february to may 2017. study design and treatments the study was conducted in a factorial randomized complete blocks design (rcbd) where treatments were replicated thrice. the treatment structure con­ sisted of five different pruning methods (factor one) evaluated on two watermelon cultivars (factor two). the five pruning methods were designed as follows: p1=no pruning at all (control), p2=pruning to four vines with two fruits per vine, p3=pruning to four vines with one fruit per vine, p4=pruning to three vines with two fruits per vine, and p5=pruning to three vines with one fruit per vine. the two studied watermelon cultivars were c1: ‘sugar baby’ and c2: ‘julie f1’. these cultivars were selected because of their high yield potential under breeder’s conditions. moreover, ‘julie f1’ is a popular hybrid among farm­ ers in east african region. in total, ten treatment com­ binations (cultivar x pruning method) were studied. trial establishment and maintenance field was ploughed twice at an interval of two weeks and was subdivided into plots of 6 m x 3 m that were mulched using dry grass. there were ten plots in each replication; and each plot received a treatment combination of cultivar and pruning method. watermelon seedlings for each cultivar were raised in biodegradable pots and transplanted 30 days after sowing at a spacing of 3 m x 2 m. pruning was achieved by pinching the apical shoot of watermelon vine after six nodes, about 18 days after ndereyimana et al. ‐ effect of vine and fruit pruning on yield attributes of two watermelon cultivars 271 transplanting (anwar et al., 2019). then, at 15 days after pinching, four vines were maintained for p2 and p3 treatments while three vines were maintained for p4 and p5. thereafter, two fruits were maintained per vine for p2 and p4 while one fruit was maintained per vine for p3 and p5. for all the treatments where pruning was carried out, one of the maintained vines was not allowed to have fruits for it to support other in feeding their fruits. apart from watermelon cultivars and pruning methods, other crop management practices such as fertilization, watering, weeding, pests and diseases management were carried out as recommended. fertilization was conducted by applying organic manure and the inorganic fertilizer n:p:k at the rates of 25 t ha­1 and 90:60:60 kg ha­1, respectively. the inorganic fertilizer npk 17­17­17 was used to supply 60:60:60 kg ha­1, which was given in two equal splits (at transplanting and at one month after transplanti­ ng); while additional 30 kg of n ha­1 was supplied in form of urea 46% and was given at two months after transplanting. where necessary, copper oxychloride and carbendazim pesticides were sprayed to control diseases while lambda­cyhalothrin and imidacloprid were used against pests. data collection and analysis observations were carried out on four randomly selected plants per each treatment combination. data were recorded on number of fruits per plant, fruit weight, fruit yield per plant and fruit yield per hectare. the number of fruits per plant was obtained at harvesting through counting all available fruits on four selected plants per treatment combination fol­ lowed by calculating the average per plant. fruit weight (kg) was achieved as the average of five fruits randomly selected from the fruits harvested on the four pre­selected observational plants. fruit yield per plant (kg) was recorded as the average weight of all fruits harvested from four observational plants in the plot under consideration. fruit yield per hectare was derived from computation using the data on fruit yield per plant. analysis of variance for the collected data was performed using genstat 14th edition soft­ ware package and the level of significance was set at p<0.05. least significant difference (lsd) test was conducted for pair­wise comparisons of means. 3. results the number of fruits per plant differed significant­ ly (p<0.05) between cultivars in both seasons (2017a and 2017b) at karama site. significant differences among pruning methods were recorded only in sea­ son 2017a at karama and in both seasons at rubona. between the two cultivars, the highest number of fruit per plant was recorded in ‘julie f1’ (3.5­4.0 fruits per plant) compared to ‘sugar baby’ (3.4­3.7 fruits per plant). for pruning method, the highest number of fruit per plant (4.1­4.8 fruits per plant) at karama site was recorded when both watermelon cultivars were pruned to four vines with two fruits per vine (table 1). a similar trend was also observed at rubona where both watermelon cultivars were pruned to four vines with two fruits per vine record­ ed the highest number of fruits per plant (4.8­5.0) (table 2). there was no significant interaction among cultivars and pruning methods on the number of fruits per plant. f r u i t w e i g h t d i ff e r e d s i g n i fi c a n t l y ( p < 0 . 0 5 ) between cultivars and among pruning methods. the highest fruit weight was recorded in plots where ‘julie f1’ was planted at both sites and it varied between 3.8­3.9 kg per fruit at karama site (table 1) and 3.4­3.8 kg per fruit at rubona site (table 2). among pruning methods, the highest fruit weight (4.2­4.3 kg at karama and 3.7­4.1 kg at rubona) was obtained when both cultivars were pruned to three vines with one fruit per vine during season 2017a and 2017b (table 1 and 2). the interaction of culti­ vars and pruning methods on fruit weight in both 2017a and 2017b seasons at rubona and at karama during the season 2017a was significantly different. however, it was not significantly different in the sea­ son 2017b at karama site. higher fruit weight (4.1 4.6 kg per fruit) was obtained under ‘julie f1’ pruned to three vines with one fruit per vine, followed by the same cultivar pruned to four vines with one fruit per vine (3.7­4.2 kg per fruit) (tables 1 and 2). fruit yield per plant and per hectare was signifi­ cantly (p<0.05) different between cultivars and among pruning methods and their interaction in both seasons 2017a and 2017b, and at karama and rubona sites was not relevant. ‘julie f1’ recorded sig­ nificantly higher fruit yield per plant (11.9­15.4 kg) and per hectare (59.6t ha­1 ­76.9t ha­1) compared to sugar baby which recorded 9.5­11.2 kg per plant and 47.5t­59.1t per hectare. among the pruning meth­ ods, the highest fruit yield per plant (13.9­15.9 kg) and yield per hectare (69.3 t ha­1 ­78.0 t ha­1) was observed under pruning to four vines with two fruits per vine at rubona site (table 2). at karama site, adv. hort. sci., 2021 35(3): 269­275 272 higher fruit yield per plant (15.8 kg) and per hectare (78.8 t ha­1) was recorded under pruning to three vines with one fruit per vine during season 2017 a (tables 1) while during season 2017b, higher fruit yield per plant and per hectare was recorded on watermelon plants that were pruned to three vines with one fruit per vine or four vines with one fruit or two fruits per vine (14.2­15.8 kg per plant and 71.1 t ha­1 ­78.9 t ha­1) (table 1). 4. discussion and conclusions among others, crops of cucurbitaceous family can be manipulated by altering the source: sink ratio through stem and fruit pruning, which affects the number of leaves per plant and consequently leaf area (source) and demand for photoassimilates (sink) (queiroga et al., 2008). in this family, more pruning studies have been carried out on other crops such as cucumber (nayak et al., 2018) and butternut (eve et al., 2016); there is limited information on this aspect as far as watermelon is concerned. therefore, the current study contributed valuable knowledge on watermelon management in a view to optimize its production. heavier fruits obtained under watermelon plants (julie f1) pruned to three or four vines with one fruit per vine fall in line with lins et al. (2013) who report­ ed that fruit thinning allows larger amounts of pro­ duced photo­assimilates to be used by few fruits causing them to attain a size demanded on market. similar to results of the current study, dhillon et al. (2017) also obtained heavier fruits on pruned cucum­ table 1 ­ performance of watermelon as affected by cultivars and pruning method, karama site c1=sugar baby, c2=julie f1, p1=no pruning, p2= pruning to 4 vines with 2 fruits per vine, p3= pruning to 4 vines with 1 fruit per vine, p4= pruning to 3 vines with 2 fruits per vine, p5= pruning to 3 vines with 1 fruit per vine, 2017a=short rains, 2017b=long rains. sed= standard error difference. * and ** =significant difference at 5% and 1% respectively, ns=not significantly different (p>0.05). means within each column followed by the same letter are not significantly different as per lsd test at p<0.05. treatments no. of fruits per plant fruit weight (kg) fruit yield (kg per plant) fruit yield ( t ha­1) season 2017a season 2017b season 2017a season 2017b season 2017a season 2017b season 2017a season 2017b factor 1: cultivar (c) c1 3.4 b 3.7 b 3.2 b 3.2 b 10.8 b 11.2 b 54.1 b 59.1 b c2 3.8 a 4.0 a 3.8 a 3.9 a 14.8 a 15.4 a 74.0 a 76.9 a significance *** *** *** *** *** *** *** *** sed 0.095 0.159 0.060 0.063 0.439 0.644 2.19 3.22 factor 2: pruning method (p) p1 3.0 c 3.4 2.8 d 2.9 e 8.6 c 10.0 c 43.2 c 49.7 c p2 4.1 a 4.8 3.2 c 3.3 d 13.5 b 15.8 a 67.3 b 78.9 a p3 3.5 b 3.4 3.8 b 3.8 b 13.4 b 13.2 b 67.0 b 65.7 b p4 3.6 b 4.0 3.5 c 3.5 c 12.8 b 14.2 ab 63.8 b 71.1 ab p5 3.7 ab 3.5 4.2 a 4.3 a 15.8 a 14.9 ab 78.8 a 74.5 ab significance *** ns *** *** *** *** *** *** sed 0.151 0.251 0.094 0.099 0.694 1.019 3.47 5.09 interaction (c x p) c1p1 2.8 3.1 2.7 f 2.7 7.7 8.5 38.3 42.4 c1p2 4.0 4.7 3.6 cd 2.9 11.5 13.7 57.4 68.3 c1p3 3.3 3.4 3.4 de 3.4 11.1 11.7 55.5 58.6 c1p4 3.3 3.8 3.1 ef 3.1 10.3 11.9 51.5 59.7 c1p5 3.6 3.4 3.8 bcd 3.9 13.5 13.3 67.7 66.6 c2p1 3.3 3.8 2.9 ef 3.0 9.6 11.4 48.0 57.0 c2p2 4.3 4.9 2.9 ef 3.6 15.4 17.9 77.2 89.5 c2p3 3.7 3.4 4.2 ab 4.2 15.7 14.6 78.5 72.9 c2p4 3.9 4.2 3.9 bc 3.9 15.2 16.5 76.2 82.6 c2p5 3.9 3.6 4.6 a 4.6 18.0 16.5 90.0 82.4 significance ns ns *** ns ns ns ns ns sed 0.213 0.355 0.133 0.140 0.981 1.441 4.91 7.20 ndereyimana et al. ‐ effect of vine and fruit pruning on yield attributes of two watermelon cultivars 273 ber plants compared to unpruned ones. these find­ ings could be further explained by the fact that plants with less branches allow more light interception and this leads to improved photosynthesis, more accu­ m u l a ti o n o f c a r b o h y d r a t e s , a n d t h u s o v e r a l l improved crop performance as compared to plants with relatively higher number of branches (feng et al., 2008). in agreement with the findings of current study, ali et al. (2016) also reported interaction effect of cultivars and pruning on yield of bottle gourd (lagenaria siceraria). in this study, fruit and vine pruning have been observed to significantly affect watermelon yield. this would be attributed to the fact that stem and fruit density is among agronomic variables associated with yield performance of vegetable crops (ayala­ tafoya et al., 2019). the number of stem per plant contributes to plant density, which affects distribu­ tion and utilization of soil nutrients and solar energy (rahmatian et al., 2014). according to campos et al. (2019), efficient solar radiation and production of photo­assimilates are the important pre­requisites for optimum watermelon production. the number of vines per plant also affects the root volume and plant vigour, which in turn influences water and nutrients’ uptake and availability. viana et al. (2008) reported that the lower the number of vine per plant, the higher the root volume and plant vigour and finally the higher yield as a result of improved nutrient and water uptake. the lower yield recorded on watermel­ on plants without pruning could result from competi­ tion for water, nutrients and light (gomes et al., 2019). similarly, muñoz­rengifo et al. (2018) argued that since watermelon has naturally many branches, table 2 ­ performance of watermelon as affected by cultivar and pruning method, rubona site c1=sugar baby, c2=julie f1, p1=no pruning, p2= pruning to 4 vines with 2 fruits per vine, p3: pruning to 4 vines with 1 fruit per vine, p4= pruning to 3 vines with 2 fruits per vine, p5= pruning to 3 vines with 1 fruit per vine, 2017a=short rains, 2017b=long rains. sed= standard error difference. * and **=significant difference at 5% and 1% respectively, ns=not significantly different (p>0.05). means within each column followed by the same letter are not significantly different as per lsd test at p<0.05. treatments no. of fruits per plant fruit weight (kg) fruit yield (kg per plant) fruit yield ( t ha­1) season 2017a season 2017b season 2017a season 2017b season 2017a season 2017b season 2017a season 2017b factor 1: cultivar (c) c1 3.4 3.6 2.8 b 3.1 b 9.5 b 11.1 b 47.5b 54.3b c2 3.5 3.8 3.4 a 3.8 a 11.9 a 14.2 a 59.6a 69.9a significance ns ns *** *** *** *** *** *** sed 0.198 0.182 0.051 0.057 0.617 0.643 3.08 3.10 factor 2: pruning method (p) p1 3.0 b 3.2 b 2.5 e 2.8 e 7.6 c 9.0 c 38.0 c 44.0 c p2 4.8 a 5.0 a 2.9 d 3.2 d 13.9 a 15.9 a 69.3 a 78.0 a p3 3.0 b 3.2 b 3.4 b 3.7 b 10.1 b 12.1 b 50.5 b 59.0 b p4 3.5 b 3.7 b 3.1 c 3.4 c 10.9 b 12.9 b 54.3 b 63.7 b p5 3.0 b 3.2 b 3.7 a 4.1 a 11.2 b 13.3 b 55.8 b 65.9 b significance *** *** *** *** *** *** *** *** sed 0.312 0.288 0.081 0.090 0.975 1.016 10.25 4.90 interaction (c x p) c1p1 2.7 2.9 2.4 f 2.7 f 6.5 7.7 32.3 37.4 c1p2 5.0 5.0 2.6 ef 2.9 ef 12.9 14.3 64.3 69.9 c1p3 3.0 3.2 3.0 d 3.3 d 9.0 10.8 45.0 52.5 c1p4 3.3 3.6 2.7 e 3.0 e 9.1 10.8 45.5 52.9 c1p5 3.0 3.2 2.4 c 3.7 c 10.1 12.1 50.5 59.0 c2p1 3.3 3.6 2.6 ef 2.9 ef 8.7 10.4 43.7 50.6 c2p2 4.7 5.0 3.2 cd 3.6 cd 14.9 17.6 74.3 86.1 c2p3 3. 3.2 3.7 b 4.2 b 11.2 13.4 56.0 65.5 c2p4 3.7 3.9 3.4 c 3.8 c 12.6 14.9 63.2 74.5 c2p5 3.0 3.2 4.1 a 4.5 a 12.2 14.6 61.0 72.9 significance ns ns * * ns ns ns ns sed 0.442 0.407 0.115 0.127 1.379 1.437 6.90 6.93 274 adv. hort. sci., 2021 35(3): 269­275 pruning is advised to keep adequate number of branches, leaves and fruits to enable them to share efficiently the plant resources. in agreement with findings of the current study, douglas et al. (2001) and palada and chang (2003) also reported a signifi­ cant effect on yield of cucumber and bitter gourd through pruning by removal of lateral shoots. based on results of the current study, pruning to three vines with one fruit per vine is therefore, recom­ mended for optimum yield with big­sized fruits of both watermelon cultivars (‘julie f1’ and ‘sugar baby’). acknowledgements we thank the government of rwanda for sup­ porting this study through the project “development of market­responsive plant varieties and seed sys­ t e m s t o r e d u c e d e p e n d e n c e o n i m p o r t a ti o n ” . a u t h o r s a r e t h a n k f u l t o d a ti v e n i y o m u f a s h a , i n n o c e n t n d u ti y e , e r i c s i b o m a n a , i m m a c u l é e mukabanana, and raurence mukanziza for their enthusiastic involvement in field work. references ali m.r., halim g.m.a., mehrajc g.h.h., 2016 ­ stages of vine pruning for vine production of bottle gourd vari‐ eties and lines in summer season. ­ 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14 adv. hort. sci., 2011 25(1): 14-20 received for publication 15 september 2010. accepted for publication 14 january 2011. radiation-induced chromosomal aberrations in grape phylloxera h. makee, n. tafesh, i. idris department of biotechnology, atomic energy commission of syria, po box 6091, damascus, syria. key words: chromosome aberrations, grape phylloxera, irradiation, reproduction. abstract: chromosomal aberrations in phylloxera females induced by different doses of gamma irradiation were detected. the results showed that the chromosomes of all tested embryos of irradiated phylloxera had aberrations, regardless of dose. when phylloxera nymphs were irradiated, the chromosomal number on the metaphase plate of some embryos’ cells was increased. the result indicated that the chromosomal aberrations influenced the mortality, longevity and reproduction of phylloxera. eight autosomal chromosomes were identified according to their length. additionally, the karyotype of irradiated and unirradiated populations of local phylloxera strain was defined. 1. introduction grape phylloxera, daktulosphaira vitifoliae fitch (homoptera: phylloxeridae), an aphid-like gall-forming parasite, is one of the most destructive insect pests of cultivated grape vitis vinifera l. world wide. grape phylloxera causes direct damage to grapevine by forming damaging root galls. these galls are metabolically active organs suited to match the nutritional requirements of phylloxera and can support populations with high reproductive rates. granett et al. (1985) reported that there was frequent decline of commercial vineyards as result of this pest, and consequently losses of quality and yield of grapes. the use of resistant rootstocks is the most common and effective means of managing phylloxera. our previous studies showed that some rootstocks were more resistance than others to grape phylloxera (makee et al., 2003). however, for unknown reasons, the resistance of some rootstocks may break down and farmers must replant vineyards (granett et al., 1983; song and granett, 1990; de benedictis and granett, 1993). therefore, additional ways to control this pest should be considered. sanitation and quarantine can be considered as required procedures to prevent the movement of this serious pest. insecticides and hot water dip treatments are used as quarantine treatments (granett et al., 2001). ionizing radiation has been recognized as an alternative method for treating agricultural products to overcome quarantine barriers in trade (fao, 2003). irradiation treatment does not influence the quality of many commodities; it is reasonably safe to the consumers and environment. previously, several authors have presented reviews of this subject (hallman, 1998; johnson and marcotte, 1999; hallman, 2000, 2001). irradiation has been successfully used for the control of many insect pests such as codling moth cydia pomonella (l.), beetle prionolplus reticularis white (lester et al., 2000), apple maggot, rhagoletis pomonella (walsh), the borer eucosma notanthes meyrick (lin et al., 2003), coconut scale aspidiotus destructor signoret (follett, 2006), the weevil sternochetus mangiferae (f.) (follett, 2001), cigarette beetle, lasioderma serricorne (f.) (hu et al., 2002), and the rice weevil sitophilus zeamais motschulsky (hu et al., 2003). makee et al. (2008) proposed that gamma irradiation could be economically very useful in quarantine treatments against phylloxera. the results showed that the percentage of matured phylloxera females and fecundity were markedly reduced when higher doses of gamma irradiation were used (makee et al., 2008). however, to our knowledge efforts of associate performance parameters of irradiated phylloxera with chromosomal rearrangements have not yet been studied. therefore, the purpose of the present research was to detect chromosomal aberrations in phylloxera females induced by different doses of gamma irradiation. the influence of such chromosomal aberrations on longevity and reproduction of phylloxera was examined. moreover, determination of the number and length of autosomal and sex chromosomes was undertaken. such 15 study will allow definition of the karyotypes of irradiated and unirradiated populations of the local phylloxera strain. 2. materials and methods establishment of phylloxera colony grape phylloxera was originally collected from field-infested roots of the local grape varieties in southern parts of syria. all insects were reared on fresh and healthy pieces of local grape variety balady roots, 4-7 mm in diameter and 5-7 cm long as outlined in makee et al. (2003). the experiments were conducted at 25±1°c with 70±5% rh, and 24 hr darkness. egg sterilization was carried out as described by makee et al. (2003). a co60 source (issledov gamma irradiator, techsnabexport co. ltd., moscow, russia) delivering a dose rate of 60 gy/min was used to treat the insects. effect of irradiation on matured females, fecundity, and oviposition period new phylloxera eggs were placed on fresh root pieces and left until hatching. a group of three-weekold feeding phylloxera nymphs was taken. nymphs were irradiated at different doses: 0-10-20 and 30 gy (n= 25 nymphs at each dose). irradiated and unirradiated nymphs were kept at 25±1°c with 70±5% rh, and 24 hr darkness. a daily microscopic inspection of all phylloxera stages at each applied dose was carried out. the number of feeding nymphs, which were able to develop to adult stage, was observed to determine the percentage mortality at each dose. female fecundity and longevity was determined at each tested dose. chromosome preparations mitotic metaphase chromosomes were obtained from 24 to 36-hr-old embryos. at each dose, 10 eggs were taken and placed in a 1.5 ml tube. the eggs in each tube were fixed in carnoy’s fixative (ethanol:chloroform:acetic acid 6:3:1) and shaken for 10 min. then a drop was taken and transferred onto a clean slide. shortly before drying, a drop of 60% acetic acid was added and macerated for 2-3 min with fine tungsten needles. then the specimen was spread on the slide using a heating plate at 45°c to allow evaporation of the acetic acid. the preparation was then stained and mounted in lactic acetic orcein for 5 min; redundant stain was removed with a piece of filter paper. the cover glass was sealed with nail polish. chromosome preparations were examined in phase contrast micrographs. chromosome measurement the lengths of the chromosomes from 12 well spread orcein-stained metaphase chromosomes were measured in digital images using digitizier software, version 1 (developed by the mathematics department, atomic energy commission of syria). chromosome lengths were ranked for each cell nucleus and means and standard deviations (sd) were calculated. relative chromosome lengths were calculated as percentages of the total length of all chromosomes in the diploid set. 3. results effect of irradiation on mortality, longevity and fecundity our results show that the percentage mortality of irradiated phylloxera nymphs was significantly higher than that of unirradiated ones. a regression line was fitted to present the relationship between gamma irradiation and percentage mortality of phylloxera (fig. 1), showing that the percentage mortality was positively correlated with dose (t= 7; p=0.05). the lowest percentages of mortality were recorded at 10 gy, after which the percentages started to increase. only about 16% of phylloxera nymphs were able to reach matured female stage at 30 gy. % m or ta lit y fig. 1 effect of gamma irradiation on percentage mortality of grape phylloxera. table 1 effect of different doses of gamma irradiation on mean number of eggs and longevity of phylloxera dose (gy) mean no. eggs(±se) mean longevity (d) (±se) 0 10 20 30 60.0±4.0 a 24.5±2.7 b 10.7±1.0 c 0.7±0.2 d 14.6±0.75 a 10.0±0.88 b 7.0±0.69 c 0.6±0.01 d means followed by different letters (columns) are significantly different at p< 0.05 (tukey hsd test). table 1 illustrates that phylloxera longevity and number of eggs were considerably influenced by the applied dose (table 1). the mean value for longevity and the mean number of eggs were significantly reduced by increasing the dose (f=75.67; df=3, 96; p=0.05 and f=106.78; df=3, 96; p=0.05, respectively). 16 chromosomal analysis under a light microscope from matured phylloxera females, 24 to 36-hr-old embryos, which contain a higher proportion of dividing cells, were taken to analyze the metaphase chromosomes. it was revealed that the wild-type metaphase karyotype of phylloxera consists of 10 chromosomes. there were eight autosomal chromosomes and sex chromosomes (xx) (fig. 2 a). a karyotype of 2n=9 was also found (fig. 2 b). based on our observations, phylloxera metaphase chromosomes appeared like thick condensed rods. in some cells, an additional very small chromosome was detected (fig. 3). a b 1 µm 1 µm fig. 2 normal metaphase kryotype from embryonic cells of grape phylloxera, daktulosphaira vitifoliae fitch: a) 2n= 10; b) 2n=9 + small chromosome (the arrow). 1 µm ý fig. 3 normal metaphase kryotype from embryonic cells of grape phylloxera: 2n= 10 + small chromosome (the arrow). to study the effect of different doses of gamma irradiation on phylloxera chromosomes, 24 to 36-hr-old embryos were examined at each tested dose, and it was found that the chromosomes of all tested embryos of irradiated phylloxera had aberrations, regardless of dose. at all different doses, sticky chromosomes were observed in the embryo cells (fig. 4). when 20 gy was applied, inter-chromosome translocations were clearly visible (fig. 5). however, increasing in the chromosomal number on the metaphase plate in some cells was noticed in embryos when phylloxera nymphs were irradiated (fig. 6). a b 1 µm 1 µm c d 1 µm 1 µm e 1 µm fig. 4 mitotic metaphase chromosomes from embryos of treated grape phylloxera with gamma ray, showing sticky chromosomes at deferent doses: a-b at dose 10 gy; c) at dose 20 gy, d-e) at dose 30 gy. fig. 5 orcein-stained preparations from embryos of irradiated phylloxera at dose 20 gy showing mitotic metaphase chromosomes with a translocation on the large chromosome (the arrow). ý 1 µm 17 chromosomes measurement it was possible to identify eight autosomal chromosomes according to their length when different metaphase chromosomes were investigated. table 2 illustrates the mean length and relative length for each identified chromosome. the total mean length of complete metaphase chromosomes was 12.17±1.97 µm. chromosome pair no. 1 is an extra large chromosome with an average total length of 2.31±0.3 µm and relative length of 18.18%; chromosome pair no. 2 is a large chromosome with an average total length of 1.33±0.3 µm and relative length of 10.5%; chromosome pair no. 3 is a medium chromosome (1.12±0.65 µm); and chromosome pair no. 4 is a short chromosome (0.90±0.160 µm) (table 2). the sex chromosome (xx) was clearly recognized. the sex chromosome is the shortest chromosome in the metaphase complements with an average total length of 0.69±0.1 µm and relative length of 5.51%. the mean length of the additional chromosome, which was observed in several cells, was only 0.5±0.1 µm. considerable variation was noted among the species tested within these orders (makee and saour, 1999; bakri et al., 2005; follett et al., 2007). in fact, a few studies were carried out to determine the radioresistance of hemiptera (scales, mealy bugs, aphids, and whiteflies). a previous study showed that the egg hatch of phylloxera decreased when eggs were subjected to high doses of gamma irradiation and the percentage of matured phylloxera females significantly increased as older nymphs and lower doses were used (makee et al., 2008). on the contrary, fecundity was markedly reduced when older nymphs and higher doses were employed. however, a relationship between chromosomal aberrations induced by irradiation and phylloxera biology was not determined in the study. phylloxera can be considered a cytogenetically exciting insect species because of abnormal features related to its cyclical parthenogenesis, and because it has holocentric chromosomes (chromosomes that lack a localized centromere). phylloxera populations can consist totally of parthenogenetic (thelytokous) females. several studies showed that grape phylloxera mainly reproduces asexually (parthenogenesis): an egg cell can develop into offspring without fertilization by a sperm. thus, the offspring and its siblings are assumed to be genetically identical to the mother (vorwerk and forneck, 2006). parthenogenetic reproduction of phylloxera has been observed in the field and can be easily maintained under constant conditions in the greenhouse or in vitro. this type of reproduction allows phylloxera populations to be replicated several fold, thus several asexual generations can be analyzed within a short period. once a year, xx parthenogenetic phylloxera females, like aphids, produce one egg that develops as an xo male, having lost half its x chromatin during the single maturation division (blackman and hales, 1986; blackman, 1987). the current study has shown that phylloxera nymph mortality increased with irradiation (fig. 1), confirming the results reported by makee et al. (2008). correspondingly, dohino et al. (1998) found that the survival of aphids was significantly decreased when they were treated with doses of 400-600 gy. the high death rate, especially when phylloxera nymphs were exposed to higher doses of gamma irradiation, can be attributed to the effects of the dominant lethal mutations induced in phylloxera nymphs’ chromosomes by irradiation (lachance, 1967). when low doses were applied, a small portion of irradiated nymphs successfully completed development and produced matured females, survival which was due to the holokinetic nature of phylloxera chromosomes. irradiation can cause fragmentation but the resulting fragments are still able to move on the mitotic spindle so that chromosome breakage does not lead automatically to the loss of genetic material (hughes-schrad and ris, 1941). our results reveal that the fecundity and longevity fig 6 orcein-stained preparations from embryos of irradiated phylloxera showing: a) at dose 20 gy: a cell with about 15 chromosomes which some of them are stuck together; b) at dose 30 gy: a cell with 14 chromosomes. a b 1 µm 1 µm table 2 the mean length and relative length (%) of phylloxera chromosomes no. chromosome mean length (µm±sd) relative length (%) 1 1 2 2 3 3 4 4 x x total mean length 2.31±0.3 2.31±0.3 1.33±0.3 1.33±0.3 1.12±0.65 1.12±0.66 0.90±0.16 0.90±0.16 0.69±0.1 0.69±0.1 12.17±1.97 18.18 18.18 10.5 10.5 8.81 8.81 7.1 7.1 5.41 5.41 100 4. discussion and conclusions several studies demonstrated that dipteran, coleopteran and hemipteran species tend to be more radiosensitive than lepidopteran species. however, 18 of surviving matured females, irradiated as nymphs, were greatly impacted by irradiation (table 1). comparable results were reported when crawls and nymphs of mealybug, maconellicoccus hirsutus (green), were irradiated (jacobsen and hara, 2003). therefore, at low doses some matured phylloxera females were recorded, but they laid only few eggs and lived for a short period of time. it could be that the induced chromosomal aberrations in irradiated nymphs prevent the normal process of mitotic division, which leads to egg production. therefore, the matured females were unable to produce a normal number of eggs. to study the effect of different doses of gamma irradiation on phylloxera chromosomes, 24 to 36-hr-old embryos were examined at each tested dose. it was noted that the chromosomes of all tested embryos of irradiated phylloxera had aberrations, regardless of dose. we noticed sticky chromosomes, inter-chromosome translocations, and increases in the chromosomal number on the metaphase plate in some cells. all these chromosomal aberrations in the embryos were expected as the phylloxera, like lepidptera species, has holocentric chromosomes. it is reported that irradiation causes fragmentations and translocations in many species of lepidptera (traut et al., 1986; makee and tafesh, 2006, 2007). and because the chromosomes are holocentric when a break occurs, the fragments are usually not lost and can still be attached on the spindle. in the present study on phylloxera, a lot of chromosomal breaks occurred during the formation of eggs in the ova of the nymphs which gave sticky chromosomes and inter-chromosome translocations in the cells of the embryos. it can also be said that spindles were affected by the irradiation which caused an increase in the chromosomal number on the metaphase plate in some embryo cells. in this work, the embryos of laid eggs varied greatly in their chromosomal rearrangements (figs. 2, 3, 4 and 5). however, such rearrangements allow the formation of embryos but it is unknown if they will permit the development of embryos until egg hatch. in mealybug only embryos with an approximately normal amount of paternal chromosomal material were able to survive (nelson-rees,1962). the current study has shown that the metaphase complement of the syrian strain of the phylloxera female consists of 10 chromosomes, representing eight autosomal and two sex chromosomes, confirming the results presented by forneck et al. (1999) and maillet (1957). we found also normal karyotype with 2n= 9 in the embryonic cells coincidently. forneck et al. (1999) found one karyotype containing 2n = 9 in the somatic cells of phylloxera and they interpreted it as a male sexual phylloxera, although they did not find any spermatides during their study, which leads us to think that maybe this deficiency in chromosome number is a kind of variety of the karyotype. when defining the karyotype of six phylloxera populations from germany, forneck et al. (1999) noticed extra chromosomes. similarly, in our study an additional very small chromosome was observed in some examined cells of the syrian phylloxera strain. the detection of supernumerary chromosomes was reported in aphids as well (blackman, 1976; wilson et al., 2003). such supernumerary or accessory chromosomes are not essential for the life of a species and are lacking in most of the individuals; they do not carry genes necessary for basic growth, but may have some functional significance such as to increase asymmetry chiasma distribution or increase variation by increasing crossing over and recombination frequencies. in aphid, blackman (1980, 1981) suggested that the differences in chromosome numbers might be due to dissociations or fusions involving elements of the normal diploid set, or to the presence of supernumerary b chromosomes. the centromeric activity of holocentric chromosomes, dispersed along its full length, allows the broken chromosomal fragments to segregate during mitosis (ris, 1942). moreover, blackman (1980) proposed that thelytokous reproduction of aphids is a factor that permits karyotype variation within populations of the same species. the phylloxera karyotype consists of 10 chromosomes. the total complement length is about 12ìm and the chromosomes range in length from 0.7 to 2 ìm. when mitosis of agallia constricta (leafhopper) was examined, the metaphase chromatin appeared to be a 2-3 ìm wide (rieder et al., 1990). based on embryo metaphase, the chromosomes of phylloxera females could be sorted into five different size-dependent groups: extra long, long, medium, short and extra short (table 2). however, in some cells a dot-like chromosome, that represents the additional chromosome, was observed. forneck et al. (1999) classified phylloxera chromosomes into two classes: one pair of large chromosomes and four pairs of shorter chromosomes. nevertheless, in their study they did not mention the exact length of each pair. the x chromosome is the shortest one in phylloxera karyotype forneck et al. (1999). however, blackman et al. (2003) reported that in most aphids species the x chromosomes could be identified as the longest or second longest pair. on the contrary, in some aphids the x chromosome was the shortest pair (blackman, 1986; blackman et al., 2003). the present study confirms the efficiency of cytogenetic techniques in analyzing the karyotype and chromosomal length of phylloxera, as well as tracing chromosome aberrations in irradiated phylloxera populations. this investigation is a contribution to the search for genetic variation of phylloxera behaviour and development from different populations and provides useful information that can be taken into account in pest management and quarantine measurements against phylloxera. however to apply irradiation technology more comprehensive studies are still needed. 19 references bakri a., heather n., hendrichs j., ferris i., 2005 50 years of radiation biology in entomology: lessons learned from ididas. ann. entomol. soc. am., 98: 1-12. blackman r.l., 1976 cytogenetics of two species of eureraphis (homoptera, aphididae). chromosoma, 56: 393-408. blackman r.l., 1980 chromosome numbers in the aphididae and their taxonomic significance. syst. entomol. 5: 7-25. blackman r.l., 1981species, sex and parthenogenesis in aphids, pp. 75-85. in: forey p.l. 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in asexual aphids (aphidoidea). biological journal of the linnean society, 79: 115-135. 32 1. introduction changes in consumers’ social environment represent a constraint to vegetable and fruit consumption and lead to convenience orientation (candel, 2001). a wide assortment of minimally processed vegetables and fruits (chunks, mousse, smoothies) has been developed to meet consumer needs for ‘‘quick’’ and convenient products, and to benefit from the healthy image of fruit and vegetables (ahvenainen, 1996). one of the main factors that influences quality of fresh-cut products is the enzymatic browning that occurs on product surfaces after cutting (garcia and barret, 2002). thus, cultivar study should be aimed at identifying cultivars which are less susceptible to browning, but also have higher nutritional and sensorial quality, covering all the season, and lead to consumer satisfaction and repeat purchase. cultivar survey represents an important step when developing a new product (cabezas-serrano et al., 2009 a, b) allowing identification of genotypes that better respond to postharvest handling and to minimal processing. peaches and nectarines are nutritionally important because they contain meaningful amounts of carotenoids including β-carotene (especially yellow-fleshed fruits), lutein and β-cryptoxanthin (gil et al., 2002). although their availability is limited by season they are one of the most important fruit commodities consumed worldwide (cantin et al., 2009). many studies on minimally processed vegetables and fruits focus on microbiological quality, safety, processing and packaging issues (foley et al., 2002; amodio and colelli, 2008) but still little information is available on varietal susceptibility. in this light, the aim of the present work was to investigate the suitability of peach varieties to be processed as fresh-convenience products throughout the peach season. 2. materials and methods from june to september 2010, 26 varieties of peach fruits from the apulia region (italy) were collected, including peaches (prunus persica l.), nectarines (prunus persica l. var nectarina) and clingstones peaches (prunus persica l. batsch). based on the ‘redhaven’ peach maturing date, peach fruits were divided into three groups: early maturing (group a), middle maturing (group b) and late maturing (group c), as shown in table 1. fruits were harvested at a commercial maturity stage, typical for each cultivar, as established by the growers. peach fruits were then transported to the postharvest laboratory (university of foggia) influence of quality attributes of early, intermediate and late peach varieties on suitability as fresh-convenience products f. colantuono, m.l. amodio, f. piazzolla, g. colelli dipartimento di scienze delle produzioni e dell’innovazione nei sistemi agro-alimentari mediterranei (prime), università degli studi di foggia, via napoli, 25, 71122 foggia, italy. key words: clingstone peaches, fruit puree, harvest season, mechanical damage, nectarines, sensorial analysis, soluble solids. abstract: fresh convenience products represent a category of minimally processed fruit and vegetables (chunks, mousse, smoothies) that respond to the changes in consumer attitudes. thanks to the image of convenience (time-saving, snack sizes, no waste, smart packaging) and healthiness their sales are steadily increasing. in this study 26 varieties (including peach, nectarines, and clingstone peaches) from apulian production were divided into three groups according to harvest dates in early (a), middle (b) and late (c) maturing. physical, chemical and sensorial analyses were performed in order to select high quality fruits for minimal processing according to the harvesting season. a multivariate principal component analysis was applied to discriminate different varieties for quality attributes. within group a, ‘honey kist’ showed the lowest acidity and intermediate susceptibility to mechanical damage. for group b, ‘stark red gold’, ‘zee glo’ and ‘venus’ resulted different in sensorial evaluation, while ‘loadel’ and ‘eolia’ were more susceptible to browning. for group c, ‘tardi belle’ and ‘baby gold7’, although more sensitive to mechanical damage, were differentiated for flavor. results of this work confirm the extreme variability among varieties in terms of sensorial quality, susceptibility to browning and to mechanical damage, and the importance of assessing varietal screening for selection of most suitable varieties for minimal processing. adv. hort. sci., 2012 26(1): 32-38 received for publication 29 september 2011 accepted for publication 22 february 2012 33 and kept under controlled temperature and humidity (5°c and 95% rh) for one day before processing. fruits were washed in a naocl solution (100 ppm), rinsed in clear water and then gently dried with a paper tissue. twenty fruits were used to assess mechanical damage, while the remaining fruits were divided into three replicates of 20 fruits and used to evaluate quality attributes. physical parameters were evaluated mainly on the whole fruit (with the exception of puree viscosity), while chemical and sensorial analysis were performed on the fruit puree. table 1 peach varieties grouped in peach, nectarine and clingstone peach type peach nectarine clingstone royal glory big bang loadel prince diamond big top eolia red elegance honey kist baby gold7 rome star amiga marilyn bigi lara zee lady ambra o’henry laura tardi belle spring bright fire top maria camilla zee glo guerriera venus stark red gold lidy star physical and chemical analysis for each variety the resistance to mechanical damage, in particular the susceptibility to impact bruising, was assessed on 20 fruits. fruits were individually impacted on one side from a fixed height (30 cm) using a free-falling steel ball (40 g and 21.4 mm diameter), and held at room temperature for 48 hr before measuring. the extension and depth of the bruise (in mm) after peeling was measured. in addition, for each variety the incidence of mechanical bruising (%) was calculated as the ratio between the number of fruits damaged and the total number of fruits considered. peach fruit firmness was assessed for each replicate, measuring the force (in n) required by a 8-mm probe to penetrate the peeled surface in two opposite regions of the fruit mesocarp, using a digital penetrometer (tr, italy). flesh fruit color was measured with a colorimeter (cm 2600d, konica minolta, japan) in the cie l*a*b* mode, taking two measurements per fruit after removing the peel. hue angle (h°) was calculated as arctg b*/a*. in order to carry out analysis on the fruit puree, fruits from each cultivar were divided into three groups of 15 fruits each, corresponding to three replicates and from each replicate three subsamples were analyzed. fruits were peeled, de-stoned, cut into big chunks and blended for 90 s. the purees were promptly transferred to sealed glass jars and stored at 5°c. a few drops of peach puree were used to measure the total soluble solids content (tss) with a digital hand refractometer (atago, japan). a small amount of fruit puree was transferred to a falcon tube and centrifuged with a centrifuge (pk 121r, thermo electron corporation, france) at 4°c for 5 min. five grams of surnatant were then used to measure the titratable acidity (ta), with an automatic titrator (titromatic 1s, crison, spain) measuring the volume of naoh 0.1n to reach a final ph of 8.1. results were expressed as percent of citric acid referred to the juice. at the same time, the ph was also measured for each puree sample. to determine the dry matter content, puree samples were desiccated in an oven at 105°c up to constant weight and the dry matter content was calculated as difference from initial weight. peach fruit puree viscosity was determined on samples at 20°c by means of a consistometer (bostwick, usa); the final results were expressed in cm * 30 s-1. sensorial analysis sensorial evaluations were carried out on the puree samples, kept at 5°c, within 3 hr after processing. puree of each variety was distributed into clear plastic cups labeled with a three-digit code. the sensorial test was performed in a sensorial laboratory with 10 trained panelists. to evaluate the intensity of aroma, freshness, sweetness and sourness, an hedonic scale, from 1 to 5, labeled 1=less intense to 5=very intense, was used. for overall quality the scale reference was 1= really poor and 5= excellent. for the level of browning, scale reference was 1= severe browning and 5= not browned. statistical analysis standard deviation was calculated on mean values for each quality attribute. principal component analysis (pca) was performed on the data. the biplot technique was used to display the relative positioning of quality attributes and cultivars according to the first two pcs. 3. results and discussion analysis of fruits in terms of physical, chemical, and sensorial attributes, revealed significant variation among the cultivars. evaluation of physical and chemical attributes the resistance to mechanical damage showed a different incidence among the cultivars. percentage of incidence to mechanical damage is shown in table 2: it was not related to the fruit harvest dates but only to the cultivar, even if cultivars in group b seemed to be more resistant to mechanical damage than the others. within group a, most 34 of the varieties showed a bruise incidence around 55%; ‘amiga’ was the most damaged variety (73.7%) whereas ‘spring bright’ did not show any evidence of damage. within group b only the clingstone peach ‘loadel’ showed a high bruise incidence (80%) whilst for the other varieties the average incidence was less than 30%. in group c all the varieties evaluated were characterized by a high bruise incidence, especially for ‘tardi belle’ (100%). no differences among the cultivars were found in bruise depth (data not shown) and only the results related to bruise extension are reported. cultivars from group a showed a great variability (table 2), while in group b the average value of bruise size was 6 mm with the exception of ‘guerriera’ and ‘marilyn’ that showed the highest (9 mm) and the lowest (2 mm) values, respectively. within group c, ‘baby7’ and ‘tardi belle’ showed the same bruise size, which was significantly higher than that of ‘o’henry’. results confirmed that bruise size and incidence of mechanical damage does not correlate with flesh firmness, as reported by mitchell and kader (1989). in fact, within the range of commercial maturity studied (table 2), varieties with the same firmness (i.e. ‘big bang’ and ‘spring bright’) showed different response to mechanical damages (55 and 0% respectively), confirming the great variability within the same peach type and among cultivars. fruit flesh color, expressed as hue angle value (table 2), was not statistically different among cultivars in groups b and c, while in group a ‘royal glory’ presented a significantly greener value (hue angle greater than 90°, in the second quadrant of l*a*b* color space) than ‘bing bang’, ‘amiga’, ‘ambra’, and ‘laura’, although showing a very low flesh firmness and acidity. fruit puree viscosity was different among the studied varieties; values ranged from 1.5 (in ‘eolia’) to 9 cm * 30 s-1 (in ‘ambra’), with the most frequent values between 4 and 5.5 cm * 30 s-1 (table 2). the high variability among cultivar viscosity was related to the maturity level; indeed in some cases it seemed influenced by fruit firmness. group a presented cultivars with higher values compared table 2 chemical and physical parameters evaluated on peach, nectarine and clingstone peach varieties for group a (early maturing), group b (middle maturing) and group c (late maturing) ripening group cultivar bruise incidence % bruise extension (mm) firmness (n) hue angle (°) viscosity (cm*30 s-1) ph ta (% citric acid) tss (°brix) a big bang 55 6±0.1 37.6±6.7 75.9±6.4 4.4±0.1 3.77±0.04 0.55±0.70 8.7±1.6 big top 50 11±0.5 43.3±9.1 85.5±8.1 4.6±0.1 3.65±0.13 0.75±0.15 11.7±0.9 honey kist 36 8±0.4 28.0±10.1 87.7±8.2 7.4±0.3 4.00±0.09 0.65±0.09 16.3±2.4 amiga 74 10±0.5 19.5±9.0 71.6±11 6.5±0.3 3.37±0.21 1.22±0.13 10.6±0.1 bigi lara 50 12±0.3 25.4±7.7 86.4±5.4 7.5±0.1 3.55±0.03 1.00±0.09 10.9±0.3 ambra 60 7±0.2 21.0±7.6 77.8±7.9 9.1±0.5 3.57±0.15 1.19±0.08 10.9±0.3 laura 29 4±0.4 18.6±8.2 79.8±3.5 8.4±0.3 3.63±0.09 1.03±0.06 10.4±0.4 spring bright 0 0±0.0 37.9±10.5 83.2±4.4 5.5±0.2 3.69±0.16 1.18±0.20 11.1±0.8 royal glory 65 11±0.3 18.8±9.5 91.4±5.0 3.5±0.2 3.85±0.10 0.68±0.11 9.9±1.0 fire top 25 3±0.1 31.2±11.0 82.8±10 3.4±0.2 3.50±0.06 1.41±0.13 11.7±0.5 b maria camilla 11 6±0.2 25.9±9.1 87.7±6.0 4.6±0.3 3.39±0.05 1.21±0.16 10.2±0.9 zee glo 25 6±0.1 16.8±4.3 85.4±3.8 7.1±0.2 3.43±0.03 1.32±0.38 11.4±1.2 guerriera 22 9±0.5 13.2±5.7 82.1±5.7 5.1±0.1 3.49±0.05 1.08±0.10 12.1±1.0 diamond princess 15 8±0.3 16.5±7.2 81.7±4.1 4.8±0.1 3.38±0.13 0.88±0.08 11.7±0.5 red elegance 25 6±0.3 61.9±13.9 84.1±2.7 2.1±0.1 3.44±0.02 0.93±0.08 12.2±0.4 rome star 0 0±0.0 40.7±11.6 81.7±4.2 4.3±0.2 3.52±0.03 0.88±0.03 12.3±0.4 loadel 80 6±0.3 32.7±6.0 83.1±4.5 4.5±0.1 3.59±0.06 0.76±0.08 13.4±0.9 eolia 25 5±0.2 42.1±14.2 79.0±4.3 1.5±0.2 3.81±0.14 0.77±0.06 12.2±0.4 venus 20 6±0.4 37.4±13.2 80.2±5.3 4.1±0.1 3.35±0.07 1.25±0.15 13.3±0.6 stark red gold 20 9±0.8 17.3±4.5 71.4±10 5.5±0.2 3.38±0.04 1.24±0.05 13.5±0.6 lidy star 30 4±0.2 13.0±3.10 78.4±4.8 5.1±0.2 3.76±0.10 0.82±0.06 13.0±0.5 marilyn 6 2±0.0 27.6±12.7 82.7±8.2 4.0±0.3 3.53±0.05 0.96±0.13 13.5±0.6 zee lady 20 4±0.2 25.8±10.6 81.5±2.7 3.5±0.2 3.46±0.07 0.96±0.09 13.1±0.3 c o’herny 64 4±0.2 62.6±8.7 80.3±4.1 7.0±0.3 3.43±0.06 0.78±0.03 13.4±0.7 tardi belle 100 5±0.1 42.9±9.3 81.6±4.3 7.5±0.1 3.59±0.07 0.69±0.08 12.8±0.4 baby gold 7 87 5±0.1 41.5±5.3 78.6±3.5 4.0±0.3 3.67±0.06 0.52±0.03 12.9±1.2 mean values±standard deviation. 35 to the other two groups, in particular ‘ambra’ and ‘laura’ (9.1 and 8.4 cm * 30 s-1 respectively ) that were characterized by a low flesh firmness (21 and 18.6 n), whereas ‘red elegance’ that showed low viscosity (2.1 cm * 30 s-1) had a high flesh firmness (61.9 n). viscosity is an important technological parameter for the formulation of smoothies and fruit purees, due to its influence on product smoothness (or thickness) which may have an effect on the mouth feel of the product. as for chemical attributes (table 2), within group a ‘amiga’ showed the lowest ph value (ph 3.37) that was significantly different from ‘honey kist’ (ph 4), which indeed were characterized by a different ta: higher for ‘amiga’ (1.22% citric acid) and lower for ‘honey kist’ (0.65% citric acid). in terms of total soluble solids (tss), ‘honey kist’ showed the highest soluble solid content (16.3°brix) even though its flesh firmness was similar to the other cultivars. the high tss value confirms its nonacid characteristics, showing the highest tss:ta ratio as well (25); this ratio is commonly used as a quality index because it is related to taste perception (byrne et al., 1991). other researchers (liverani et al., 2003) indicated that the tss:ta ratio at commercial harvest in non-acid cultivars is three to four times higher than in acid cultivars. ‘royal glory’ showed high ph value (ph 3.85) together with a low ta (0.68% citric acid) and low tss (9.9 °brix); this cultivar was significantly different from ‘fire top’ and ‘bigi lara’ that had similar ph values (ph about 3.5) and similar tss contents (11.7 and 10.9°brix respectively) but a different ta content (1.41 and 1.00% citric acid respectively). peach fruit acidity is controlled by several factors such as the cultivar, environmental conditions, canopy position, crop load and fruit maturity (crisosto et al., 1997; castellari et al., 2006). cultivars with the same flesh firmness as ‘spring bright’ and ‘big bang’ showed similar ph values (ph 3.69 and 3.77 respectively) but different ta values (1.18 and 0.55% citric acid) and tss (11.1 and 8.7°brix); this implies that at harvest not only the firmness but also the other chemical attributes must be taken into account to select the right maturity stage for harvesting. within group b, ‘maria camilla’ resulted different from the other cultivars since it showed a lower tss content (10.2°brix) with ph 3.39 and high ta (1.21% citric acid). even if it was in the range of maturity for consumption (firmness 25.9 n) it would have had a low potential impact on consumer preference due to the very low tss:ta ratio (8.4). ‘eolia’ and ‘lidy star’ showed the highest ph values (ph 3.81 and 3.76 respectively) and they were similar in ta (0.77 and 0.82% citric acid) and tss (12.2 and 13°brix). ‘eolia’ was significantly different from ‘venus’ and ‘stark red gold’ that had lower ph together with higher ta (1.25 % citric acid) and same tss (13.3 °brix). with regard to harvest date, it has been reported that medium and late season cultivars have a greater capacity to accumulate sugars compared to early season cultivars, and this is due to the non-interruption of the growing process, sugar accumulation, acid degradation and aroma synthesis (byrne, 2002). among late maturing varieties (group c), ‘o’henry’ showed lower ph value (ph 3.43) than the others, indeed it also had higher ta (0.78) than ‘baby gold7’ and no differences in terms of tss were found among them. the lowest ta value in ‘baby gold7’ gave a higher tss:ta ratio value (24.8, data not shown) with a potential high consumer preference. dry matter includes both soluble (largely sugars) and insoluble solids (mainly the structural carbohydrates and starch). as a large proportion of the dry matter at harvest is starch plus soluble sugars, its value can be related to the soluble sugars that will be contained in the ripe fruit. indeed in accordance with tss values, dry matter contents ranged between 8.6% for ‘big bang’ and 16.4 % for ‘honey kist’. burdon et al. (2004) proposed dry matter in kiwifruit as being both a maturity indicator for timing harvest and also as a predictor of the sensory quality of the fruit once ripe. results obtained in the present study on peach fruits were in accordance with this theory since peach fruit cultivars (‘honey kist’, ‘lidy star’, ‘stark red gold’, ‘baby gold7’) with high dry matter content showed a higher value of tss and were also preferred for sweetness by panelists during sensorial tests. sensorial analysis it is well documented that in peach organic acids and soluble sugars are the major determining factors of fruit taste and, together with the volatiles (responsible for the aroma), have an impact on the overall eating quality of the fruit (iglesias and echeverría, 2009). among the cultivars tested, ‘honey kist’ was the most preferred from group a together with ‘big bang’, ‘laura’ and ‘ambra’ as indicated by the overall evaluation score (table 3). the puree obtained with ‘honey kist’ was described as sweet and fresh, and received a high overall evaluation (score 4.4). varieties in group b showed differences for aroma, freshness, sweetness, and overall evaluation: ‘zee glo’, ‘guerriera’, ‘diamond princess’, ‘stark red gold’, ‘venus’, ‘lidy star’ and ‘loadel’ resulted the most pleasant with a score between 3 (intense or fair) and 4 (good). moreover, ‘maria camilla’, ‘zee glo’, ‘diamond princess’, and ‘stark red gold’ were evaluated positively in terms of color, with a score of 3.5 (slightly browned). in group c ‘tardi belle’ and ‘o’henry’ were evaluated well balanced on freshness and aroma while ‘baby gold7’ was considered the sweetest, most probably because of its high tss:ta ratio. in general, panelists disliked those varieties that were less sweet and more sour, rating them negatively (score 2-2.5) since ta plays an important role at low tss levels (<10%). when tss and ta are low even with a high tss:ta ratio (‘royal glory’), the perception of sweetness is low, as reported by crisosto et al. (2006). moreover, in the selection of new varieties, low acid content (non-acid) and a sweet taste are desirable traits, which give an acceptable flavor and result in better quality for consumers (nicotra and conte, 2003). the nectarine ‘honey kist’, a new variety, was the most appreciated due to its tss content (16.4°brix), higher than 36 the optimum level (11-12%) suggested by hilaire and mathieu, (2004) for consumer satisfaction. principal component analysis each sample from groups a and b was plotted using the first and second pc factors, which retained 69% of the total variance, while in group c the first and second pc factors retained 99% of total variance, but in this case only three cultivars were used. grouping of component loadings separated quality attributes into three groups for all maturing groups (well displayed by the biplot graphs in figure 1). fig. 1 biplot graphs for group a (early maturing) and group b (middle maturing) varieties. grouping of peaches, nectarines and clingstone peaches according to their physical, chemical and sensorial attributes determined by pca. table 3 sensorial parameters evaluated on purees of peach, nectarine and clingstone peach varieties for group a (early maturing), group b (middle maturing) and group c (late maturing) ripening group cultivar color aroma freshness sweetness sourness overall evaluation a big bang 3.7±0.5 4.4±0.8 4.3±1.1 2.6±0.8 2.9±0.9 3.0±0.8 big top 1.4±0.5 2.6±0.8 2.0±0.7 2.8±0.8 1.8±0.4 2.8±0.8 honey kist 2.4±0.9 3.8±0.8 4.2±0.8 4.0±0.0 1.6±0.5 4.4±0.5 amiga 4.2±0.8 3.6±0.5 3.2±0.4 1.6±0.5 3.4±1.5 2.4±1.0 bigi lara 2.0±1.0 3.2±0.8 2.8±0.8 2.4±0.9 1.8±0.8 2.4±0.9 ambra 5.0±0.0 4.4±0.9 3.6±0.9 2.2±0.8 3.2±0.4 3.2±0.8 laura 4.2±0.4 4.2±0.4 3.0±0.7 2.4±0.9 3.4±0.9 3.0±1.2 spring bright 2.2±0.8 2.8±0.4 2.8±1.3 2.4±0.9 3.4±1.5 2.2±0.8 royal glory 2.0±0.0 3.6±0.5 3.6±0.5 2.0±0.7 3.2±0.4 2.4±0.5 fire top 1.5±0.5 2.6±1.0 2.8±1.0 2.6±1.5 2.7±1.2 2.3±0.9 b maria camilla 3.5±1.5 3.3±0.9 3.3±1.0 3.0±1.2 2.8±0.6 3.5±0.8 zee glo 3.5±1.5 3.3±0.9 3.3±1.0 3.0±1.2 2.8±0.6 3.5±0.8 guerriera 1.6±0.5 3.7±1.0 3.2±0.7 3.4±0.9 1.8±0.7 3.3±0.9 diamond princess 3.5±0.4 3.1±1.1 3.5±0.8 2.7±0.5 3.0±1.0 3.1±0.6 red elegance 1.7±0.8 2.1±0.8 2.5±1.4 2.4±0.5 2.8±0.8 2.4±1.1 rome star 1.3±0.5 2.2±0.7 2.8±0.4 2.5±0.8 2.3±1.0 2.8±0.8 loadel 2.2±0.7 3.5±0.5 3.2±1.5 3.3±0.4 1.9±0.7 3.6±0.5 eolia 1.2±0.4 2.5±0.8 2.8±1.2 3.4±0.6 2.0±0.7 3.2±0.7 venus 2.7±1.0 2.7±0.8 2.9±1.0 2.6±0.6 3.2±0.7 2.8±0.8 stark red gold 3.5±1.0 3.0±1.2 3.6±0.9 2.7±1.0 2.7±1.0 2.8±0.7 lidy star 3.0±1.0 3.0±1.0 3.0±1.4 3.5±1.2 2.3±1.1 3.5±1.2 marilyn 1.2±0.4 3.1±1.2 2.6±1.2 2.4±0.6 2.9±0.7 2.1±0.6 zee lady 2.8±0.9 3.2±0.7 2.9±0.6 3.1±0.7 2.6±0.5 3.0±0.7 c o’herny 3.4±0.8 3.7±0.9 3.4±0.5 2.9±0.8 2.7±1.1 3.3±0.5 tardi belle 3.0±1.0 3.3±0.6 3.7±0.6 2.0±0.0 3.0±1.0 2.7±0.6 baby gold 7 2.7±0.4 3.6±0.9 3.7±1.0 4.0±0.7 1.6±0.5 3.7±0.6 intensity of aroma, freshness, sweetness and sourness scored from 1=less intense to 5=very intense; overall quality scored from 1= really poor to 5= excellent; color scored from 1= severe browning to 5= not browned. mean values±standard deviation. 37 in the early maturing cultivar biplot graph, the first group associates acidity and sourness, the second tss, sweetness, dry matter and overall evaluation, and the third viscosity, color, bruising susceptibility, aroma, and freshness. based on this positioning, opposite relationships between acidity and sensorial evaluation, and between sourness and sweetness were found. specifically, pca analysis in this study indicated that in early maturing cultivars, ‘fire top’, ‘spring bright’, ‘royal glory’, ‘big top’ and ‘bigi lara’ were positioned in the upper quadrant along with acidity since ta values were higher than for other varieties. ‘honey kist’ was in the lower right quadrant along with tss, sweetness, dry matter and overall evaluation. this positioning is in accordance with the chemical and sensorial analysis results; ‘honey kist’ showed the highest tss value (16°brix) associated with a high impact on consumer sweetness perception. cultivars ‘laura’, ‘ambra’, ‘amiga’, and ‘big bang’ were spread in the lower left quadrant according to sourness, color, bruising susceptibility, viscosity, and freshness; in particular ‘ambra’ obtained a high sensorial score for aroma and color. it could be concluded that for group a, nectarine ‘honey kist’ showed the highest tss content, the lowest acidity, and an intermediate susceptibility to mechanical damage, with a potential positive impact on consumer preference together with ‘laura’ and ‘ambra’ that retained good color after processing. in the middle maturing cultivar biplot graph the first group was associated by tss, sweetness, sensorial evaluation and bruising, the second by viscosity, freshness, color and ta, the third by sourness. within this group, an opposite relationship between sweetness and sourness, and between dry matter content and ta was observed. ‘eolia’ was in the left upper quadrant along with dry matter in accordance with chemical analysis. ‘lidy star’ and ‘guerriera’ were in the upper right quadrant for sweetness and sensorial evaluation, ‘stark red gold’ was associated with aroma due to the high overall score, while ‘loadel’ differentiated for bruising due to the high incidence of mechanical damage. ‘zee glo’, ‘diamond princess’, ‘venus’ and ‘maria camilla’ were spread around freshness, ta and color. ‘marilyn’, ‘zee lady’, ‘rome star’ and ‘red elegance’ formed a third group positioned in the lower left quadrant between sourness and dry matter, due to their similar dry matter content (13%) and to the high score received for sourness. within this group ‘stark red gold’, ‘zee glo’, ‘venus’, and ‘diamond princess’ resulted different from the others because of a higher overall evaluation and a lower susceptibility to bruising, indicating their suitability to minimal processing. on the contrary, clingstone peaches ‘loadel’ and ‘eolia’ were more susceptible to browning after blending, and although pleasant for sweetness and overall quality, their use for fresh convenience products would not lead to promising results. as for group c, the principal component analysis is not really meaningful due to the low number of cultivars. from the biplot graph (not shown) it was observed that ‘tardi belle’ was in the upper right quadrant associating with bruising susceptibility, due to the very high incidence to mechanical damage (100%). although it was the most sensitive to mechanical damage, ‘tardi belle’ differentiated from the other varieties for the well balanced flavor. ‘o’henry’ positioned in the lower right quadrant between ta and overall evaluation, while ‘baby gold7’ was in the left quadrant along with sweetness, aroma, freshness, and color, according to the sensorial results obtained. in conclusion, this work confirms the extreme variability existing among genotypes in terms of sensorial quality, susceptibility to browning and to mechanical damage, and the importance of assessing screening to select the most suitable varieties for minimal processing. from these preliminary data, the best suited cultivars for minimal processing were ‘honey kist’, ‘laura’ and ‘ambra’ from group a (early maturing), ‘stark red gold’, ‘zee glo’, ‘venus’ and ‘diamond princess’ from group b (middle maturing); cultivars from group c (late maturing) did not show many differences although this may be due to the limited number of varieties evaluated. further studies are needed in order to better understand the biochemical and technological behavior and to extend the screening to other potentially interesting varieties. references ahvenainen r., 1996 new approaches in improving the shelf life of minimally processed fruit and vegetables. trends in food science & technology, 7: 179-187. amodio m.l., colelli g., 2008 effect of thermal treatment and dipping on quality and shelf-life of fresh-cut peaches. adv. hort. sci., 22(1): 21-26. burdon j., mcleod d., lallu n., gamble j., petley m., gunson a., 2004 consumer evaluation of “hayward” kiwifruit of different at harvest dry matter contents. postharvest biology and technology, 34: 245-255. byrne d., 2002 peach breeding trends: a worldwide perspective. acta horticulturae, 592: 49-59. byrne d.h., nikolic a.n., burns e.e., 1991 variability in sugars, acids, firmness, and colour characteristics of 12 peach genotypes. j. am. soc. hort. sci., 116(6): 1004-1006. cabezas-serrano a.b., amodio m.l., cornacchia r., rinaldi r., colelli g., 2009 a suitability of five different potato cultivars (solanum tuberosum l.) to be processed as fresh-cut products. postharvest biol. technol., 53: 138-144. cabezas-serrano a.b., amodio m.l., cornacchia r., rinaldi r., colelli g., 2009 b screening quality and browning susceptibility of 5 artichoke cultivars for freshcut processing. j. sci. food agric., 89(15): 2588-2594. candel m.j.j.m., 2001 consumers’ convenience orientation towards meal preparation: conceptualization and measurement. appetite, 36: 15-28. cantin c.m., moreno m.a., gogorcena y., 2009 evaluation of the antioxidant capacity, phenolic compounds, and vitamin c content of different peach and nectarine [prunus persica (l.) batsch] breeding progenies. j. of agric. and food chemistry, 57: 4586-4592. 38 castellari l., malavolti a., colombo r., rondinelli g.p., 2006 l’impiego dei ‘‘panel test’’ nella valutazione qualitativa di alcune nettarine emiliano-romagnole. rivista di frutticoltura, 7-8: 60-63. crisosto c.h., crisosto g., neri, f., 2006 understanding tree fruit quality based on consumer acceptance. acta horticulturae, 712: 183-189. crisosto c.h., scott johnson r., dejong t., day k.r., 1997 orchard factors affecting postharvest stone fruit quality. hortscience, 32(5): 820-823. foley d.m., dufour a., rodriguez l., caporaso f., prakash a., 2002 reduction of escherichia coli o157:h7 in shredded iceberg lettuce by chlorination and gamma irradiation. radiation physics and chemistry, 63: 391-396. garcia e.l., barrett d.m., 2002 preservative treatments on fresh-cut fruits and vegetables, pp. 276-303. in: lamikanra o. 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20-25. impaginato 41 adv. hort. sci., 2023 37(1): 41­48 doi: 10.36253/ahsc­13910 quality of cold stored lemon fruit from orchards consociated to ancient olive trees m. allegra (*), f. ferlito, b. torrisi, s. trovato, g. cicciarello, m.c. strano consiglio per la ricerca in agricoltura e l’analisi dell’economia agraria (crea), centro di ricerca olivicoltura, frutticoltura e agrumicoltura, corso savoia, 90, 95024 acireale (ct), italy. key words: agroecology, agroforestry, cold storage, consociation, lemon fruit, postharvest quality. abstract: in the hilly area of gioia tauro (calabria, southern italy), lemon orchards are grown in consociation with centuries­old olive trees. lemons are partially shaded by olive canopies and the microclimate at the level of their canopies is suitable for plants growth and quality productions. under these conditions, lemon trees are grown even without irrigation, providing, despite this limitation, a quality product. this study aimed to i) investigate the qualita­ tive characterisation of two clonal selections of the lemon cultivar femminello, f. siracusano (s) and f. zagara bianca (zb), from the described intercropping, on irrigated (i) and non­irrigated (ni) crops; ii) assess the quality preservation during cold storage, in order to evaluate the availability of lemons for market­ ing in a period of shortage such as the summer season. fruits were harvested at commercial maturity, and cold stored at 10±1°c and rh 85­90%, for 60 days. decay incidence, physiological disorders, weight loss, and the main physical­ chemical parameters were assessed at harvest (t0) and every 15 days (t15, t30, t45, t60). the absence of decay and physiological disorders was observed throughout the 60­day storage period, in both clonal selections under the two management conditions. the weight loss was greater in fruits from irrigated lemon groves of both s and zb. s_i showed significantly lower fruits weight and higher titratable acidity than s_ni. total soluble solids and titratable acidity were statistically lower for zb_i than for ni fruits. 1. introduction several research have highlighted that some agri­food models are not sustainable from an economic, social and environmental point of view (ipes, 2016; fao, 2017). for many local agricultural contexts, the main perspective to survive is the design and set up of agricultural systems more at the service of the right to food (food sovereignty), enhancing ter­ ritoriality and reducing ecosystems degradation (loker and francis, 2020; ciaccia et al., 2021). in some cases these objectives are reached by the cultural consociation, adopted among crops with the aim of fostering (*) corresponding author: maria.allegra@crea.gov.it citation: allegra m., ferlito f., torrisi b., trovato s., cicciarello g., strano m.c., 2023 ­ quality of cold stored lemon fruit from orchards consociated to ancient olive trees. ­ adv. hort. sci., 37(1): 41­ 48. copyright: © 2023 allegra m., ferlito f., torrisi b., trovato s., cicciarello g., strano m.c. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 31 october 2022 accepted for publication 28 december 2022 ahs advances in horticultural science https://doi.org/10.36253/ahsc-13910 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2023 37(1): 41­48 42 agro­ecological services thus providing both environ­ mental and economic benefits (las casas et al., 2022). this represents an agroforestry model, mixed and multifunctional by definition, that should be pro­ moted, empowering marginalized actors and farmers by creating something different (rossi, 2020) and fea­ sible only by a holistic approach that embrace a long­ term vision, such as agroecology (barrios et al., 2020). these models are not purely productive, as the presence of trees also provides environmental services such as soil improvement, surface runoff reduction, and conservation of biodiversity. at the same time, agroforestry systems provide aesthetic services as well (katsoulis, 2022). in the hilly area of gioia tauro (calabria, southern italy), lemon orchards are planted in consociation with centuries­old olive trees over 20 meters high. the general distance between olives is wide (10x10 m) according to their large and rarely pruned canopy, ensuring that the lemons are planted both in intra­ and inter­rows, in a 5x5 m design. with this regular agroforestry model, olives play a key role in terms of the agroecological service, characterizing the land­ scape and preserving the fragile area, preventing soil erosion, and increasing biodiversity. moreover, thanks to its long history in the area and manage­ ment, compared to the other intensive woody crops, does not require high external inputs, thus contribut­ ing to reducing environmental pollution (de graaff and eppink, 1999). the microclimate and the ade­ quate lighting conditions at the level of the lemon canopy is suitable for the growth of this culture and for obtaining high quality productions. under these conditions, lemon trees can be grown rainfeed, although irrigation may influence fruit quality at har­ vest and during the postharvest process (asrey et al., 2018; tadayon and hosseini, 2020). to the best of our knowledge no studies were carried out about the effect of the consociation of olive­lemon cultivations and on postharvest performance of lemon fruit, which quality could also be prolonged by proper cold storage (strano et al., 2022, 2023). the extension of the availability to market of high quality fruit, in a period of shortage such as the summer season, would allow to obtain the maximum economic profit for the growers. this study aimed to assess i) the physical­chemical parameters of two clonal selec­ tions of lemon cv femminello: f. siracusano (s) and f. zagara bianca (zb), with high commercial value, consociated with olive cultivations, from irrigated (i) and non­irrigated (i.e., rain­fed, ni) crops; ii) the qual­ itative response to the cold storage of lemons com­ ing from the two different management techniques. 2. materials and methods study site and fruit sample fruits of lemon (citrus limon (l.) osbeck) cultivar femminello, clones siracusano (orchard 1) and zagara bianca (orchard 2), both grafted on sour orange (c. aurantium l.), planted in a 5x5 m design on a loam soil, from irrigated and non­irrigated crops of the corresponding orchard, were grown in the hills of gioia tauro (calabria, italy; lat. 38,32 n; long. 15,97 e; elevation 200 m a.s.l.). according to the usda (2017) texture triangle, soil of the two orchards had a loam texture (orchard 1: 462 g kg­1 sand, 317 g kg­1 silt, 222 g kg­1 clay; orchard 2: 602 g kg­1 sand, 231 g kg­1 silt, 167 g kg­1 clay), and an organic matter content of 97 (orchard 1) and 88 g kg­1 (orchard 2). plants of irrigated crops were fully irrigated, corresponding to 95­98% of crop demand, while non­irrigated crops had no irrigation system, relying on rainfall for water (ferlito et al., 2014). fruits were harvested at commercial maturity in april 2022, transported to crea laboratories (acireale, sicily, italy), selected accordingly to their uniformity in size, absence of defects and alterations (lesions and/or rot symptoms) and, washed with tap water. three replicates of 100 fruits for each sample, were randomly placed in plastic boxes, for a total of 300 fruits, then air­dried at 20°c and stored for 60 days at 10±1°c and 85­90% relative humidity (rh). physical‐chemical changes fruits were evaluated at harvest (t0), for the fol­ lowing parameters: weight, rind thickness, carpel axis, peel and pulp color, firmness, juice yield, total soluble solids (tss), ph, titratable acidity (ta), matu­ rity index (tss/ta) and, after 15 (t15), 30 (t30), 45 (t45) and 60 (t60) days of storage, also for weight loss, decay incidence and severity of physiological disorders (chilling injury and aging). fruit weight loss, expressed as percentage, was evaluated on 30 fruits per sample (10 fruits/replicate) by weighting the same fruit at t0 and at each control. the results were calculated as follows: = [(m0­m1) / m0] × 100; where: m0 = the initial weight; m1 = the weight measured at each control. the juice was extracted using an electric citrus fruit squeezer and the pooled juice of five fruits per replicate was analyzed. the juice yield was calculated allegra et al. ‐ lemon‐olive tree consociation: effect of lemon quality 43 as follows: juice yield (%) = (juice weight/fruit weight) x 100. tss content was determined with a digital refractometer (atago rx­5000, atago, japan) and results expressed as °brix. titratable acidity (ta), expressed as % (w/v) of citric acid equivalent, was d e t e r m i n e d b y p o t e n ti o m e t r i c ti t r a ti o n ( t 5 0 automatic titrator, mettler toledo) with 0.1 n sodi­ um hydroxide solution (aoac, 1995; ladaniya, 2008). vitamin c (ascorbic acid, mg·100 ml­1) concentration w a s d e t e r m i n e d b y l i q u i d c h r o m a t o g r a p h y (rapisarda and intelisano, 1996). the technological index ‘ti’ was calculated according to kluge et al. (2003) by the equation: ti = (total soluble solids x juice percentage) / 100. ti is an important variable linked to the quality of the juices destined for the citrus industry. higher ti val­ ues correspond to better quality (chitarra and chitarra, 1990). decay incidence (%) was determined by a visual evaluation of fruit infected by rots. the severity of chilling injury was determined by the visual examina­ tion of the fruit pericarp, using a four­grade scoring system to estimate the damage of the rind surface: 0 = none, 1 = less than 5% (light), 2 = 5% to 25% (mod­ erate), and 3 = over 25% (severe). the incidence of the fruits affected by aging (%) was determined by the presence of fruits with a necrotic area of rind tis­ sue around the stem­end button. firmness measurement f i r m n e s s w a s t e s t e d b y a t e x t u r e a n a l y s e r (zwick/roell do­fb0.5 ts model 2002, genoa, italy) using an 8 mm flat probe (mitcham et al., 1996). two measurements were made on two opposite of the equatorial zones of 15 fruits per sample. the results were reported as the peak force in newton (n) (nasrin et al., 2020). colour evaluation fifteen fruits for each sample were taken, at each time interval, for the determination of peel and pulp colour by cielab coordinates l* (lightness), a* (red­ green component) and b* (yellow­blue component) using a minolta spectrophotometer cm­2500d (minolta, milan, italy). three color measurements were made for each sample fruit and the results were expressed as citrus colour index (cci) = (1000 × a*)/(l* × b*) (jiménez­cuesta et al., 1981). the effect of cold storage at 10°c on the color of lemon fruits was also evaluated, at each time inter­ val, through the chroma (c*), which defines the color intensity (higher c* values indicate brighter yellow color); the hue angle (h°), with values closer to 90° for the more yellow fruits and increasing for the greener fruits; and browning index (bi), both for peel and pulp (kluge et al., 2003). the browning index (bi) was used as an indicator of the intensity of brown discoloration. bi was calculated as follows (palou et al., 1999; olivas et al., 2007): bi = [100(x−0.31)] / 0.172 where: x = [(a* +1.75)] / [5.646 l + a* ­3.012 b*]. statistical analysis o n e ­ w a y a n d f a c t o r i a l a n a l y s i s o f v a r i a n c e (anova) was performed using statistica 6.0 soft­ ware (stat soft italia srl) according to a completely randomized experimental design. data are means of three independent determinations. means compar­ isons were performed by tukey’s hsd test at p≤0.05, 0.01, 0.001, based on the f­test significance. 3. results physical‐chemical changes observation of the fruits revealed the absence of decay and physiological disorders in both clonal selections under the two management conditions (i and ni) throughout the 60­day storage period (data not showed). as shown in figure 1, the weight loss was signifi­ cantly greater in fruits from irrigated lemon groves of both clonal selections, with average values of 23.7% fig. 1 ­ weight loss percentage of 30 lemon fruits from each clonal selection (femminello siracusano and f. zagara bianca) and irrigation management (i: irrigated; ni: non­ irrigated) monitored during cold storage at 10±1°c and rh 85­90%. textured bars represent total weight loss from t0 to t60 (p≤0.001 ***). means of the same time interval indicated by different letters are significantly dif­ ferent (lowercase p≤0.05, uppercase p≤0.001) based on tukey’s hsd test. error bars show the standard deviation. adv. hort. sci., 2023 37(1): 41­48 44 (i) vs 12.9% (ni), in the interval from t45 to 60 days of cold storage, although it remained 3.6% (i) and 3% (ni) up to t45. the response to irrigation management resulted clonal selection­specific. the response of each clonal selection over time is reported. the width of the carpellar axis did not reveal significant differences in the comparison between the irrigation managements of each clonal selection, except at t15 in the case of s, and at t30 in the case of zb, in favor of ni (fig. 2). no significant difference between managements in the percentage of juice and tss for s. only at t60, the juice yield of zb_i was significantly higher than zb_ni, while tss already differed at t0 up to t30 in favor of zb_ni (fig. 2). the titratable acidity had a dif­ ferent behavior for the two clonal selections, with i statistically higher for s at t15, t45 and t60, and ni for zb at t15 and t45 (fig. 2). tss/ta was statistically higher for s_ni, compared to s_i, while no difference was recorded for zb (fig. 2). peel percentage and peel to pulp ratio did not show significant differences. only at the end of the storage (t60) zb_ni showed significantly higher val­ ues (fig. 3). fig. 3 ­ response of the physicochemical parameters (peel, % w/w; peel to pulp ratio; technological index, ti; peel and pulp citrus color index, cci) monitored during cold stor­ age at 10±1°c, rh 85­90%, of lemon fruits from two clon­ al selections (femminello siracusano and f. zagara bian­ ca) to irrigation management (i= irrigated; ni= non­irri­ gated). means of the same time interval indicated by dif­ ferent letters are significantly different (lowercase p≤0.05*, uppercase p≤0.01** and p≤0.001***; ns, no significant differences) based on tukey’s hsd test. error bars show the standard deviation. fig. 2 ­ response of the physicochemical parameters (carpellar axis, mm; firmness, n; juice, %; total soluble solids, tss °brix; titratable acidity, ta %; tss/ta ratio) monitored during cold storage at 10±1°c, rh 85­90%, of lemon fruits from two clonal selections (femminello siracusano and f. zagara bianca) to irrigation management (i= irrigated; ni= non­irrigated). means of the same time interval indi­ cated by different letters are significantly different (low­ ercase p≤0.05*, uppercase p≤0.01** and p≤0.001***; ns, no significant differences) based on tukey’s hsd test. error bars show the standard deviation. allegra et al. ‐ lemon‐olive tree consociation: effect of lemon quality 45 vitamin c values were significantly higher for s, while no significant differences emerged between i and ni management (fig. 4). 4. discussion and conclusions the modern woody crops systems are generally characterized for a strong modification on agro­ ecosystem structure and functioning. in a climate change scenario also a modification of the related fig. 5 ­ response of the colour parameters of fruit peel and pulp (chroma, c*ab; hue angle, h°; browning index, bi) moni­ tored during cold storage at 10±1°c, rh 85­90%, of lemon fruits from two clonal selections (femminello siracusano and f. zagara bianca) to irrigation manage­ ment (i= irrigated; ni= non­irrigated). means of the same time interval indicated by different letters are significant­ ly different (lowercase p≤0.05*, uppercase p≤0.01** and p≤0.001***; ns, no significant differences) based on tukey’s hsd test. error bars show the standard deviation. fig. 4 ­ variation of the vitamin c content during cold storage at 10±1°c, rh 85­90%, of lemon fruits from two clonal selections (femminello siracusano ‘s’ and f. zagara bian­ ca ‘zb’) and two irrigation management (i= irrigated; ni= non­irrigated). means observed at each time interval relative to each irrigation management of the clonal selections indicated by different letters are significantly different (p≤0.001) based on tukey’s hsd test. textured bars represent the results of the factorial anova (p≤0.05*; ns, no significant differences). error bars show the standard deviation. firmness measurement no significant difference during storage regarding firmness for zb, while s showed higher values for i as early as t0 (fig. 2). colour evaluation while the color parameters and indices relating to the pulp very rarely showed significant differences between irrigation managements i and ni (fig. 5), those relating to the peel show in some cases signifi­ cant, and often antithetical, differences between the two clonal selections. this is the case of cci, chroma and bi, for which s_ni had statistically higher values than s_i, while the opposite occurred for zb. the peel h° had a specular behavior with respect to what was described for the other indices (fig. 5). differences at harvest (t0) occurred in the case of s, for firmness (fig. 2), peel cci (fig. 3) and peel bi (fig. 5) with higher values for i, which was probably later in ripening, as the lower tss/ta value seems to demonstrate (fig. 2). zagara bianca showed differences already at har­ vest (t0) in tss (fig. 2) and peel h° (fig. 5), higher in ni, while peel cci (fig. 3), c* and bi (fig. 5) were higher in i. 46 adv. hort. sci., 2023 37(1): 41­48 agronomical processes such as the water and mineral availability, the solar radiation and the spontaneous flora growth could be affected (ciaccia et al., 2019). in the present work the response of the particular consociation between the lemon and the old­century olives” in calabria was investigated, assuming that it could allow to lengthen the lemon quality traits and shelf life throughout the response to long periods of cold storage (fung et al., 2019). furthermore, the response of irrigated and non­irrigated lemon groves was compared. understanding these linkages, the obtained data can predict how the effect of a single agronomic practice at the micro and macro­area scales can reduce the water input for irrigation. moreover, the lemon storage could drastically reduce the lengthen of the irrigation season. moreover, from the econom­ ical point of view, the proposed study can be an evaluable way for the marketing of fresh lemons. in fact, as observed by ciriminna et al. (2020), the fresh lemons have a constant demand from consumers which in some seasons cannot be entirely satisfied by italian production. finally, the consociated model could be diversified also adopting other species that are well adapted to shade such as berries (cicala et al., 2002). the microclimatic effects induced by the presence of the olive trees are reflected on the preservation of the fruit quality, on the protection from natural adversities such as excessive insolation or hail, and seems to result in a postponement of ripening, par­ ticularly in non­irrigated lemon groves (brunori et al., 2019). the results of this study seem to support the hypothesis of the effectiveness of the shading effect and of the microclimate induced by the presence (cover, protection) of the olive trees, on the quality of the production of both clonal selections studied, and on the sustainability of non­irrigated crops, given the comparability of many of the qualitative parame­ ters with those of irrigated crops. therefore, despite the considered period is not too long to draw general conclusions the preliminary results of the research show that the agroecological system and the use of the non­economical valuable olive as an agro­ecolog­ ical service crop could realize a mutual relationship into the agroecosystem. storage at 10°c avoided the development of decay and physiological disorders regardless of clonal selection and irrigation management. management strategies that increase the produc­ tivity of existing agricultural land are increasingly needed (salmon et al., 2015). the diffusion of the consociation here studied could allow the exploita­ tion of large tracts of land that host centuries­old olive groves, which must be preserved for their envi­ ronmental and cultural importance, but no longer usable at a production level, given the very high man­ agement and harvest costs (brunori et al., 2019). additionally, the possibility of eliminating or reducing the water supply makes this intercropping particular­ ly interesting. increased agricultural productivity is generally achieved by increasing inputs. irrigation, which cur­ rently accounts for about 70% of global freshwater withdrawals, is one of the most important and wide­ spread means of achieving this goal (lobell et al., 2009). rain­fed croplands, also called dryland farm­ ing, include all cropland where no water from any storage or delivery mechanism is utilized, but crops are not flooded, and where harvesting occurs at least once per year (salmon et al., 2015). as a result of the present work, the comparable quality of the fruits at harvest, both with and without irrigation, and their qualitative maintenance with cold storage would allow to have a good product on the market even in periods of shortage. the comparison with lemon groves without olive trees will be investigated in order to estimate any qualitative improvement induced by the two crops consociation. references aoac, 1995 ­ official methods of analysis. ­ in: cuniff p. 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https://onlinelibrary.wiley.com/action/dosearch?contribauthorraw=fidalgo%2c+alexandra https://onlinelibrary.wiley.com/action/dosearch?contribauthorraw=scurria%2c+antonino https://onlinelibrary.wiley.com/action/dosearch?contribauthorraw=sciortino%2c+marzia https://onlinelibrary.wiley.com/action/dosearch?contribauthorraw=lino%2c+claudia https://onlinelibrary.wiley.com/action/dosearch?contribauthorraw=meneguzzo%2c+francesco https://onlinelibrary.wiley.com/action/dosearch?contribauthorraw=ilharco%2c+laura+m https://onlinelibrary.wiley.com/action/dosearch?contribauthorraw=pagliaro%2c+mario adv. hort. sci., 2023 37(1): 41­48 48 shallow irrigation wetted area and application of organ‐ ic mulch on citrus decline amelioration. ­ adv. hort. sci., 34(2): 213­221. usda, 2017 ­ soil survey manual. ­ soil survey division staff, soil conservation service, u.s. department of agriculture, handbook, 18. 199 1. introduction summer pruning of apples (malus x domestica) is a practice used primarily to enhance fruit quality through the manipulation of tree physiology and alteration of the canopy environment, particularly light. the majority of studies of summer pruning in apple have focused on physiological impacts such as photosynthate partitioning, winter hardiness, return bloom, fruit coloring and post-harvest quality. however, summer pruning can have impacts on apple diseases, because it alters canopy microclimate, can remove diseased tissue, improves deposition of fungicides and other chemicals, and in altering tree physiology may change resistance to disease. summer pruning may enhance disease management, but in some cases may increase the risk of disease. of the few studies that have investigated interactions between summer pruning and apple diseases, most were done on semi-dwarf trees, and very few have looked at high-density systems. in semi-dwarf trees, summer pruning offered both horticultural and disease management benefits. this is illustrated by an example from the mid-1980’s in the northeastern us, where summer pruning was used to compensate for the loss of daminozide in commercial apple production (autio and greene, 1990). the predominant cultivar produced in the region at that time was ‘mcintosh’, which frequently drops fruit to the ground before they are sufficiently colored for harvest. daminozide (alar®) prevented this premature fruit drop, allowing development of full fruit color. when the manufacturers of daminozide removed the registration for apple use, it created potentially devastating crop loss through premature drop in ‘mcintosh’. as a response, growers were encouraged to summer prune, primarily to accelerate development of fruit color through increased light penetration in the canopy. the practice was successful, to a large extent compensating for daminozide treatments. it also had a side-benefit, in that incidence of the summer disease complex sooty blotch and flyspeck (sbfs) decreased in summer-pruned trees, primarily as the result of reduced humidity and improved fungicide penetration in the canopy (cooley et al., 1997). at this time, the most recent major review of summer pruning was written 25 years before this review, focusing exclusively on effects on tree growth, yield, flowering, and fruit development (saure, 1987). since then, commercial apple production has seen wide-spread adoption of highdensity planting systems in which the methods and impacts of summer pruning would be expected to differ substantially from those used in semi-dwarf (e.g. m.7, ca. 5 m tall) trees, but few studies have examined the impacts of summer pruning on apple diseases in high-density trees. in larger, semi-dwarf trees, summer pruning does not have an impact on the tree scaffold, but focuses on small branches and is largely intended to increase light penetration and summer pruning of apple: impacts on disease management d.r. cooley, w.r. autio department of plant, soil and insect sciences, university of massachusetts amherst, amherst, ma, usa. key words: apple scab, black rot, canopy management, cultural control, fire blight, malus x domestica, microclimate, nectria canker, powdery mildew, sanitation, sooty blotch and flyspeck, white rot. abstract: pruning, including summer pruning, of apples can have a positive impact on disease management in two basic ways: through removal of dead tissue and inoculum, and through alteration of the canopy microclimate. summer pruning can also increase diseases if it is done when disease risk is high. however summer pruning is used almost exclusively as a horticultural tool to improve fruit quantity and quality. as orchard planting and training systems have moved from semi-dwarf trees to high-density, fully-dwarf trees, very few summer pruning studies have looked at impacts related to disease, yet summer pruning in high-density systems may have important disease management effects. growers should avoid summer pruning practices which will increase disease risks, and use those that offer both horticultural and disease management benefits. more research in this area is needed, as cultural components of apple disease management will become increasingly important in sustainable production systems. this review looks at important apple diseases, including apple scab, fire blight, sooty blotch and flyspeck, black rot, white rot, nectria canker and powdery mildew, and uses dormant pruning studies plus knowledge of the epidemiology of the diseases to suggest ways that summer pruning would be expected to impact disease management. adv. hort. sci., 2011 25(3): 199-204 received for publication 20 june 2011 accepted for publication 27 july 2011 200 air circulation, beneficial to both fruit quality and disease management. however, in modern high-density production systems a primary goal is light penetration, and overall tree training maintains a relatively small, open canopy. in such trees, summer pruning may not significantly increase air circulation, improve drying in the canopy, or improve pesticide deposition, and it is unclear whether it has benefits in terms of disease management. instead, pruning cuts during the growing season may increase the risk of infection. alternatively, pruning and removing diseased tissue, sanitation, may reduce disease impacts and future risks, but such cuts may conflict with desired tree architecture in a high-density system. yet as restrictions on chemical use in plant disease management increase, cultural controls such as sanitation and inoculum destruction become more important. much of this review must extrapolate from studies on semi-dwarf trees, dormant pruning, and the epidemiology of apple diseases to identify potential benefits and risks of summer pruning related to disease. it outlines types of summer pruning used in high-density systems, and then looks at important apple diseases that may be impacted by summer pruning and how pruning for sanitation may be useful. 2. impacts on tree growth, yield, flowering and fruit development summer pruning can take on various forms from simple watersprout removal only to significant reductions in canopy density. much study of summer pruning came from an interest in enhanced light penetration into the summer canopy, thus improving fruit color development. vincent (1917), preston and perring (1974), stiles (1980), lord and greene (1982), marini and barden (1982), morgan et al. (1984), autio and greene (1990), schupp (1992), and ystaas (1992) all showed increased fruit redness as a result of summer pruning. decreases in fruit size, however, have also been reported in some studies (stiles, 1981; marini and barden, 1982; greene and lord, 1983; myers and ferree, 1983) but not all and not consistently. li et al. (2003) modeled tree physiology as a result of summer pruning and found reductions in carbohydrates, potentially leading to a carbohydrate shortage after summer pruning. the potential for shortage was greater as the intensity of summer pruning increased. fruit size impacts of that shortage can be mediated by improved water status resulting from reduced transpiration. additionally, varying responses may be partially explained by the location of the summerpruning treatment. greene and lord (1983) suggested that, as the severity of pruning increased or as the distance between the cut and the fruit decreased, the potential for a size reduction is enhanced. timing of summer pruning also is an important consideration. in general, regrowth during the period following summer pruning is greater the earlier the summer pruning is performed. autio and greene (1990) showed a linear decrease in the amount of regrowth as the pruning was performed from early (~45 days after full bloom) to late summer (~105 days after full bloom). zamani et al. (2006) described a similar response from summer pruning from 30 to 90 days after full bloom. such data indicate that summer pruning acts as a stress on apples. if a stress is severe enough it can predispose woody plants to disease particularly from canker pathogens, but plants will recover from light to moderate stress (schoeneweiss, 1981). at the same time, abiotic stresses can induce disease resistance in plants, including apples, though the physiological mechanisms behind induced resistance are not well understood (hammerschmidt, 1999; poupard et al., 2003). developing a better understanding of the detrimental and positive impacts of pruning stress at the physiological level is an area in need of more research. 3. impacts on disease pruning apple trees, including summer pruning, can impact disease in several ways: by altering microclimate and architecture of the canopy, by removing inoculum and infected tissue, and by creating wounds that pathogens can invade. in their experiment in an organic orchard, simon et al. (2006) observed that centrifugal training decreased apple scab and key insect pests in an organic orchard, and listed five explanatory hypotheses: 1) removal of inoculum and arthropods with the removal of fruiting spurs; 2) change in canopy microclimate, particularly better aeration; 3) decreased shoot density and increased distance between growing shoots slowing transmission; 4) changes in tree physiology inducing resistance or otherwise changing tissue susceptibility; 5) decreased canopy density improving pesticide penetration and deposition. pruning for sanitation is specifically designed to remove inoculum, thereby delaying or slowing epidemics and decreasing disease incidence and severity. for example, pruning removes primary inoculum of apple powdery mildew and fire blight, and is routinely recommended as part of the management programs for these diseases (covey and fischer, 1990; xu, 1999; steiner, 2000; holb, 2005). however, pruning for sanitation often requires pruning cuts that do not conform to horticultural goals, and in most cases is performed in winter or early spring rather than summer. summer pruning may remove inoculum, though it is not the primary purpose of the practice. changing the canopy density alters microclimate thereby impacting infection and disease development. microclimate factors, particularly those related to moisture, have a major effect on plant diseases (huber and gillespie, 1992), and altering canopy density, as summer pruning does, influences canopy drying and disease (gubler et al., 1987; cooley et al., 1997; sentelhas et al., 2005; batzer et al., 2008). leaf wetness duration (lwd) is a particularly important factor in plant disease epidemics and is often used in disease forecast models (gleason et al., 2008). for example, lwd has a major impact on whether or not apple scab (venturia inaequalis) infections occur (mills, 1944; 201 machardy and gadoury, 1989; stensvand et al., 2005; xu and robinson, 2005). high humidity and lwd also increase disease incidence and severity of sbfs (cooley et al., 2011), cedar-apple rust (gymnosporangium juniperivirginianae) (aldwinckle et al., 1980), black rot (botryosphaeria obtuse) (arauz and sutton, 1989), white rot or bot rot (botryosphaeria dothidea) (sutton and arauz, 1991), nectria canker (nectria galigena) (krahmer, 1981; xu et al., 1998), and fire blight (erwinia amylovora) (steiner, 2000). of these diseases, apple scab, sbfs, black rot, white rot and bitter rot drive most of the fungicide applications made in the eastern us, and account for substantial fungicide use in many apple production areas around the world (cooley, 2009). by maintaining an apple canopy that dries relatively quickly, lwd periods are shortened, which may allow decreases in fungicide use. the relationship between plant growth and the rate of an epidemic is complex, depending on tissue susceptibility, existing infections and the density of susceptible tissue, among other factors (ferrandino, 2008). for apple scab, the relative risk of infection is affected by the amount of leaf tissue available for infection interacting with increasing ontogenic resistance and inoculum availability, which all change over time (ficke et al., 2002). summer pruning removes target tissue as well as sources of inoculum, and hence should slow development of apple scab epidemics. the timing of tissue removal should affect disease, and in general removal early in the growing season should reduce disease more than midor late-season removal. still the interaction between pruning and the different epidemiological factors is complex. in the scab example, if early pruning stimulates vegetative growth, the rapid development of relatively large amounts of young, susceptible tissue may erase any earlyseason disease suppression. holb et al. (2004) observed that heavy winter pruning suppressed foliar scab, but the impact on fruit at harvest was not significant. for fire blight, it is recommended that flower clusters be removed from non-bearing trees before bloom, because the flowers are an important infection court for e. amylovora (steiner, 2000). pruning relatively non-productive flower clusters, as in centrifugal training, may reduce risk of fire blight. pruning also usually improves penetration of fungicides and other disease controlling chemicals (sutton and unrath, 1984; travis et al., 1987; cooley et al., 1997; cross et al., 2003). both summer (cooley et al., 1997) and winter pruning (ocamb-basu et al., 1988; holb, 2005) have been shown to improve spray penetration relative to non-pruned trees, though a comparison between two high density pruning methods in which centrifugal training reduced scab relative to original solaxe pruning did not show a difference in spray deposition (simon et al., 2006). apple scab scab is probably the most important diseases threatening apples in humid production regions, but only one published study has been done on the impacts of summer pruning on the disease. disease incidence in apple scab is strongly related to the duration of wetting periods and the amount of inoculum available in an orchard, with longer wetting periods and more inoculum increasing disease incidence and severity (machardy, 1996). simon et al. (2006) showed a decrease in scab in high-density plantings pruned using centrifugal pruning relative to original solaxe pruning. they suggested that centrifugal pruning shortened wetting periods in the canopy thereby reducing the number and/or intensity of infection periods, though they do not present data on canopy microclimate. a study by holb (2005) looked at different levels of winter pruning on scab in high-density organic orchards, and concluded that heavy pruning significantly reduced the area under the disease progress curve for foliar and fruit scab on susceptible cultivars. they hypothesized that suppression of scab epidemics were caused by a reduction of inoculum overwintering in apple buds, improved fungicide deposition in pruned trees, and modification of the in-canopy microclimate, though microclimate factors did not consistently vary among pruning regimens. fire blight fire blight is an increasingly serious disease of apples worldwide. the disease affects all apple tissues, but is most damaging when it migrates from primary infections, commonly in blossoms and young shoots, to limbs and trunks. scaffold limb and trunk infections are particularly damaging, cutting production over several seasons and often killing trees (van der zwet and beer, 1995; steiner, 2000; thomson, 2000). primary infections may also occur when trees are damaged mechanically by hailstones or other means, allowing the bacterial pathogen to enter the plant. in the case of fire blight, pruning for horticulture purposes also may provide entry points for the pathogen. an early study of effects of summer pruning on fire blight in apple showed that it markedly increased infections (lake et al., 1975). hence pruning when fire blight models (e.g. (steiner and lightner, 1996; smith, 1999) indicate risk of infection is high should be avoided if possible, or a treatment of streptomycin or other chemical prior to pruning be made if it is not. for example, mechanical hedging as practiced in fruiting walls would be expected to open multiple sites to fire blight infection for several days, and would be analogous to a hailstorm in terms of generating risk of infection from fire blight. fire blight epidemics often force growers to prune in an attempt to stop the progress of infections and remove inoculum. recommendations for such pruning generally suggest cutting back to a healthy branch union approximately 25 cm below visible infections; disinfection of pruning tools with alcohol or bleach between cuts may also be recommended (van der zwet and beer, 1995; steiner, 2000; toussaint and philion, 2008). in apples it has generally been recommended diseased tissue be pruned out as soon as symptoms are observed, and pruning continued at frequent intervals thereafter in order to slow and stop epidemics (covey and fischer, 1990; steiner, 2000; toussaint 202 and philion, 2008). shtienberg et al. (2003) found that factors related to the host, pathogen and environment should all be taken into account when determining whether and how to prune fire blight in pears, and elements of this approach may be useful in apples. sooty blotch and flyspeck summer pruning has been shown to decrease sbfs in apples, primarily because it reduces relative humidity and improves fungicide penetration and coverage in the canopy (cooley et al., 1997). this study was conducted on freestanding semi-dwarf apple trees approximately 5 m tall by 3 m diameter. it is not known whether similar results would be obtained in systems using fully dwarf trees in dense plantings. however a trial in the us showed that it took over 450 hours lwd for sbfs symptoms to develop in fully-dwarf, well-maintained trees while large trees with dense canopies developed signs at 225 hours lwd (ellis et al., 1999). the extent to which fully-dwarf trees in high-density systems may benefit from summer pruning has not be studied. mummified fruit have been shown to harbor inoculum for sbfs pathogens, and removing these mummies can reduce disease incidence (gleason et al., 2011). black rot and white rot black rot and white rot are fungal diseases that can attack fruit, foliage and woody tissue of apples. sutton (1981) showed that much of the inoculum for these diseases comes from prunings in or near production blocks, and it is recommended that prunings be removed or chopped so that they rapidly disintegrate so as to remove inoculum from the orchard. removing mummified fruit is also recommended as a cultural control. in that rates of these diseases on fruit is related to wetness duration and can be controlled with fungicides, pruning that reduces canopy humidity and improves fungicide coverage would be expected to enhance their management (sutton, 1981; arauz and sutton, 1989; arauz and sutton, 1990; sutton and arauz, 1991; parker and sutton, 1993). nectria canker nectria can cause infections on fruit and woody tissue in apple. pruning wounds are susceptible to the disease, and hence summer pruning can have an impact on canker incidence (krahmer, 1981; xu et al., 1998). studies have consistently shown that new wounds, including pruning cuts, are readily colonized by n. galigena. while this is another example of a disease that can be exacerbated by summer pruning, unlike fire blight, there are no forecast models to predict when summer pruning is less risky. there are fungicidal chemicals that can be very effective in reducing canker incidence if applied right after pruning, including the organically accepted slaked lime (heijne et al., 2005). powdery mildew since the early 20th century, dormant pruning has been recommended as a control measure against powdery mildew (fisher, 1920). more recent studies from eastern europe suggest that summer pruning that targets infected shoots can significantly reduce the disease, and even eliminate the disease (e.g. berbekov et al., 2006; holb and abonyl, 2007). disease reduction is probably primarily the result of inoculum removal, though reduction in canopy humidity may play some role as well (xu, 1999). 4. conclusions while the role of summer pruning in modern highdensity apple orchards is not disease management, the practice does alter the canopy architecture in ways that may reduce moisture levels and wetting period duration. for some diseases, notably fire blight and nectria canker, summer pruning can increase the risk of infection by causing wounds. while some recent studies show that summer pruning can reduce risk for major apple diseases, such as scab and powdery mildew, virtually no studies have been done outside europe. there is a need to assess the horticultural and disease management benefits and costs of summer pruning across a broader range of climates and highdensity production systems, in order to determine whether summer pruning can be an element in economically and environmentally sustainable apple production. 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(ed.) fire blight: the disease and its causative agent, erwinia amylovora. cabi, oxon, wallingford, uk, pp. 370. toussaint v., philion v., 2008 natural epidemic of fire blight in a newly planted orchard and effect of pruning on disease development, pp. 313-320. in: johnson k.b., and v.o. stockwell (eds.) proceedings of the eleventh international workshop on fire blight. international society horticultural science, leuven 1, belgium. travis j.w., skroch w.a., sutton t.b., 1987 effect of canopy density on pesticide deposition and distribution in apple trees. plant disease, 71: 613-615. van der zwet t., beer s.v., 1995 fire blight its nature, prevention, and control: a practical guide to integrated disease management. cornell university, usda agricultural information bull. no. 631, pp. 91. vincent c.c., 1917 winter versus summer pruning of apple trees. idaho agric. expt. sta. bul. university of idaho, usa. xu x.m., 1999 modelling and forecasting epidemics of apple powdery mildew (podosphaera leucotricha). plant pathology, 48: 462-471. xu x.m., butt d.j., ridout m.s., 1998 the effects of inoculum dose, duration of wet period, temperature and wound age on infection by nectria galligena of pruning wounds on apple. eur. j. plant pathol., 104: 511-519. xu x.m., robinson j., 2005 modelling the effects of wetness duration and fruit maturity on infection of apple fruits of cox’s orange pippin and two clones of gala by venturia inaequalis. plant pathology, 54: 347-356. ystaas j., 1992 effects of summer pruning on yield, fruit size, and fruit quality of the apple cultivar ‘summerred’. acta horticulturae, 322: 277-282. zamani z., saie a., talaie a., fatahi, r., 2006 effects of summer pruning on growth indices of two important iranian apple cultivars ‘golab’ and ‘shafi-abadi’. acta horticulturae, 707: 269-274. 232 1. introduction a key to success for the planting of new trees, both in open-field nursery and in the urban environment, is the protection of young plants from non-crop plant species (including some hardwoods, shrubs, grasses, and forbs). these fast-growing plants often kill or greatly suppress desired trees by competing with them for light, water, and nutrients. as a result, nurserymen, arborists and urban forest managers generally use herbicides to suppress noncrop vegetation. however, the use of herbicides in the urban environment may be limited or even banned in certain countries and/or municipalities. as a consequence, to protect young trees, environmentally sound, effective, cost-efficient, and socially acceptable techniques for managing non-crop vegetation are needed. in this scenario, we focused on the need for the establishment of environmentally friendly and low cost management methods for nurseries and urban green areas. mulching and its skilled use can contribute to such a development by improving organic matter content in the soils and by affecting other soil characteristics (harris et al., 2004). for tree management in the urban landscape, especially in the first years after planting, mulching with organic materials can be advantageous. organic mulching with different materials (mainly shredded wood, chipped wood, pine bark and composted materials), when skillfully applied, is an environmentally friendly way of establishing, protecting and managing young trees at a low cost in a new plantation. a recent review compared the costs and benefits of landscape mulches as reported in the technical and scientific literature, underlining how plants and soil can both benefit in terms of weed suppression, evaporation reduction and other environmental modifications (chalker-scott, 2007). even if mulching is a world-wide practise in urban green areas and different materials (as noted above) can be used for this purpose (rakow, 1989), little research has been done in italy to determine its effectiveness. positive effects following organic mulch application have been obtained in previous research, demonstrating beneficial effects on soil physical and chemical properties (fraedrich and ham, 1982; litzow and pellett, 1983; watson, 1988; appleton et al., 1990; himelick and watson, 1990; smith and rakow, 1992; iles and dosmann, 1999; dahiya et al., 2007; tiquina et al., 2007) and on plant growth and physiology (watson, 1988; green and watson, 1989; appleton et al., 1990; himelick and wateffects of mulching with compost on growth and physiology of acer campestre l. and carpinus betulus l. a. fini, f. ferrini dipartimento di scienze delle produzioni vegetali, del suolo e dell’ambiente agroforestale, università degli studi di firenze, viale delle idee, 30, 50019 sesto fiorentino (fi), italy. key words: chlorophyll fluorescence, leaf gas exchange, mulching, soil respiration, soil temperature, spad. abstract: the aim of this work was to evaluate the effects of mulching with compost on growth and leaf gas exchange of two widely-used ornamental trees in comparison to local nursery management standards. in addition, effects on soil respiration, soil temperature and water evaporation from soil were determined. an equal number (180 each) of uniform hedge maple (acer campestre l.) and 180 hornbeam (carpinus betulus l.) were planted in an experimental plot located in pistoia. treatments compared were: 1) chemical weeding by herbicides; 2) natural grass cover, mowed twice per year; 3) harrowing once a year; and 4) mulching with mixed compost (50% green+50% from household waste, 5 to 10 cm thick). over a two-year period, stem diameter, shoot extension and leaf gas exchange were measured. in the second year leaf chlorophyll content, chlorophyll fluorescence, soil respiration, soil evaporation content and soil temperature were also recorded. mulching with compost influenced shoot extension and stem diameter growth of field maple and white hornbeam. plants grown with natural grass cover had, generally, smaller stem diameters and shoot growth than the other treatments. leaf gas exchange, chlorophyll content and chlorophyll fluorescence were influenced by the different treatments. soil respiration was unaffected by the different treatments while soil temperature was significantly lower in mulched plots. adv. hort. sci., 2011 25(4): 232-238 received for publication 5 august 2011 accepted for publication 9 november 2011 233 son, 1990). however, sometimes the results from mulching are variable as they are affected by different environmental conditions and different tree species (whitcomb, 1979; iles and dosmann, 1999). moreover, if the quality of the mulching materials supplied by the producers is not satisfactory, tree performances can be affected in a negative way. this fact can be related either to quality or misuse, i.e adding too much material which can negatively affect soil oxygen content (gilman and grabosky, 2004; hanslin et al., 2005), although watson and kupkowski (1991) found no detrimental effect from the application of 0.45 m of wood chip mulch over soil in which the roots of trees were growing. the application of bark mulch can sometimes decrease growth in the first year, but the effects on plant growth are positive when examined in the long term (samyn and de vos, 2002). this can be due to a temporary nitrogen depression until the microorganisms are able to decompose a sufficient amount of organic material to provide the needed nitrogen (craul, 1992). in fact, although this temporary depression mainly affects the interface between mulch and soil, it has been shown that fine roots tend to grow into the organic mulch layer (watson, 1988; watson and kupkowski, 1991), where nconcentration is temporary depressed. as far the composted material as concerned, it has to be remarked that it needs to be well characterised for nutrient values, stability and other properties for the support of tree growth and effect against weeds. in a review of the use of composts for mulching and soil amendments, sæbø and ferrini (2006) suggest designing the composts to fit the specific effects that are wanted. for example composts for mulching should consist of layers of compost of different particle sizes, so that nutrients can be supplied and weeds are not given good germination conditions. the purpose of the present study was to investigate the use of mulching materials and their possible influence on growth and physiology of two shade tree species widely grown in the urban environment. in addition, effects on soil respiration, soil temperature and water evaporation from soil were assessed in the second year of the experiments. 2. materials and methods one hundred eighty uniform acer campestre l. and 180 carpinus betulus l. two-year-old seedlings 1.20 m in height from container were planted in an experimental plot located in pistoia (43°56’ n; 10°54’ e). planting density was 1.80 m in-row and 2.50 m between rows. mean temperature and rainfall over the last 50 years are 14.3°c and 1257 mm/year respectively. however, during the years of the current experiment, a decrease in rainfall and an increase in mean temperatures were recorded: rainfall in the first and second year was 971 and 903 mm, respectively; while in both the first and second year, an extended dry period with no rainfall was recorded from early june to the end of july. plants were irrigated the first year after planting to help establishment. from the second year no irrigation or fertilization were applied to plants. after two years from planting, when trees had overcome transplant phase, the experiment was started and the following thesis were compared: 1) weeding by herbicides using glyphosate twice a year (w); 2) natural grass cover, which was mechanically cut twice per year (g); 3) tillage by harrowing at a depth of 15 cm, once per year (t); and 4) in-row mulching with green compost (layer 5-10 cm thick and 1.5 m wide) and natural grass cover soil between the rows (m). the experimental design was a randomized complete block with six blocks and 30 plants per block. the following year (three years after planting and one year after treatments), biometric and physiological analyses were performed. stem diameter was measured 5 cm above the root flare on all plants at the end of the growing season. at the same time shoot extension was determined on 10 shoots per plant on three plants per block (18 plants per treatment). during the growing season, leaf gas exchange was measured with an infrared gas analyzer (ciras-2, pp-system, new hertfordshire, uk) on six leaves per block (36 leaves per treatment x species) twice during the first growing season and four times during the second. measured parameters were net assimilation (a, µmol*m2*s-1) and transpiration (e, mmol*m-2*s-1). measurements were taken between the hours 8.00 and 13.00 on the first fully expanded leaf from the shoot apex. measurements were taken under fixed co 2 concentration (360 ppm) and saturating irradiance (1300 µmol*m-2*s-1) provided with a built-in red light emitting diode radiation source. water use efficiency (wue) was calculated as the ratio between a and e as described in a previous work (ferrini et al., 2008). leaf greenness index was determined in the second growing season with a minolta spadmeter (spectrum technologies, plainfield, il, usa) on six leaves per block (36 leaves per treatment x species). for each leaf, the spad value was obtained averaging three different measurements made in different points of the leaf blade. this parameter is a good indicator of leaf chlorophyll, n and carotenoid content (percival et al., 2008). chlorophyll fluorescence was measured three times in the second year with a portable plant efficiency analyzer handypea (hansatech ins., king’s lynn, uk) on six leaves per block (36 leaves per treatment x species). fv/fm values were obtained by placing leaves in darkness for 30 min by attaching light-exclusion clips to the leaf surface. fv/fm is the maximum quantum yield of the psii and it is a reliable indicator of the occurrence of environmental stress (maxwell and johnson, 2000). soil respiration and evaporation were measured with a soil respiration chamber (src, pp-system, new hertfordshire, uk) at a depth of 5 cm below soil surface. measurements were taken twice in july around midday on soil exposed to full sunlight (not shaded by the trees). at least 30 days had passed since the last remarkable rainfall 234 (fig. 1). four measurements per treatment per species and per replicate were made. before measuring respiration and evaporation, the mulch was removed from an area of about 25 cm2 and the chamber was placed on the soil beneath. after taking the measurements, mulch was spread again. soil temperature was measured with a temperature probe at a depth of 10 cm below soil surface four times per treatment per species and per replicate. all data were analyzed with glm using the spss statistical package for windows (spss inc., chicago, il, usa). effects of soil management technique and species were analyzed with a random model two-way anova. when no significant interaction between factors was found, differences among soil management techniques were tested with duncan’s multiple range test (p≤0.05 and p≤0.01). parameters which showed significant interaction between factors were plotted separately in order to compare each level of factor a (soil management) for each level of factor b (species) (chew, 1976). data on leaf gas exchange, fv/fm and soil parameters were analyzed per single sampling date, merged together and processed again to obtain an average value on annual basis. 3. results and discussion mulching with compost (m) affected shoot growth of acer campestre and carpinus betulus (table 1). significant interaction between species and soil management technique was found for shoot growth (table 1). in the first year of measurements, mulched plants of both species had higher shoot growth than the other treatments (table 2). in maple, no difference was found among treatments w (herbicide), t (tilling) and g (ground cover). in hornbeam, shoot growth was lower in g than in t and w. in the second year, m carpinus plants had greater shoot growth than w and t which, in turn, had longer shoots than g. in the second year, m and t acer had greater shoot growth than w. again, the shortest shoots were found in g maples. in the first year and second years, g plants had lower stem ditable 1 summary table of two-way anova for the investigated parameters parameter measurement unit management technique species management x species shoot growth (1 year) cm ** ** ** shoot growth (2 year) (g) ** ** ** stem diameter (1 year) (g) ** ns ns stem diameter (2 year) (g) ** ns ns leaf greenness index spad unit ** ** ns fv/fm ** ns ns a (1 year) μmol m-2 s-1 ** ** ns a (2 year) μmol m-2 s-1 ** ** ns e (1 year) mmol m-2 s-1 ns ** ns e (2 year) mmol m-2 s-1 ** ** ns wue (1 year) μmol co2/mmol h2o ns ns ns wue (2 year) μmol co2/mmol h2o ** ** ns soil temperature (2nd year) ° c ** ns ns soil respiration (2nd year) μmol m-2 s-1 ns ns ns soil evaporation (2nd year) mmol m-2 s-1 ** ns ns * and ** indicate significant differences between treatments at p<0.05 and p<0.01, respectively. table 2 effect of soil management technique on shoot extension in acer campestre and carpinus betulus in the first and second years shoot growth (cm) acer campestre carpinus betulus treatment 1 year 2 year 1 year 2 year chem. weeding 70.2 b 58.3 b 70.0 b 44.7 b tillage 70.0 b 57.5 b 67.0 b 53.4 a grass cover 69.6 b 45.5 c 57.1 c 35.6 c mulching 86.0 a 72.0 a 74.0 a 54.4 a different letters within the same column indicate significant differences at p≤ 0.01. fig. 1 rainfall from 1 june (day 1) to 31 july (day 61) in 2006 and 2007 measured in the experimental centre where the research was carried out. 0 10 20 30 40 50 60 1 2 3 4 5 6 7 8 9 1st year 2nd year weeks m m o f ra in 235 ameter than the other treatments (table 3); no differences in stem diameter were found between species (table 3). there are several reports on how turf or natural grass cover decrease plant growth (garrity and mercado, 1994; stork and jerie, 2003; yao et al., 2005; chalker-scott, 2007). in the present experiment, natural grass cover decreased shoot growth and stem diameter in both the species studied. also, carpinus betulus, whose growth was affected both in the first year and second year, is probably a worse competitor with turf than acer campestre. mulch increased growth of both species, and mulched plants had shoot growth and stem diameter similar or higher than plants grown in tilled soil. greater plant growth in response to mulching has been observed by many authors (sæbø and ferrini, 2006; chalker-scott, 2007; ferrini et al., 2008). some authors found that mulching decreased growth in the first year after application and most of the authors attributed this reduced growth to temporary immobilization of soil n due to the high c/n ratio of the mulch (ferrini et al., 2009). the mulch applied in this experiment had relatively low c/n ratio (about 30), so no nitrogen immobilization occurred and growth was enhanced even in the first year after application, in agreement with that reported in previous works (tilander and bonzi, 1997; erhart and hartl, 2003; sonsteby et al., 2004; granatstein and mullinix, 2008). leaf greenness index was affected by the different treatments and species (table 4). regardless of the species, mulched plants showed higher values than the other treatments. the lowest readings were found in w and g plants, whereas t plants performed intermediately. higher spad readings following low c/n mulch application were also found by granatstein and mullinix (2008), who found a soil n-enrichment due to mulch mineralization. we did not consider the effect of management technique on soil n, however according to percival et al. (2008), the higher spad-reading of m plants may reflect a better nutritional status generated by mulch application and its decomposition. leaf greenness index was also affected by species, with field maple having higher values than hornbeam (table 4). fv/fm was affected by soil management technique but not by species. fv/fm measurements of healthy, unstressed plants are associated with values ranging from 0.76 to 0.85 (percival, 2004; percival et al., 2006). regardless of the treatment, all plants in this experiment were subjected to some degree of stress because of the very low rainfall during the summer (fig. 1), since fv/fm values were lower than 0.75. in any case, m and t plants had a significantly higher fv/fm than g plants (table 4). fv/fm is very sensitive to oxidative stresses, and to drought stress in particular (angelopulos et al., 1996; maxwell and johnson, 2000; rong-hua et al., 2006). thus, the lower spad and fv/fm observed in g plants must be attributed to grass competition for nutrients and water. leaf gas exchange was affected by soil management technique and species (table 5). in the first year, net assimilation was higher in m plants than in the other treatments. in the second year, m and t had greater assimilation than g and w. transpiration was not affected by management technique in the first year, while in the second year t plants had greater transpiration than the other treatments. water use efficiency was not affected by soil management technique in the first year. in the second year, m and w had greater wue when compared to t and g. leaf gas exchange was also affected by species: hedge maple always showed higher values than hornbeam (table 5). a significant quadratic relation was found for acer (p<0.01; r2=0.855) and carpinus (p<0.01; r2=0.298) between leaf greenness index and net assimilation (fig. 2). leaf greenness index has shown to be accurate in predicting leaf n content (follett et al., 1992; wood et al., 1992). in our experiment, the relationship between spad and a was much stronger in hedge maple than in hornbeam, suggesting a n limitation to photosynthesis in the case of maple and a less n-dependant limitation in hornbeam. soil temperature was affected by management technique: in july (second growing season) plots mulched with compost were 13°c, 10.8°c and 7.2°c cooler than bare soil, tilled and turf plots respectively (table 6). this is consistent with previous works, which found that mulching is more effective than cover crops and tillage to reduce extreme summer temperatures and that, in dry climates, mulching can lead to a reduction in soil temperature of up to 10°c (martin and poultney, 1992; zhang et al, 2009). table 3 effects of soil management technique and species on stem diameter growth in the first and second years stem diameter (cm) 1 year 2 year effect of different treatments chem. weeding 6.5 a 7.5 a tillage 6.1 a 7.6 a grass cover 5.6 b 6.7 b mulching 6.2 a 7.9 a effect of species a. campestre 6.1 7.4 c. betulus 6.1 7. 4 different letters within the same column indicate significant differences at p≤0.01. table 4 effects of soil management technique and species on leaf greenness index and fv/fm in the second year greenness index (spad) fv/fm effect of different treatments chem. weeding 38.7 c 0.721 ab tillage 40.2 b 0.731 a grass cover 38.8 c 0.701 b mulching 42.3 a 0.743 a effect of species a. campestre 43.0 a 0.728 c. betulus 40.0 b 0.719 data are the average of two measurement campaigns made in the second year. different letters within the same column indicate significant differences at p≤ 0.01. 236 the significant reduction in soil temperature contributed to create a more favourable environment for root growth: soil temperature in mulched plots did not exceed 35°c, which is considered the threshold temperature above which root growth is hampered and root mortality increases (coder, 1996; fini and ferrini, 2007). the reduction of soil temperature under mulch is due to the low albedo and thermal conductivity of woody mulches (montague and kjelgren, 2004). this finding means that the radiation reaching the mulch was not reflected, but the mulch acted as insulation and prevented energy from being conducted to the soil. soil temperature under natural grass cover was lower than tilled and bare soil, but higher than mulch. the cover crop acted as a barrier which absorbed solar energy and shaded soil surface, but it also transpired soil water, reducing soil water content and, by consequence, its buffering capacity (zhang et al., 2007, 2009). this is confirmed by water evaporation from soil (table 6). evaporation was measured 30 days after the last rainfall. in the mulched plot, the mulch layer was temporary removed and the measurement taken on the soil below. the lowest evaporation was found in g plots and the highest value was found in m plots. in absence of irrigation and natural rainfall, the higher value measured in mulched soil provides further confirmation of the effectiveness of mulching to reduce evaporation: after 30 days of drought, soil was very dry fig. 2 relationship between leaf greenness index (chl, spad-units) and net assimilation (a, mmol*m-2*s-1) in acer campestre and carpinus betulus. table 5 effect of soil management technique and species on net assimilation (a, µmol*m-2*s-1), transpiration (e, mmol*m-2*s-1) and water use efficiency (wue, µmol co 2 /mmol h 2 o). data are the average of two (the first year) and four (the second year) measurement campaigns a (μmol m-2 s-1) e (mmol m-2 s-1) wue (μmol co2/ mmol h2o) 1 year 2 year 1 year 2 year 1 year 2 year effect of different treatments chem. weeding 8.2 b 6.3 b 2.5 2.0 b 3.6 3.5 a tillage 8.4 b 7.5 a 2.5 2.6 a 3.8 2.9 b grass cover 7.7 b 6.3 b 2.2 2.1 b 3.8 3.1 b mulching 9.3 a 7.5 a 2.5 2.0 b 4.0 3.7 a effect of species a. campestre 10.3 a 8.3 a 2.9 a 2.4 a 3.8 3.6 a c. betulus 6.5 b 5.5 b 1.9 b 1.9 b 3.7 2.9 b different letters within the same column and factor indicate significant differences at p<0.01. table 6 effect of the different treatments on soil temperature (measured 10 cm below soil level), soil evaporation (measured 5 cm below soil level), and soil respiration (measured 5 cm below soil level). data are the average of two measurement campaigns made in the second year treatment soil temperature (°c) soil evaporation (mmol m-2 s-1) soil respiration (µmol m-2 s-1) chem. weeding 43.4 a 28.0 b 0.3 tillage 41.2 a 29.7 b 0.4 grass cover 37.6 b 18.3 c 0.3 mulching 30.4 c 94.2 a 0.5 different letters within the same column indicate significant differences at p≤ 0.01. 237 in treatments g, w and t and no further evaporation was possible. on the contrary, moisture was still available under the mulch so that, after mulch removal, evaporation was higher than in the other treatments. soil respiration was somewhat higher in m and t plots, but differences were not significant (p=0.172). most of the parameters measured showed that plants performed better when mulching was used and there are probably multiple causes which determined these results. the effect of mulching in reducing soil temperature which can allow a higher root growth is probably the most important under the conditions of this study, also because it might have prevented dehydration. compost mineralization might also have increased soil nutrient content. these effects could have allowed a greater root growth and, as a consequence, greater water and nutrients absorption. unfortunately we did not analyze the soil and this might be a potential shortcoming of this research. 4. conclusions the use of compost as mulching material had great impact on plant growth and physiology. in agreement with the results obtained in a previous work (ferrini et al., 2008), the present study confirms that mulching with compost is a useful practice to improve plant growth, leaf gas exchange and leaf chlorophyll content. positive effects of organic mulching on soil organic matter, soil water holding capacity and weed suppression have already been revealed in previous works (sæbø and ferrini, 2006; chalker-scott, 2007; granatstein and mullinix, 2008; mulumba and lal, 2008). the present investigation provides the evidence that mulching also has positive effects on plant growth and physiology, comparable or even superior to tillage. despite being inexpensive and very effective in weed control, the use of chemical herbicides reduced shoot growth and gas exchange when compared to mulching. an even greater reduction in growth and carbon assimilation and a significant increase of oxidative stress on psii was found in plants growing with natural grass cover, mechanically cut twice per year. considering that mulching is cheaper than tillage and mechanical weeding (zhang et al., 2009), it can be considered an environmentally-friendly and sustainable alternative for managing plants in the forest, in the nursery and in the urban environment. acknowledgements we 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bird g.w., abawi g.s., thies j.e., 2005 orchard floor management practices that maintain vegetation or biomass groundcover, stimulate soil microbial activity and alter soil microbial community composition. plant & soil, 271: 377-389. zhang s., lövdahl l., grip h., jansson p.e., tong y., 2007 modelling the effects of mulching and fallow cropping on water balance in the chinese loess plateau. soil till. res., 93: 283-298. zhang s., lövdahl l., grip h., tong y., yang x., wang q., 2009 effects of mulching and catch cropping on soil temperature, soil moisture and wheat yield on the loess plateau of china. soil till. res., 102: 78-86. 170 1. apricot in apricot, growth habit and fruiting behavior are strongly inter-related. accordingly, the varieties of apricot can be classified into five groups (guerriero and xiloyannis, 1975 a; bassi et al., 2003). since some changes in shoot morphogenetic gradient during rest period were evident in response to dormancy and chilling (guerriero and scalabrelli 1982), any classification of branch habit should be strictly associated with a specific environment. depending on the type of bud (floral or vegetative) and shoot (sylleptic, long brindle, brindle or spur) the chilling requirement may be very different and, eventually, can be a factor in regulating branch habit and fruiting behavior (guerriero and xiloyannis 1975 b; guerriero and viti, 1997). to decide how to manage pruning, the shoot should be identified by its specific growth rate after bud break. for this purpose, its physiological behavior should be constantly monitored. there is some evidence that the emergence of sylleptic shoots is highly probable when a threshold level of growth rate is exceeded (zucconi, 2003). this means that in some conditions apical dominance is not able to inhibit the growth of lateral meristems, which thus originate sylleptic (anticipated) shoots rather than buds (fig. 1). generally, the buds (once formed) become rapidly dormant (fig. 2) and will only grow in the following spring after a specific amount of chilling, and as a result they will originate proleptic shoots. it is also possible that, depending on growing conditions, sylleptic shoot formation can take place more than once (fig. 1) along the shoot growth. thus buds that are formed on sylleptic shoots at different times compared to proleptic ones may have a different fruiting performance and time of flowering: this is frequently reported by growers. consequently the date of pruning, whether during the vegetative period (summer pruning, early or late) or during the winter before bud break, can have a strong influence in controlling the fruiting of different varieties. moreover, the knowledge of how the shoots grow is very important spring and summer pruning in apricot and peach orchards d. neri, f. massetani dipartimento di scienze agrarie, alimentari e ambientali, università politecnica delle marche, via brecce bianche, 60131 ancona, italy. key words: catalonian open vase, early and late pruning, plant architecture, shoot heading back, shoot thinning. abstract: spring and summer pruning are based on the possibility to manipulate the physiological control of axillary sylleptic growth and carbon allocation in the shoot through alteration of the apical dominance and light distribution in the canopy. the practical result in modern orchards is a higher flower bud differentiation for apricot and an easier training system maintenance for peach with more efficient use of labor. cultivated apricot varieties show diverse tree architectures, habit and fruiting branches. the effect of pruning intensity at different times during spring and summer seasons is specific for the singular growth habits. differences among the peach varieties are less evident than in the apricot. the ease with which peach water sprouts produce axillary sylleptic shoots makes the use of mechanical topping possible in the first two years of intense growth in order to train the tree as a bush and then to open it as a vase with manual pruning (catalonian open vase). in all modern peach orchards, pruning in late summer results very useful to obtain a better light distribution in the canopy and a more efficient carbon allocation to fruiting shoots, preventing and reducing the need for winter pruning. adv. hort. sci., 2011 25(3): 170-178 received for publication 9 june 2011 accepted for publication 17 september 2011 fig. 1 shoot growth rate and sylleptic shoot formation in apricot. this model assumes that the growth rate threshold for inducing sylleptic growth decreases during the season. left: the threshold was overcome only at the end of the season. right: the critical growth rate was overcome twice in the season. 171 for effective pruning, improving the chance of acclimatizing a cultivar that may be productive in a specific environment. pruning for apricot should be modulated both in intensity and timing according to the interaction between the variety and its environment. finally, the specific shoot physiology and architectures of fruiting branches of apricot varieties will determine very dissimilar regimes for pruning. for this reason there should be in each growing area a classification of the varieties, according to their precise branch habit, fruiting behavior and need for pruning. the most common classification in northern italy is arranged into three groups: a with very vigorous and spreading habit, and a tendency to fruit on spurs, brindles and sylleptic shoots; b with less vigorous, assurgent, or semi-spreading habit, fruiting on spurs and vigorous shoots; c with very vigorous, assurgent, or mixed spreading habit, and ability to fruit on all kinds of shoot (neri, 2003; pirazzini, 2004; neri et al., 2010). therefore, for each apricot variety, it is important to predict the response (in terms of the number and type of lateral shoots) to head back pruning of shoots and branches in different periods of the spring and summer seasons. pruning intensity and cultural techniques (fertilization, irrigation, soil management, and eventually forcing and protection conditions) play an extraordinary role in determining the final result and the possible optimal training system (neri et al., 2011). spring shoot heading back and thinning the intensity of head back pruning of growing shoots can be performed within these two extremes: short pruning (leaving half of the shoot or only the basal portion of it with three to five buds, as a spur) and long pruning (which reduces the apical portion of the shoot by pinching or cutting a few centimeters below the tip). generally, these pruning techniques are limited to the spring with fast growing shoots. the time of pruning can be intended as early or late spring pruning, in which the early pruning induces the formation of long sylleptic shoots, while the latter induces nil growth or the formation of few, short, sylleptic brindles with, likely, a higher flower differentiation aptitude. after spring heading back, shoot vigour is strongly reduced and the number of sylleptic shoots is generally increased. short head back pruning was generally less effective than long heading back in inducing flowering brindles, with the exception of weak varieties which need to improve shoot growth. the response to pruning is always higher in fertile and irrigated soil. apparentely the pruning in late spring induces a better response if it is limited to the terminal part of the long shoot (long pruning) (fig. 3). delaying spring pruning (late spring pruning) reduces the number of sylleptic shoots per single cut and also the fig. 2 left: apical dominance intensity along the growing shoot in early spring. centre: apical dominance and bud dormancy intensity along the growing shoot in late spring. right: apical dominance and bud dormancy intensity along the shoot in late summer. fig. 3 number of sylleptic shoots per single cut in response to short and long pruning delaying the operations from early to late spring. 172 flowering intensity. flowering intensity is higher when pruning is applied in early spring (may) in different varieties (fig. 4), probably because more sylleptic brindles were produced. apparently late pruning in june induces a better response if it is performed on the terminal part of the shoot (long pruning). shoot thinning is generally carried out a few weeks before harvest to improve fruit quality in very vigorous trees. it is devoted to eliminating overcrowded and malpositioned shoots. the final goal is to have better light distribution inside the canopy and less carbon directed to water sprouts and suckers which cannot be used for fruit production in the future management of the branches. summer shoot heading back and thinning summer shoot head back pruning aims to increase flower differentiation but in apricot this is possible only if there is new shoot growth, which can be induced by water supply after summer drought or by heavy cuts such as late summer heading back. summer shoot thinning can be performed with the aim of improving the quality of shoots as a consequence of better light penetration and carbon allocation. this practice reduces the need for winter pruning and can be useful in areas where frost damage may challenge flowering and therefore winter pruning must be delayed until after fruit set. different pruning intensity is meant to stimulate more vegetative vigour when the shoot is suddenly cut very short, but to induce the formation of flowering brindles when it remains quite long (fig. 5). under northern italian continental climate, the vegetative response to pruning is always greater in fertile and irrigated soils, resulting in greater shoot vigour. finally, sylleptic shoots tended to bloom later than the rest of the plant (a very important advantage in climates where late frost is common) but to bear fruits of small size, at least in some varieties (pirazzini, personal communication). varietal differences in response to spring heading back pinkcot. early short heading back induces numerous, equally balanced and productive sylleptic shoots. sylleptic shoot growth is very active when it is stimulated on vigorous shoots (water sprouts), which by the end of the season are well ramified. at blooming the number of flowers on sylleptic shoots is higher with long heading back than with short. short heading back resulted in more uniform distribution of brindles and spurs along the original branch. sweetcot. growth is greater after early shoot heading back: nevertheless only few sylleptic shoots were formed, they are not too long and the flower number is increased. in non-irrigated soils late pruning does not improve shoot ramification, while growth is short and rich in flowers. robada. in fertile soil, early pruning generally induces a few sylleptic shoots, often only one as an extension growth from the terminal bud, even though the number of flowers on all type of shoots is high. with late pruning there is a certain number of sylleptic shoots (brindles and fig. 4 flowering estimated entity in 8 varieties in response to pruning applied in may (early pruning), or in june. fig. 5 orange rubis forms shoots after early long-heading back (left): a high number of sylleptic shoot with good flower differentiation is formed. shoot after late short-heading back (right): sylleptic growth and flower differentiation is visible. 173 spurs) but fewer flowers per shoot. in non-irrigated soil, short heading back does not induce any good growth, neither for shoots nor flowers. orange rubis. long early shoot heading back is very effective in stimulating sylleptic growth with high flower differentiation, while short heading back is useful only with weak shoots. heading back of water sprouts, especially when late, does not favor flowering formation. kioto. the number of brindles and spurs is dramatically increased by early pruning, with very high flower differentiation along the old wood. late pruning is positive only on vigorous lateral shoots. flower differentiation is good in all the shoots after early pruning, and intermediate in sylleptic brindles in vigorous shoots after late pruning. flavorcot. both short and long early pruning induce weak growth in comparison with the vigorous cultivars; the sylleptic shoots are limited in number and growth even in water sprouts. late pruning does not induce good sylleptic ramification, but the flower induction is enhanced in brindles and spurs in all the plants. after early pruning, flowers are scarce in all the sylleptic shoots; after late pruning, flower production is much better in the old wood and in sylleptic brindles. zebra. early long head back pruning induces positive sylleptic shoot growth; short heading back is less effective even in water sprouts; flower differentiation confirmed this result. late heading back was negative and induced only few flowers. pieve. early heading back is generally positive and, especially with long pruning, the number of sylleptic brindles is higher and flower differentiation is good. late pruning reduces the branching of shoots, and induces a very limited number of flowers. pisana. for this low fertile variety, it is worth noting that the terminal shoot on the intact branch showed less growth than the second one below, as opposed to the heading back causing the terminal shoot to become the most vigorous of the branch. this means that in low fertility conditions the varieties of this group need to be stimulated by winter pruning instead of weakened by summer pruning. bella di imola. this variety shows very high productivity on the one-year shoot, the terminal portion being the most productive. growth was greater at the terminal position of the branch as well, revealing a much stronger acrotony than pisana, and greater vigor. for this reason it is important to avoid any pruning which induces a vegetative response which is too strong. in fact in orchards with low fertilization, growth was not excessive even with winter pruning; flower differentiation was high in any case. it can be hypothesized that in more fertile soils vigor can be too strong, and so late summer pruning can be widely utilized (neri et al., 2010). pruning in different training systems for apricot actual training systems for apricot are specific for each production area. the two most widely diffused training systems in the northern part of italy are free open vase (with several variations, from delayed open vase to bush) for low density, hilly orchards and spindle for high density systems in flat fertile lands with low vigorous rootstocks. the date and intensity of pruning effectively determines the branch architecture and fruiting potential of each cultivar. these observations lead us to conclude that for apricot, summer pruning is a basic practice in modern orchards but it must be adapted to local conditions and genetic material. shoot physiology, theoretically modeled on the basis of growth rate, can help in choosing the best period and most effective intensity for the pruning of each new cultivar in the different training systems of a particular growing area. we can generalize that summer pruning reduces vigor and induces greater flower production. early long shoot heading back is more effective with high vigor varieties and fertile soil conditions, whereas short shoot and branch head back pruning is favorable for weak and spreading varieties, although the latter habit could be more easily controlled by winter pruning than the former. varieties of group a, such as some of the new varieties, benefit from early summer pruning (early heavy shoot heading back) in order to induce the formation of sylleptic shoots; and summer pruning (without heading back but possibly with shoot thinning) to encourage shoots to be more lignified. group b performs best after winter pruning (shoot thinning and heading back of twoto three-year-old branches). group c may be pruned in late summer or at the end of winter (shoot thinning, heading back the branches), depending on local growing conditions. in order to limit the development of sylleptic shoots, which only bear small fruits, it is better to carry out heading back in late summer instead of in the winter. in any case, every pruning strategy must be tested on each variety before it is adopted throughout commercial orchards. this is due to the possible very specific influence of varietal differences in chilling requirements, and specific shoot and flower differentiation physiology. 2. peach in modern peach orchards, application of spring and summer pruning is increasing (from 20% up to 60% of the total amount of pruning), depending on the training system, production area and farm management (giovannini et al., 2010). peach shows good ability to form sylleptic shoots and strong epinastic control that makes the sylleptic shoot insertion angles wider moving from the top to the basal part of the shoot. these features require an appropriate shoot pruning technique and finally, if well managed, lead to dwarf the tree with the open habit of mature peach plants. during training of modern intensive orchards, spring pruning is therefore applied more than summer pruning (and obviously of winter pruning), in order to address the inclination of vigorous growing shoots and to anticipate formation of the skeleton structure of the canopy. the re174 moval of mal-positioned water sprouts and stimulation of a higher number of well positioned shoots (ferree et al., 1984; lanzellotti et al., 1998) finally dwarf the trees (kappel and bouthillier, 1995; hossain and mizutani, 2008). the second goal of a greater use of spring and summer pruning is to reduce the vegetative unproductive phase and enhance early bearing in all new training systems (giovannini et al., 2010; neri et al., 2010). summer pruning is applied to shorten the not-fruiting initial phase, improve light distribution on fruiting shoots and strengthen the future scaffold branches with a higher number of fruiting shoots (miller, 1987). nevertheless, pruning time in less intensive orchards is often determined by farm organization, depending on the availability of labor and arrangement of the working schedule, rather than on the plant physiology (chalmers et al., 1981; marini and barden, 1987; sansavini and neri, 2005). pruning can be also applied in the spring for biological reasons. in fact for some cultivars pruning intensity can be adjusted near blooming time, when flower buds enlarge and become more visible, depending on the quantity of buds that were damaged by frost during the winter. this kind of spring pruning can thus augment fruit set per tree. if the risk of frost damage is extended to blooming time, pruning can be carried out precisely during the fruit set period. in this case “winter pruning” is completely substituted by an early spring pruning which eliminates the excess shoots, based on the rate of fruit set, by heading back two-year-old branches. late spring pruning is commonly used for training, but in modern orchards it is not very common to control production if trees are mature and equilibrated. also early summer pruning, before harvest time, is used only to thin the water sprouts and to improve light distribution in the canopy, whenever the vigor is too high, to increase fruit color and quality and to prevent diseases. however when this pruning is too heavy or too early it can negatively affect fruit development. summer pruning after harvest can better manage excess vegetative growth and change the distribution of assimilates (rom and ferree, 1984; marini, 1985; mizutani et al., 1997; hossain et al., 2004). it improves bud differentiation and branch hierarchy organization. during summer, shortening branches results in a more regular sprouting in the following spring, with less vigorous shoots and high quality flower buds. in some cultivars, when it is necessary to cut big branches, the summer period is useful also because it induces a more rapid and efficient wound healing. this sort of pruning can also be considered when it is necessary to mitigate severe water stress (lopez et al., 2006; marsal et al., 2006). late summer pruning in august-september is important and widespread in all environments because it contributes to reduce canopy volume and allows shoot hardening. it partially prevents winter pruning (therefore it is called pre-winter pruning) and strongly reduces the need for it. furthermore, it is more selective than winter pruning as the best buds for fruit set are chosen in advance. summer pruning for peach training systems in mediterranean climate depending on the training system, both spring and summer pruning are applied to favor the branch inclination of shoots through pinching or cutting the upper part of the shoot to induce sylleptic ramification (fig. 6), also more than once per season as is done for the catalonian vase (monserrat and iglesias, 2011). removal of excess and mal-positioned shoots is also practiced to give a regular shape to the spindle and to the small open vase (neri et al., 2010). in any case, the pruning intensity is minimal and eventually some spring interventions are postponed from the first to the second year, and/or continued in the third if tree vigor is too weak. in mediterranean areas, with long growing season and early ripening cultivars, also vase training systems (i.e. low open vase) are commonly managed with the application of spring-summer pruning. in fact small vase formation can be improved using summer shoot cuts to direct vegetative growth to the well displaced lateral sylleptic and proleptic shoots. modern systems derived from the vase are characterized by a low scaffold (0.5 m above ground), low tree height (2.5 m), and free growth during the first years (bush type to enhance early bearing). fig. 6 catalonian vase during the first growing season. left: first manual topping when the shoot exceeds 100 cm from the soil. centre: second topping (manual or mechanical) when the shoots exceeds 150 cm from the soil. right: the final growth at the end of the first year (redrawn from monserrat and iglesias, 2011). 175 the catalonian vase, which originated in spain, is the most recent and wide-spreading vase system. spring-summer pruning is repeatedly employed to form and dwarf the trees during the first two years of training. mechanical topping is applied every 50 cm till the final size of the plant is reached (2.5 m) at the end of second year (figs. 6 and 7). topping involves removing a few apical buds per shoot, thus inducing suppression of apical dominance and increasing the possibility for growth of the external sylleptic shoots. this sets off a temporary strong competition among all growing shoots, but in peach the external shoots are privileged, and thus the main shoots are naturally oriented toward the external direction. finally, the whole plant height is lower but several shoots are well positioned to become the future branches of the vase. during the second year, or third in the case of low fruiting cultivars, the vase shape can be completed by thinning the primary branches and cleaning the central part, mainly in late summer. in mature orchards, pruning labor in this system can require less than 100 hr/ha (60% during vegetative season), and the fruiting winter pruning (the remaining 40%) completes the late summer pre-winter pruning (fig. 8). specific summer pruning is required also in the “y” trellis system. inclined branches with angles wider than 40-45° promote the growth of vertical shoots in the internal portion of the canopy and late spring and early summer pruning are necessary to remove them during the initial years. the high density planting of this system, the possibility to use long pruning, without eliminating shoot apex of primary branches, and the reliability of pruning during the vegetative season provide early fruiting. nevertheless, the “y” system is well performing only where climate conditions provide a high level of light, allowing the reduction of branch angles close to 30°, and thus reducing water sprout formation. this angle promotes a balanced vegetative growth and fruit production in all the lateral and basal portions of the two fruiting walls. “v” systems with double tree density and less vigorous rootstocks can be used to reduce further the need for spring and summer pruning. in this case, defining the two oblique productive planes is easier and more rapid but the cost of planting is very high and there is risk for overcrowding in the internal part of the canopy, which could induce an exponential increase in the need for pruning. spring and summer pruning in peach training systems for continental climate in the northern part of italy, where there are short growing seasons and high risk of frost in the spring, with midseason varieties high hedgerow systems (palmette and central leader, 4 m high) are still popular. this is because late spring frost may dramatically damage the production in the bottom part of the tree (first 2 m from the ground). in this condition pruning may be delayed after blooming (when fruit set is already complete). thus winter pruning becomes an early spring pruning, while early summer fig. 7 catalonian vase during the second growing season. left: first mechanical topping when the shoots exceed 200 cm from the soil. right: second mechanical topping when the shoots exceed 250 cm from the soil, and manual pruning to thin the main branches down to four to five in number, opening the centre in very late summer. this last pruning is done only if the variety has a very high productivity (redrawn from monserrat and iglesias, 2011). fig. 8 catalonian vase during the fourth growing season at blooming in the sibari area. the skeleton is completed and the plant is dwarf and equilibrated. 176 pruning is not frequent and limited to lightening the upper part of the canopy (if necessary). during the training period to reduce shoot vigor of the mal-positioned water sprouts, turning and partially crashing them can improve their fruiting aptitude. late summer pruning is used to maintain the shape of the trees and to increase lignification of the shoots that otherwise would be too shaded. the hedgerow made by trees trained as “u” or candelabras along the row requires less green pruning than palmette or central leaders because of less vigor of each vertical branch. for training high density peach orchards, well feathered scions from the nursery must be used to obtain fruit production in the second year. in mature orchards, green pruning is mandatory in two specific seasons: i) in early spring before harvest to eliminate water sprouts and to induce a higher number of productive shoots in well defined positions; after fruit set it is also possible to thin the shoots proportionally to the amount of fruit set; and ii) in late summer after harvest to anticipate the winter pruning (pre-pruning). in this case the water sprouts are eliminated and the vigorous shoots on the main branches are cut only if there is excess flower differentiation; the top part of the branches can be reduced to better permit light distribution in the canopy. if the prepruning in late summer is well executed with light shoot thinning, winter pruning can be avoided. in this way labor can be saved and/or better organized. in low bush open vase (delayed open vase) (sansavini and neri, 2005), for the first three-four years the training of the trees is free with only a few pruning cuts; green pruning is not important. late summer pruning becomes important in the third to fourth year to cut the central leader and to open the centre of the vase. in the fourth to fifth year, the main branches are headed back and the tree is completely formed as a vase. finally, pre-winter pruning is necessary to manage fruit shoot quality and quantity when production becomes important (starting from the third year). production pruning when the tree is well mature and fully formed, spring pruning is less important and must be carried out only in very specific cases when excess vigor of the growing shoots can compete with the growing fruits, interfering with the fruit quality and flower induction for the next year. peach production is located on one-year shoots (brindles, fruiting shoots and in some varieties also in water sprouts) and in a very limited quantity on the spurs (these are important only in clingstone peaches and in some nectarines with low fertile shoots). because of this specific fruiting behavior, it is very important to control shoot growth to form highly specific shoots in each variety (day et al., 1989). if the vigor is very high (generally in early ripening varieties) it is necessary to execute the first pruning before harvest in late spring in order to eliminate mal-positioned water sprouts and to improve light distribution in the canopy. whereas pruning in late summer is very helpful both in early and late ripening varieties to improve the quality of the fruiting shoots, favoring shoot hardening and carbon allocation. to improve light distribution in the canopy it is important to thin the shoots and to head back the branches. this pruning in late summer anticipates winter pruning, which consequently can be delayed at blooming to determine the final number of flowers per plant. winter pruning can even be eliminated and early spring pruning after fruit set can be applied to determine more precisely the number of fruits per plant. it is important to remember that avoiding heavy shading is important to obtain homogeneous distribution of the shoots along a branch. in fact when the shoots are shaded they can be damaged during winter and necrotize. because peach trees do not produce adventitious meristems and do not maintain latent buds for long, winter pruning is not able to recover new adventitious shoot growth and finally the shaded area of the canopy is lost. to avoid this dramatic loss of efficiency of the internal part of the canopy, springsummer pruning is mandatory in modern orchards. this problem is even more accentuated in high density planting systems in which early spring pruning can be associated with fruit thinning to reduce the impact of self shading and inter shading on shoot and fruit quality. traditional low density orchards under continental conditions are mainly pruned using precise winter pruning because once the open vase is well formed it provides high quality and constant fruit production. 3. conclusions in apricot each group of varieties has its own optimum season and intensity for pruning, according to fruiting aptitude and branch habit. for high density orchards, heavy late spring pruning may be used to reduce vigor and improve flower differentiation during the summer. for free open vase, use of late summer pruning only can be suggested to obtain a better carbon partitioning towards the fruiting shoots and a more uniform light distribution in the canopy. the higher the flower differentiation aptitude, the greater the possibility to use different seasons for pruning. with low aptitude, manipulation of spring growth of the shoots and light distribution in the canopy through springsummer pruning is mandatory. apricot varieties can be classified into groups characterized for different vegetative and reproductive habits, accordingly and depending on the fertility of the soil, they can be pruned in summer (if soil fertility is high) or in winter (if soil fertility is low). some of the new low productive varieties benefit from early summer pruning (early or late spring shoot heading back) to induce the formation of sylleptic shoots and to obtain good flower differentiation. weak varieties perform the best after winter pruning (branch heading back). other, very productive varieties may be pruned in late summer or at the end of winter (shoot or branch heading back) depending on local growing conditions (pirazzini, 2004). 177 in peach, habit is mainly influenced by assurgent growth in low chilling varieties that are well adapted to the mild mediterranean climate with low chill in winter, and by the widespread habit of the varieties, suitable for more continental climatic conditions with cold winters and tolerant to late frost in the spring. among these groups of varieties, flower aptitude is another factor which influences the type and intensity of spring and summer pruning. in modern peach orchards, late summer pruning is widely diffused as a common practice to manage light distribution and carbon allocation and finally shoot quality. this technique is applied in substitution or to reduce the amount of the winter pruning. late spring pruning is applied only if necessary when tree vigor is too high, while early spring pruning is used less and limited only to cases of unpredictable fruit set due to erratic climatic conditions. in any case, the labor for all kinds of pruning (spring, summer, and winter pruning) takes not more than 100 hours per hectare per year. with some very productive varieties and appropriate training systems (delayed open vase and free spindle) it is possible to reach an amount of labor for all the manual operations during the season of about 15 hours per ton of fruit (giovannini et al., 2010; neri et al., 2010). during training of catalonian vase in the first two years, spring and summer pruning can be mechanized with a moving machine to further reduce labor. late summer pruning may start in the second year for heavy producing varieties and in the third year for the less productive ones. in conclusion, spring and summer pruning increase the efficiency of labor (both for the ease and speed of the work and for the capability of the tree to rapidly compensate for errors and incorrect interventions) and improve fruit quality. late summer pruning can particularly improve modern orchard management efficiency. acknowledgements the authors wish to acknowledge the regione emilia romagna for financing part of the work (crpv 2008 impiantistica e gestione dei fruttiferi). references bassi d., guerriero r., guerrieri f., monteleone p., rizzo m., viti r., 2003 apricot, pp. 24-49. in: bassi d. (ed.) growth habit in stone-fruit trees. il divulgatore, bologna, italy. chalmers d.j., mitchell p.d., van heek l., 1981 control of peach tree growth and productivity by regulated water supply, tree density and summer pruning. j. am. soc. hortic. sci., 106: 307-312. day k.r., dejong t.m., hewitt a., 1989 postharvest and preharvest summer pruning of ‘firebrite’ nectarine trees. hortscience, 24: 238-240. ferree d.c., myers s.c., rom c.r., taylor b.h., 1984 physiological aspects of summer pruning. acta horticulturae, 146: 243-252. giovannini d., neri d., di vaio c., sansavini s., del vecchio g., guarino f., pennone c., abeti d., colombo r., 2010 efficienza gestionale degli impianti di pesco in un confronto nord-sud. frutticoltura, 7/8: 16-26. guerriero r., scalabrelli g., 1982 relationship between bud dormancy and growing and fruiting behaviour on different apricot varieties along the tuscan coast line. a) changes of one year shoot morphogenetic gradient during rest period. acta horticulturae, 121: 85-92. guerriero r., viti r., 1997 problemi relativi alla biologia fiorale e di fruttificazione dell’albicocco. italus hortus, 4: 29-36. guerriero r., xiloyannis c., 1975 a comportamento produttivo di alcune cultivar di albicocco nel litorale toscano. proceeding of “problemi e prospettive della coltura dell’albicocco”, imola, italy, pp. 83-92. guerriero r., xiloyannis c., 1975 b rapporti tra andamento climatico, dormienza habitus vegetativo e produttivo nell’albicocco. proceeding of “problemi e prospettive della coltura dell’albicocco”, imola, italy, pp. 35-44. hossain a.b.m.s., mizutani f., 2008 dwarfing peach trees and fruit quality development by using summer pruning as physiological changed dwarfing component. austr. j. of basic and appl. sci., 2: 844-849. hossain a.b.m.s., mizutani f., onguso j.m., 2004 effects of summer pruning on maintaining the shape of slender spindle bush of peach tree grafted on vigorous rootstock. j. of the jap. soc, of agric. tech. manag., 11: 55-62. kappel f., bouthillier m., 1995 rootstock, severity of dormant pruning, and summer pruning influences on peach tree size, yield, and fruit quality. canadian j. of plant sci., pp. 491-496. lanzelotti j., gonzalez b.a., boragno o.a., 1998 different dates of pruning in peach tree. acta horticulturae, 465: 629-635. lopez g., mata m., arbones a., solans j.r., girona j., marsal j., 2006 mitigation of effects of extreme drought during stage iii of peach fruit development by summer pruning and fruit thinning. tree physiology, 26: 469477. marini r.p., 1985 vegetative growth, yield and fruit quality of peach as influenced by dormant pruning, summer pruning and summer topping. j. am. soc. hortic. sci., 110: 133139. marini r.p., barden j.a., 1987 summer pruning of apple and peach trees. horticultural reviews, 9: 351-375. marsal j., lopez g., mata m., girona j., 2006 branch removal and defruiting for the amelioration of water stress effects on fruit growth during stage iii of peach fruit development. scientia horticulturae, 108: 55-60. miller s.s., 1987 summer pruning affects fruit quality and light penetration in young peach trees. hortscience, 22: 390-393. mizutani f., rabbany a.b.m.g., amano s., hino a., akiyoshi h., kadoya k., 1997 -effect of summer pruning and gibberellin application on reduction of flower bud formation in “saotome” peach (prunus persica batsch.) 178 trees grafted on p. japonica rootstocks. bulletin of the experimental farm college of agriculture, ehime university, japan, 18: 1-8. monserrat r., iglesias i., 2011 i sistemi di allevamento adottati in spagna: l’esempio del vaso catalano. frutticoltura, 7/8: 18-26. neri d., 2003 ipertesto potatura. crpv, cesena, italy. neri d., giovannini d., massai r., di vaio c., sansavini s., del vecchio g., guarino f., mennone c., abeti d., colombo r., 2010 efficienza produttiva e gestionale degli impianti di pesco in un confronto nord-sud. italus hortus, 17(3): 46-62. neri d., massetani f., giorgi v., 2011 la potatura. edagricole, bologna, italy, pp. 370. neri d., morini f., massetani f. pirazzini p., 2010 pruning: how to manage shoot growth. acta horticulturae, 862: 355-363. pirazzini p., 2004 osservazioni sulla potatura di produzione di alcune varietà di albicocco. frutticoltura, 1: 36-38. rom c.r., ferree d.c., 1984 the influence of summer pruning current season shoots on growth, floral bud development and winter injury of mature peach trees. hortscience, 19: 543-545. sansavini s., neri d., 2005 forme di allevamento e potatura, pp. 115-143. in: fideghelli c., and s. sansavini (eds.) pesco. edagricole, bologna, italy, pp. 259. zucconi f., 2003 nuove tecniche per i fruttiferi. edagricole, bologna, pp. 246. 245 1. introduction the potential impact of atmospheric deposition of pollutants such as sulphur dioxide on vegetation has been the subject of many papers (tingey and olszyk, 1985; darral, 1989; mesanza et al., 1996; johnson et al., 1999). plant injury caused by air pollution is most common near large cities and industrial enterprises, and damage in isolated areas occurs when pollutants are spread over long distances by wind currents (cicek, 2003). some studies have also begun to surface from developing countries (hassan et al., 1995; wahid et al., 1995), where yield losses of cereals and legumes have been attributed to both so 2 and ozone. exposure to so 2 has been reported to decrease or inhibit photosynthesis (silvius et al., 1975; ziegler, 1975; carlson, 1983; rao et al., 1983; katainen et al., 1987; kropff, 1987; sheu, 1994; lorenc-plucińska, 1998; ranieri et al., 1999) and pollen tube growth (karnosky and stairs, 1974; varshney and varshney, 1981). even if the so 2 sensitivity of grapevine is poorly documented (ishikawa, 1972; weinstein, 1984; garciahuidobro et al., 2001), the species is considered sensitive to chronic so 2 exposure, and adverse effects on growth or yield were presumed possible in the field (daines, 1968; fujiwara, 1970). development of black and brown lesions was observed especially on leaves (cicek, 2003); shoot growth reduction and, in some cases, leaf abscission were reported (shertz et al., 1980). the interaction of so 2 with ozone is evident. the combination of the two gases led to an increase in oxidant stipple in leaves (forsline et al., 1983). in other species this interaction has not occurred (kreess et al., 1986), suggesting the role of the genotype in resistance to the pollutant. interaction so 2 /carbon black was also suggested by ionescu et al. (1971) in the industrialized copsa mica zone of romania, where the damaging effects of sulphur-containing effluents were enhanced by the presence of carbon black. fujiwara (1970) found, with increasing so 2 concentration, that leaf abscission, in cv. fredonia, began earlier and progressed at a greater rate. effects of sulphur dioxide were also found on the stomatal apparate of vitis labrusca l. cv. ives, where so 2 induced both stomatal closure and a higher stomatal resistance (rosen et al., 1978). unlike other species (lorenc-plucińska and ziegler, 1989; maurousset and bonnemain, 1990), there is not, for the vine, specific knowledge on the effects of so 2 on shortand long-term effect of sulphite on sucrose transport in grapevine (vitis vinifera l.) leaves. an electrophysiological study e. rinaldelli, r. bandinelli, m. pagano dipartimento di scienze delle produzioni vegetali, del suolo e dell’ambiente agroforestale, sezione coltivazioni arboree, laboratorio di elettrofisiologia, università degli studi di firenze, viale delle idee, 30, 50019 sesto fiorentino (fi), italy. key words: membrane potential, sucrose transport, sulphite, sulphur dioxide, vitis vinifera l. abstract: sucrose is the main carbohydrate translocated in grapevine and its transport may be restricted or inhibited by a number of factors such as the pollutant sulphur dioxide. the present study investigated, for the first time in grapevine, the effects of sulphite on membrane electrical response of sucrose transport in the mesophyll cells. co-transport of sucrose across the membrane is linked to the free energy in a electrochemical proton gradient. without the pollutant, electrophysiological traces displayed a metabolic-dependent sucrose electrical response in which an initial depolarization was followed by complete repolarization. in the presence of sulphite, instead, there were different trends depending on time of contact with the tissue of the pollutant. in the short-term, a slower repolarization was observed and in the long-term (after 6 and 12 h) the extent of depolarization (δ mv) was also reduced. transmembrane electrical potentials, measured in the presence of sulphite, became significantly (p < 0.01) more positive with increasing time of incubation of the tissue. on the whole, electrophysiological results highlight a direct or indirect effect of the pollutant on the activity of proton pump h+/atp ase. since carbohydrate translocation has a central role in the balance between source and sinks in the plant, the results of the research suggest that sulphite can modify the above balance with negative implications for the export of carbohydrate from the leaves. adv. hort. sci., 2011 25(4): 245-252 received for publication 4 november 2011 accepted for publication 24 november 2011 246 sugar transport in leaves. in phaseolus vulgaris and ricinus communis so 2 inhibits assimilate translocation and this inhibition seems to depend on damage to the sucrose transport system or proton pump (noyes, 1980; teh and swanson, 1982; lorenc-plucińska and ziegler, 1987 a, b, 1988). according to a more recent and detailed study on purified plasma membrane vesicles of ricinus communis, the decreased uptake of sucrose may be attributed to a dissipation in the transmembrane ph gradient (russell et al., 1999). sucrose transport in leaves is a secondary active transport (co-transport) in which solute (sucrose) translocation across the membrane is linked to the free energy available in a proton electrochemical potential difference (bush, 1993). it is widely known in the leaves of many plants (giaquinta, 1979; delrot, 1981; delrot and bonnemain, 1981; huber and moreland, 1981), and also in the vine is assumed to be so (mullins et al., 1992). since sucrose transport involves the entry of protons into the cell, it gives rise to changes in transmembrane potential. thus electrophysiological techniques are very useful for following, in real time, the transport of solutes. regarding the effect of sulphite on sucrose transport, the problem has been studied mainly on a biochemical level, while there is little information on the electrophysiological aspects. to our knowledge, only one paper (maurousset and bonnemain, 1990) reports some information about these aspects, however it does not include combined electrophysiological tests with sucrose and sulphite. therefore we felt it was interesting to investigate, for the first time in grapevine, how sucrose membrane electrical response was influenced by so 2 supplied as sulphite. 2. materials and methods plant material electrophysiological tests were carried out on leaves of vitis vinifera l., cv. sangiovese clone ss-f9-a5-48, removed from two-year-old plants grown in a container and grafted on rootstock 420 a. preliminary tests were performed on whole leaves, while the subsequent tests were on leaf segments (3 x 7 mm), cut with a scalpel under b.s. solution. chemicals for electrophysiological experiments, the substance to be tested has to be dissolved in the solution bathing the tissue (treatment solution). sulphur dioxide is highly soluble in water but, being gaseous, it is not easy to dose the exact amount to be dissolved. therefore, in this research, na 2 so 3 was employed, which at ph 5.5, is found mainly in the form of bisulfite ion (hso3-). previous research conducted on isolated chloroplasts of spinacia oleracea l. showed that so 2 and bisulphite ions, at the same equimolecular concentration, have a parallel mode of action in the inhibition of photosynthetic oxygen evolution (silvius et al., 1975). moreover the effects of so 2 on chloroplast enzyme systems were studied by means of hydration products of bisulphite and sulphite (ziegler, 1975). the components of the various treatments were added to the basal solution (b.s.) for the electrophysiological experiments (table 1). in all treatments in which na 2 so 3 alone was employed, even the b.s. contained the same amount of sodium supplied as na 2 so 4 . carbonyl cyanide m-chloro phenyl hydrazone (cccp), a protonophore and uncoupler of oxidative phosphorilation (marrè et al., 1973) was used in 0.05% ethanol, starting from a stock solution of 0.5 mol m-3. controls, in the different trials, also contained the same percentage of ethanol in the treatments. the salts present in the basal solution were only in sulphate form. the presence of clanions was avoided since their entry by symport with h+ can influence cytoplasmic ph (bellando et al., 1995). table 1 composition of the basal solution (b.s.) and concentration of the components in the treatments basal solution treatment components* k2so4 2.5 sucrose 20.0 caso4 0.5 na2so3 1.0 mes 5.0 cccp 10-2 all solutions were adjusted to ph 5.5 by way of tris or dilute h 2 so 4 when na 2 so 3 was present. mes: 2-n-morpholinoethane-sulphonic acid (sigma); cccp: carbonyl cyanide m-chlorophenylhydrazone (sigma); tris: 2-amino-2-hydroxymethyl-1,3-propanediol (fluka). *concentrations are in mol m-3. electrophysiology before beginning the electrophysiological experiments, whole leaves and/or leaf segments were incubated in basal solution in the light for 2 h (short-term). during this period, the solution was renewed twice and constantly aerated. subsequent tests were performed after incubation of leaf segments in sulphite for 6 and 12 h (long-term). even in this case the solution was constantly aerated and renewed every hour. membrane potential (em) was measured according to standard electrophysiological technique as previously adopted (rinaldelli and bandinelli, 1999; rinaldelli, 2005) with some modification. in brief, whole leaves and/or leaf segments were mounted on a poly(methyl methacrylate) chamber secured to a microscope stage. the chamber for whole leaves was slightly inclined and wide enough to accommodate a grapevine leaf, while the one for the segments was small (3 ml) and placed horizontally. continuously aerated b.s. or treatments were permitted to perfuse through the chamber at a flow rate of 10 ml min-1. they reached the tissue by gravity, each through its own adductor channel controlled by a manual valve. in the case of whole leaf, only the stomatal area around the insertion point and the petiole were perfused. a small glass thermometer placed inside the chamber allowed verification of the temperature of the solution. heating or cooling of the solution was obtained by way of a peltier-effect heat pump located along the solution conduit before the poly(methyl methacrylate) chamber; it was electronically controlled. 247 the measuring electrodes used were micropipettes (tip diameter <1 µm) obtained from single-barrelled borosilicate capillaries (w.p.i., usa) by way of a vertical homebuilt puller. the micropipettes and the reference electrode were filled with 500 mol m-3 kcl. the electrodes were connected, by ag/agcl wires, to a high input impedence electrometer (ad 549, 1015 ω). the output signals from the electrometer, before being transmitted to a chart recorder, were passed through a low-pass filter (10 hz) in order to eliminate possible noise. the insertion of microelectrodes into the cells of the central part of the mesophyll took place under two different magnifications. in the case of whole leaves the electrode passed through a stoma and a magnification of 500x was necessary, obtained with a 50x long working distance objective (mitutoyo, japan). in the case of leaf segments, instead, a magnification of 250x, obtained with a 25 x long working distance objective (wpi, usa), was sufficient. the insertion of the microelectrodes was, in both cases, at an angle of about 45°, and took place by way of a very stable, manual, three-axis, homebuilt micromanipulator. treatments started after em stabilized for 5 min. all experiments were performed under faraday cage. the complete electrophysiological set-up is presented in figure 1. except where indicated otherwise, preincubation and electrophysiological tests were carried out at +22°c (± 0.5) in the light (30 watt/m2). fig. 1 schematic electrophysiological set-up for membrane potential measurements. 1. faraday cage. 2. digital cameras. 3. microscope tube. 4. measuring microelectrode. 5. reference electrode. 6. probe. 7. microscope stage. 8. vibration-free table. 9. lamp. 10. micromanipulator. statistical analysis the data relating to transmembrane potentials, measured at different times, in bs and sulphite, were subjected to statistical analysis of variance (anova). comparisons were carried out using duncan’s test. statistical significance of differences were accepted when p <0.01. in the electrophysiological traces, depolarizations (δ mv) are presented as mean ± se. 3. results preliminary experiments on whole leaves and leaf segments since the cuticle is often a barrier to organic molecules (rinaldelli and bandinelli, 1999; rinaldelli, 2005), before working on a tissue it is necessary to verify its permeability to the solutions that will be employed. thus, preliminary tests were performed on whole leaves and leaf segments. they showed that both were permeable to sulphite (fig. 2, a, c) but not to sucrose. in whole leaves, in fact, sucrose depolarization was almost one-quarter that in leaf segments (fig. 2, b, d). based on this evaluation, all subsequent tests were performed on leaf segments. fig. 2 effects of treatments with sucrose and sulphite on whole leaves and leaf segments. all measurements were carried out in light. numbers preceded by are negative mv. electrophysiological traces are representative of five equivalent measurements. δ is the depolarization (mv) as the mean of five measurements ± se. 30 min 248 sucrose transport and its metabolic support as co-transport of sucrose in grapevine leaves was assumed (mullins et al., 1992) but not electrophysiologically investigated, initial tests were carried out to verify this assumption. in the light, in the presence of the uncoupler cccp, sucrose depolarization was less than half, compared to the same test without the inhibitor (fig. 3 a, b). subsequent repolarization was also very slow. in the dark, instead, sucrose depolarization was lower than in the light, and it was completely abolished by the uncoupler (fig. 3 c, d). the different extent of depolarization, in light and dark, reflects metabolic support to the operation sucrose/ h+ symport. as discussed later, cccp only inhibited oxidative phosphorylation and had incomplete or no effect on photophosphorylation. at 5°c sucrose depolarization was nearly annulled (fig. 3 e) and a similar response occurred when the ph of the treatment solution was brought to 8.0 (fig. 3 f). shortand long-term experiments under sulphite in the short-term tests, sucrose depolarization, in the light, was about 20 mv (fig. 3 b) whereas in the dark it was much less (fig. 3 c). also, in the presence of sulphite, differences in extent of depolarization, in light and dark, remained approximately the same as previous tests without pollutant, but a slower repolarization was observed (fig. 4 a, b). based on this evidence, in the short-term sulphite affected only in part the membrane electrical response of sucrose transport. figure 5 shows the electrical responses to sucrose after leaf segments were preincubated in b.s. (control) or sulphite, for 6 and 12 h. since shortand long-term treatments in the dark showed the same trends, only the first are reported. sulphite already reduced the sucrose depolarization after 6 h of incubation (fig. 5, b), but the result was more evident after 12 h where the extent of depolarization defig. 3 effects of chemical and physical treatments to verify energetic support of sucrose transport. except where indicated otherwise, measurements were carried out in light. solution at ph 8.0 was buffered with 2 mol m-3 hepes [n-(2-hydroxyethyl)piperazine-n’-(2-ethanesulfonic acid] -tris. numbers preceded by are negative mv. electrophysiological traces are representative of five equivalent measurements. δ is the depolarization (mv) as the mean of five measurements ± se 249 creased by almost half as compared to the control (fig. 5d). also the repolarization, after 6 h, and even more after 12 h, was strongly slowed. it should be noted that even with increasing incubation time there is a progressive decrease in the extent of depolarization. also the transmembrane potentials decreased (they became more positive) with increasing incubation time (table 2). these effects were evident under both, b.s. and sulphite. under sulphite, however, they were more marked. table 2 membrane potentials (mv) recorded at different times of incubation, in b.s. and sulphite incubation time (h) membrane potentials b.s. (no sulphite) 1 mol m-3 na2so3 0 -160.2 ± 2.61 a -153.0 ± 2.05 b 6 -152.2 ± 2.04 b -139.7 ± 2.45 d 12 -149.0 ± 2.44 c -125.1 ± 2.33 e each value is the mean ± sd of ten experiments. different letters denote statistically significant differences between membrane potentials (p<0.01). 4. discussion and conclusions energetic support to sucrose transport cccp is an uncoupler of oxidative phosphorilation which, in agreement with the chemiosmotic hypothesis (mitchell, 1961), dissipates the electrochemical gradient fig. 4 short-term treatments in light and dark. numbers preceded by are negative mv. electrophysiological traces are representative of five equivalent measurements. δ is the depolarization (mv) as the mean of five measurements ± se. fig. 5 long-term treatments, after 6 h and 12 h of incubation in b.s. and/or sulphite. all measurements were carried out in light. numbers preceded by are negative mv. electrophysiological traces are representative of five equivalent measurements. δ is the depolarization (mv) as the mean of five measurements ± se. 30 min 30 min 250 of h+ ions preventing atp synthesis. consequently, active transport systems (primary and also, indirectly, secondary), lacking energy, are deactived. under cccp, membrane electrical response of sucrose showed, in light and dark, two different results (fig. 3 a, d). this finding suggests that the metabolic support of the operation of sucrose/h+ symport comes from both respiration and photosynthesis. in dark, since there is no photosynthesis, atp production is only of respiratory origin and it is blocked by the uncoupler cccp. in light, instead, some energetic support appears because cccp does not inhibit, or only partially inhibits, the photophosphorylation. this may be the case, taking into account the variable effects of cccp on photophosphorylation as a function of leaf greening (oelze-karow and butler, 1971; butler et al., 1972) or light intensity (saha et al., 1970; prins et al., 1980). since chemical inhibitors may simultaneously affect many different processes of cells (khalilov et al., 2002), we also tested the effect of low temperature. treatment with sucrose at 5°c nearly led to the annulment of the depolarization. this suggests a metabolic dependence of sucrose transport on the activity of a plasmalemma atpdriven proton pump. at 5°c, since the metabolic energetic support is highly limited (mengel and shubert, 1985; rinaldelli and bandinelli, 1999; rinaldelli, 2000; 2004), the activity of the pump is inhibited. the use of sucrose solution buffered at ph 8.0 strongly reduced depolarization. under these conditions, the lack of protons in the apoplast would explain the annulment of h+ co-transport. this response has also been observed for nitrate (ullrich and novacky, 1981; mcclure et al., 1990) and urea co-transports (rinaldelli, 2004). all these results are considered sufficient to support a metabolic and ph-dependent sucrose co-transport. membrane electrical responses under sulphite sucrose is the main carbohydrate translocated in grapevine. from the leaves, where it is produced, it moves to the various sinks according to the specific needs of the plant (coombe and mccarthy, 2000; hunter and ruffner, 2001). during the annual vegetative and reproductive cycle, it plays a preeminent role in regulation of the carbon/nitrogen ratio (rodriguez-lovelle and gaudillère, 2002). sucrose, being a non-polar molecule, does not have direct effects on membrane potential when it enters the cell, but only indirect, because its transport is related to the flow of protons that move along an electrochemical gradient. this co-transport results in an initial depolarization observed under both b.s. and sulphite (figs. 2 b, d; 3, 4, 5). under sulphite, it has to be excluded that the depolarization could be due to na+ or the accompanying anion, since sucrose and perfusing solutions contained an equal amount of na 2 so 3 . repolarization of the membrane, which sometimes exceeded the starting level (overshoot), is proposed to depend on stimulation of the h+-atpase, caused by either changes in the cytoplasmic ph or the membrane potential itself. the h+-atpase has a ph optimum at 6.6, i.e. well below the physiological ph of the cell cytoplasm (usually around 7.2-7.5). thus, whenever protons start accumulating in the cytoplasm, the activity of the pump increases to remove excess protons from the cell (michelet and boutry, 1995). this response is comparable, as cause and effect, to the one caused by permeant weak acids (marrè et al., 1983; rinaldelli and bandinelli, 1999). sulphite alone, at the concentration used, induced a slight depolarization followed by partial or complete recovery. this is visible in both whole leaves (fig. 2 a) and leaf segments (fig. 2 c). maurosset and bonnemain (1990) found that the depolarization by sulphite, does not depend on the concentration used. effect of the pollutant on the membrane would be, initially, that of “exciting agent”. instead, the repolarization would depend, according to the same authors, on the concentration of sulphite in the treatment. in our study we tested sulphite concentrations below and above 1 mol m-3 (data not shown) finding, however, less marked differences compared to the aforementioned authors. however the comparison is difficult because the study of maurosset and bonnemain (1990) did not include our same electrophysiological tests. their tests, in addition, were conducted on a different species (vicia faba l.) and this may also affect the reported results. when the treatment with sucrose was under sulphite, however, repolarization slowed and decreased gradually moving from shortto long-term. this is the first and most important effect of sulphite on sucrose transport. since repolarization is dependent on proton pump activity, the direct or indirect effect of sulphite should be placed at this level. in the long-term, under b.s. and sulphite, sucrose depolarization was lower (fig. 5) and the transmembrane electropotentials became significantly more positive (table 2). since these effects are evident even in b.s., they should be attributed, at least in part, to phenomena of senescence which start when leaves are detached or segments are cut (thimann et al., 1977; gepstein, 1982; malik, 1982). however, since the variations under sulphite were greater, there is clear evidence, in addition to senescence, of a direct or indirect effect of pollutant on proton pump activity. proton pumps, in fact, are considered the primary motors that build up transmembrane electrochemical proton gradients (felle, 2001). the complex of electrophysiological tests performed in this research highlights a clear membrane electrical response of sucrose transport to sulphite. two points stand out clearly enough: a) in grapevine leaves sucrose membrane electrical response is supported by metabolic energy; b) sulphite alters the above response by acting on the proton pump and/or energetic metabolism, from which the pump draws energy to extrude h+ ions outside the cell. an effect of sulphite on respiration and photosynthesis cannot be excluded, especially in the long-term. the link may be there because both slowed repolarization and more positive transmembrane potentials under sulphite reflect a partial inhibition of the proton pump, whose activity is supported by respiratory and photosynthetic atp. 251 the results obtained constitute an interesting acquisition in grapevine physiology, but it should be taken in account that conditions for the so 2 effect, in the vineyard ecosystem, may differ from those adopted in the laboratory due to the high number of cultural, climatic and pedological variables. acknowledgements this study was supported by university funds, ex 60%. references bellando m., marrè m.t., sacco s., talarico a., venegoni a., marrè e., 1995 transmembrane potential-mediated coupling between h+ pump operation and k+ fluxes in elodea densa leaves 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aromatic profiles of tulbaghia simmleri beauv. edible flowers during cold storage i. marchioni 1, b. najar 1, 2 (*), a. copetta 3, b. ferri 2, b. ruffoni 3, l. pistelli 2, l. pistelli 1, 4 1 dipartimento di scienze agrarie, alimentari e agro‐ambientali (disaaa‐a), università di pisa, via del borghetto, 80, 56124 pisa, italy. 2 dipartimento di farmacia, università di pisa, via bonanno pisano, 12, 56126 pisa, italy. 3 crea centro di ricerca orticoltura e florovivaismo, corso inglesi, 508, 18038 sanremo (im), italy. 4 centro interdipartimentale di ricerca nutraceutica e alimentazione per la salute (nutrafood), università di pisa, via del borghetto, 80, 56124 pisa, italy. key words: antioxidant activity, enzyme activity, low temperature, postharvest, secondary metabolites, sweet wild garlic, volatile organic com­ pounds. abstract: edible flowers are appreciated due to their aesthetic features, nutri­ ti o n a l v a l u e a n d a n ti o x i d a n t p r o p e r ti e s . t u l b a g h i a s i m m l e r i b e a u v . (amaryllidaceae family) flowers are characterized by a pleasant garlic taste and are consumed both as fresh and dried products. the aim of this work was to assess the effect of chilling temperature (+4°c) on the visual quality, nutritional content, and aroma profile of t. simmleri flowers after two (t2) and six (t6) days of storage. colorimetric analysis highlighted a reduction in petal bright­ ness at t6 and hence their darkening, due to a significant increase in a* coordi­ nate and the decrease in the b* one. total polyphenols and flavonoids content remained unchanged until the end of the experiment, while total anthocyanins increased at t2. flowers antioxidant activity (dpph assay) decreased progres­ sively during cold storage, while catalase (cat) and ascorbate peroxidase (apx) activities increased. the aroma profile was analyzed by hs­spme associated with gc­ms, underlining that fresh flowers were dominated by high content in monoterpenes (around 80%), with 1,8­cineol as main compound (53.1%). cold storage reduced this class of volatiles while sesquiterpenes and non­terpenes increased; between them, benzyl benzoate reached 12%. 1. introduction edible flowers (efs) are traditionally consumed since ancient times (mlcek and rop, 2011). some of them are commonly recognised as veg­ etables (e.g. artichokes, broccoli, capers), while others are still considered (*) corresponding author: basmanajar@hotmail.fr citation: marchioni i., najar b., copetta a., ferri b., ruffoni b., pistelli l., pistelli l., 2023 ­ phytonutritional and aromatic profiles of tulbaghia simmleri beauv. edible flowers during cold storage. ­ adv. hort. sci., 37(1): 25­32. copyright: © 2023 marchioni i., najar b., copetta a., ferri b., ruffoni b., pistelli l., pistelli l. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 14 october 2022 accepted for publication 13 december 2022 ahs advances in horticultural science https://doi.org/10.36253/ahsc-13857 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2023 37(1): 25­32 26 “unusual food” (reviewed in pires et al., 2019). efs straights rely on their colours, shapes, flavours, tastes, and nutrients (e.g. carbohydrates, proteins, vitamins, phytochemical compounds with antioxidant and healthy properties) (fernades et al., 2017). their market is constantly expanding, and new species with attractive sensorial features and good storage attitude are required. tulbaghia (common name: wild garlic) is a genus of monocotyledonous plants (amaryllidaceae family) indigenous to south africa (lyantagaye, 2011). herbaceous perennial bulbs, corms or rhizomes char­ acterize its species. tulbaghia spp. flowers, held in umbels in groups of ten or more, are strongly fra­ grant and characterised by tubular shape (zschocke and van staden, 2000). a raised crown­like structure or a fleshy ring at the centre of the flower tube are distinctive features of this genus (vosa, 2000). the colours are different, mainly white, pink or mauve. flowers and rhizomes produce cysteine­derived sul­ phur compounds (e.g. marasmicin), which confer to this organs a pleasant alliaceous smell, especially when bruised or during senescence (aremu and van staden 2013; kubec et al., 2013). the peculiar aroma and the pungent garlicky taste of flowers make sever­ al tulbaghia spp. interesting for the food industry (kubec et al., 2013). t. simmleri beauv. is mainly known as ornamental plant, which flowers consist of six tepals and a cen­ tral crown of six lobes, fused for more than a third of their length to form a tube. the lobes have pointed tips, giving the crown a fringed edge (vosa, 2000). in the southern hemisphere, its period of blooming ranges between april to october, even though, with particular climate conditions, it could be extended until early spring (zschocke and van staden, 2000). in the northern hemisphere, however, its period of blooming ranges between october to april. several bioactive compounds characterize this plant, since it is used to treat fever, colds, headaches, asthma, and tuberculosis in south african traditional medicine (zschocke and van staden, 2000). t. simmleri has been severely neglected when compared to the most common t. violacea, for which several culinary uses are known, also concerning flowers (aremu and van staden, 2013; rivas­garcía et al., 2022). further investigation on t. simmleri worth to be performed, since this species produce deep mauve, long lasting edible flowers, which period of bloom does not over­ lap the one of t. violacea (not available in autumn and winter). this will ensure the availability of efs with garlic taste for most of the year. moreover, takaidza et al. (2018) highlighted good total polyphe­ nolic and flavonoid content, and hence good antioxi­ dant activity, in t. simmleri plants, in comparison with other seven tulbaghia species, t. violacea included. postharvest technologies are common methods to extend efs shelf­life, as it is generally rather short (2­ 10 days) (fernandes et al., 2019, 2020). flowers are high value products, which must be picked with care, p a c k a g e d p r o p e r l y t o p r o t e c t t h e m f r o m a n y mechanical damage, and stored at proper tempera­ ture until consumption (fernandes et al., 2020). improperly handled/stored edible flowers suffer tis­ sue browning, flower wilt, dehydration, petal discol­ oration, and abscission. the senescence process is a s s o c i a t e d w i t h p h y s i o l o g i c a l c h a n g e s a n d catabolism, which are linked to accelerated respira­ tory levels, weight reduction, and/or plant hormone response (kou et al., 2012; landi et al., 2018). to address these concerns, fresh edible flowers are often stored under low temperatures, generally at chilling ones (4­5°c) (fernandes et al., 2020). since different efs species showed different behaviour at cold storage (landi et al., 2018; marchioni et al., 2020 a, 2020 b), postharvest studies should be per­ formed for each flower, in order to elucidate their physiological response to low temperature and hence their shelf­life. the aim of this work was to evaluate the phytonu­ tritional and aromatic profile of t. simmleri efs s t o r e d a t 4 ° c f o r 0 , 2 a n d 6 p o s t h a r v e s t d a y s . spectrophotometric and chromatographic analyses were performed in order to highlight any changes in polyphenolic content (flavonoids and anthocyanins included), antioxidant activity, and volatile organic compounds (vocs) during cold storage. 2. materials and methods plant material and postharvest conditions tulbaghia simmleri plants were provided by the chambre d’agriculture des alpes­maritimes (cream, nice, france) and were grown at research centre for vegetable and ornamental crops (crea, sanremo, imperia, italy, gps: 43.816887, 7.758900). details on plant cultivation is reported in najar et al. (2019). full open flowers were picked in april, weighed and cold stored as described in marchioni et al. (2020 b), for two (t2) and six (t6) postharvest days. fresh flowers marchioni et al. ‐ changes of t. simmleri edible flowers during cold storage 27 were considered as control (t0). weight loss and colour determination flowers weigh was measured (ohaus® analytical standard series™ model as60s, ohaus corporation, florham park, n.j. usa) before cold storage (t0) and at the end of each experimental point (t2 and t6) to calculate their weight loss (formula reported in fernandes et al., 2018). once flowers had been weighed, their colour was evaluated with a spec­ trophotometer sp60 series (x­rite incorporated, michigan, usa). l* (lightness), a* (redness) and b* (yellowness) colour coordinates (cielab scale, cie 1976) were measured in different point of at least ten flowers, in order to best describe their colour variations. biochemical analyses biochemical analyses were performed using frozen samples. total phenolic, flavonoid and antho­ cyanins content were determined as reported by marchioni et al. (2020 b). data were reported as mg gallic acid equivalents (gaeq)/g fresh weight (fw) (polyphenols), mg catechin equivalents (ceq)/g fw (flavonoids), and mg malvin chloride equivalents (meq)/g fw (anthocyanins). radical scavenging activ­ ity (dpph assay) of each sample was determined as described by brand­williams et al. (1995). data was expressed in ic50, which represent the concentration of the sample able to inhibit by 50% the radical dpph. all absorbance were read in a uv­1800 spec­ trophotometer (shimadzu corp., kyoto, japan). enzymatic activities frozen flowers (200 mg) were pulverized and homogenized in 2 ml of extraction buffer, consisting of 50 mm sodium phosphate buffer (ph 7.0), 1 mm edta, 1 mm phenylmethylsulfonyl fluoride (pmsf), and 2% (w/v) insoluble polyvinylpolypyrrolidone (pvpp), as reported by pistelli et al. (2017). samples were centrifuged at maximum speed for 30 min at 4°c and the supernatant was used for enzyme activi­ ties. the soluble protein content was determined according to bradford (1976) using bovine serum albumin as standard. catalase (cat, ec 1.11.1.6) activity was measured by monitoring the decomposition of hydrogen perox­ ide (h2o2), recording the decline in absorbance per minute at 240 nm (zhang and kirkham, 1996). the reaction started by adding 20 μl of extract to 980 μl of 8.8 mm h2o2 solution in 50 mm sodium phosphate buffer. one unit of cat is determined as the amount of enzyme required to detoxify 1 μmole of h₂o₂ (ε= 394 m­1 cm­1) per minute. data were expressed as unit of cat per mg of soluble proteins (μmol min­1 mg­1). ascorbate peroxidase (apx, ec 1.11.1.11) activity w a s d e t e r m i n e d b y f o l l o w i n g t h e d e c r e a s e i n absorbance at 290 nm (ε = 2.7 mm­1 x cm­1) due to enzymatic ascorbate oxidation (nakano and asada, 1981). the reaction started by the addition of 50 mm h 2o 2 solution to the reaction mixture (20 μl of extract, 0.15 mm disodium edta and 0.37 mm ascor­ bic acid in 50 mm sodium phosphate buffer). a unit of apx is defined as the amount needed to oxidize 1 μ m o l e o f a s c o r b i c a c i d p e r m i n u t e . d a t a w e r e expressed as unit of apx per mg of soluble proteins (μmol min­1 mg­1). spontaneous emission analysis the spontaneous emission analysis was per­ formed as reported in our previous work (marchioni et al., 2020 b). briefly, and after the chosen storage time had elapsed (0, 2 and 6 days at 4°c), 1g of t. simmleri was properly weighted to be sealed in a 25 ml glass flask and kept at laboratory temperature (around 21°c) for 15 min (equilibration time). once the time expired, the 100 μm polydimethylsiloxane pdms fiber (supelco, bellefonte, pa, usa), was exposed to the flask headspace for 10 min, to be than transferred into the gc­ms instrument. statistical analysis the normal distribution of the residuals and the homogeneity of variance was determined and then data were statistically analyzed by one­way analysis of variance (anova) (past3, version 3.15), using tukey honestly significant difference (hsd) with a cut­off significance of p<0.05 (letters). 3. results and discussion weight loss and chromatic changes during cold stor‐ age the visual quality of t. simmleri flowers has been almost entirely maintained up to the sixth days of cold storage (t6) (fig. 1, table 1). the main changes observed during postharvest treatment were the decrease in flowers fresh weight, brightness (l*) and bluish parameter (b*), along with the increase in the reddish parameter (a*) (table 1). taken together, these variations resulted in a slight darkening of the petals at the end of the experiment, without any evi­ adv. hort. sci., 2023 37(1): 25­32 28 dent loss of flower firmness. the decrease in fresh weight is due to the loss of cell turgor, which is correlated to flower shape. significant water loss can determine decreased floral diameter, as well as petals curling and crumpling (kou et al., 2012; ahmad and thair, 2016; marchioni et al., 2020 b). nevertheless, the weight loss in t. simmleri flowers was very limited (around 7%), show­ ing, therefore, a good aptitude to cold storage. moreover, the latter was observed to reduce the brightness of seven different efs (landi et al., 2018), as well as t. simmleri flowers (table 1). this decrease in l* values is indicative of tissue darkening, com­ monly associated with the oxidation of phenolics and their polymerization into dark brown pigments, as a result of the activities of polyphenol oxidase (ppo), peroxidase and phenylalanine ammonia lyase (pal) (landi et al., 2018; hu and shen, 2021). the same process could also be responsible for the changes in the color coordinates a* and b*, which turn towards darker hues (table 1). antioxidant compound and enzyme activities polyphenols are considered as the most important and widest natural compounds with antioxidant activity (cavaiuolo et al., 2013). thanks to their bioactive potential, these molecules can help to pre­ vent chronic degenerative diseases, cardiovascular disorders, and different types of cancer (pires et al., 2019; skrajda­brdak et al., 2020). postharvest treat­ ment should maintain unaltered flowers polyphenols concentration to guarantee health benefit until flow­ ers consumption. our results satisfied this statement, because no changes were observed up to t6 for p o l y p h e n o l a n d fl a v o n o i d s a m o u n t s ( t a b l e 2 ) . indeed, a short increase in the total anthocyanins content was quantified already after 2 days (t2) that could be correlated to the interchange between bluish and reddish parameters (table 1). despite this positive trend, it should be noted that t. simmleri fresh flowers are characterized by low amount of phenolic compound than other well­known and cur­ rently consumed efs (li et al., 2014; chen et al., 2018). moreover, higher quantities of polyphenols and flavonoids were also reported in other species of the same genus, such as t. cominsii and t. violacea, probably connected to the use of different extraction methods (landi et al., 2018; rivas­garcía et al., 2022). nevertheless, maintaining the levels of pheno­ lic compounds in t. simmleri flowers could indicate that this species did not show substantial signs of decay up to the end of the experiment. as regards total anthocyanins content, their increase was previ­ table 1 ­ weight loss and chromatic changes of t. simmleri flowers at 0 (t0), 2 (t2), and 6 (t6) postharvest days (storage at 4°c) data are reported as mean ± standard error (weight loss, n = 4; l*, a*, b*, n = 15). different letters indicate statistically signifi­ cant differences (p<0.05; tukey’s hsd test). table 2 ­ antioxidant compounds, radical scavenger activity (dpph assay), catalase (cat) and ascorbate peroxidase (apx) activities of t. simmleri flowers at 0 (t0), 2 (t2), and 6 (t6) postharvest days (storage at 4°c) data are reported as mean ± standard error (n = 6). different letters indicate statistically significant differences (p<0.05; tukey’s hsd test). parameters days 0 2 6 weight loss (%) 0 c 3.21 ± 0.04 b 7.34 ± 0.69 a l* 56.01± 1.25 a 55.54 ± 1.19 a 49.45 ± 0.68 b a* 22.71 ± 0.64 c 25.78 ± 0.65 b 27.84 ± 0.46 a b* ­14.16 ± 1.08 a ­19.73 ± 0.69 b ­20.02 ± 0.51 b parameters days 0 2 6 total polyphenols (mg gaeq/g fw) 1.22 ± 0.01 a 1.30 ± 0.04 a 1.32 ± 0.03 a total flavonoids (mg ceq/g fw 0.30 ± 0.01 a 0.32 ± 0.01 a 0.29 ± 0.01 a total anthocyanins (mg meq/g fw) 0.21 ± 0.02 b 0.29 ± 0.01 a 0.24 ± 0.02 a dpph assay (ic50 mg/ml) 4.20 ± 0.28 a 3.46 ± 0.19 a 5.22 ± 0.09 b cat activity (µmol min­1 mg­1) 12.68 ± 0.41 b 9.05 ± 0.24 c 21.25 ± 0.27 a apx activity (µmol min­1 mg­1) 0.66 ± 0.04 b 0.64 ± 0.04 b 0.83 ± 0.04 a fig. 1 ­ visual appearance of t. simmleri flowers after different times of cold storage (4°c): freshly picked flowers (a); after 2 days of cold storage (t2) (b); and after 6 days of cold storage (t6) (c). bar scale: 1 cm. marchioni et al. ‐ changes of t. simmleri edible flowers during cold storage 29 ously observed also in other efs stored at low tem­ perature, but the regulatory mechanisms in flowers are still under debate (shvarts et al., 1997; landi et al., 2015; marchioni et al., 2020 b). senescence and flowers exposure to low tempera­ tures are tightly associated with a rise in reactive oxy­ gen species (ros) level in the cells, whose production is accompanied by the activation of several enzymes involved in ros scavenging (cavaiuolo et al., 2013; darras, 2020). polyphenolic compounds also take part to this process, as demonstrated by the reduc­ tion of flowers antioxidant activity observed at t6 (table 2). in this work, the attention was paid to the ros scavenging enzymes that use hydrogen peroxide (h2o2) as substrate, namely catalase (cat) and ascor­ bate peroxidase (apx). t. simmleri flowers showed that cat activity is higher than the one of apx (table 2), suggesting a greater involvement of cat in h2o2 inactivation. moreover, both the enzymes increased their activity at t6 (table 2). to the best of our knowledge, very few papers investigated ros scav­ enging enzymes activity in efs stored at chilling tem­ perature as single postharvest treatment. in fact, chrysargyris et al. (2018, 2019) combined the conser­ vation at 5°c with preharvest salinity treatment and modified atmosphere packaging to observe the stor­ age aptitude of tagetes patula and petunia × hybrida flowers. nevertheless, in agreement with our results, apx activity was lower than the one of cat in t. pat‐ ula flowers, after both 7 and 14 postharvest days (chrysargyris et al., 2018). cat activity was also investigated by rizzo et al. (2019), highlighting differ­ e n t t r e n d d e p e n d i n g o n t h e s p e c i e s a n d t h e polypropylene (pp) film used. in the control thesis (comparable with our experiment), cat activity increases significantly after 6 days of cold storage only in half out of the four studied flowers (malva sylvestris and papaver rhoeas), similarly to what we observed for t. simmleri. aroma profile monoterpenes were the main class of compounds, regardless the storage time and their percentage, that represented at least 50% of the identified fraction ( t a b l e 3 ) . i n t e r e s ti n g t o n o t e i s t h e d r a s ti c a l l y decrease in oxygenated hydrocarbons content which was of 77% (passing from 0­ to 2­day conservation) and 60% (passing from 0­ to 6­day conservation) respectively. on the contrary, this decrease was some­ how compensated by the increase in the monoter­ pene hydrocarbons after 2­day storage (an increase of about 2­folds) and by non­terpene compounds after 6­day storage (an increase of about 2.5­folds). in detail of composition, the fresh flower (t0) was rich in linalool and 1,8­cineol and these compounds almost completely disappear after 2 days of storage. a decrease of linalool content was observed also in papaya “golden” fruit stored at low temperature (gomes et al., 2016). interestingly is also the increase of limonene content, about 5­folds, from t0 and t2 (3.01% vs 14.78%, respectively), the same compound conserved the latter percentage even at t6. worthy to note, the presence of benzyl­benzoate in the flow­ ers is only noticeable after 2­ and 6­days of refrigera­ tion, and its quantity is tripled during this time. this work reported for the first time the chemical composition of spontaneous emission of the studied species. also noteworthy is the absence of sulfur compounds. almost similar behavior has been seen in t. violacea, where such compounds were present in a negligible amount, which were around 1.2% in leaves and do not exceed 4% in roots detected using the same analysis technique (hs­spme) (staffa et al., 2020). rhizomes’ essential oil (eo) of a south african species of t. violacea was also reported to be rich in 2,4­dithiapentne, which represent more than the half of the identified fraction (soyingbe et al., 2013). hydrocarbons were the major compounds in the hexane extract of t. violacea calli from cairo (egypt) (55.0%), while the flowers were rich in oxygenated compounds (74.6%) (eid and metwally, 2017). on the contrary, the eo from the same species studied by the same team but published two year before under­ line the prevalence of sulfur compounds in both leaves and flowers and represented 79.7% and 57.5%, respectively (eid, 2015). 4. conclusions cold storage can reduce some biochemical reac­ tions, although stress conditions increase the reactive s p e c i e s o f o x y g e n ( r o s ) i n s i d e p l a n t ti s s u e s . tulbaghia simmleri flowers maintain almost unal­ tered their visual quality, and their content in antioxi­ d a n t c o m p o u n d s , u p t o 6 p o s t h a r v e s t d a y s . moreover, cells counteract ros production increas­ ing cat and apx activity. the aroma profiles changed during the cold treatment, even if monoterpenes remained the most represented class of volatile com­ pounds. looking at the main characteristics of the flowers we can conclude that t. simmleri showed a good aptitude to chilling temperature, suggesting the need to test longer period of storage. 30 adv. hort. sci., 2023 37(1): 25­32 table 3 ­ aroma profile of t. simmleri flowers detected by headspace solid phase microextraction (hs­spme) at 0 (t0), 2 (t2), and 6 (t6) postharvest days n° class component l.r.i days 0 2 6 1 nt (e)­3­hexen­1­ol 866 2.37 ± 0.10 2 mh α­thujene 932 0.20 ± 0.00 tr 3 mh α­pinene 939 0.19 ± 0.02 3.36 ± 0.83 1.78 ± 0.12 4 mh camphene 953 0.38 ± 0.08 0.19 ± 0.01 5 nt benzaldehyde 961 0.93 ± 0.18 6 mh sabinene 976 0.53 ± 0.11 0.26 ± 0.00 7 nt 1­octen­3­ol 978 4.89 ± 0.16 8 mh β­pinene 980 1.18 ± 0.27 0.59 ± 0.02 9 nt 3­octanone 988 2.90 ± 0.16 10 om 2,3­dehydro­1,8­cineole 991 0.96 ± 0.08 11 mh myrcene 992 1.48 ± 0.61 12 nt 3­octanol 993 2.09 ± 0.13 0.80 ± 0.21 13 mh δ­3­carene 1011 0.44 ± 0.00 14 mh α­terpinene 1018 0.18 ± 0.04 1.03 ± 0.37 0.84 ± 0.08 15 mh p­cymene 1026 0.12 ± 0.01 7.26 ± 2.92 4.72 ± 0.70 16 mh limonene 1031 3.10 ± 0.08 14.74 ± 6.42 14.80 ± 1.13 17 om 1,8­cineole 1033 53.10 ± 0.08 10.38 ± 0.25 18 om (z)­β­ocimene 1033 0.20 ± 0.06 0.14 ± 0.03 19 mh (e)­β­ocimene 1040 0.62 ± 0.00 0.38 ± 0.08 20 nt phenyl acetaldeyde 1043 1.30 ± 0.04 21 mh γ­terpinene 1062 0.63 ± 0.08 5.74 ± 0.12 3.82 ± 0.02 22 om cis­sabinene hydrato 1068 0.82 ± 0.16 23 mh terpinolene 1088 0.32 ± 0.15 1.23 ± 0.39 1.05 ± 0.02 24 mh linalool 1098 15.51 ± 0.10 1.32 ± 0.68 0.92 ± 0.04 25 nt phenyl ethyl alcohol 1110 1.31 ± 0.03 26 om trans­limonene oxide 1139 1.24 ± 0.06 27 om trans­pinocarveol 1140 0.83 ± 0.10 28 om camphor 1143 1.89 ± 0.44 1.95 ± 0.14 29 om menthone 1154 0.46 ± 0.03 0.51 ± 0.01 30 om isomenthone 1164 0.40 ± 0.18 0.22 ± 0.01 31 om borneol 1165 0.76 ± 0.06 0.59 ± 0.02 32 om δ­terpineol 1167 tr 33 om trans­linalool oxide 1172 0.47 ± 0.05 0.32 ± 0.02 34 om neo­menthol 1174 0.76 ± 0.14 35 om cis­pinocamphone 0.71 ± 0.06 36 om 4­terpineol 1177 0.24 ± 0.08 1.57 ± 0.40 1.19 ± 0.09 37 om α­terpineol 1189 5.27 ± 0.08 0.79 ± 0.15 0.26 ± 0.04 38 nt decanal 1204 0.64 ± 0.06 39 om verbenone 1205 0.30 ± 0.05 40 om lilac alcohol b 1210 1.08 ± 0.11 41 nt methyl 4­nonenoate 0.36 ± 0.14 42 om trans­carveol 1217 0.32 ± 0.08 43 om methyl carvacrol 1244 0.68 ± 0.30 0.68 ± 0.12 44 om linalyl acetate 1257 2.14 ± 0.33 1.95 ± 0.35 45 om isobornyl acetate 1285 2.11 ± 0.49 1.98 ± 0.35 46 om myrtenyl acetate 1325 1.43 ± 0.16 0.30 ± 0.00 47 om methyl perillate 0.15 ± 0.07 48 sh α­cubebene 1351 0.23 ± 0.00 49 sh α­longipinene 1352 1.98 ± 0.35 50 sh α­copaene 1376 0.63 ± 0.11 0.27 ± 0.07 51 sh β­caryophyllene 1418 0.99 ± 0.06 2.48 ± 0.49 2.89 ± 0.21 52 sh α­guaiene 1439 0.35 ± 0.04 53 sh aromandrene 1442 0.12 ± 0.00 0.17 ± 0.02 54 ac­12 (e)­geranyl acetone 1453 tr data are reported as mean ± standard deviation (sd) (n=2). ... to be continued marchioni et al. ‐ changes of t. simmleri edible flowers during cold storage 31 acknowledgements t h i s w o r k w a s s u p p o r t e d b y a g r a n t f r o m european union in the frame of interreg alcotra v­a france­italy antea project n.1139 ­ attività inno­ vative per lo sviluppo della filiera del fiore edule/ fleurs comestibles: innovations pour le development d’une filière transfrontalière, and interreg alco­ t r a v ­ a f r a n c e ­ i t a l y a n t e s p r o j e c t n . 8 3 3 6 ­ capitalizzazione di progetti antea e essica. the authors want to thank mrs. arianna cassetti for the technical support of the preparation of figure 1. references ahmad s.s., tahir i., 2016 ­ increased oxidative stress, lipid peroxidation and protein degradation trigger senescence in iris versicolor l. flowers. ‐ physiol. 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attributes of tomato (solanum lycopersicum l.) fruits a.o. oduntan 1 (*), o.a. oyetunde 1, b.a. shobo 2, j.g. bodunde 3 1 department of crop production and horticulture, lagos state polytechnic, ikorodu, lagos state, nigeria. 2 babcock university, ilishan‐remo, ogun state, nigeria. 3 federal university of agriculture, abeokuta, nigeria. key words: 1­mcp, kmno4, postharvest, rra, zeolite. abstract: tomato fruit is highly perishable because of the characteristic high rate of ethylene production and respiration during ripening. delayed ripening could be achieved through the use of ripening remediation agents (rra) that either absorb or block ethylene binding to the fruit receptor. the effects of ripening remediation agents on shelf life and biochemical quality attributes were evaluated on tomato fruits harvested at three maturity stages (breaker, turning and full­ripe). in 2018 and 2019, harvested fruits were stored under seven ripening remediation treatments: 0.1 µl/l 1­mcp, 0.3 µl/l 1­mcp, 0.5 µl/l 1­mcp, 5% kmno4, 10% kmno4, 10 g of zeolite and 20 g of zeolite and an open shelf condition as the control. at the end of the storage period, fruits were assessed for shelf life as well as total soluble solids (tss), titratable acids (ta), ascorbic acid, and lycopene contents. there was significant (p≤0.05/0.01) influence of ripening remediation treatments on fruits for all the measured parameters. fruits stored with rras consistently out­performed those stored in the open shelf. rras 0.3 µl/l1­mcp, 0.5 µl/l1­mcp and 5% kmno4 solution media had longer shelf life and higher values of total soluble solids, titratable acidity, lycopene and ascorbic acid contents. the use of 1­mcp and 5% kmno4 is recommended as effective scavenger of ethylene for extending the shelf life and maintaining some quality attributes of stored tomato fruits. 1. introduction tomato (solanum lycopersicum l.) is one of the most important fruit vegetables crops in the world. it plays an important role in human diet, being mostly used as a vegetable in the preparation of soup, salad, pick­ les, ketchup, puree, sauces and in many other ways. it is also a rich source of phytochemicals and vitamins that provide protection against chronic diseases, different types of cancers, cardiac vascular diseases and age­ related ailments because of its anti­oxidant, anti­carcinogenic and anti­ (*) corresponding author: oadebusolami@gmail.com citation: oduntan a.o., oyetunde o.a., shobo b.a., bodunde j.g., 2022 ­ effect of harvest maturity stage and ripening remediation agents on the shelf life and biochemical quality attributes of tomato (solanum lycopersicum l.) fruits. ­ adv. hort. sci., 36(4): 255­263. copyright: © 2022 oduntan a.o., oyetunde o.a., shobo b.a., bodunde j.g. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 21 march 2022 accepted for publication 1 september 2022 ahs advances in horticultural science adv. hort. sci., 2022 36(4): 255­263 256 mutagenic properties (chaudhary et al., 2018). regardless of the health and nutritional relevance of tomato, its production and value chain potentials realization is constrained by post­harvest losses of quantity and quality of produce available to con­ sumers. one of the impacts of post­harvest losses results in reduction of food that is accessible for human consumption, which is worsened by increas­ ing demand for food (kikulwe et al., 2018). the toma­ to fruit is highly perishable, and a climacteric rise in respiration takes place during ripening, which is con­ sidered a turning point in the life of the fruits as regards quality. being a climacteric fruit, a marked increase of respiration rate and ethylene production during ripening process occurs that reduces the shelf life of the fruit, which may constitute a major chal­ lenge in the value chain (arah et al., 2015). the pres­ ence of this gas accelerates fruit ripening and quality deterioration by shortening the shelf life of the fruit. ripening is a natural phenomenon that involves a series of biochemical changes that are responsible for the textural changes, starch breakdown, change of color, pigment formation, volatile and aroma d e v e l o p m e n t a n d fi n a l l y a b s c i s s i o n o f f r u i t s (maduwanthi and marapana, 2019). in tomato, ripening involves different dramatic biochemical and physiological changes of the fruit which are charac­ terized by lycopene accumulation, chlorophyll loss, softening, and changes in aroma and other composi­ tional properties. the regulation of these changes, thus, has been a major corcern for research aimed t o w a r d s i m p r o v i n g f r u i t q u a l i t y a n d s h e l f l i f e (yasuhiro, 2016). increasing the postharvest life of tomato is an important aspect in view of its huge postharvest loss­ es. the onset of ripening in tomato is governed by an increase in ethylene production and it is highly dependent on continuous presence of ethylene and ethylene­mediated actions (zhao et al., 2021), there­ fore, the need to prevent the build­up of the gas around the produce. this has been found effective in delaying ripening in bananas (zewter et al., 2012) and was achieved through the use of substances that either absorb or block ethylene binding to its recep­ tor and these substances could be termed ripening remediation agents (rra). because of the important role of ethylene and ethylene­mediated actions in the onset and progres­ sion of ripening in tomato (paul et al., 2002), pre­ venting the buildup of the gas around the produce had been used to delay ripening in bananas. this has b e e n a c h i e v e d t h r o u g h t h e u s e o f r i p e n i n g remediation agents (rras) which either absorb or block ethylene binding to its receptor. concerning the latter, some rras inhibits ethylene’s role in ripening by their presence at the ethylene­binding sites so that ethylene would not be able to bind and cause subsequent signal translation and transduction in the ripening process (zewter et al., 2012). others act by removing unwanted ethylene gas through the oxidation process, converting it to carbon dioxide and water, thereby halting the ripening process and ensuring the quality of freshness of the product in the packaged environment (sen et al., 2012). also some act as ethylene adsorbers as they have great potential in the agro­industry to remove ethylene due to their cation exchange capacity, high porosity and surface area of uptakes (yin et al., 2020). tomato, being a perishable crop due to its high moisture content, has a short shelf life under tropical conditions (arah et al., 2015). this makes it impor­ tant to develop strategies for the development of handling technologies that reduce or remove the eth­ ylene production of the storage environment, while at the same time sustaining the quality. in view of these, the present study was carried out to compare the effects of different ripening remediation agents on shelf life and some biochemical quality attributes of stored tomato fruits. 2. materials and methods plant materials the tomato fruits (var. beske) used for the experi­ ment were obtained from the experimental field at the teaching and research farm, directorate of university farms, federal university of agriculture, abeokuta (funaab), ogun state, nigeria in 2018 and 2019 where they were grown under field conditions. harvesting was done at three harvest maturity indices following the usda tomato colour chart. harvested fruits were taken to the laboratory of the d e p a r t m e n t o f h o r ti c u l t u r e a n d l a n d s c a p e management, college of plant science and crop p r o d u c ti o n , f e d e r a l u n i v e r s i t y o f a g r i c u l t u r e , abeokuta, ogun state, nigeria for storage. the fruit samples were sorted and graded according to unifor­ mity of size. thereafter, fruits were carefully visually observed and only those that were wholesome were finally used in the experiment while those with defects or signs of diseases were discarded. odutan et al. ‐ postharvest handling of bell pepper 257 treatments and experimental design tomato fruits were harvested at three maturity stages identified by the usda tomato colour chart, viz breaker, turning, full ripe (usda, 2005), and were washed in distilled water to reduce microbial popula­ tion and remove adhering dirt and dust. thereafter, 500 g of fruits in the different maturity stages were exposed to seven (7) rras and an open shelf (that is, without any rra, serving as the ‘control’). seven rras were employed which were in three categories: e t h y l e n e a b s o r b e r [ p o t a s s i u m p e r m a n g a n a t e (kmno4)], ethylene absorber (zeolites) and ethylene inhibitor [1­methylcyclopropene (1­mcp)]. the amounts of rras used were: 0.1 µl/l, 0.3 µl/l and 0.5 µl/l for 1­mcp; 5% and 10% for kmno4 solution; 10 g and 20 g of zeolite. thus, 24 treatments were com­ posed by combining three harvest maturity indices and and eight exposures (rras and control). gaseous 1­mcp was prepared from smartfreshtm (agrofresh inc.) commercial powder (0.14% of active ingredient). the application was done in air­tight plastic contain­ ers (capacity of 1 m3) applying 0.1, 0.3 and 0.5 µl/l of 1­mcp for 24 h, at a temperature of 25°c and 85­90% rh. potassium permanganate (kmno4) solutions of 5% and 10% concentrations were prepared by dis­ solving 5 g and 10 g kmno4 powder in 100 ml of dis­ tilled water and put into small containers to be placed beside fruit samples. also, 10 g and 20 g of zeolite, in granular form, were also put into small containers to be placed beside fruit samples. fruits with the treatments applied were stored at room temperature. control treatment fruits were also stored at room temperature, but without any rra. each treatment had 500 g of whole and healthy tomato fruits stored in perforated plastic containers. untreated fruits (control) were kept in similar con­ tainers and placed in an open shelf. the experiment was laid out in completely randomized design with three replications. after the storage, five fruits were sampled randomly per treatment in each replication and were evaluated for shelf life and some quality parameters, viz total soluble solids, ascorbic acid, titratable acidity and lycopene. assessment of shelf life and some fruit quality attrib‐ utes tomato fruits were stored at an average tempera­ ture between 30­32°c and a relative humidity of 78­ 80% in both years. the shelf life (days) of the tomato fruits was determined by visually observing the inci­ dence and extent of spoilage with respect to storage days. this was determined from the time they were stored to the time they became unsuitable for con­ sumption. for the analysis of total soluble solids con­ tent (tss) and titratable acidity (ta) of each sample, tissue sap was squeezed out from fresh fruit materi­ als with a press. in this juice, tss were determined with an atago handheld refractometer in brixº. titratable acid (ta) content was determined by titrat­ ing method and calculating the result as grams of malic acid per 100 g fresh weight (%). ascorbic acid content of the samples was determined according to the recommended method of aoac (2000) using 2, 6­dichlorophenol indophenol and expressed as mg k g ­ 1. l y c o p e n e c o n t e n t w a s e s ti m a t e d u s i n g a s p e c t r o m e t e r b y e x t r a c ti o n w i t h h e x a n e a n d absorbance measurement at 503 nm and expressed in mg kg­1. fruit firmness was determined using hand­ pi model gy­series penetrometer. fruit firmness in a sample was measured by pushing the central probe against the equatorial plane of the fruit until the cen­ tral probe flattened. the flattening of the probe caused the needle in the instrument to deflect and the number where the needle stopped was recorded as the value for the fruit firmness (kitinoja and hussein, 2005). statistical analysis data were subjected to 2­way analysis of variance (anova) and significantly different means were sep­ arated at 5% probability level. correlation between all pair­wise traits were estimated for measured t r a i t s . a l l a n a l y s e s w e r e p e r f o r m e d u s i n g t h e statistical analysis system, sas, version 9.3 (sas institute, 2012). 3. results harvest maturity index significantly (p≤0.05) affected the shelf life of stored tomato fruits, and fol­ lowed the same trend in both years of the study. in 2018 and 2019 respectively, fruits harvested at breaker stage had the longest shelf life of 37 and 40 days, followed by those harvested at turning stage with 36 and 35 days shelf life while fruits harvested at full ripe stage had the shortest shelf life of 35 and 32 days (table 1). the shelf life of stored tomato fruits was signifi­ cantly (p≤0.05) affected by the ripening remedia­ tion treatments and this has comparable trends in both years of the study (table 2). in 2018, fruits exposed to 0.3 µl/l had the longest shelf life of 45 days, immediately followed by fruits stored with 0.5 adv. hort. sci., 2022 36(4): 255­263 258 µ l/l 1­mcp and 5% kmno4 solution media with 43 and 42 days shelf life respectively. furthermore, fruits stored with 0.1 µl/l 1­mcp had a shelf life of 37 days while those stored in 10% kmno4 solution and 20 g of zeolite media had the same shelf life of 36 days. untreated fruits however, had the shortest shelf life of 18 days. in the second year of the experiment, a similar trend was observed in the effect of rras on the shelf life of the stored fruits. fruits exposed to 0.3 µl/l and 0.5 µl/l 1­mcp and those stored in 5% kmno4 solution medium had shelf life of 46, 43 and 39 days respectively, followed by fruits under 0.1 µl/l 1­mcp with a shelf life of 37 days. fruits stored in 10% kmno4 solution and 20 g of zeolite medium had com­ parable shelf life of 32 days while fruits left in the open shelf had the shortest shelf life of 18 days. it was noted that 0.3 µl/l1­mcp, 0.5µl/l1­mcp and 5% kmno4 solution were the most effective in extending the shelf life of tomato. as displayed in table 3, total titratable acidity (tta) was significantly (p≤0.05) affected by the ripen­ ing remediation treatments in both 2018 and 2019. in years 2018 and 2019, the highest tta of 0.43 and 0.42 g/l respectively were recorded for fruits treated with 0.3 µl/l and 0.5 µl/l 1­ mcp and those in 5% kmno4 solution medium while fruits left on the open shelf had the lowest average tta concentration of 0.37 g/l. the lycopene content of the fruits was signifi­ cantly affected by the ripening remediation treat­ ments (table 3). in both years of the experiment, fruits kept in open shelf condition recorded the high­ est lycopene contents of 401.40 µg/100 g (in 2018) and 392.53 µg/100 g (in 2019) which were compara­ ble with the values obtained for fruits treated with 10% kmno4 solution (405.55 µg/100 g in 2018), 10 g zeolite medium having lycopene content of 403.20 and 392.45 µg/100 g in 2018 and 2019 respectively. the lowest lycopene contents of 389 µg/100 g in 2018 and 365.19 µg/100 g in 2019 were observed for 5% kmno4 and 0.5 µl/l 1­mcp respectively. further­more, ascorbic acid content of the fruits was significantly (p≤0.05) influenced by the ripening table 1 ­ effect of harvest maturity index on shelf life of tomato fruits in years 2018 and 2019 means followed by the same letters in the same column are not significantly different at 5% probability level of dmrt. table 2 ­ effect of ripening remediation agent on shelf life of tomato fruits in years 2018 and 2019 ripening remediation agent shelf lfe (days) 2018 2019 0.1 µl/l 1­mcp 36.63 b 36.75 ab 0.3 µl/l 1­mcp 43.25 a 42.75 ab 0.5 µl/l 1­mcp 41.88 a 37.88 ab 5% kmno4 44.63 a 45.74 a 10% kmno4 36.38 b 32.10 b 10 g of zeolite 27.75 c 30.25 cb 20 g ofzeolite 35.50 b 32.40 b open shelf (control) 18.25 d 17.88 c means followed by the same letters in the same column are not significantly different at 5% probability level of dmrt. table 3 ­ effect of ripening remediation substances on some nutritive traits of tomato fruits in 2018 and 2019 means followed by the same letters in the same column are not significantly different at 5% probability level of dmrt. ethylene remediation treatments tta (g/l) lycopene (µg/100 g) ascorbic acid (mg/100 g) tss (%) 2018 2019 2018 2019 2018 2019 2018 2019 0.1 µl/l 1­mcp 0.42 a 0.40 ab 390.79 ab 381.41 ab 18.26 a 18.55 a 5.73 ab 5.78 ab 0.3 µl/l 1­mcp 0.43 a 0.42 a 392.21 ab 372.06 b 17.71 ab 19.15 a 5.89 a 5.85 a 0.5 µl/l 1­mcp 0.43 a 0.42 a 392.99 bc 365.19 b 17.79 ab 18.80 a 5.80 a 5.89 a 5% kmno4 0.43 a 0.42 a 389.00 b 375.36 b 17.78 ab 18.07 a 5.80 a 5.85 a 10% kmno4 0.39 b 0.40 ab 405.55 a 384.29 ab 17.34 ab 18.09 a 5.50 b 5.61 b 10 g zeolite 0.38 b 0.40 ab 403.20 a 392.45 a 16.69 b 16.74 b 5.50 b 5.49 b 20 g zeolite 0.41 ab 0.38 b 398.29 ab 387.28 ab 17.01 ab 17.29 ab 5.50 b 5.52 b open shelf (control) 0.37 b 0.37 b 407.40 a 392.53 a 16.31 b 17.51 ab 5.25 bc 5.22 bc harvest index shelf life (days) 2018 2019 breaker 37.99 a 40.70 a turning 35.73 b 34.70 b full ripe 31.08 c 32.18 c odutan et al. ‐ postharvest handling of bell pepper 259 remediation treatments in both years (table 3). in 2018, the ascorbic acid content ranged from 16.31 mg/100 g for fruits stored in the open shelf to 18.26 mg/100 g for fruits stored with 0.1 µl/l 1­mcp. substantial amounts of ascorbic acid were also observed in fruits with 0.5 µl/l 1­mcp, 5% kmno4, 0.3 µl/l 1­mcp, 10% kmno4, and 20 g zeolite in decreasing order. in 2019, ascorbic acid content of stored fruits ranged from 16.74 mg/100 g for 10 g z e o l i t e t o 1 9 . 1 5 m g / 1 0 0 g f o r 0 . 3 µ l / l 1 ­ m c p . s u b s t a n ti a l a s c o r b i c a c i d c o n t e n t s w e r e a l s o observed for 0.1 µl/l 1­mcp, 0.5 µl/l 1­mcp, 10% kmno4, 5% kmno4, open shelf and 20 g zeolite, in decreasing order. the total soluble solids (tss) of stored fruits was also significantly (p≤0.05) affected by the ripening remediation treatments (table 3). fruits kept in the open shelf condition, however, recorded the highest tss in both years of the study. in 2018, the tss content recorded in fruits exposed to 0.1 µl/l and 0.5 µl/l 1­mcp and 5% kmno4 solu­ tion medium was lower compared to those stored in zeolite medium and those kept in the open shelf. in 2019, fruits stored in 10 g and 20 g of zeolite medi­ um had higher tss compared to those exposed to 1­ mcp concentrations and those stored in 5% and 10% kmno4 solution (table 3). as reported before, tomato fruits harvested at the breaker and turning stages recorded significantly longer shelf life than fruits harvested at the full ripe stage except for fruits harvested at the turning stage and stored with zeolite (table 1). comparing fruits from the same stage of maturity in both 2018 and 2019, fruits stored with 0.3 µl/l 1­mcp had the longest shelf life for fruits harvested at the breaker stage, while 5% kmno4 effected the longest shelf life for fruits picked at the turning stage while full ripe fruits had the longest shelf life when stored with 0.3 µl/l 1­mcp and 5% kmno4 (table 4). there was a general decrease in tta for all the treatments. however, fruits harvested at breaker and turning stages recorded higher tta contents with exposure to 0.1 µl/l, 0.3 µl/l and 0.5 µl/l 1­ mcp and those stored in 5% kmno4 solution medium when compared to other treatments (table 5). lycopene content was higher for fruits harvested at full ripe kept in the open shelf while there was low lycopene content for those harvested at breaker stage with stored with 1­mcp and 5% kmno4 solution medium. in the same vein, fruits harvested at breaker and turning stages had higher ascorbic acid contents when exposed to 1­mcp and 5% kmno4 solution medium while those kept in the open shelf had lower ascorbic acid content comparable with those stored with 10% kmno4 solution and zeolite. on the other hand, fruits harvested at the breaker stage and exposed to 1­mcp or kmno4 solution media recorded significantly lower tss than the full­ripe fruits kept in the open shelf as shown in table 5. significant (p≤0.05/0.01) levels of association, comparable for both 2018 and 2019 experiments, were observed in the relationship among shelf life and measured biochemical parameters of tomato table 4 ­ harvest maturity index and ripening remediation agents on shelf life of tomato fruit in years 2018 and 2019 means followed by the same letters in the same column are not significantly different at 5% probability level of dmrt. shelf life (days) 0.1 µl/l 1­mcp 0.3 µl/l 1­mcp 0.5 µl/l 1­mcp 5% kmno4 10% kmno4 10 g zeolite 20 g zeolite open shelf (control) breaker 2018 37 ab 46 a 37 ab 42 a 40 a 27 b 40 a 19 c 2019 31 ab 42 a 36 ab 45 a 36 ab 36 ab 36 ab 18 c mean 34 44 36.5 43.5 38 31.5 38 18.5 turning 2018 36 ab 37 ab 33 ab 41 a 40 a 25 b 36 ab 19 c 2019 33 ab 40 a 37 ab 42 a 33 ab 28 b 36 ab 19 c mean 34.5 38.5 35 41.5 36.5 26.5 36 19 full‐ripe 2018 34 ab 40 a 31 ab 40 a 37 ab 31 ab 39 a 16 c 2019 37 ab 39 ab 34 ab 39 ab 28 b 33 ab 37 ab 13 c mean 35.5 39.5 32.5 39.5 32.5 32 38 14.5 260 adv. hort. sci., 2022 36(4): 255­263 fruits in this study (table 6). in 2018 and 2019, titrat­ able acidity had positive and significant correlation with ascorbic acid (r = 0.64 and 0.63 respectively) and shelf life (r= 0.70 and 0.74 respectively) but shared negative and significant correlation with lycopene (r = ­0.86 and ­0.83 respectively) and total soluble sug­ ars (r= ­0.94 and ­0.82 respectively). in 2018 and 2019, lycopene content had positive and significant association with total soluble sugars (r = 0.80 and table 5 ­ interaction of harvest maturity index and ripening remediation agents on biochemical quality attributes of tomato fruits in years 2018 and 2019 *, ** significant at 5 and 1% probabilities, respectively. harvest maturity index ripening remediation treatments tta (g/l) lycopene (µg/100g) vitamin c (mg/100g) tss (%) 2018 2019 2018 2019 2018 2019 2018 2019 breaker 0.1 µl/l 1­mcp 0.41 a 0.42 a 382.26 ab 381.38 ab 16.67 a 16.87 a 5.59 b 5.57 b 0.3 µl/l 1­mcp 0.41 a 0.42 a 384.93 ab 371.42 b 16.43 a 16.39 a 5.56 b 5.52 b 0.5 µl/l 1­mcp 0.41 a 0.42 a 382.18 ab 378.93 b 16.78 a 16.36 a 5.54 b 5.52 b 5% kmno4 0.42 a 0.42 a 388.67 ab 384.94 ab 16.21 a 16.11 a 5.53 b 5.54 b 10% kmno4 0.36 b 0.40 a 399.10 ab 396.71 ab 15.21 ab 15.22 ab 5.67 b 5.62 b 10 g zeolite 0.37 b 0.38 b 397.89 ab 411.30 a 15.83 ab 15.93 ab 5.75 ab 5.75 ab 20 g zeolite 0.38 ab 0.38 b 396.71 ab 411.23 a 15.38 ab 15.01 ab 5.75 ab 5.75 ab open shelf (control) 0.35 b 0.35 b 412.73 a 407.36 a 15.91 ab 15.96 ab 5.95 a 5.93 a turning 0.1 µl/l 1­mcp 0.39 ab 0.39 ab 394.77 ab 391.22 ab 16.91 a 16.91 a 5.64 b 5.69 b 0.3 µl/l 1­mcp 0.39 ab 0.40 a 386.42 ab 389.41 ab 16.71 a 16.86 a 5.69 b 5.66 b 0.5 µl/l 1­mcp 0.39 ab 0.40 a 386.99 ab 388.50 ab 16.82 a 16.50 a 5.66 b 5.64 b 5% kmno4 0.40 a 0.40 a 389.63 ab 389.71 ab 16.38 a 16.31 a 5.66 b 5.63 b 10% kmno4 0.37 ab 0.38 ab 400.51 a 402.38 a 15.98 ab 15.92 ab 5.72 ab 5.77 ab 10 g zeolite 0.37 ab 0.38 ab 396.18 ab 403.78 ab 15.78 ab 15.61 ab 5.75 ab 5.79 ab 2 0g zeolite 0.37 ab 0.39 ab 389.28 ab 391.18 a 15.86 ab 15.89 ab 5.71 ab 5.71 ab open shelf (control) 0.34 b 0.34 b 410.91 a 413.67 a 14.71 b 14.62 b 5.99 a 5.98 a full­ripe 0.1 µl/l 1­mcp 0.38 ab 0.38 ab 392.51 ab 392.73 ab 15.43 ab 15.93 ab 5.77 ab 5.78 ab 0.3 µl/l 1­mcp 0.39 ab 0.39 ab 389.42 ab 392.11 ab 15.56 ab 15.45 b 5.72 ab 5.78 ab 0.5 µl/l 1­mcp 0.39 ab 0.39 ab 384.66 ab 389.81 ab 15.77 ab 15.86 ab 5.72 ab 5.78 ab 5% kmno4 0.39 ab 0.39 ab 387.48 ab 389.82 ab 15.39 ab 15.93 b 5.74 ab 5.74 ab 10% kmno4 0.40 a 0.37 ab 402.38 a 404.86 a 14.73 b 14.78 b 5.82 a 5.89 a 10 g zeolite 0.37 b 0.37 ab 403.56 a 405.36 a 14.48 b 14.14 b 5.94 a 5.92 a 20 g zeolite 0.37 b 0.37 ab 399.83 ab 396.74 ab 14.97 b 14.76 b 5.95 a 5.95 a open shelf (control) 0.34 b 0.37 ab 413.56 a 411.86 a 13.92 b 13.82 b 5.99 a 5.99 a means followed by the same letters in the same column are not significantly different at 5% probability level of dmrt. table 6 ­ pearson correlation coefficients of the relationship among shelf life and measured biochemical quality components of tomato fruits stored with ripening remediation agents in 2018 (lower diagonal) and 2019 (upper diagonal) parameter measured titratable acidity lycopene ascorbic acid total soluble sugars shelf life titratable acidity 1 ­0.86 ** 0.64 ** ­0.94 ** 0.70 ** lycopene ­0.83 ** 1 ­0.70 ** 0.80 ** ­0.65 ** ascorbic acid 0.63 ** ­0.71 ** 1 ­0.76 ** 0.51 * total soluble sugars ­0.82 ** 0.85 ** ­0.80 ** 1 ­0.70 ** shelf life 0.74 ** ­0.68 ** 0.42 * ­0.69 ** 1 0.85 respectively) and shared negative and signifi­ cant association with ascorbic acid (r= ­0.70 and ­ 0.71 respectively) and shelf life (r= ­0.65 and ­0.68 respectively). furthermore, there was negative and significant correlation between ascorbic acid and total soluble sugars with r= ­0.76 and ­0.80 while sharing positive and significant association with shelf life with r= 0.51 and r 0.42 for 2018 and 2019 respec­ tively. total soluble sugars also had negative and sig­ nificant correlation with shelf life (r= ­0.70 and r= ­ odutan et al. ‐ postharvest handling of bell pepper 261 0.69 in 2018 and 2019 respectively). 4. discussion and conclusions the ripening remediation treatments considerably affected the shelf life of stored tomato in both years of the study and the extended shelf life could have been as a result of the efficacy of these treatments to delay the conversion of starch to sugars thus reduc­ ing the ethylene production and peroxidase activity of the fruits. similar results of delay in conversion of starch to sugars for extended shelf life of tomato were observed with the use of gibberellic acid as reported by srividya et al. (2014). the identified rras could also have been able to extend the shelf life of the fruits due to their ability to control respiratory metabolism, thus maintaining the produce for a longer period as suggested by nath et al. (2015). during storage, acidity decreased with ripening as the organic (malic and citric) acids in the fruits got metabolized. the loss of tta during storage period could be related to higher respiration rate as ripening advances, where organic acids are used as substrate in the respiration process. exposure of the tomato fruits to the rras in this study delayed the consump­ tion of the tta, with 1­mcp and 5% kmno4 being the most reliable in achieving this. regassa et al. (2012) reported the sequential disappearance of malic and citric acids in ripening tomato fruits leading to reduc­ tion in the amount of tta. lycopene content of tomato fruits were different­ ly affected by rra but exposure to 1­mcp concentra­ tions and 5% kmno4 solution treatments delayed the accumulation of lycopene in the fruits for both years. this might be due to decrease in respiratory rate, inhibiting ethylene activity, consequently reducing metabolism of the fruit (nath et al., 2015). the delay in lycopene development in this study could have been as a result of the efficacy of the ripening reme­ diation treatments in suppressing the production of ethylene in fruits thus delaying lycopene accumula­ tion. the restrictive effect of 1­mcp on lycopene accumulation in this study supports the previous reports of taye et al. (2019). the treatment with 1­mcp concentrations and kmno4 had comparable patterns of effect on ascorbic acid content. generally, fruits treated with 1­mcp and 5% kmno4 had higher ascorbic acid contents compared to other treatments. the efficacy of 1­mcp concentrations in this study corroborates the obser­ vations of sabir et al. (2012) that 1­mcp had signifi­ cant effect on ascorbic acid content by decreasing ethylene content of tomato fruit thereby increasing ascorbic acid content. generally, this study indicated that there was a decrease in ascorbic acid content of tomato fruits which showed significant decrease dur­ ing storage as reported by ahmed et al. (2018). generally, as reported by tilahun et al. (2019), tomato fruits harvested at the matured green and breaker stages had lower tss level, while fruits har­ vested at light­red stage of full­ripe had the highest tss. however, in this study, the efficacy of the ripen­ ing remediation agents was evident in slowing down the breakdown of carbohydrates into soluble sugars (fructose and glucose) or excessive moisture loss that aids the hydrolysis of cell wall polysaccharides. the fact that fruits kept in the open shelf recorded the highest tss in both years can be attributed to faster advancement in ripening than those treated with rras as previously specified by ahmed et al. (2018). the increase as influenced by the ripening remedia­ tion treatments may have occurred as a result of breakdown of carbohydrates into soluble sugars, or excessive moisture loss that aids the hydrolysis of cell wall polysaccharides. however, beckles (2012) earlier noted that 1­mcp may increase, reduce or leave unchanged, the development of tss depending on fruit species. 1­mcp at 0.3 µl/l concentration and 5% kmno4 were the most effective in extending the shelf life of fruits harvested however, those left in the open shelf consistently had the shortest shelf life implying that rra application was effective in extending the mar­ ketable life of the fruits. the longer shelf life recorded by the fruits at the breaker and turning stages may be attributed to the ability of the ripening remediation agents to control respiratory metabolism, thus main­ taining the produce for a longer period as suggested by nath et al. (2015). harvesting fruits at the proper maturity stage has a great influence on the nutrient content as well as shelf life of any fruit. however, in this study, the ripening remediation agents had great influence in slowing down the action of the ripening hormone that could accelerate the decline in the ascorbic acid content of the tomato fruit. it is cumbersome to consider multiple traits in a selection scheme. information on the relationship among various traits with shelf life would thus be beneficial to designing an efficient storage system. the significant correlation of shelf life with all the measured nutritional quality attributes coupled with adv. hort. sci., 2022 36(4): 255­263 262 the interrelationship among the attributes presents the possibility of extending the shelf life of tomato fruits through designing an effective storage system that focuses on manipulating the production and/or accumulation of titratable acids, total soluble solids, lycopene and ascorbic acid. comparable findings in the relationship among nutritional quality traits of tomato, including titratable acids, total soluble solids, and lycopene have earlier been reported by singh et al. (2018) and shobo et al. (2020). the use of rras was effective in increasing the shelf life and maintaining the nutritional properties of tomato fruits in storage. however, the rras dif­ fered in their effectiveness in both capacities. rras 0.3 µl/l1­mcp, 0.5µl/l1­mcp and 5% kmno4 solu­ tion media had longer shelf life and higher values of total soluble solids, titratable acidity, lycopene and ascorbic acid contents. the use of 1­mcp and 5% kmno4 is recommended as effective scavenger of ethylene for extending the shelf life and maintaining some quality attributes of stored tomato fruits. acknowledgements the authors are grateful to agro­fresh tm for pro­ viding the 1­mcp that was used for the study. we a l s o t h a n k t h e s t a ff o f t h e l a b o r a t o r y o f t h e department of horticulture, college of plant science a n d c r o p p r o d u c ti o n , f e d e r a l u n i v e r s i t y o f agriculture, abeokuta, nigeria, for assistance ren­ dered during the conduction of the study. references ahmed 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counter­season supply of horticultural products is of increasing demand. consumers are demanding annual supply of raspberries, which has historically been challenging due to their seasonal summer supply and charac­ teristically high metabolism resulting in a short shelf life and limited period of availability. however, the development of freezing technologies for increasing the length of storage of raspberries offers an opportunity for continual supply of premium quality raspberries. we investigated berry quality after freezing whole fresh raspberries, comparing conventional freezing methods with a mod­ ern cryogenic freezing method over a period of six months. significant increases in total soluble solids, titratable acidity, hue and chroma were found when raspberries were frozen compared to fresh raspberries. no overall difference in berry quality was observed between freezing methods for any parameter assessed. when assessed at time intervals, total soluble solids, ph, titratable acidity, chroma and hue were consistent between freezing methods for all durations of time frozen. these findings provide decision support for producers and distributors pursuing a novel counter season supply chain. 1. introduction raspberries (rubus ideaus l.) are a perennial plant that produce a compound fruit composed of many drupelets (wang et al., 2009). raspberries grown for fresh production typically have a unique bright red colour with distinctive aroma and taste, as well as health benefits due to their antioxidant properties (liu et al., 2002). raspberries are a non­cli­ macteric fruit with a high metabolism, making them perishable with a very short shelf life (tezotto­uliana et al., 2014). the ability to maximise fruit quality whilst harvesting and during post­harvest is imperative to commercially viable raspberry production. the development and adoption of innovative technologies has allowed conventional agricultural systems to pursue increased efficiency, yield, quality, food safety, and sustainability with reduced costs (sunding and zilberman, 2001). in the raspberry industry, freezing technologies for post­harvest storage offer producers the ability to extend the market win­ dow whilst preserving or increasing fruit quality standards. additionally, freezing horticultural products allows access to diversified markets and (*) corresponding author: oliver.gales@seh.ox.ac.uk citation: gales o., jones j., swarts n., 2022 ­ an analy‐ sis on the impacts of cryogenic freezing on rasp‐ berry quality. ­ adv. hort. sci., 36(4): 293­301. copyright: © 2022 gales o., jones j., swarts n. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 29 september 2022 accepted for publication 4 november 2022 ahs advances in horticultural science https://doi.org/10.36253/ahsc-13824 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2022 36(4): 293­301 294 supply chains, creating increased economic feasibility and prosperity (bora et al., 2021; safari et al., 2021). demand by consumers for out­of­season supply of produce has driven research into the impacts of dif­ ferent storage methods on fruit and berry quality parameters (boorse et al., 1998; gonzález et al., 2002; chassagne­berces et al., 2010; ceballos et al., 2012; alhamdan et al., 2018). the perishable charac­ teristics of raspberries caused by high rates of metab­ o l i s m a n d l i m i t e d s e a s o n a l p r o d u c ti o n , m a k e s counter season supply problematic for producers (adobati et al., 2015). despite extensive literature investigating variances in quality parameters for hor­ ticultural products post conventional and cryogenic (liquid nitrogen) freezing, there is currently a gap in research comparing the impacts of the two freezing methods on the quality of whole fresh raspberries (alhamdan et al., 2018; otero et al., 2000). some notable issues with freezing of berry fruit have been identified including ice crystal nucleation and subse­ quent cell membrane rupture and electrolyte leakage which compromises the eating quality of frozen fruit (silva et al., 2008). the mouth feel of frozen fruit is related to cell turgor pressure which is driven by each cell having a separate functional semi permeable membrane that splits when ice nucleation occurs, resulting in soft textures in comparison to crisp fresh fruit (grout et al., 1991). to reduce the damage to cell membranes by crystal nucleation during the freezing of berries, there have been several develop­ ments in freezing methodologies. liquid nitrogen (~­ 80°c) applied to each berry individually reduces the thermal gradient and creates a snap freeze process that reduces the loss of quality through cell mem­ brane damage caused by slower and higher tempera­ ture conventional freezing, resulting in a higher quali­ ty product (ceballos et al., 2012). studies have demonstrated that the loss of quality, particularly tis­ sue damage and subsequent texture and colour from freezing, is impacted by the rate of freezing. fruit frozen at lower temperatures (­40°c) have been shown to display better quality parameters than f r u i t s f r o z e n a t h i g h e r t e m p e r a t u r e s ( ­ 1 4 ° c ) (chassagne­berces et al., 2010). whilst the demand for frozen horticultural prod­ ucts has seen considerable investment into studies on the impacts of freezing on vegetables and fruits, there is comparatively limited evidence on the specif­ ic effects of freezing on raspberry quality to support industry investment. gonzález et al. (2002) found that berry variety, harvest timing and quality at har­ vest are critical factors influencing the impact freez­ ing has on raspberry fruit quality. de ancos et al. (2000) showed an increase in anthocyanin content during freezing; however, this conclusion was deter­ mined to be a result of anthocyanin extraction effica­ cy after ice nucleation, rather than a biological i n c r e a s e i n a n t h o c y a n i n c o n t e n t . c o n v e r s e l y , kampuse et al. (2001) highlighted a decrease in anthocyanins in frozen raspberries, with varietal dif­ ferences influencing the degree of anthocyanin depletion. cryogenic freezing technology has the potential to improve the quality of frozen berries. this study investigated if the application of freezing technolo­ gies can lead to improvements in raspberry fruit quality when frozen. specifically, using a traditional commercially grown variety and a new propriety vari­ ety, we asked ­ does freezing method (conventional and cryogenic) and freezing duration (2, 4, and 6 months) influence raspberry quality? the findings of this research are discussed in the context of industry p u r s u i n g c o n ti n u o u s s u p p l y t o a m a r k e t t h a t d e m a n d s h i g h q u a l i t y a n d a ff o r d a b l e p r o d u c e throughout the year. 2. materials and methods experimental site t h e e x p e r i m e n t a l s i t e w a s l o c a t e d a t t h e westerway raspberry farm (42° 47’ 26.34” s, 146° 47’ 26.34” e), an established 60­acre raspberry and m i x e d b e r r y f a r m a t w e s t e r w a y , i n s o u t h e r n tasmania. the westerway raspberry farm is situated on an alluvial soil deposit adjacent to the tyenna river. the raspberry canes utilised for the experi­ ment were oriented in a ne sw direction and were trained in a commercial vertical trellis system. westerway receives an average annual rainfall of 764 m m ( b o m , 2 0 1 9 ) . t h e t r a d i ti o n a l a n d p o p u l a r ‘willamette’ raspberry variety and a newly developed proprietary variety were investigated for the purpose of this experiment. the proprietary variety has been developed for the purpose of machine harvesting, with characteristics of diseases resistance, fruit firm­ ness and low release force from plant. sample collection harvest coincided with the commercial raspberry season on 27 december 2018. fruit was hand har­ vested from randomly selected rows within the block gales et al. ‐ cryogenic freezing on raspberry quality 295 with berries collected randomly along each row. consistent with commercial practice, samples were collected into individually labelled commercial plastic 400 g punnets and stored in a commercial cool room (7°c) on site for five hours until transportation. treatments and storage conditions for both raspberry varieties there were two dif­ ferent treatments (post­harvest storage technique and duration frozen) with five replicates for each treatment combination. treatments were estab­ lished as a fully factorial trial with two freezing tech­ niques (conventional and cryogenic) and three lengths of storage (2, 4 and 6 months) tested with two varieties of raspberries. punnets containing fruit were immediately frozen on site in either a commercial conventional ­8°c freezer (gunter, germany) or snap frozen in a liquid nitrogen (ln) tunnel at ­80°c (linde cryoline mt 5­ 600 quick­freezing (iqf) tunnel, munich, germany) depending on treatment. for ln tunnel freezing, the berries were tipped out of the punnets and passed through the ln tunnel individually on the inbuilt con­ veyor belt and collected into labelled punnets imme­ diately after freezing. all berries from both treat­ ments were then stored in the commercial ­8°c freezer for the duration of the storage period. during storage, all samples were stored in the commercial grade plastic punnets with lids and stacked into crates holding 50 punnets per crate, aligned with industry practice. frozen samples remained in the freezer for three prescribed periods of up to six months. laboratory analysis samples were transported from the farm freezing facilities back to the laboratory at the tasmanian institute of agriculture (university of tasmania) for analysis in an ice chilled eski. the samples were then stored in a cool room (4°c) during the laboratory analyses, which were completed within 48 hr after being removed from the freezer. the ph for every replicate was measured using a diluted sample in an auto­titrator (metrohm 702 sm titrino, herisau, switzerland). a subsample of 50 berries from each sample was juiced by squeezing berries through a microporous cloth (30­micron mesh filter cloth; allied filter fabrics p/n m0032, sydney, australia). a sample of the juice (5 ml) was pipetted (brantech 10 ml transfer pipette s, san francisco, america) into a 25 ml beaker and com­ bined with 15 ml of distilled water. the solution was analysed for initial ph by electrode probes used in the auto­titration. the same solution used for ph was analysed for titratable acidity by the metrohmauto­ titrator (702 sm titrino). the solution was mixed using a magnetic stirrer and combined with 1m sodi­ um hydroxide to determine the titratable acidity (titration to ph = 8.2). berry titratable acidity was expressed in g/l of citric acid. total soluble solids (tss) was determined using a portion of the juice (~3 ml) and a digital refractometer (atago pocket refractometer pal­1). the sample of juice was analysed immediately after juicing. using a hand dis­ posable plastic pipette, the sample was placed into the measuring chamber and analysed with the tss figure (%) recorded manually. the chamber was cleaned between each sample using paper towels and distilled water to remove any residue. the total anthocyanin content (mg cyanidin­3­glu­ coside equivalents/g berry fresh weight) was deter­ mined using the ph differential method (lee et al., 2005). subsamples (30 berries) were homogenised for 30 seconds at 7000 rpm using a retsch grindomix gm200 (haan, germany). homogenised subsamples of 10 g were weighed into 50 ml centrifuge tubes and combined with 40 ml of acidified 70 % methanol [700 ml methanol, 300 ml distilled h2o, 0.1 ml con­ centrated hcl (0.01%v/v)]. the tubes were then placed into an ultrasonic bath (grant xuv digital ultrasonic bath (royston, uk) in darkness for 30 min­ utes at 10°c. the tubes were then dried and placed into a hettich benchtop centrifuge (universal 320 r model tuttlingen, germany) and centrifuged for 10 minutes at 1520 g and 4°c. a 0.5 ml sample of the clear supernatant solution was then mixed with each of the buffers; buffer 1 solution being 0.025 m potas­ sium chloride and buffer 2 being 0.4 m sodium acetate. the dilution used was a 1:10 dilution (0.5 ml supernatant to 4.5 ml of buffer). the solutions were then left to equilibrate for 30 minutes. using a benchtop spectrophotometer (thermo scientific genesys 10s uv­vis, waltham, america), the absorp­ tion of each solution was measured at 530 nm and 700 nm. this allowed the absorbance of the diluted sample to be calculated using the following equation (lee et al., 2005): absorbance = (a530 ­ a700) ph1 ­ (a530 ­ a700) ph 4.5 where a530 is the spectral wavelength absorption measurement at 530 nm, and a700 is the spectral wavelength absorption at 700 nm. adv. hort. sci., 2022 36(4): 293­301 296 u s i n g t h e c a l c u l a t e d a b s o r b a n c e v a l u e , t h e monomeric anthocyanin content was calculated using the equation (lee et al., 2005): monomeric anthocyanin pigment (mg/l) = (a x mw x df x 1000)/ (e x 1) where a = absorbance, mw = 449.2 g/mol for cyani­ din­3­glucoside, df = dilution factor determined (1 in 10 dilution), 1000 = factor for conversion from g to mg, e = 26,900 molar extinction coefficient, in l x mol­1 x cm­1, for cyd­3­glu and 1 = path length in cm. max used e = 30200. the final value from the monomeric anthocyanin pigment (mg/l) was converted to the commonly expressed mg per 100 g fresh weight unit. this con­ version was completed by knowing the total amount of anthocyanin in a litre and the initial fresh mass of raspberry sample used. anthocyanin (mg per 10 g fresh weight) = (0.04 x anthocyanin mg/l value) x 100 g/fresh weigh (g) where 0.04 = proportion of 40 ml of a litre, 100 g = final unit, anthocyanin mg/l value = answer from m o n o m e r i c e q u a ti o n a n d f r e s h w e i g h t = 1 0 g weighed initially using the homogenised samples, homogenate colour was measured using a colourimeter (konica minolta chroma meter cr­400, new york, usa) (edgley et al., 2019). spectrophotometer tubes were filled with homogenate from each sample and placed in the colourimeter. the values for l*, a* and b* were recorded manually and gave a three­dimensional colour space with each value interpreted as: l* a measure from opaque (0) to completely black (100), a positive a* indicates redness whilst negative a* indicates greenness and a positive b* indicates yel­ lowness whilst negative b* indicates blueness on the hue­circle (voss, 1992; gonçalves et al., 2007). chroma was obtained by the formula: chroma = (a*2 + b*2)2 and hue from the formula: hue= arctg b*/a* (gonçalves et al., 2007). statistical analysis analysis of variance and statistical significance of quality parameters (tss, ph, titratable acidity, antho­ cyanins, hue and chroma) between treatment inter­ actions and treatment main effects were analysed using the 2019 ibm spss statistics package. analysis of mean variance was determined using univariate linear models for each differing continuous variable. post hoc tests were completed using tukey’s range test of statistical significance. 3. results there were no three­way interactions between variety, freezing method and length of duration frozen for all quality parameters assessed. however, there were two­way interactions for freezing method and variety, duration frozen and variety, and dura­ tion frozen and freezing method for a range of the quality parameters assessed. freezing method and variety raspberries of both proprietary and ‘willamette’ varieties had significantly (p<0.05) increased tss val­ ues after freezing, regardless of method, in compari­ son to their fresh tss values (fig. 1a). conventionally frozen proprietary berries had significantly (p<0.05) lower ph values in comparison to fresh, however they were not significantly (p>0.05) different to cryo­ g e n i c a l l y f r o z e n p r o p r i e t a r y b e r r i e s ( f i g . 1 b ) . cryogenically frozen ‘willamette’ berries and frozen fig. 1 ­ tss (a), ph (b), titratable acidity (c), anthocyanin content (d), hue (e) and chroma (f) values for fresh and frozen propriety (grey bars) and ‘willamette’ raspberries (black bars). errors bars denote two times standard error. letters above bars indicate significant differences from the mean at p<0.05. gales et al. ‐ cryogenic freezing on raspberry quality 297 proprietary berries had statistically similar ph values in comparison to their respective fresh berries (fig. 1b.) freezing both ‘willamette’ and proprietary rasp­ berries significantly increased (p<0.05) titratable acidity (g/l citric acid), whilst the method of freezing made no significant (p>0.05) difference for either variety (fig. 1c). anthocyanin content of berries of both varieties were the same whether fresh or frozen, regardless of freezing method (fig. 1d). freezing both ‘willamette’ and the proprietary vari­ ety significantly increased (p<0.05) both the hue and chroma values for berry colour, whilst the method of freezing did not have a significant impact on each variety individually (fig. 1e and 1f). proprietary rasp­ berries exposed to cryogenic freezing however had significantly greater (p<0.05) hue and chroma values than ‘willamette’ exposed to cryogenic and conven­ tional freezing (figs. 1e and 1f). duration frozen and variety the tss (%) for the proprietary raspberry variety was not significantly influenced (p>0.05) within the first two months of freezing, however tss increased significantly (p<0.05) after four and six months frozen c o m p a r e d t o t h e f r e s h s a m p l e s ( f i g . 2 a ) . t h e ‘willamette’ variety had significantly increased (p<0.05) tss two and six months after freezing, how­ ever tss for the four­month frozen sample was not significantly different (p>0.05) from the fresh sam­ ples (fig. 2). once frozen, there was no significant dif­ ference (p>0.05) in tss for berries frozen for two, four or six months for either variety. t h e ‘ w i l l a m e tt e ’ v a r i e t y h a d n o s i g n i fi c a n t (p>0.05) change in ph for the duration frozen. ph of the proprietary variety berries continuously declined at each assessment date but was only significantly lower after being frozen for six months when com­ pared to fresh berries (fig. 2b). ta (g/l citric acid) of both the ‘willamette’ and proprietary variety significantly (p<0.05) increased once frozen but did not significantly change through the duration of freezing (two, four and six months) (fig. 2c). the change in titratable acidity to tss ratio was statistically similar across duration frozen. the colour (hue units) of both ‘willamette’ and the proprietary variety raspberries also significantly (p<0.05) increased when frozen (fig. 2d). once frozen, hue of the proprietary variety did not change for the duration of frozen storage, whilst hue for the ‘willamette’ berries significantly (p<0.05) reduced at the six­month assessment compared to the second month assessments (fig. 2d). duration frozen and freezing method berries that were initially cryogenically frozen had significantly (p<0.05) higher tss values at four months compared to fresh raspberries, but no signifi­ cant (p>0.05) difference at six months in comparison to fresh berries (fig. 3a). for berries that were initial­ ly frozen in a conventional freezer, the two­ and four­ month frozen treatments were not significantly dif­ ferent from fresh raspberries in tss, but at six months there was significantly (p<0.05) greater tss fig. 2 ­ brix (a), ph (b), titratable acidity (c) and hue (d) for both the proprietary (black bars) and ‘willamette’ (grey bars) variety of raspberry after being frozen for up to six months. errors bars denote two time standard error. letters above bars indicate significance differences from the mean at p<0.05 of time frozen values. fig. 3 ­ tss (%) (a), ph (b), titratable acidity (c) and hue (d) for both the conventional (grey bars) and cryogenic (black bars) methods of freezing raspberries. error bars denote two times standard error. letters above bars indicate significance differences from the mean at p<0.05. 298 adv. hort. sci., 2022 36(4): 293­301 compared to fresh berries (fig. 3a). cryogenically frozen berries had no significant (p>0.05) differences in ph value for the duration frozen in comparison to fresh berries (fig. 3b). however, conventionally frozen berries at six months had significantly (p<0.05) lower ph compared to fresh raspberries. there was a significant increase in ta once frozen, but at each assessment interval (two, four and six months), there was no significant difference in freez­ ing method on the ta concentration (fig. 3). for both conventional and cryogenic freezing, there was no significant change in ta between two, four and six months frozen (fig. 3). for hue, there was no significant difference between freezing method at any single duration measured for the duration of the experiment (fig. 3). both freezing methods did exhibit a significant increase in hue colour units initially after freezing, increasing by ~25 % between 0 and two months frozen (fig. 3). 4. discussion and conclusions consumer demand for counter season supply of premium horticultural products is driving the need and innovation of storage techniques whilst main­ taining premium quality produce (kuchler and arnade, 2015; martindale and schiebel, 2017). the results of this study found significant differences in quality parameters for whole raspberries after they w e r e f r o z e n c o m p a r e d t o f r e s h b e r r i e s . measurements of total soluble solids, titratable acidi­ ty, hue and chroma all significantly increased when berries were frozen. this study found no difference however in berry quality once frozen between freez­ ing methods for any parameter assessed. total solu­ ble solids, ph, titratable acidity and hue were consis­ tent between freezing methods for all durations of time frozen. these results improve the understand­ ing of the impacts of post­harvest storage on rasp­ berry quality and provides critical information and decision support for producers to ensure optimal raspberry quality for consumers. raspberries are no exception in the unrelenting d e m a n d f r o m c o n s u m e r s f o r p r e m i u m q u a l i t y counter season supply of horticultural products, however, the metabolic characteristics that result in raspberries being highly perishable makes the long­ term storage of raspberries comparatively difficult (gonçalves et al., 2018). for both the ‘willamette’ and proprietary varieties, tss increased once frozen regardless of freezing method. this finding of initial t s s i n c r e a s e a ft e r f r e e z i n g i s c o n s i s t e n t w i t h gonzález et al. (2002) who showed a similar initial increase in tss after freezing ‘heritage’, ‘autumn bliss’, ‘zeva’ and ‘rubi’ raspberries. the treatments of two, four­ and six­months freezing duration showed differences for tss between varieties when fresh, with the ‘willamette’ variety having greater than 20% higher tss than the proprietary variety. varietal difference as shown here is consistent with shamaila et al. (1993) who found differences for fresh berries for the quality parameters ph, tss, ta and anthocyanins between ‘chilcotin’, ‘chilliwack’, ‘meeker’, ‘skeena’ and ‘tulameen’ varieties. this sig­ nificant difference between varieties persisted for all freezing periods. however, once frozen, tss values did not significantly change for both varieties for the duration of the trial, and the freezing method, con­ ventional vs cryogenic, also had no impact on tss. this finding contrasts with the gonzález et al. (2002) study described earlier, which showed that tss con­ tinued to significantly increase during duration frozen. the findings of consistency in tss levels once frozen for the proprietary and ‘willamette’ variety suggest the quality of these varieties once frozen is more stable compared to varieties such as ‘heritage’, ‘zeva’ and ‘rubi’ used by gonzález et al. (2002). this finding is likely a result of differing tss profiles between varieties having differing stability when frozen, resulting in a variation in tss level changes. titratable acidity contributes to the mouth feel a consumer experiences when stimulated by the multi­ ple acids including citric, ascorbic and phosphoric acid found in raspberries (haffner et al., 2002; marsh et al., 2004). freezing is the most common form of long term storage for fresh produce, with the reduc­ tion in metabolism and respiration when frozen allowing raspberries to be stored for long periods of time (chaves and zaritzky, 2018). cryogenic freezing facilitates the rapid transition into the frozen state which reduces membrane damage (and therefore maintains raspberry quality) from slow water crystal­ lization and subsequent membrane damage that occurs during conventional freezing (cao et al., 2018). consistent with findings for changes in tss, there was a significant increase in titratable acidity for frozen berries compared to fresh berries for both varieties, but there was no significant difference resulting from freezing method for either variety. gales et al. ‐ cryogenic freezing on raspberry quality 299 titratable acidity significantly increased when initially frozen but did not change with length of duration frozen. these results are consistent with findings of increased total and conjugated acid in frozen rasp­ berries by mullen et al. (2002). however, de ancos et al. (2000) who quantified ellagic acid concentrations, found a significant decrease (14­21%) in ellagic acid content when raspberries were frozen, highlighting that the increase in titratable acidity in this investiga­ tion may be due to increase in other acids found in raspberries. raspberry quality is derived from multiple para­ meters with each contributing to the consumer’s sen­ sory experience and as such, the ratio of tss with ta is a major contributor to consumer experience. despite possible variances of specific acids during freezing, it is the ratio of sugar to acid that influences the consumer’s tasting experience (shamaila et al., 1993). the increase in sugar when frozen is propor­ tional to the increase in acidity and showed no signif­ icant difference between freezing methods and dura­ tion, and it is this consistency in ratio that is critical to consumer experiences. the consistency in the ratio between tss and titratable acidity whilst frozen is also broadly consistent with research by gonzález et al. (2002) who used different raspberry varieties and durations frozen. no physiological mechanism or chemical changes induced by freezing/thawing has been described for the increase in titratable acidity post­harvest for raspberries. the measurement of increased titratable acidity in this study may be a physiological response to storage or an increase in extraction efficiency post storage, and until further investigation quantifies the reason for the change, the implications cannot be fully understood. in straw­ berries, retention of quality parameters including acids, colour and anthocyanin content was associat­ ed with the thawing process, where rapid thawing was shown to be more favourable (holzwarth et al., 2012). the impact of freezing method and duration on the colour of raspberries is important for influencing consumer demand. a colour that is perceived to be preferable by the consumer increases both the accep­ tance and perception of other quality parameters such as taste and texture (clydesdale, 1993). both varieties had similar hue and chroma values when fresh, however once frozen, colour values increased significantly. when frozen conventionally, both vari­ eties had similar coloured fruit; however, when frozen cryogenically, the proprietary variety had significantly higher hue and chroma unit values than ‘willamette’ raspberries (fig. 1). this was the only quality parame­ ter analysed that showed similarities between vari­ eties for fresh berries but when frozen showed a sig­ nificant difference between varieties. this highlights a d i ff e r e n c e i n t h e i m p a c t s o f f r e e z i n g m e t h o d s between the varieties and the subsequent impact on colour. the contrasting influence of freezing between v a r i e ti e s i s c o n s i s t e n t w i t h fi n d i n g s b y o t h e r researchers. gonzález et al. (2002), for example, showed that for the varieties ‘heritage’ and ‘autumn bliss’, chroma and hue was not different when fresh, but after six and nine months of being frozen, hue and chroma values differed between varieties. t h e s i g n i fi c a n t c h a n g e i n h u e a n d c h r o m a between fresh and frozen berries was not accompa­ nied by a change in anthocyanin levels. this is incon­ sistent with findings by han et al. (2004), who attrib­ uted the darker colour of frozen raspberries to anthocyanin synthesis. however, for strawberries, holcroft and kader (1999) showed that ph affects the colour expression of anthocyanins, concluding that the red flavylium cation only remains stable in acidic conditions, and strawberries lose their red colour (become pale) when the ph increases. therefore, if the anthocyanin stability and subsequent redness of raspberries is associated with the amount of acids present (acidity), the increase in titratable acidity observed in this study, may be associated with increasing the amount of stable red flavylium cations, causing the raspberries to be darker when frozen. this investigation used commercial standards of raspberry quality as a benchmark to compare innova­ tive freezing methods for post­harvest management of raspberry fruit. in this investigation, consistent quantitative results for the quality parameters, benchmarking the commercial standard, provides support for the application of new technology in pro­ ducing a consistent high­quality product. however, the commercial implications of a non­consistent (sig­ nificantly different) result are not known, as it may be of better or worse quality in the perception of the consumer. therefore, results that highlight significant (p<0.05) differences present opportunities for further studies and evaluations of quality as well as potential for changing practices. the results of this investigation broadly demon­ strated consistent quality parameters between con­ ventional freezing and the modern, innovative and comparatively expensive technique of cryogenic freezing. we suggest that based on these findings, adv. hort. sci., 2022 36(4): 293­301 300 there is evidence­based support for the commercial retention of the cheaper conventional freezing over cryogenic freezing for post­harvest storage of rasp­ berries. the study has provided critical decision sup­ port for post­harvest management of commercial raspberry production. this is particularly important for the uptake of horticultural technology that aims to access new markets and innovate logistics in sup­ ply chains. the findings of this study provide the framework and basis for the uptake and refinement of innovative technologies used in the raspberry industry, and allows freezing method choices to be evidence­based. the changes in titratable acidity dur­ ing the freezing duration trial is unexplained here nor a n y p u b l i s h e d l i t e r a t u r e t o d a t e . d e t e r m i n i n g whether a physiological increase of titratable acidity occurs or if an increase in extraction efficiency of titratable acidity is the reason behind the results of this study would provide additional insights into the impacts of freezing on raspberries. the quantitative results of the current study also provide impetus for a sensory analysis to investigate consumer percep­ tions in parallel with the quantitative laboratory evi­ dence provided in this study. acknowledgements this research did not receive any specific grant from funding agencies in the public, commercial, or not­for­profit sectors. the assistance of professor dugald close, dr max edgeley and ms caroline claye from the university of tasmania was greatly appreci­ ated. the authors greatly appreciated the support of the westerway raspberry farm (tasmania) for this research. references adobati a., limbo s., uboldi e., piergiovanni l., 2015 ­ active packaging in master bag solutions and shelf life extension of red raspberries (rubus ideus l.): a reliable strategy to reduce food loss. ­ italian j. food sci., 1: 107­110. alhamdan a., hassan b., alkahtani h., abdelkarim d., younis m., 2018 ­ cryogenic freezing of fresh date fruits for quality preservation during frozen storage. ­ j. saudi soc. agric. sci., 17: 9­16. bom, 2019 ­ daily rainfall ‐ westerway (leesons road) ­ b u r e a u o f m e t e r o l o g y , a u s t r a l i a n g o v e r n m e n t , http://www.bom.gov.au/jsp/ncc/cdio/weatherdata/av ?p_nccobscode=136&p_display_type=dailydatafile&p _startyear=&p_c=&p_stn_num=095075. boorse g.c., bosma t.l., meyer a.c., ewers f.w., davis s.d., 1998 ­ comparative methods of estimating freezing temperatures and freezing injury in leaves of chaparral shrubs. ­ int. j. plant sci., 159: 513­521. bora j., tongbram t., yaseen m., malik m., hanan e., 2021 ­ characterization of modern cold storage for hor‐ ticulture crops, pp. 1­26. ­ in: alam t. 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(ed.) frozen food science and technology. blackwell, bristol, uk, pp. 355. sunding d., zilberman d., 2001 ­ chapter 4. the agricul‐ tural innovation process: research and technology adoption in a changing agricultural sector, pp. 207­261. ­ in: barrett c.b., and d.r. just (eds.) hanbook of agricultural economics. elsevier, uc berkeley, usa, pp. 4562. tezotto­uliana j.v., fargoni g.p., geerdink g.m., kluge r.a., 2014 ­ chitosan applications pre‐ or postharvest prolong raspberry shelf‐life quality. ­ postharvest biol. techn., 91: 72­77. voss d.h., 1992 ­ relating colorimeter measurement of plant color to the royal horticultural society colour chart. ­ hortscience, 27: 1256­1260. wang s.y., chen c.­t., wang c.y., 2009 ­ the influence of light and maturity on fruit quality and flavonoid con‐ tent of red raspberries. ­ food chemistry, 112: 676­684. 43 adv. hort. sci., 2022 36(1): 43­52 doi: 10.36253/ahsc­10449 phytoprotective film for resistance induction, growth, and yield of organic strawberries i. dos santos pereira (*), f. grecco da silva porto, l.e. corrêa antunes, â. diniz campos brazilian agricultural research corporation, embrapa, brasília, brazil. key words: colletotrichum spp., enzymatic activity, fragaria x ananassa, induced systemic resistance, k2hpo4. abstract: the objective of this work was to evaluate a phytoprotective film of chitosan­pyroligneous extract in promoting growth, productivity, induction of systemic resistance in strawberry cultivars managed in an organic production system. treatments consisted of rates (0, 25, 50, and 100 ml l­1) of chi­pyro­ film and a reference resistance inducer (dipotassium hydrogen phosphate ­ k2hpo4), evaluated in three strawberry cultivars (‘albion’, ‘san andreas’ and ‘portola’). growth, yield, anthracnose incidence, and enzymatic activity were evaluated. the experimental design was a randomized block design with four replications. chi­pyro­film increases the growth, yield, and anthracnose resis­ tance of strawberry plants. the best concentration of chi­pyro­film varies between 50 and 60 ml l­1, according to strawberry cultivar. 1. introduction strawberry (fragaria x ananassa duch.) is the most planted red fruit in brazil. the high demand is due to its sensory characteristics such as color, texture, aroma, and taste (ventura­aguilar et al., 2018). however, according to a report by the brazilian health surveillance agency (anvisa) of 2016, 26% of strawberry samples collected between 2013 and 2015 presented nonconformities with pesticide residues. pesticides detected not authorized for the crop, captan stood out, detect­ ed in 20.4% of the samples analyzed, among others such as dithiocarba­ mates, pyrimethanil, carbendazim, tebuconazole, iprodione, and azoxys­ trobin (anvisa, 2016). most of these fungicides aim to control or prevent anthracnose (colletotrichum spp.), a disease that is considered main in strawberry fields in brazil (kososki et al., 2001; capobiango et al., 2016). currently, there is a search for technologies, which make agriculture sustainable and “smart”, with practices and ways to minimize the exces­ sive use of chemicals (grewal et al., 2018). in this context, the use of resistance inducing products activates the plant’s natural defenses, enabling disease control in the organic production system. (*) corresponding author: ivanspereira@gmail.com citation: dos santos pereira i., grecco da silva porto f., corrêa antunes l.e., diniz campos â., 2022 ­ phytoprotective film for resistance induction, growth, and yield of organic strawber‐ ries. ­ adv. hort. sci., 36(1): 43­52. copyright: © 2022 dos santos pereira i., grecco da silva porto f., corrêa antunes l.e., diniz campos â. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 10 february 2021 accepted for publication 25 february 2022 ahs advances in horticultural science adv. hort. sci., 2022 36(1): 43­52 44 the resistance is associated with various defense responses, such as protein and phytoalexin synthesis, cell wall changes, and increased activity of various enzymes defense­related (durrant and dong, 2004). responses that are associated with changes in the activity of various enzymes such as peroxidase (pox, ec 1.11.1), polyphenol oxidase (ppo, ec 1.14.18.1), and phenylalanine ammonia­lyase (pal, ec 4.1.3.5) (prasannath et al., 2014; prasannath, 2017). poxs have been implicated in many defense processes, such as hypersensitive response, lignifica­ tion, phenolic and glycoprotein cross­linking, suber­ ization, and phytoalexin production (thakker et al., 2 0 1 2 ; p r a s a n n a t h , 2 0 1 7 ) . p p o s a r e a g r o u p o f enzymes that catalyze the oxidation of hydroxyphe­ nols to their quinone derivatives, which have antimi­ c r o b i a l p r o p e r ti e s ( p r a s a n n a t h , 2 0 1 7 ) . p a l (e.c.4.1.3.5) is the major enzyme in the phenyl­ propanoid pathway and acts in the synthesis of defense­related secondary compounds such as phe­ nols and lignins (hemm et al., 2004; vanitha et al., 2009). among the compounds that have activating prop­ erties of defense mechanisms in plants are the chi­ tosan and pyroligneous extract (di piero and garda, 2008; grewal et al., 2018; souza et al., 2018). chitosan is a polycationic β­1,4 polymer bound to d­glucosamine chemically derived from crustaceans and soluble in organic acids and known to be a natur­ al elicitor and triggers various physiological and bio­ chemical responses in plants that act in the growth, production, and protection against disease (chandra et al., 2015; katiyar et al., 2015; pichyangkura and chadchawan, 2015). chitosan has several characteris­ tics that make this polymer advantageous for many applications: (1) has a defined chemical structure; (2) may be chemically and enzymatically modified; (3) is p h y s i c a l l y a n d b i o l o g i c a l l y f u n c ti o n a l ; ( 4 ) i s biodegradable and biocompatible with many organs, tissues and cells; (5) can be processed into various products including flakes, powders, membranes, fibers and films (badawy, 2012; van den broek et al., 2015; porto et al., 2019). the pyroligneous extract is a liquor with strong smoke flavors, is a crude and acid condensate pro­ duced from the distillation of the smoke generated in the carbonization of wood. it consists of a complex mixture of compounds derived from the chemical decomposition of wood components through the condensation of vapors and gases generated during pyrolysis in a low oxygen concentration (campos, 2018; pimenta et al., 2018). the pyroligneous extract is composed of water (80­90%) and more than 200 species of organic com­ pounds (10­20%) (theapparat et al., 2018). the pres­ ence of phenolic compounds in the pyroligneous extract confers growth­promoting and antifungal p r o p e r ti e s , a s f o u n d i n t h e l i t e r a t u r e o n helminthosporium sativum, cochliobolus sativus, waltz, colletotrichum orbiculare, alternaria mali (jung, 2007; baimark and niamsa, 2009; wei et al., 2010 a; grewal et al., 2018). in this context, the cationic character of chitosan in acidic conditions offers the possibility to establish electrostatic interactions with other negatively charged compounds, for example with the pyrolig­ neous extract, considered a raw material obtained from renewable sources, and is a good solvent for chitosan (campos et al., 2012; van den broek et al., 2015; porto et al., 2019). the phytoprotective film of chitosan­pyroligneous extract (chi­pyro­film) consists of the chitosan dilut­ ed in the pyroligneous extract, and its characteristics are the formation of a film with photoprotection capability against radiation (uv­b and uv­c), fungi toxic action, and inducing systemic resistance in plants (campos et al., 2012). the objective of the present work was to evaluate the effect of different concentrations of phytoprotec­ tive film formulated with chitosan and pyroligneous extract (chi­pyro­film) and dibasic potassium phos­ phate (k2hpo4) on growth promotion, resistance induction to anthracnose, yield, and defense enzyme activity in strawberry cultivars in the organic produc­ tion system. 2. materials and methods the study was carried under field conditions, at embrapa temperate climate experimental station, in pelotas city, rio grande do sul state, brazil (31°40’49 “s 52°26’18” o, at 60 m altitude) (fig. 1 a). the cli­ mate of the region, according to the köppen classifi­ cation is cfa type, temperate and humid, with hot summers. raw materials the pyroligneous extract was obtained through an extraction procedure of eucalyptus grandis proposed by campos (2018) and the distillation process was performed according to that described by porto et al. dos santos pereira et al. ‐ resistance induction in strawberries cultivars 45 (2019). chitosan was supplied by nutrifarm™, with a 97% degree of deacetylation, determined by proton magnetic resonance (porto, 2011). plant material the seedlings of neutral­day strawberry cultivars, ‘albion’, ‘san andreas’ and ‘portola’, were planted in may and were grown under a low tunnel system with mulching and drip irrigation. the spacing between lines and between plants was 0.30 m, with three lines per bed. the area had a history of many years with severe anthracnose incidence in the strawberry plants (fig. 1b, 1c). liming and fertilization were per­ formed according to the recommendation for straw­ berry organic production. climatic data from the experiment period are shown in figure 1d. treatments the treatments of induction of systemic resis­ tance consisted of four concentrations of the chi­ pyro­film and a reference treatment with dibasic potassium phosphate (k2hpo4). the chi­pyro­film was registered in the field of green chemistry at the national institute of intellectual property in brazil (pct/br2013/000597), united states (us201503 36854a1) and germany (de112013006230t5) as a phytoprotective for agriculture use. the chi­pyro­film with a concentration of 30 g l­1 (fig. 2) was diluted with distilled water in different concentrations (0, 25, 50, and 100 ml l­1). treatment with k2hpo4 was applied at a concentration of 50 mm (orober et al., 2002; aleandri et al., 2010). this compound has shown efficacy against e.g. powdery mildew on barley, cucumber, pepper, and tomato (blumeria graminis f. sp. hordei, sphaerotheca fulig‐ inea, leveillula taurica, and erysiphe oronti, respec­ tively), anthracnose (colletotrichum lagenarium) on cucumber, rust (puccinia sorgi) and leaf blight (exserohilum turcicum) on maize and rice blast (pyricularia oryzae), mildew (sphaerotheca fuliginea) (reuveni et al., 1996; reuveni and reuveni, 1998; manandhar et al., 1998; reuveni et al., 2000; ehret et al., 2002; orober et al., 2002; hamza et al., 2017). the spraying of treatments started at the begin­ ning of fruiting, and the application dates were indi­ cated in figure 1d. the spray volume was 4.5 ml per plant (500 liters per hectare), applied through conical jet nozzles, observing a total coverage of the plants until close to the runoff point. analysis of plant growth, damage by anthracnose, and fruit yield vegetative growth evaluations consisted of dry mass (g plant­1) of crown, root, and leaf, by drying to constant weight (65°c) of three plants collected in each experimental unit at the end of the production cycle, 256 days after seedlings transplanting. the pro­ ductive variables measured were fruit yield (g plant­1) and fruit weight (g fruit­1), obtained by evaluating the total fruits harvested in each experimental unit. the percentage of fruits with anthracnose was obtained by counting the fruits with symptoms at each harvest (fig. 1c). the harvests were carried out three times a week between october and january. enzymatic activity analysis biochemical evaluations were realized by determi­ nation of the specific activity of peroxidase (pox), fig. 1 ­ experiment under field conditions (a), anthracnose symptoms in strawberry plants (b) and fruits (c), and cli­ matic data from the experiment period (d). fig. 2 ­ electron micrograph of the phytoprotective film of chi­ pyro­film, after spraying on a smooth surface at a tem­ perature of 18 to 25°c (a and b). chitosan strawberry leaf covered with a film formed by chi­pyro­film (c). adv. hort. sci., 2022 36(1): 43­52 46 3. results chi­pyro­film concentrations had a significant effect on the growth, yield, anthracnose resistance induction, and enzymatic activity of strawberry plants. growth and development the dry mass of the plant showed factors interac­ tion. the three cultivars showed a quadratic response to chi­pyro­film concentrations (fig. 3), but the high­ est efficiency concentration was different. ‘albion’ and ‘portola’ had the highest efficiency concentration estimated at 60 ml l­1 of chi­pyro­film, while for ‘san andreas’ the concentration was 50 ml l­1 (fig. 3). the treatment with reference resistance inducer, k2hpo4, had also different according to cultivars. in ‘albion’, it provided a leaf mass slightly higher than the control treatment and similar to the 100 ml l­1 chi­pyro­film concentration, but it was lower than 25 and 50 ml l­1 (fig. 3). the yield also was significantly influenced by chi­ pyro­film, with a quadratic response to isolated effect of the film concentration factor (fig. 4). regardless of cultivar, the highest efficiency concen­ tration was estimated at 60 ml l­1. regarding the ref­ erence treatment (k2hpo4), it was observed that it had a performance similar to 100 chi­pyro­film and higher than control (0 ml l­1 of chi­pyro­film), but less than the 50 ml l­1 (fig. 4). between cultivars fig. 3 ­ dry mass of leaves of albion, portola and san andreas cultivars, in response to chi­pyro­film concentrations and reference treatment (k2hpo4, 50 mm). interaction effect between factors (cultivar and film concentration). *, **, ***, significant at p<0,05, p<0,01, p<0,001, respec­ tively. polyphenoloxidase (ppo), and phenylalanine ammo­ nia­lyase (pal) enzymes, in strawberry leaf samples collected immediately before application (ba) and 48 hours after application (aa) of chi­pyro­film concen­ trations and reference treatment (k2hpo4). for the extraction of pox and ppo, was used 500 mg of ground tissue below 4°c in 10 ml of 0.05 m phos­ p h a t e b u ff e r ( p h 7 . 0 ) c o n t a i n i n g 1 m g o f polyvinylpyrrolidone­10. subsequently, centrifuga­ tion was performed at 4,000 g for 30 minutes under refrigeration. the supernatant was preserved on ice and used for determinations according to campos et al. (2003). the pox and ppo extraction was carried o u t b y g r i n d i n g t h e l e a v e s w i t h 2 0 m g polyvinylpyrrolidone (sigma­aldrich). the enzyme extract obtained after filtration (whatman 1) and centrifugation (5,600 gn, 15min) were used to test the activity. pox activity was determined in the enzyme extract mix with a phosphate­citrate buffer com­ posed of 0.2 m sodium phosphate solution and 0.1 m citric acid (ph 5.0). the mixture was homogenized in vortexed for 15 seconds. pox activity was determined according to campos et al. (2004). ppo activity was determined in the enzyme extract with 3.6 ml of 0.05 m phosphate buffer (ph 6.0) and 0.1 ml of 0.1 m catechol. ppo activity was determined according to campos et al. (2004). pal activity was determined in crude leaf extracts according to the methods described by hyodo and yang (1971) and hyodo et al. (1978) modified by campos et al. (2003). for extraction, 500 mg of tissue was macerated (below 4°c) with 8 ml of 50 mm sodium borate buffer (ph 8.5) containing 25 g l­1 of polyvinylpyrroli­ done­10 and 4 ml l­1 of mercaptoethanol. the pro­ tein extract obtained after filtration (whatman 1) and centrifuged (5,600 gn, 30 min) under refrigera­ tion (below 4°c). was used to assay the pal activity. protein in the extracts was determined by the bradford method (bradford, 1976). experimental design and statistical analyses the experimental design was randomized blocks with four replications of nine plants. the results were submitted to variance analysis and the means of the variables with significant effect were compared using the tukey test (cultivars) or regression analysis (con­ centrations) at 5% error probability. dos santos pereira et al. ‐ resistance induction in strawberries cultivars 47 effect, ‘portola’ showed the highest yield than ‘albion’ and ‘san andreas’ (table 1). results similar to those observed by carini et al. (2015) in a study of evaluation of strawberry cultivars in an organic sys­ tem, in which ‘portola’ was also more productive than ‘albion’ and ‘san andreas’. the fruit weight was not influenced by chi­pyro­ film treatments. however, there was an effect of the cultivar factor, with ‘san andreas’ producing fruits of a higher mass (table 1). corroborating with carini et al. (2015), which evaluated the same three cultivars, found a higher fruit weight of ‘san andreas’, but with the same weight that ‘albion’. the variable fruits with anthracnose showed an isolated effect of the factor chi­pyro­film concentra­ tions, that is, all cultivars had the same response to the application of chi­pyro­film, with a quadratic reduction in the number of fruits attacked, with the maximum efficiency concentration estimated at 60 ml l­1 chi­pyro­film (fig. 5). the reference treatment with k2hpo4 was similar to the 100 ml l ­1 of chi­pyro­ film, but lower than the 25 ml l­1 and 50 ml l­1 (fig. 5). among the cultivars, ‘portola’ was the most sensi­ tive to the occurrence of anthracnose in fruits, with no difference between ‘albion’ and ‘san andréas’ (table 1). enzymatic activity in the present study, there was a significant effect of chi­pyro­film concentrations on the activity of pox, ppo, and pal enzymes (figs. 6 and 7). an inter­ action effect between film concentration and sam­ pling time indicated that 48 hours after application, there was a quadratic effect of film concentrations on the activity of the three enzymes studied. in the case of pox and ppo, an activity reduction effect was obtained up to the estimated concentrations of 54 ml l­1 and 50 ml l­1, respectively, followed by an increase (fig. 6a and 6b). however, pal activity increased until the estimated concentration of 36 ml l­1, with a subsequent reduction (fig. 6c). about the fig. 4 ­ yield of strawberry, in response to chi­pyro­film concen­ trations and reference treatment (k2hpo4, 50 mm). isolated effect of film concentration (average of cultivars responses). *, **, ***, significant at p<0,05, p<0,01, p<0,001, respectively. table 1 ­ yield, fruit weight and fruits with anthracnose in albion, portola and san andreas cultivars (z) (z) means followed by different lowercase letters in the row, differ by tukey test at 5% error probability. the averages correspond to the isolated effect of cultivar factor to studied variables. variables cultivar c.v. (%) albion portola san andreas yield (g plant­1) 131.90 ± 7.93 c 221.35 ± 12.87 a 170.4 5± 9.30 b 20.18 fruit weight (g fruit­1) 8.60 ± 0.67 b 8.50 ± 0.57 b 13.33 ± 1.09 a 72.71 fruits with anthracnose (%) 5.29 ± 1.71 b 9.13 ± 1.53 a 5.83 ± 1.49 b 107.57 fig. 5 ­ percentage of fruits with anthracnose in response to chi­ pyro­film concentrations and reference treatment (k2hpo4, 50 mm). isolated effect of film concentration (average of cultivars responses). *, **, ***, significant at p<0,05, p<0,01, p<0,001, respectively. 48 adv. hort. sci., 2022 36(1): 43­52 reference treatment, with k2pho4, it induced activi­ ties similar to chi­pyro­film in the concentration of 100 ml l­1 for pox, ppo, and pal. the enzymatic activity also had an interaction effect between treatments of resistance induction and strawberry cultivars. pox activity decreased to concentrations of 49 ml l­1 and 57 ml l­1 in albion and portola, respectively (fig. 7a). for san andreas, although there was a similar trend, it was not signifi­ cant (fig. 7a). fig. 6 ­ specified activity of peroxidase­pox (a), polyphenoloxi­ dase­ppo (b) and phenylalanine ammonia lyase­pal (c) in the leaves, before (ba) and after application (aa) of chi­pyro­film concentrations and reference treatment (k2hpo4, 50 mm). interaction effect between film con­ centration and sampling time (before and 48 hours after application). *, **, ***, significant at p<0,05, p<0,01, p<0,001, respectively. fig. 7 ­ specified activity of peroxidase­pox (a), polyphenoloxi­ dase­ppo (b) and phenylalanine ammonia lyase­pal (c) in leaves of albion, portola and san andreas, in response to chi­pyro­film concentrations and reference treatment (k2hpo4, 50 mm). interaction effect between film con­ centration and cultivars. *, **, ***, significant at p<0,05, p<0,01, e p<0,001, respectively. dos santos pereira et al. ‐ resistance induction in strawberries cultivars 49 the cultivars albion and san andreas also had a quadratic response to chi­pyro­film, with a decrease in ppo activity up to the estimated concentrations of 54 ml l­1 and 55 ml l­1 (fig. 7b). concerning portola, there was a linear increase up to a 100 ml l­1 concen­ tration of chi­pyro­film (fig. 7b). concerning pal, there was a quadratic response in albion, with an increase up to the 38 ml l­1 rate of chi­pyro­film, as well as a linear reduction in portola (fig. 7c). the reference treatment with k2hpo4, showed a response similar to the 100 ml l­1 film concentration for the enzymes ppo and pal in the three cultivars studied (figs. 7b and 7c). for the pox in albion and san andreas, the reference treatment was similar to the 25 ml l­1 concentration, while in portola, it was similar to the 50 ml l­1 film (fig. 7a). 4. discussion and conclusions in general, the results indicate that chi­pyro­film contributed to the increase of vegetative growth and yield, as well as, to the anthracnose damage reduc­ tion in strawberry fruits. results that are in agree­ ment with those found in the literature, where it is observed an increase in vegetative, productive, and health variables, such as height, number of leaves, leaf area, yield and reduction of incidence of diseases in plants that received chitosan or pyroligneous extract (el­miniawy et al., 2013; masum et al., 2013; mungkunkamchao et al., 2013; mukta et al., 2017; kumaraswamy et al., 2018; ventura­aguilar et al., 2018). the effect of chi­pyro­film on strawberry plant growth can be attributed to the role of chitosan as a non­toxic and biodegradable plant growth promoter (salachna and zawadzińska, 2014; ahmed et al., 2020). some authors suggest that foliar application of chitosan enhances the endogenous concentration of phytohormone such as gibberellic acid and auxin (uthairatanakij et al., 2007; ahmed et al., 2016). but the increase in macro and micronutrient accumula­ tion and improved the content of photosynthetic pig­ ments, provided by chitosan are also related to its influence on plant growth (shehata et al., 2012; ahmed et al., 2016). on the other hand, pyroligneous extract contributes to plant growth by its phytopro­ tective effect against pathogens, especially fungi. was reported antipathogenic effects of the pyrolig­ n e o u s e x t r a c t o n p l a n t p a t h o g e n i c f u n g i l i k e helminthosporium sativum, cochliobolus sativus, valsa mali, colletotrichum orbiculare, and alternaria mali (jung 2007; wei et al., 2010 a). this antifungal activity has been related to the presence of furalde­ hydes and phenols in pyroligneous extract (grewal et al., 2018). the efficiency of spraying with chi­pyro­film can be attributed to the effects of pyroligneous extract and chitosan individually, and to an interaction effect between both. according to porto et al. (2019), who studied physicochemical properties of chi­pyro­film, the film showed a semicrystalline structure, which is smooth and stable up to 50°c, being persistent in environmental conditions; it is permeable to water vapor and has high hygroscopicity, in addition to being able to efficiently block incident uvb and uvc radiation. the coverage presented by the chi­pyro­ film, as well as its persistence on the leaf surface (fig. 2), probably provide several days of action, per­ haps a large part of the application interval (15 days). the main effect of chitosan and the pyroligneous extract is attributed to resistance induction by increased defense enzyme activity and accumulation of phenolic compounds acting on reactive oxygen species (ros) (wei et al., 2010 b; katiyar et al., 2015; pichyangkura and chadchawan, 2015; grewal et al., 2018). this effect is in line with the behavior verified b y t h e p a l a c ti v i t y i n t h i s s t u d y ( f i g s . 6 c , 7 c ) . however, the activity of the pox and ppo enzymes responded differently, with a reduction in activity in the concentrations estimated between 50 and 60 ml l­1, 48 hours after application of treatments (figs. 6a 6b), especially in the albion and san andreas culti­ vars (figs. 7a, 7b). this film rate (50­60 ml l­1) had a better performance in the dry mass accumulation, yield, and incidence of anthracnose. t h e r e s u l t s i n d i c a t e t h a t i n a d d i ti o n t o t h e increase in systemic resistance, suggested by the pal response, there is a direct effect of phytoprotection and a reduction of stress condition. some aspects that can be associated with this second aspect, maybe the block that the film exerts concerning uva and uvb radiation, as well as its potential action on pathogens, a hypothesis that corroborates the results obtained by porto et al. (2019). in this way, it can be suggested that chi­pyro­film has a complex performance, acting both on metabo­ lism with increased plant resistance and reducing cel­ lular damage caused by physical (radiation) and bio­ logical stress (pathogens), as well as forming a phyto­ protective film that inhibits the direct action of stress adv. hort. sci., 2022 36(1): 43­52 50 agents, such as uva and uvb radiation and inhibiting the attack of pathogens. in general, chi­pyro­film showed greater efficien­ cy in promoting growth, yield, and resistance to anthracnose than the reference treatment (k2hpo4), mainly in concentrations between 25 and 50 ml l­1. k2hpo4 performed similarly to the 100 ml l ­1 of the film. different studies indicate the effect of k2hpo4 in the resistance induction of several species (reuveni and reuveni, 1998; kashiap and dhiman, 2009; aleandri et al., 2010; hamza et al., 2017; el­tanany et al., 2018). according to orober et al. (2002), foliar application of k2hpo4 results in the activation of sys­ temic resistance mechanisms. the positive effect of k2hpo4 is associated with salicylic acid involved in t r i g g e r i n g p l a n t c e l l d e f e n s e a n d s e n s i ti z a ti o n responses for a faster and stronger response to sub­ sequent pathogen attack (mauch­mani and métraux, 1998; orober et al., 2002). the 100 ml l­1 concentration of chi­pyro­film may b e h i g h f o r t h e s t r a w b e r r y c r o p . p r o b a b l y t h e increase in film thickness, which according to porto et al. (2019), can significantly reduce the film’s per­ meability to water vapor. an aspect that can make some physiological processes such as the flowing water, the absorption of nutrients, and photosynthe­ sis less efficient. this study provides indicates that the phytopro­ tective film (chi­pyro­film) is effective as a growth promoter and inducer of systemic resistance to anthracnose, resulting in increased growth and fruit production in strawberry plants of different cultivars. the optimal concentration of chi­pyro­film ranges between 50 and 60 ml l­1, depending on the straw­ berry cultivar. we believe that the tested film can be an impor­ tant tool for production systems, especially agroeco­ logical and organic systems, which require alterna­ tives for disease control. but also, due to its physical­ chemical properties and great stability, we believe that the film can be combined with other compo­ nents, such as nutrients, biostimulants, and plant hormones, to enhance its effect. however, such com­ binations need to be studied. acknowledgements this study was financed by the coordenação de aperfeiçoamento de pessoal de nível superior ­ brasil ( c a p e s ) , e m p r e s a b r a s i l e i r a d e p e s q u i s a a g r o p e c u á r i a ( e m b r a p a ) a n d b y t h e p r o j e c t “avaliação de biofilmes estabilizados a base de quito­ sana associada a diferentes moléculas, para uso em sistemas de produção orgânico”. references ahmed a.h.h., nesiem m.r.a.e., allam h.a., el­wakil a.f., 2016 ­ effect of preharvest chitosan foliar applica‐ tion on growth, yield and chemical composition of washington navel orange trees grown in two different regions. ­ afr. j. biomed. res., 10: 59­69. ahmed k.b.m., khan m.m.a., siddiqui h., jahan a., 2020 ­ chitosan and its oligosaccharides, a promising option for sustainable crop production ‐ a review. ­ carbohydr. polym., 227: 115331. aleandri m.p., reda r., tagliavento v., magro p., chilosi g., 2010 ­ effect of chemical resistance induc‐ ers on the control of monosporascus root rot and vine decline of melon. ­ phytopathol. mediterr., 49: 18­26. anvisa, 2016 ­ relatório das análises de amostras monito‐ radas no período de 2013 a 2015. ­ agência nacional de vigilância sanitária, portugal, pp. 246. 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natural resources, animals and environment, via dell’università, 16, 35020 legnaro (pd), italy. 3 marvil engeneering, zona produttiva schwemm, 8, 39040 magré sulla strada del vino (bz), italy. key words: dynamic controlled atmosphere, erf, low oxygen, malus domestica, postharvest. abstract: dynamic controlled atmosphere (dca) is beneficial in maintaining specific quality parameters but, due to the extreme oxygen levels applied, can cause adverse effects on the fruit by inducing excessive anaerobic metabolism and the production of off­flavors. the metabolic adaptation and responses of apples (malus domestica borkh.) cv. red delicious to static or dynamic oxygen concentrations (0.3 and 0.8%, with sequential shifts) during cold storage for 7 months were studied by monitoring quality parameters and the expression of genes involved in sugar, fermentative metabolism, and ethylene physiology. ethanol content reached the highest levels (around 400 mg/kg fw) under 0.3% oxygen concentration and fruit firmness appeared to be reduced in samples accumulating the highest levels of ethanol. the oxygen switch was effective in reducing the ethanol concentrations with timing­dependent variable effects. the expression of fermentative (alcohol dehydrogenase, lactate dehydroge‐ nase, pyruvate decarboxylase) and sugar metabolism (β‐amylase; phosphofruc‐ tokinase; sucrose synthase) genes resulted to be differently affected by the hypoxic conditions imposed, in particular during the early stages of storage. sucrose synthase expression appeared to be highly sensitive to changes in low oxygen concentration. ethylene biosynthesis (acc synthase and oxidase) genes showed marked differences in their expression in relation to the static and dynamic protocols and the hypoxic conditions, as well as six ethylene responsive factors (erf) genes, some of them possibly involved in the oxygen sensing mechanism operating in fruit tissues. (*) corresponding author: stefano.brizzolara@santannapisa.it citation: salamé e., brizzolara s., rodrigues m., iob m., tonutti p., ruperti b., 2023 ­ ethanol fermentation‐ and ethylene physiology‐related gene expression profiles in red delicious apples stored under variable hypoxic conditions and protocols. ­ adv. hort. sci., 37(1): 89­99. copyright: © 2023 salamé e., brizzolara s., rodrigues m., iob m., tonutti p., ruperti b. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 12 january 2023 accepted for publication 18 february 2023 ahs advances in horticultural science https://doi.org/10.36253/ahsc-14180 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2023 37(1): 89­99 90 1. introduction dynamic controlled atmosphere (dca) represents one of the latest technical innovations for the long storage of apples (and few other fruit crops) (tonutti, 2015). with this technology, fruits are kept under extremely low oxygen concentrations (0.4 kpa or lower) that are beneficial for maintaining specific quality parameters (e.g., flesh firmness, acidity). however, by activating anaerobic metabolism, an accumulation of ethanol takes place. low concentra­ tions of ethanol are desirable in terms of improving organoleptic traits, reducing the incidence of chilling injuries (e.g., superficial scald) and limiting ethylene biosynthesis (dixon and hewett, 2000; scott et al., 2000; weber et al., 2020). yet, the accumulation of excessive ethanol results in the appearance of off­fla­ vors and physiological disorders (pedreschi et al., 2009). thus, based on different stress indicators (chlorophyll fluorescence ­cf­, respiratory quotient ­ rq­, and ethanol concentration), oxygen must be promptly adjusted (increased) to reach “safe” con­ centrations. the imposed extreme hypoxic conditions induce selective responses of apple tissues starting from the modulation of gene expression involved in particular in primary metabolism and hormone (mainly ethylene) physiology (cukrov et al., 2019). in granny smith, one of the apple cultivar most fre­ quently stored in dca, differential expression of sucrose synthase (susy), alcohol dehydrogenase (adh) and pyruvate­related metabolism (lactate dehydrogenase, ldh, pyruvate decarboxylase, pdc, and alanine aminotransferase, alaat) genes was detected when comparing 0.4 with 0.8 kpa oxygen concentration (cukrov et al., 2016). when, according to the dca protocol, oxygen level is increased from the lowest applied concentrations, molecular and m e t a b o l i c r e a r r a n g e m e n t s r a p i d l y o c c u r , w i t h changes in both primary and secondary metabolism (brizzolara et al., 2019), indicating that highly reac­ tive mechanisms and oxygen sensors are present in apple cortex. the expression of genes involved in fer­ mentative metabolisms (e.g., adh), in secondary metabolism (e.g., phenylpropanoid pathway), hor­ monal responses and regulatory mechanisms (ethyl­ ene biosynthesis, erfs) resulted to be affected by the oxygen switch. the duration of the storage and the oxygen con­ centrations applied obviously play a key role in deter­ mining the fruit metabolic responses and the dynam­ ics of fermentative metabolite accumulation. in addi­ tion, apple varieties react differently to extremely low oxygen conditions during storage, in particular in terms of fermentation, ethanol production and accu­ mulation (thewes et al., 2019; brizzolara et al., 2020; thewes et al., 2021 a; park et al., 2022). zanella and stürz (2015) showed that, differently from eight other varieties, ‘red delicious’ apples react signifi­ cantly and accumulate higher ethanol levels under hypoxia, and in a specific comparison between granny smith and red delicious (brizzolara et al., 2017), it was reported that the latter considerably accumulated ethanol under both ulo (0.9 kpa oxy­ gen) and dca (0.2­0.55 kpa oxygen) conditions, as also observed by lumpkin et al. (2014). among important commercial apple varieties, the responses of red delicious to controlled atmosphere (ca) and dca still need to be compared and clarified, which makes this cultivar a genotype of interest in terms of both applied aspects and physiological stud­ ies related to dca conditions and different oxygen regimes and concentration adjustment protocols. 2. materials and methods experimental design and sampling organic apple (malus domestica borkh., cv. red delicious) fruit were harvested in correspondence of an average tss value of 10.8°brix. fruit were selected f o r t h e i r u n i f o r m i t y a n d a b s e n c e o f p h y s i c a l defects/decay and then kept for 3 days of acclima­ tion at low temperature (0°c). control atmosphere storage was applied by divid­ ing the fruit into two groups in two different cold chambers. the first group (about 300 fruit) was ini­ tially stored under oxygen concentration of 0.3% (0.3ox) while the second group (60 fruit) was stored under the safer oxygen concentration of 0.8% (0.8ox) for a total period of 218 dia (days in atmosphere). samples were collected at harvest and t0 sampling was carried out after 24h in atmosphere (1 dia). to simulate the dynamic changes in oxygen concentra­ tions applied during a dca storage, at t1 (10 dia) 60 fruit originally stored under 0.3ox were shifted to 0.8% oxygen level and kept under these conditions for the whole period of storage (218 dia). these sam­ ples were called shift 1 and were sampled successive­ ly at the following time points. at t2 (20 dia), anoth­ er 60 fruit were moved from 0.3% to 0.8% oxygen and were called shift 2. shift 3 was performed at t3 (31 dia) and shift 4 took place at t4 (110 dia). a salamé et al. ‐ dca storage of red delicious apples 91 schematic diagram of the experimental design is reported in figure 1. at each sampling point, three biological replicates taken from three different fruit for each treatment were considered. samples of cor­ tex tissue were collected, immediately frozen in liq­ uid nitrogen, and stored at ­80°c. flesh firmness, total soluble sugars content and ethanol quantification flesh firmness was measured at harvest (t0) and a t t h e e n d o f t h e s t o r a g e a t t 5 ( 2 1 8 d i a ) . measurements were taken at the two opposite sides of the equatorial part using a fruit penetrometer (mod. ft 327, 3­27 lbs) with a large plunger tip (11 mm­diameter) after removing 1 mm of the peel. total soluble solid content (tss, °brix) was deter­ mined in the flesh juice of apples using a portable refractometer (sinergica soluzioni, pescara, italy); measurements were performed at harvest and the end of the storage at t5 (218 dia) on pulp juice sam­ ples taken from the opposite sides of the fruit. ethanol content was measured by using a tectronik (tectronik, padova, italy) senzytec analyser, following the instructions of the manufacturer and using 100µl of juice obtained by collectively pressing portions (approximately 1/3) of the cortex of three apples rep­ resenting the biological replicate. rna extraction and cdna synthesis total rna was isolated from cortex tissue using ‘sigma ­aldrich’ rna extraction kit following the manufacturer instructions. total rna was quantified (ng/μl) using uv spectrophotometry calibrated with rnase­free water. rna purity was assessed by evalu­ ating the absorbance ratio at 260/280 and 260/230 nm. ribosomal rna bands integrity was verified using gelred™­stained 1% agarose gel (aranda et al., 2012). rna was reverse­transcribed into first­strand c d n a u s i n g 4 µ l r e a d y s c r i p t ™ ( s i g m a , r d r t ­ 500rxn), starting from 400 ng of total rna in a final volume of 20 µl using depc treated water. the reac­ tion was incubated at 25°c for 5 minutes, then at 46°c for 20 min and then heated at 95°c for 1 min and finally at 4°c for 15 min. the synthetized cdna was diluted 1:5 by adding sterile water. gene expression analysis by real‐time pcr quantitative real­time pcr was performed using three biological replicates and two technical repli­ cates for each sample. based on the paper by cukrov e t a l . ( 2 0 1 6 ) , p r i m e r p a i r s o f g e n e s r e l a t e d t o sucrose/starch metabolism (β‐amylase, mdbam; phosphofructokinase, mdpfk; sucrose synthase, mdsusy), the fermentative/pyruvic acid metabolism (alcohol dehydrogenase, mdadh; lactate dehydroge‐ nase, mdldh; pyruvate decarboxylase, mdpdc; ala‐ nine aminotransferase, mdalaat), ethylene biosyn­ thesis (acc synthase, mdacs; acc oxidase, mdaco), and 6 ethylene responsive factors (erfs) were used (table 1). actin was used as a housekeeping gene. reaction mixtures were prepared, under sterile conditions, for the target and reference genes, con­ taining each 5 μl of 2xsybr® green qpcr readymix™ (sigma), 1 μl of each primer (forward and reverse) (10 µm), 2 µl rnase­free water and 1 μl of cdna. the automated thermal cycler was programmed according to the following conditions: initial denatu­ ration of 95°c for 30 sec followed by 40 cycles of: denaturation at 95°c for 10 sec, primer annealing (according to primer tm) for 30 sec and extension at 72°c for 30 sec. finally, melt­curve stage at 65°c for 0.5 sec followed by 95°c for 0.5 sec. the ct­values generated were used to evaluate the results of the gene expression levels comparing the expression of each target gene to the housekeep­ ing gene (actin). for samples under static 0.8ox, data were expressed with the 2­δδct method (livak and schmittgen, 2001) and normalized to the correspond­ ing sample at t1 (1 dia). data of samples under 0.3ox and the shifts were expressed as fold change of the expression level using the formula: fc=log2(2 ‐δδct) normalized to the corresponding sample of 0.8ox at each time point starting from 10 dia. statistical analysis for gene expression, data analysis was performed on six replicates (3 biological x 2 technical replicates). rstudio was used with external package “pcr” to cal­ culate the relative expression and fold change. for fig. 1 ­ red delicious dca storage experimental design. the numbers indicate the days in atmosphere (dia), ▽ indicates the sampling with color coded for different samples, 0.3ox (blue), 0.8ox (purple), sh1 (red), sh2 (orange), sh3 (yellow) and sh4 (light blue). adv. hort. sci., 2023 37(1): 89­99 92 fruit firmness, tss and ethanol content 3 biological replicates were used. internal statistical functions and external package “agricolae” were used to ana­ lyze the data (kronthaler and zoellner, 2021). all data were analyzed using t‐test for samples at 1 dia to compare 0.3 to 0.8ox samples. one­way anova and mean comparison with least significant differ­ ence (lsd) post­hoc test (p≤0.05) was used to com­ pare different samples of shifts to the corresponding 0.8ox at each time point starting from 10 dia. a kruskal­wallis test was applied to non­parametric data (p≤0.05). 3. results flesh firmness, tss and ethanol production at the end of the storage period and after five days of shelf life at room temperature no physiologi­ cal disorders or external/internal defects were observed in all analysed samples collected from the different protocols. concerning technological parameters, table 2 reports apple firmness and tss values for samples taken at harvest (t0) and at the end of the trial at 218 dia. results showed that samples stored for 30 table 1 ­ primers pairs used in the rt­qpcr analysis the table reports the genes nomenclature according to https://iris.angers.inra.fr/gddh13/, the primers sequence, length, gc content and melting temperature (tm) genes primer sequence 5'­3' length (bp) gc content (%) tm (°c) acc synthase md15g1302200 f aagtggcgaactggagtcga 20 55 67.2 r ggtttgatgggttcgtgacc 20 55 66.4 acc oxidase md10g1328100 f cagtcggatgggaccagaa 19 57.8 66.3 r gcttggaatttcaggccaga 20 50 66.3 pyruvate decarboxylase md04g1160100 f caaggcagtaaagccggtta 20 50 63.9 r aaatcggtccagcaaacaag 20 45 63.9 alcohol dehydrogenase md10g1014200 f ggaagcactgaagccatgat 20 50 64.2 r ctccacgacagagggaatgt 20 55 64.2 lactate dehydrogenase mdp0000143956 f cataaaactccttcaggctcca 22 45.4 64.1 r gtgsggtcttgggtgaggat 22 55 63.9 beta‐amylase 6 md09g1103800 f ctatgtgccgatcttcgtga 20 50 63.8 r actgcttgaaacacgctcct 20 50 63.9 sucrose synthase md15g1223500 f tccgtgttcactgctacgag 20 55 64.1 r gcctcaagaaggtccaacag 20 55 63.8 phosphofructokinase md04g1042400 f agtcgtggagtggtggaatc 20 55 64.1 r tagagggtgagggcttcaga 20 55 63.9 alanine aminotransferease md09g1173000 f tgctgtccgaggtgaaatcgtc 22 54.5 70.6 r agcccggattcgcctttaactc 22 54.5 69.9 ethylene response factor md11g1306500 variant f cggtggtgctataatctccg 20 55 64.2 r ggaattgagtcggtgtgagtagtt 24 45.8 64.3 ethylene response factor md11g1306500 f ctcccttcgccaagttcg 18 61.1 65.9 r ttgagtcggtgcgattaacc 20 50 64.9 ethylene response factor md16g1162900 f ccagaagcccaaaccatcag 20 55 66.7 r ttcctcggcggtgttgta 18 55.5 65.4 ethylene response factor md13g1163300 f ggtggggaaatgtatgctaaga 22 45.4 63.7 r gtcatccagcatccacagg 19 57.8 64.4 ethylene response factor md17g1152400 f cttctgcaaagcgttctgtg 20 50 63.7 r ggcaggatcggatggag 17 64.7 64.5 ethylene response factor md09g1174400 f ttctgcaaagcgttccatc 19 47.3 64 r ttcattggcagggaaggtg 19 52.6 66 actin md04g1127400 f tgaccgaatgagcaaggaaatta 25 40 67.4 r tactcagctttggcaatccacatc 24 45.8 68 salamé et al. ‐ dca storage of red delicious apples 93 (sh3) and 110 (sh4) days under 0.3% oxygen before being shifted to 0.8% oxygen showed the lowest val­ ues of firmness at the end of the trial. while 0.8ox, sh1 and sh2 samples maintained firmness values not significantly different from those detected in t0 sam­ ples. no significant difference (p=0.414, alpha level ≤ 0.05) was recorded for tss levels over time or between the different applied protocols. ethanol levels have been monitored along the entire experimental period to assess the activation of fermentative metabolism under static and dynamic ca storage (fig. 2). 0.3ox samples already showed higher levels than those of the 0.8ox sample at 10 dia, and a further increase from 10 to 20 dia with values around 400 mg/kg fw up to 110 dia (last sampling time for this specific treatment). on the other hand, 0.8ox samples showed a similar trend in terms of ethanol accumulation but with significantly lower amounts compared to 0.3ox samples and a decreasing trend after the highest concentrations detected at 20 and 31 dia. samples subjected to par­ tial re­oxygenation at different time points (sh1, sh2, sh3 and sh4) showed a different behaviour: sh1 did not show significant difference from 0.3ox at 20 dia , but displayed a reduced amount at 31 and at 110 dia as also observed for sh2. interestingly, at 110 dia ethanol levels were simi­ lar in all shifted and in 0.8ox samples. this was also observed at 218 dia, except for sh4 apples that dis­ played still significant higher levels (fig. 2). effect of different protocols on sugar metabolism‐ and fermentation‐related gene expression for gene expression data analysis, 0.8ox samples, kept under a static concentration throughout the experiment (from 1 to 218 dia), were considered as a reference for the other storage protocols (0.3ox, sh1, sh2, sh3 and sh4). consequently, the gene expres­ sion levels of these latter samples were expressed as fold change in relation to 0.8ox. considering sugar metabolism, the expression lev­ els of three genes were monitored throughout stor­ age (fig. 3). in 0.8ox samples, mdbam gene revealed a significant up regulation, compared to t0, at 31 and 110 dia. a significantly lower expression level was recorded in 0.3ox samples at 10 and 110 dia. sh1, sh2, sh3 and sh4 samples had significantly lower lev­ els of expression at 110 dia. at the last sampling time, 218 dia, all shifted samples, except sh3, revealed significantly higher expression values com­ pared to 0.8ox. mdpfk gene expression showed a steady state in samples stored at 0.8ox. a lower expression level of this gene was detected at 10 dia in 0.3ox samples. sh1 samples showed higher expres­ sion at 31 dia, sh2 had lower expression level at 31 dia and higher at 110 and 218 dia, while sh3 sam­ ples showed higher expression levels at 218 dia. considering mdsusy, in 0.8ox apples the expression showed a significant induction at 10 and 20 dia, fol­ lowed by a basal expression level at all the other s a m p l i n g p o i n t s . s a m p l e s s t o r e d u n d e r 0 . 3 o x revealed two marked and significant peaks of induc­ tion, at 10 and 110 dia. interestingly, sh1 showed significantly reduced levels of expression at 20 dia, while sh2 and sh4 revealed significantly higher expression at 218 dia. concerning the gene related to the fermenta­ tive/pyruvic acid metabolism (fig. 4), mdldh expres­ sion showed, in 0.8ox samples, a significant increase fig. 2 ­ ethanol content (mg/kg fw). samples stored in 0.3ox (blue), 0.8ox (purple), sh1 (red), sh2 (orange), sh3 (yellow) and sh4 (light blue) analysed from 10 to 218 days in atmosphere (dia). different letters indicate sig­ nificant differences among samples (anova, lsd post­ hoc test (p ≤ 0.05). different letters indicate significant differences among samples (anova, lsd post­hoc test (p≤0.05). table 2 ­ firmness and total soluble solids (tss). mean values (±se) of apple samples at harvest (t0) and after 218 dia of static (0.8ox) and dynamic atmosphere storage (shift 1­4) are reported in the table samples dia firmness (n) tss (°brix) at harvest 0 75.10 ± 0.96 a 10.85 ± 0.45 static 0.8ox 218 67.67 ± 2.85 ab 11.40 ± 0.06 shift 1 218 62.52 ± 3.51 ab 11.76 ± 0.32 shift 2 218 67.91 ± 0.73 ab 11.53 ± 0.24 shift 3 218 58.10 ± 6.76 b 10.86 ± 0.13 shift 4 218 61.30 ± 5.83 b 11.40 ± 0.06 94 adv. hort. sci., 2023 37(1): 89­99 of expression only at 10 dia. compared to 0.8ox sam­ ples, 0.3ox treatment induced higher expression at 10 and 110 dia, and a general up­regulation in shift­ ed samples at 218 dia. mdpdc, involved in the pro­ duction of acetaldehyde, increased its expression at 2 0 a n d 3 1 d i a i n 0 . 8 o x s a m p l e s . 0 . 3 o x a p p l e s revealed an earlier increase in the expression of mdpdc gene, significant at 10 dia. among samples that underwent partial re­oxygenation, a general lower expression, always compared to 0.8ox, was observed at 110 dia, followed by increased levels at 218 dia. acetaldehyde can be further converted to fig. 4 ­ relative expression of genes related to fermentative/pyruvic acid metabolism, lactate dehydrogenase, pyruvate decarboxylase, alcohol dehydrogenase and alanine aminotransferase. for samples 0.3ox (blue), sh1 (red), sh2 (orange), sh3 (yellow) and sh4 (light blue) the expression level is reported from 1 to 218 days in atmosphere (dia) as log2 fc normalized on 0.8ox expression level at each time point. the red line represents gene relative expression in 0.8ox samples. black asterisks indicate significant dif­ ferences (anova, lsd post­hoc test (p≤0.05) comparing each sample to 0.8ox level at the same sampling time. red asterisks indicate significant differences (anova, lsd post­hoc test (p ≤ 0.05) between 0.8ox samples at the specific time points and 0.8ox apples at 1 dia. fig. 3 ­ relative expression of genes related to sugar metabolism and energy production β­amylase, phosphofructokinase, and sucrose synthase. for samples 0.3ox (blue), sh1 (red), sh2 (orange), sh3 (yellow) and sh4 (light blue) the expression level is reported from 1 to 218 days in atmosphere (dia) as log2 fc normalized on 0.8ox expression level at each time point. the red line repre­ sents gene relative expression in 0.8ox samples. black asterisks indicate significant differences (anova, lsd post­hoc test (p≤0.05) comparing each sample to 0.8ox level at the same sampling time. red asterisks indicate significant differences (anova, lsd post­hoc test (p≤0.05) between 0.8ox samples at the specific time points and 0.8ox apples at 1 dia. salamé et al. ‐ dca storage of red delicious apples 95 ethanol under low oxygen levels by the enzyme coded by mdadh genes, and this is a crucial reaction in apple tissue under hypoxia. compared to t0 sam­ ples, the selected mdadh gene showed a significant increase only at 218 dia in 0.8ox sample. the applica­ tion of 0.3% oxygen resulted in general higher levels of expression until 31 dia, significant at 10 and 31 dia. at 110 dia, a significant decrease in mdadh expression was recorded for 0.3ox apples as well as for all shifted samples. mdalaat is involved in the reversible transfer of an amino group from glutamate to pyruvate, which in turn forms 2­oxoglutarate and alanine. in 0.8ox sam­ ples, this gene showed a peak of expression at 110 dia. the expression in 0.3ox samples is highly induced at 10 dia, while it strongly decreased at 110 dia, similarly to sh2 and sh3. higher expression of this gene was detected at 218 in sh4 sample (fig. 4). ethylene biosynthesis and erfs gene expression the expression of two genes involved in ethylene biosynthesis, namely mdacs and mdaco, has been analysed (fig. 5). these two genes appeared to be highly affected during storage under 0.8% oxygen c o n c e n t r a ti o n . t h e e x p r e s s i o n o f b o t h g e n e s increased with time, constantly for mdaco, which also reached the highest recorded levels of expres­ sion, while, in the case of mdacs, a peak at 110 dia was detected. regarding mdacs gene expression, 0.3ox samples showed increased values (compared to 0.8ox) at 10 dia, but lower levels at 20, 31 and 110 dia, when also sh2 and 3 showed low expression lev­ els. mdaco gene revealed marked higher levels in 0.3 ox samples at 10 dia and in sh1 samples at 20, 31, and 110 dia. lower expression levels were detected in 0.3ox apples at 20, 31 and 110 dia. interestingly, sh1 samples showed higher levels of expression, compared to 0.8ox, at 20, 31, and 110 dia. all shifted samples had lower expression level than 0.8ox apples at 218 dia. t h e e t h y l e n e s i g n a l l i n g a n d r e s p o n s e pathway includes ethylene response factors (erfs), which belong to the transcription factor family apetala2/erf that plays important roles in stress­ related responses. the effect of the different applied storage protocols on the expression level of six erf genes has been investigated throughout the experi­ ment (fig. 6). in general, samples showed relatively similar responses in terms of erf expression. overall, considering 0.8ox samples a general increase of erf genes expression was observed up to 110 dia, which was significant at different time points for the differ­ ent analysed erfs (md09g1174400 gene at 110 dia; m d 1 3 g 1 1 6 3 3 0 0 g e n e a t 1 0 , 2 0 a n d 3 1 d i a ; m d 1 1 g 1 3 0 6 5 0 0 g e n e a t 1 0 , 2 0 a n d 3 1 d i a ; m d 1 6 g 1 1 6 2 9 0 0 g e n e a t 1 0 , 3 1 a n d 1 1 0 d i a ; md11g1306500 variant gene at 10, 20 and 31 dia; fig. 5 ­ relative expression of ethylene biosynthesis genes acc­synthase and acc­oxidase. for samples 0.3ox (blue), sh1 (red), sh2 (orange), sh3 (yellow) and sh4 (light blue) the expression level is reported from 1 to 218 days in atmosphere (dia) as log2 fc normalized on 0.8ox expression level at each time point. the red line represents gene relative expression in 0.8ox samples. black asterisks indicate significant differences (anova, lsd post­hoc test (p≤0.05) comparing each sample to 0.8ox level at the same sampling time. red asterisks indicate significant differences (anova, lsd post­hoc test (p≤ 0.05) between 0.8ox samples at the specific time points and 0.8ox apples at 1 dia. adv. hort. sci., 2023 37(1): 89­99 96 md17g1152400 gene at 10 and 110 dia). the expression of erf genes in 0.3ox samples was instead characterized by significant lower levels at the different time points with only two exceptions: md09g1174400 gene at 10 dia and md11g1306500 gene at 20 dia, when significantly higher levels of expression than those of 0.8ox apples were recorded. on the other hand, considering apples subjected to partial re­oxygenation samplings, lower levels of expression were generally detected up to 31 dia, with only two exceptions (md11g1306500 and md16g1162900) concerning sh2 samples at 31 dia, when a significantly higher level of expression was observed. after 110 dia, the different samples revealed vari­ able patterns. concerning md09g1174400 and md13g1163300 genes, sh1 and sh3 showed signifi­ cantly lower levels of expression, while sh2 had sig­ nificantly higher levels. for these two genes at 218 dia only sh2 revealed significantly higher levels of expression compared to 0.8ox samples, despite a general increasing trend in all apples subjected to partial re­oxygenation. as far as the md11g1306500 gene is concerned, significant higher expression was detected for both sh1 and sh2 at 110 dia, while only sh3 had signifi­ cantly higher levels at 218 dia. md16g1162900 gene expression in shifted samples revealed significant lower levels at 110 dia, while only sh4 had signifi­ c a n t l y h i g h e r l e v e l s a t 2 1 8 d i a . i n t h e c a s e o f md11g1306500 variant gene sh1 and sh2 had higher expression levels at 110 dia, while only sh2 and sh4 showed significantly higher levels at 218 dia. lastly, md17g1152400 gene had a significantly high­ er level of expression in samples of sh1 and sh2 at 110 dia, while sh3 apples had significantly lower fig. 6 ­ relative expression of genes belonging to ethylene response factor (erf) gene family. for samples 0.3ox (blue), sh1 (red), sh2 (orange), sh3 (yellow) and sh4 (light blue) the expression level is reported from 1 to 218 days in atmosphere (dia) as log2 fc normalized on 0.8ox expression level at each time point. the red line represents gene relative expression in 0.8ox samples. black asterisks indicate significant differences (anova, lsd post­hoc test (p≤0.05) comparing each sample to 0.8%ox level at the same sampling time. red asterisks indicate significant differences (anova, lsd post­hoc test (p≤0.05) between 0.8ox samples at the specific time points and 0.8ox apples at 1 dia. salamé et al. ‐ dca storage of red delicious apples 97 expression for this gene. on the other hand, all shift­ ed samples showed significantly higher expression of this gene at 218 dia compared to 0.8ox apples. a general trend was identified for all samples subjected to partial re­oxygenation: in general the expression level of the erf gene was induced at 218 dia. 4. discussion and conclusions a n i n ­ d e p t h u n d e r s t a n d i n g o f l o w o x y g e n responses in fruits is of fundamental importance for the optimisation of storage approaches and for the development of protocols aimed at maintaining opti­ mal quality while preventing the occurrence of physi­ ological disorders associated with long term storage. metabolic adaptation responses to hypoxic condi­ tions have only recently started to be clarified in apple fruits and are gaining increasing interest since apples are routinely stored for very long periods of time thanks to the adoption of low oxygen (0.8 kpa oxygen, ultra low oxygen, ulo) or dynamic con­ trolled atmosphere (dca, 0.4 or lower kpa oxygen) protocols. primary metabolism and ethylene physiol­ ogy are markedly affected by hypoxia with differ­ ences depending on the oxygen concentration and modulation, and the genotype. in this study we char­ acterized the ethanol accumulation and the expres­ sion pattern of sugar/fermentative metabolism­ and ethylene physiology­related genes of red delicious apples in ca/dca storage. our goal was that of better understanding the behaviour and the responses (also in terms of specific quality parameters) of this apple variety in relation to two levels of low oxygen con­ centration and the variable (in terms of timing) switch from 0.3 to 0.8% oxygen levels. the storage under 0.3% oxygen resulted in an early significant accumulation of ethanol already at 10 dia and that further increased at 20 dia. this level remained rather stable as long as the fruit were kept at 0.3% oxygen atmosphere until 110 dia. although to a lesser extent, ethanol content also increased in 0 . 8 o x s a m p l e s , c o n fi r m i n g w h a t o b s e r v e d b y brizzolara et al. (2017). in these apples, ethanol con­ tent levelled off after three months of storage. the metabolization of ethanol seemed to be more sensi­ tive to re­oxygenation when apples had experienced a shorter period of dca. in fact, the longest storage under 0.3% oxygen resulted in more stable ethanol levels in the cortex at the end of the storage period (218 dia, sh4 in fig. 2). considering one of the main parameters dictating the commercial life of apples, the samples sh3 and sh4 showed the lowest values o f fl e s h fi r m n e s s a t t h e e n d o f t h e t r i a l . t h i s behaviour could be associated to the highest levels of ethanol accumulated in these samples. in braeburn apples ethanol production exceeding 472 μl·l­1 and the overproduction of anaerobic metabolites in royal gala resulted in a decrease of flesh firmness (weber et al., 2020; thewes et al., 2021 b). in persimmon, it has been observed that accelerated loss of flesh firm­ ness during storage was induced by ethanol treat­ ments applied to reduce astringency (vilhena et al., 2022). these authors observed that this event is closely related to greater parenchyma degradation during storage caused by ethanol treatment. if this cellular event also occurs in apple fruit accumulating high ethanol levels following hypoxic storage condi­ tions remains to be elucidated. it is interesting to note that even in the samples with the highest ethanol content (0.3ox) no internal physiological disorders (e.g., flesh breakdown) were detected. the gene expression data confirmed that the molecular regulation of hypoxic responses is overall conserved among apple varieties: the up­regulation of mdpdc, mdadh and mdalaat in response to extreme levels of hypoxia (0.3 oxygen concentration) is readily activated and peaks at 10 dia, after which is promptly and progressively levelled down until 110 dia, when a general low level of expression (com­ pared to 0.8ox samples) is present in 0.3ox and shift­ ed samples. this possibly suggests a negative feed­ back exerted by ethanol on its own synthesis. in agreement with these findings, one of the genes e n c o d i n g g r o u p v i i e t h y l e n e r e s p o n s e f a c t o r s (md09g1174400), with similarity to rap2 proteins involved in low oxygen signalling in model systems (licausi et al., 2011; gibbs et al., 2011), displayed an expression pattern overlapping with that of the fer­ mentative metabolic genes with a transient up­regu­ lation at 10 dia and low expression levels at 110 dia. it is well known that under energy shortage condi­ tions, such as those induced by low oxygen condi­ tions, plant tissues and organs (including fruit) instead of using invertases and hexokinases to pro­ duce hexose­phosphates to form sucrose, an atp consuming process, can use sucrose synthase as alternative energy saving pathway (mustroph et al., 2014). the activation under hypoxic conditions of sucrose synthase was already reported by cukrov et al. (2016) in granny smith apples, and our expression adv. hort. sci., 2023 37(1): 89­99 98 data (showing a high induction at 10 dia in 0.3ox and a prompt reduction of expression level in sh1 apples at 31 dia) confirm that this gene can be considered highly sensitive to oxygen levels also in cv. red delicious. a cultivar­specific behaviour is, instead, observed regarding beta­amylase and phosphofruc­ tokinase. in fact, these genes in granny smith apples follow a similar expression pattern compared to mdsusy (cukrov et al., 2016), not observed in the present trials on red delicious. as far as ethylene biosynthesis is concerned, the expression pattern of acc oxidase detected in 0.8ox red delicious samples mirrors that observed in granny smith (cukrov et al., 2016), while the tran­ sient higher expression levels observed for both acc synthase and oxidase at 10 dia under 0.3% oxygen appear to be a specific response of cv. red delicious apple. interestingly, the transcription of both genes appeared to be re­activated exclusively in the first and second shift to 0.8ox (sh1 and sh2), performed after 10 and 20 dia, respectively, and showed a peak at 31 dia followed by lower expression levels. however, the increase of mdacs and mdaco tran­ script following the oxygen resupply reached a level significantly lower than that reached by apples that had been constantly kept at 0.8ox. it could be hypothesised that this may be due to the higher lev­ els of ethanol accumulated in the pulp of dca stored apples, which might exert a suppressive action on ethylene biosynthesis as previously shown by some authors in different apple varieties (pesis et al., 2005; thewes et al., 2019; weber et al., 2020; thewes et al., 2021 a). concluding, the recovery from anoxia in apple fruits is dependent on the length of exposure to the anoxic stress (wood et al., 2022). our data on ethanol accumulation and ethylene­related gene expression are in line with these findings, showing that longer periods of exposure to 0.3% oxygen result in the maintenance of higher levels of ethanol and on the prevention of transcription of ethylene biosyn­ thetic genes. the effect of low oxygen storage of red delicious apples on transcript abundance of several important genes related to hypoxic stress response in apple fruit revealed both similarity with granny smith apples stored under the same ca protocols, and spe­ cific responses of cv. red delicious. in both red delicious and granny smith apples two phases can be recognized in relation to fermentative metabolism: a first phase characterised by the activation of fermen­ tative pathways, and a second phase (from two months onward) in which a generalized de­activation of fermentative metabolism is observed. acknowledgements this study was carried out by pt and sb within the agritech national research center and received f u n d i n g f r o m t h e e u r o p e a n u n i o n n e x t ­ generationeu (piano nazionale di ripresa e resilienza (pnrr) – missione 4 componente 2, i n v e s t i m e n t o 1 . 4 – d . d . 1 0 3 2 1 7 / 0 6 / 2 0 2 2 , cn00000022). this manuscript reflects only the authors views and opinions, neither the european union nor the european commission can be consid­ ered responsible for them. sb was supported by the special project of scuola superiore sant’anna isvstrategico2019. this work has been supported by the networking activities “oxygen sensing a novel mean for biology and technology of fruit quality” (ca:18210) which is implemented under the cost action “roxy­cost,” funded by the european cooperation in science & technology (2019­2023). funding was provided by marvil engeneering srl t h r o u g h p r o j e c t “ r u p e c o m m 1 7 _ 0 1 ” a n d b y university of padova project bird173975/17. references aranda p.s., lajoie d.m., jorcyk c. l., 2012 ­ bleach gel: a simple agarose gel for analyzing rna quality. ‐ elect., 33(2): 366­369. brizzolara s., cukrov d., mercadini m., martinelli f., ruperti b., 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università, 100, 80055 portici (na), italy. (1) ** dipartimento di arboricoltura, botanica e patologia, università degli studi di napoli federico ii, via università, 100, 80055 portici (na), italy. *** servizi integrati per l’agricoltura, s. felice circeo (lt), italy. key words: diplotaxis tenuifolia (l.) d.c., raphanus sativus l., cucurbita pepo l., cultivation time, fertilization, production, nitric ion accumulation. abstract: research was carried out in latina province (italy) on rocket, radish and zucchini grown under tunnel. ten treatments, obtained by the factorial combination of two crop cycles (autumn-winter and winter-spring) and six nitrogen fertilizer forms (organic, organic-mineral, mineral in three modes, control with no nitrogen fertilization) were compared. the effects of these treatments were evaluated in terms of yield and nitrate content in the edible organs. in rocket, no significant difference in yield was detected between the autumn-winter and winter-spring crop cycles, although the former cycle resulted in a higher leaf nitrate content. the organic fertilizer treatment and the n-unfertilized control gave the lowest yields, but the mineral fertilizers caused the highest leaf nitrate accumulation. radish yield did not vary between the two crop cycles, but the hypocotyl nitrate content was higher in the autumn-winter cycle. the crops fertilized with the two highest mineral supplies produced the highest yields, compared with the organic or organic-mineral treatments. in the autumn-winter crop, the mineral n fertilization resulted in the highest hypocotyl nitrate content, whereas in the winter-spring crop only the highest mineral n dose caused a higher nitrate content compared with the organic fertilizer. the highest zucchini yield was obtained from the winter-spring cycle at the two highest mineral fertilizer supplies. in the autumnwinter crop the highest mineral nitrogen dose resulted in the highest fruit nitrate content, while in winter-spring the two highest supplies caused this effect. 1. introduction an adequate supply of nitrogen fertilizers is generally needed to achieve high yield and quality performance of vegetable crops (hochmuth, 1992), although a positive correlation between nitrogen availability and production has not always been shown (mccall and willumsen, 1998). the ratio between nitric and ammonium nitrogen in n fertilizers also affects yield: in a study on zucchini, chance et al. (1999) reported that a ratio between 1:0 and 1:3 is more effective than 3:1 in terms of production. on the other hand, an excessive supply of n fertilizer may have negative effects both on the quality of vegetables and on the environment (beretta et al., 1990). excessive nitrogen fertilization may also result in nitrate accumulation, especially in leafy vegetables and a high food nitrate content is considered to be potentially dangerous to human health. this occurs because 5-10% of the ingested nitrate is reduced by bacterial enzymes in saliva and the gastrointestinal tract into the more toxic nitrite ion (walters and smith, 1981) which can, in turn, react with amines and amides giving rise to carcinogenic n-nitrose compounds (hill, 1999). a positive correlation between drinking water nitrate levels and diabetes mellitus incidence in northern england was also reported (parslow et al., 1997). however, some beneficial effects of nitrate on human health were also recognized (duncan et al., 1997; addiscott and benjamin, 2000), while the high content in antioxidant compounds of some vegetables, like wild rocket (martinez-sanchez et al., 2008), can inhibit the formation of carcinogenic compounds (steinmetz and potter, 1991). in this respect, a contrasting report was published by vermeer et al. (1998) who found that despite the low 82 nitrate content of cauliflower, peas, beans and carrots, the presence of vitamin c and other antioxidants in the same vegetables could not prevent nitrosamine formation. therefore, the nitrate content alone of vegetable foods cannot be considered as a quality indicator. genetic, environmental and cultural factors affect nitrate uptake and accumulation in plants (bonasia et al., 2002). nitrate in the soil is readily taken up by the plant where it is reduced to nitrite and ultimately converted into organic compounds by the nitrate-reductase enzyme complex, whose presence in the leaves increases in response to light and to nitrate availability in the soil (guerrero et al., 1981). when the rate of nitrate uptake is faster than its assimilation rate, it is mainly accumulated in the plant cell vacuoles where is not toxic, differently from the ammonium ion (crawford and glass, 1998). nitrate accumulation rate depends on the vegetable species and on the plant organs, the highest concentrations being found in leafy/petiole species while lower nitrate contents are found in root/hypocotyl (meah et al., 1994, santamaria, 1999 b) or in fruit crops (quince and dvorak, 1980). the nitrate content also varies among cultivars within the same species: large differences were found in spinach (cantliffe, 1972), celeriac (delorez and vulsteke, 1985), lettuce (reinink et al., 1987), endive (reinink et al., 1994) and carrot (gutezeil and fink, 1999). in rocket, the nitrate content was found to increase under low solar radiation, as did the flavonoid content and antioxidant activity (jin et al., 2009). however, when this crop is grown under high light intensity, more rapid plant growth requires high organic nitrogen availability, which consequently prevents nitrate accumulation. in fact, according to padgett and leonard (1993), organic nitrogen compounds can replace nitrate as osmolyte and adjust the plant nitrate absorption. temperature also affects the uptake, translocation and assimilation of nitrate: nutrient solution heating stimulates nitrate absorption (malorgio et al., 1995) but excessive air temperature favours its accumulation in the plant tissues (behr and wiebe, 1992). as regards fertilization, ammonium and nitrate ions are the main nitrogen sources for plants. even though nitrate assimilation is energetically rather expensive, it is usually the plant-preferred form (salsac et al., 1987). in fact, a rapid ammonium uptake rate, exceeding its assimilation rate, would result in ammonium accumulation and toxicity (maynard and barker, 1969), while nitrate accumulation in plant tissues does not cause negative consequences. however, the preference between the two nitrogen forms depends on several factors: plant species, plant age, cultural method and the ratios between the concentrations of nitrogen/ammonium and other nutrients in the growth medium. in fact, while celery and fennel prove indifferent to the inorganic nitrogen form, chard is inhibited by ammonium nutrition (santamaria et al., 1999 b). in lettuce, nitrate content can be reduced by distributing a part of the whole nitrogen supply as ammonia form, without changing the overall yield results (van der boon et al., 1990) while in endive the exclusively ammonia form allows for no nitrate heads (elia and santamaria, 1997). moreover, rouphael and colla (2005) reported that a greater nitrate content in zucchini was found in soil-grown plants compared to a soilless cultural system. in the latter context, it was found that interrupting the nitrogen supply at an advanced stage of the crop cycle, the fruit nitrate content was significantly reduced (santamaria et al., 1998). this reduction appears to be related to the plant’s ability to use the nitrate previously accumulated in vacuoles for protein synthesis (blom-zandstra and lampe, 1983), in order to ensure growth when the substrate resources decrease (koch et al., 1988). finally, nitrapirine, a nitrification inhibitor, did not improve productive results in radish (mills et al., 1976). the present study was carried out to evaluate the effects of different nitrogen fertilization forms on yield and nitrate content of wild rocket, radish and zucchini in the pontina plain (latina, italy), grown under tunnel in two cultural cycles, autumn-winter and winterspring. 2. materials and methods wild rocket, radish and zucchini were grown on sandy soil (table 1) in fondi (pontina plain, latina province, italy) in 2003-2004. the crops were grown under a thermal pe tunnel equipped with anti-freeze irrigation, activated at a temperature of 5°c. the structural unit was 7.20 m wide, 40.00 m long and 2.00 to 3.50 m tall, respectively, from wall to roof. temperature and solar radiation values of the trial environment are reported in figure 1. the experimental protocol was planned in order to compare 10 treatments, which originated from the factorial combination of two crop cycles (autumn-winter and winter-spring) with six nitrogen fertilizer forms (organic, organic-mineral, mineral in three modes, control not fertilized with nitrogen). for the treatment distribution within each crop cycle, a randomized block table 1 soil characteristics fondi (latina, italy), 2003-2004 soil characteristics sand silt clay organic matter total nitrogen kjeldhal method available phosphorus olsen method available potassium ammonium acetate method total lime ph electrical conductivity (1:5) at 25°c % % % % % ppm ppm % ds·m-1 86.00 4.00 10.00 2.06 0.14 69.00 245.00 not detected 6.40 0.74 83 design with three replicates was followed. for each crop, the local standard agricultural practices were respected: plowing, hoeing, ridging, preplanting chemical weed control, drip irrigation and parasite control. the fertilization plans were scheduled according to the average nutrient requirements of the crops (tesi and lenzi, 2005) and to the expected production levels. crops were harvested at midday in all of the plots and random representative samples of the edible plant parts were collected. the samples were transferred to the laboratory, where nitrogen content was determined by high-performance liquid chromatography (hplc) using a waters 600e chromatographic system, managed through the millennium software version 3.05.01. the analytical column was a dionex anion exchange column (model as11, 4×250 mm, code p/n 044076) with a dionex pre-column (4×50 mm, code p/n 044078). the eluent was 21 mmol l-1 naoh at a flow rate of 1 ml min-1. the detector was a dionex pulsed electrochemical detector together with an anion selfregenerating suppressor. statistical data processing was performed by anova, using the duncan test for mean separation. wild rocket (diplotaxis tenuifolia (l.) d.c.) pre-planting fertilization was applied with 50 kg·ha-1 of p2o5 from mineral superphosphate 18/20 and 150 ofk2o from potassium sulphate 48/50. with the excep-tion of the n-unfertilized control, in all plots 90 kg·ha1 of n were supplied as follows: organic n fertilization was applied at pre-planting as roasted leather; mixed organic-mineral n fertilization was supplied with 45 kg·ha-1 at planting from roasted leather and 45 kg·ha-1 at dressing from ammonium nitrate 26/27; mineral n fertilization was supplied as 25 kg·ha-1 at planting from ammonium sulphate 20/21 plus 65 kg·ha-1 from ammonium nitrate during the crop cycle. organic-mineral n fertilization was applied in two phases, at six and three weeks before the expected second harvest, while the three mineral n treatments differed with respect to the application time at one, two or three weeks before the expected second harvest date. alveoli containing 10 plants each were transplanted on 25 september 2003 and on 15 january 2004 in the first and the second crop cycle, respectively. in plots of 12.60 m2 (3.00 x 4.20 m) three ridges were made 0.40 m apart from each other, which included six rows with spacing 0.20 m, with a density of 215 plants per m2. rocket was harvested in two phases, between 13 november and 9 january (2003-2004) in the first cycle and from 14 march to 28 april 2004 in the second cycle, according to the fertilization treatment. this crop was hand harvested by cutting the plants at about 2 cm above the soil surface when they reached 18 cm in height; crop weight was measured. at the second harvest, a random sample of 100 plants was collected in each plot and transferred to the laboratory for nitrate determination. radish (raphanus sativus l. subsp. parvus) radish cv. suprella, a commonly grown variety in the experimental area, was sown on 24 november 2003 in the first crop cycle and on 2 march 2004 in the second. the plots had a 5.20 m2 (2.60 x 2.00 m) surface area including 0.80 m wide beds, with 0.10 x 0.05 m spacing between plants. pre-planting fertilization was applied with 80 kg·ha1 of p2o5 from mineral superphosphate 18/20 and 100kg·ha-1 of k2o from potassium sulphate 48/50. in addi-tion, 100 kg·ha-1 n, corresponding to the crop requirement, was supplied to all plots with the exception of the n-unfertilized control: organic n fertilization was applied at pre-planting as roasted leather; mixed organic-mineral n fertilization was applied in two phases as 50 kg·ha-1 n as roasted leather at pre-planting plus 50 kg·ha-1 n as ammonium nitrate at 20 days after sowing. the mineral n fertilization treatments supplied 70, 100 or 130 kg·ha-1 n: one-half of the total n supply was given at pre-planting as ammonium sulphate while the remaining 50% was given as ammonium nitrate in two applications, at 12 and 24 days before the expected harvest date. therefore, the range of mineral n fertilization treatments supplied the intermediate amount (100 kg·ha-1) +/a 30% increase or reduction. radish crops were harvested 16-22 january and 611 april in 2004, depending on the crop cycle and fertilization treatment. radish plants were manually harvested when the hypocotyls reached the 30-40 mm caliber: weight, number and average weight data were recorded. moreover, a sample of 100 units was collected in each plot and transferred to the laboratory for nitrate assessment. zucchini (cucurbita pepo l.) zucchini cv. velvia, a commonly grown variety in the experimental area, was used for this study. the plants were transplanted on 23 september 2003 in the fig. 1 trend of temperature and solar radiation. fondi (latina), 20032004. 84 first cultural cycle and on 20 january 2004 in the second. each plot was 35.10 m2 (5.40 x 6.50 m) and plant density was 1.71 plants per m2 (1.80 m between double rows and 0.65 m between plants along the row). the fertilization doses were adjusted to the expected yields, which changed with the crop cycle: 60 kg·ha1 of p2o5 from mineral superphosphate 18/20 and 240of k2o from potassium sulphate 48/50 were supplied atplanting for the autumn-winter cycle; while 120 kg·ha1 of p2o5 and 480 of k2o were supplied at planting forthe winter-spring cycle. in addition, nitrogen fertilization supplied 150 or 300 kg·ha-1 n for the autumn-winter or the winter-spring cycle, respectively. n fertilization was supplied as follows: organic n fertilization was applied at pre-planting as roasted leather; organicmineral n fertilization was applied in two phases (50% at planting as roasted leather and 50% at dressing as ammonium nitrate). mineral n fertilization treatments supplied 105, 150 and 195 kg·ha-1 or 210, 300 and 390 kg·ha-1 to the autumn-winter or winter-spring crop, respectively. one-third of the total n dose was applied at planting (35, 50, 65 kg·ha-1 as ammonium sulphate in the autumn-winter cycle or 70, 100, 130 kg·ha-1 in the winter-spring crop) while the remaining two-thirds of the total n dose was applied during the crop cycle (70, 100, 130 or 140, 200, 260 kg·ha-1 as ammonium nitrate). therefore, the range of mineral n fertilization treatments supplied an intermediate rate (150 or 300 kg·ha-1) +/a 30% increase or reduction. the n fertilization at dressing was evenly distributed in three applications with a 21-day interval. fruits were hand harvested when the corolla was open, from 23 october to 22 december 2003, and from 29 march to 27 may 2004, depending on the crop cycle and fertilization treatment. fruit weight, fruit number and average fruit weight were recorded. in addition, 10 days after the last fertilizer application a sample of 20 marketable fruits was collected from each plot and transferred to the laboratory for nitrate assessment. 3. results no significant difference in rocket yield was found between the autumn-winter and winter-spring crop cycles (data not shown). in the first cycle, however, the leaf nitrate content was as much as 64.3% higher than in the winter-spring. table 2 shows that in the autumn-winter cycle there was no yield difference between the mineral and mixed organic-mineral n fertilization forms. however, the mineral and organic-mineral treatments produced better results compared with the organic n fertilization and with the n-unfertilized control. in rocket, mineral n fertilization resulted in the highest leaf nitrate content compared with the other treatments, while the n-unfertilized control resulted in the lowest. in the winter-spring cycle (table 3), the production trend was similar to that observed in the autumn-winter cycle. however, organic-mineral fertilization did not produce significantly different results from the organic n fertilization treatment. the mineral and organic-mintable 2 wild rocket under tunnel: yield results and leaf nitrate content in the autumn-winter cycle, as influenced by nitrogen fertilizer form. fondi (latina, italy), 2003-2004 treatment nitrogen fertilizer form non-fertilized control organic organic-mineral mineral 1: one week before the second harvest mineral 2: two weeks before the second harvest mineral 3: three weeks before the second harvest marketable yield (t·ha-1) leaf nitrate content (mg·kg-1 of fresh weight) 10.3 c 13.9 b 15.4 a 16.0 a 16.3 a 16.4 a 2706.7 c 3866.7 b 4240.3 b 5032.0 a 5150.0 a 5317.7 a means followed by different letters are significantly different according to the duncan test at p≤0.05. table 3 wild rocket under tunnel: yield results and leaf nitrate content in the winter-spring cycle as influenced by nitrogen fertilizer form. fondi (latina, italy), 2003-2004 treatment nitrogen fertilizer form non-fertilized control organic organic-mineral mineral 1: one week before the second harvest mineral 2: two weeks before the second harvest mineral 3: three weeks before the second harvest marketable yield (t·ha-1) leaf nitrate content (mg·kg-1 of fresh weight) 11.0 c 15.1 b 16.8 ab 17.3 a 17.4 a 17.6 a 1626.0 c 2322.8 b 2838.3 a 2842.2 a 3083.3 a 3300.0 a means followed by different letters are significantly different according to the duncan test at p≤0.05. 85 eral n fertilization treatments resulted in higher leaf nitrate content compared with the organic n fertilizer application or with the n-unfertilized control. radish yield did not vary between the two crop cycles: the hypocotyl number per unit surface area and their average weight were unaffected by the crop cycle factor (table 4). in contrast, n fertilizer form significantly affected the edible organ production: mineral fertilization at the two highest doses was more effective than the organic and the organic-mineral n fertilizer forms. the organic n supply had better effects on yield, compared to n-unfertilized control, but it was less effective than the other treatments. these results were mainly affected by the average hypocotyl weight, which changed significantly in response to the different n fertilization forms while only the unfertilized control resulted in a reduced hypocotyl number. similarly to rocket, also in radish (table 5) nitrate content was higher in the autumn-winter cycle, on average as much as 25.3% compared to the winter-spring cycle. in the autumn-winter cycle, leaf nitrate content was higher in response to organic-mineral and mineral n fertilization. instead, during the winter-spring cycle the highest mineral nitrogen supply caused a nitrate concentration increase only in comparison with the organic form. moreover, in the winter-spring cycle, the maximum mineral fertilizer dose (130 kg·ha-1 equally distributed before and after planting) resulted in a three-times higher leaf nitrate content compared with the unfertiltable 4 radish under tunnel: hypocotyl yield as a function of crop cycle and nitrogen fertilizer form. fondi (latina, italy), 2003-2004 treatment crop cycle autumn-winter winter-spring nitrogen fertilizer form non-fertilized control organic organic-mineral mineral 1: 70 kg·ha-1 mineral 2: 100 kg·ha-1 mineral 3: 130 kg·ha-1 marketable hypocotyl yield no. per m2 mean weight (g) means followed by different letters are significantly different according to the duncan test at p≤0.05. weight (t·ha-1) 26.9 27.4 ns 20.8 d 25.4 c 27.9 b 28.5 ab 30.0 a 30.5 a 139.9 137.0 ns 128.2 b 138.0 a 140.0 a 140.2 a 142.2 a 142.3 a 19.2 20.0 ns 16.2 e 18.4 d 20.0 c 20.4 bc 21.1 ab 21.5 a table 5 radish under tunnel: hypocotyl nitrate content as a function of crop cycle and nitrogen fertilizer form. fondi (latina, italy), 2003-2004 treatment nitrate content (mg·kg-1 of fresh weight) autumn-winter winter-spring nitrogen fertilizer form non-fertilized control organic organic-mineral mineral 1: 70 kg·ha-1 mineral 2: 100 kg·ha-1 mineral 3: 130 kg·ha-1 1020.3 d 1229.7 c 1431.7 b 1670.0 a 1697.0 a 1743.0 a 478.3 c 1038.3 b 1258.3 ab 1311.7 ab 1357.3 ab 1571.7 a means followed by different letters are significantly different according to the duncan test at p≤0.05. table 6 zucchini under tunnel: yield results and fruit nitrate content in the autumn-winter cycle as a function of nitrogen fertilizer form. fondi (latina, italy), 2003-2004 treatment nitrogen fertilizer form non-fertilized control organic organic-mineral mineral 1: 105 kg·ha-1 mineral 2: 150 kg·ha-1 mineral 3: 195 kg·ha-1 marketable fruit yield no. per plant fruit nitrate content (mg·kg-1 of fresh weight) 13.8 e 19.9 d 23.1 c 23.9 bc 25.8 ab 26.9 a means followed by different letters are significantly different according to the duncan test at p≤0.05. weight (t·ha-1) mean weight (g) 9.3 c 12.0 b 13.0 ab 13.3 ab 14.0 a 14.3 a 87.1 c 97.5 b 103.8 ab 105.7 ab 107.5 a 110.6 a 496.7 e 638.7 d 723.7 d 850.0 c 1022.0 b 1186.0 a in both crop cycles, the highest zucchini production was obtained with the two highest mineral fertilizer supplies (tables 6 and 7). however, in the autumn-winter cycle (table 6) the intermediate dose was not different from the lowest one and the latter was as effective as the organic-mineral treatment in both crop cycles. the control treatment resulted in lower fruit number and average weight while the mineral n fertilization produced the highest values. the winter-spring yield (table 6 and 7) was more than double the autumn-winter yield, as a consequence of the increased fruit number (+100%), while their ized control, whereas in the autumn-winter cycle a 71% increase was recorded between the same two treatments. 86 average weight was not significantly different. in the autumn-winter cycle (table 6), the maximum mineral n rate and the unfertilized control resulted in the highest and lowest fruit nitrate concentrations (1186 vs 497 mg·kg-1), respectively. furthermore, no difference was detected between the organic and organicmineral n fertilization treatments. in the winter-spring cycle (table 7), the mineral n treatments led to a greater fruit nitrate accumulation compared with the control and the organic n fertilization treatment. 4. discussion and conclusions in the present investigation rocket and radish did not show yield differences between the autumn-winter and winter-spring cycles. this is presumably due to the light and temperature requirements of these crops which allow for an equally good production in both seasons. in fact, the two crop periods are quite similar in terms of duration and day-length, though the latter decreases from the beginning to the end of the autumn-winter cycle while the opposite trend occurs in winter-spring. our results are in accordance with those reported by inada and yabumoto (1989) who found that growth of a radish crop was promoted by increasing day-length, but it was less sensitive to variations in temperature regime. differently from rocket and radish, zucchini yield was affected by the cultural cycle, as the winter-spring cycle resulted in a higher fruit production compared with the autumn-winter cycle. presumably, the increasing daylength, light intensity and temperature of the second part of the winter-spring cycle played a crucial role in improving crop productivity. similar results were reported by rouphael and colla (2005). as regards n fertilization, the wild rocket yield was favourably affected by the mineral fertilizer treatment as well as the organic-mineral treatment. a contrasting report was published by cavarianni et al. (2008) who found that an increasing mineral n supply caused the rocket yield to decrease. differently from rocket, radish showed a positive response to nitrogen increase, both in organic-mineral and in mineral form. guven (2002) reported that radish yield benefited from an increase of inorganic nitrogen table 7 zucchini under tunnel: yield results and fruit nitrate content in the winter-spring cycle as a function of nitrogen fertilizer form. fondi (latina, italy), 2003-2004 treatment nitrogen fertilizer form non-fertilized control organic organic-mineral mineral 1: 210 kg·ha-1 mineral 2: 300 kg·ha-1 mineral 3: 390 kg·ha-1 marketable fruit yield no. per plant fruit nitrate content (mg·kg-1 of fresh weight) 30.3 d 39.9 c 47.0 b 48.3 b 54.5 a 55.0 a means followed by different letters are significantly different according to the duncan test at p≤0.05. weight (t·ha-1) mean weight (g) 19.3 c 23.3 b 25.7 ab 25.7 ab 28.0 a 28.0 a 91.3 d 100.3 c 107.6 b 109.7 ab 113.6 ab 115.5 a 331.7 c 426.7 bc 443.0 bc 536.3 ab 606.0 a 678.3 a supply, while fuke et al. (2000) reported a better effect of the mixed organic-mineral fertilization compared with the exclusively mineral treatment. in the present work, the organic n fertilization was less effective on radish production than the other examined treatments, although it produced better results than the unfertilized control. yield was influenced almost exclusively by the hypocotyl average weight, which gradually decreased from the highest nitrogen rate to the unfertilized control. the number of marketable edible organs was conditioned instead only by the absence of n in the control treatment, which resulted in 9% deformed or undersized hypocotyls. the zucchini yield in both crop cycles was better affected by mineral nitrogen fertilization achieved with the intermediate or 30% increased rate, compared to the organic-mineral or organic fertilization forms. nevertheless, in the autumn-winter cycle the intermediate mineral supply did not produce better yield results than the 30% reduced treatment. zotarelli et al. (2008) reported that, in the same cultural cycle, a 50% increased n supply, compared to the crop requirement, did not modify zucchini yield but a 50% decrease was less effective. moreover, the reduced mineral nitrogen application did not result in a better production than the organic-mineral supply while, as recorded also for wild rocket and radish, the organic n treatment accelerated zucchini plant development only compared to the control. however, it should be stressed that the lower yield in the organic n fertilization treatments resulted from a combination of the production factors. in fact, these treatments did not affect the fruit emission rate nor their size. termine et al. (1987) reported that organic n fertilization did not condition the production level of leek and turnip. in this study the tested species showed a different attitude toward accumulation of nitrate in their edible organs. in particular, rocket was the only crop that in the autumn-winter cycle displayed nitrate levels above the highest ec regulation limits (4500 mg of no3-·kg-1 offresh weight), confirming also the influence of cultivation time on vegetable nitrate content. a clear tendency to accumulate more nitrates in the autumn than in the spring was shown, the latter being characterized by low cloud cover and increasing photoperiod. this is in agreement with the reports of rouphael and colla 87 (2005) on zucchini and elia et al. (1997) on broccoli rab grown in growth chamber and subjected to additional lighting. in the latter case, the leaf blade nitrate reduction was a result of nitrate-decreased absorption rather than its assimilation increase. in fact, in spinach (steingröver et al., 1986 b), parallel to the leaf blade nitrate reduction, the net nitric ion adsorption was reduced while the nitrate-reductase activity did not change except at the end of the night period. this effect would not result from direct light inhibition, but from an adsorption feedback regulation achieved by the amino acids formed in the leaf blades and then transported to the roots. moreover, it was found that under low solar radiation, typical of the winter season, the nitrate content is high even with a low nitrogen supply, whereas with high solar radiation the ion concentration increases only by supplying nitrogen (roorda van eysinga and van der meijs, 1985; santamaria et al., 1999 a). this indicates that solar radiation intensity and photoperiod length regulate the nitratereductase activity, the enzyme which reduces nitrate to nitrous ion (steingröver et al., 1986 a). in particular, the solar radiation effect is multiple, as it gives input to the nitrate-reductase synthesis and induction, supplying also the reducing power (nadh) through photosynthesis (behr and wiebe, 1992). it is believed that in the vacuole, nitrate acts as the cell turgor osmotic regulation, as an alternative to sugars and organic acids, poorly synthesized under low radiation conditions (blom-zandstra and lampe, 1985). plant nitrate concentration is known to be also subject to the available nitrogen amount and quality (citak and sonmez, 2010), and this explains why the unfertilized control plants always exhibited the lowest tissue nitrate levels. only in the zucchini fruits harvested in spring, values were not different between the control and organic or mixed treatments, confirming this species’ lower attitude. on the contrary, wild rocket displayed a remarkable tendency to accumulate nitrates in the aerial apparatus: in the unfertilized plants, a nearly triple (2.9) and more than five-fold (5.2) content was detected, compared with the corresponding control of radish and zucchini, respectively. according to quinche and dvorak (1980), the latter accumulate less nitrate because they receive organic nitrogen mainly through the phloem, which does not carry inorganic nitrogen forms. in other studies, rocket was reported to accumulate high nitrate levels both under reduced nitrogen availability (bianco et al., 1998; santamaria et al., l999 a) or increased supply (santamaria et al., 2002). organic fertilization, compared to the other supply forms, caused the lowest nitrate content in the edible organs; it was only higher than the unfertilized control (as much as 29% in zucchini and 51% in radish on average). similar results were reported for lettuce (stopes et al., 1989) and radish (ebid et al., 2008) crops fertilized with composted manure. the organic-mineral treatment resulted in nitrate content increases between 41% in zucchini and 80% in radish compared with unfertilized plants. therefore, even in such circumstance zucchini demonstrated to be a refractory species to nitrate accumulation. the mineral nitrogen fertilization caused the highest nitrate content in the edible organs: compared to the non-fertilized control, the increase was +90.2, +108.0 and +96.3% on average, respectively in rocket, radish and zucchini. santamaria et al. (1993) reported that mineral nitrogen caused a higher nitrate accumulation than the organic-mineral form in spinach. nevertheless, in the autumn-winter cycle a 30% reduction of the mineral nitrogen dose resulted in a lower nitrate accumulation, compared to the maximum and to the intermediate supply, respectively in radish and in zucchini. previously, other researchers found a direct relationship between the nitrogen rate supplied and the vegetable nitrate content (maynard et al., 1976; tesi et al., 1995; cavarianni et al., 2008). with regard to the time of fertilizer supply, in rocket, in both crop cycles, the latest n dressing application (one week before harvest) did not cause a higher leaf nitrate accumulation than the earliest one. opposite findings were reported by graifenberg et al. (1990) for lettuce: leaf nitrate content increased with the reduction of the interval between nitrogen fertilizer application and harvest. in conclusion, the studies carried out in the pontina plain (latina, italy) under tunnel, in 2003-2004, showed that the cultivation time produced a significant effect only on zucchini yield, which was better affected by the winter-spring cycle. moreover, the autumn-winter crops exhibited higher nitrate content in the edible organs. as for the fertilization treatments, in rocket n fertilization in organic-mineral form was the most appropriate, as it gave yield values as high as the exclusively inorganic supply. in addition, the organic-mineral fertilizer resulted in a lower leaf nitrate accumulation in the autumnwinter cycle, which did not exceed the ec threshold. in contrast, in radish and zucchini the two mineral supplies corresponding to nitrogen crop requirement or to its 30% increase produced the best yield results. moreover, they also caused 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of lettuce as affected by total nitrogen and chloride concentration, nh4/no3 ratio and temperature of the recirculating nutrient solution. j. hort. sci., 65: 309-321. vermeer i.t.m., pachen d.m.f.a., dallinga j.w., kleinjans j.c.s., van maanen j.m.s., 1998 volatile n-nitrosamine formation after intake of nitrate at the adi level in combination with an amine-rich diet. environ. health perspect, 106: 459-463. walters c.l., smith p.l.r., 1981 the effect of water-borne nitrate on salivary nitrite. food. chem. toxicol., 19: 297302. zotarelli l., dukes m.d., scholberg j.m., hanselman t., le femminella k., muñoz-carpena r., 2008 nitrogen and water use efficiency of zucchini squash for a plastic mulch bed system on a sandy soil. sci. hortic., 116(1): 8-16. impaginato 112 adv. hort. sci., 2011 25(2): 112-122 received for publication 24 march 2011. accepted for publication 3 june 2011. root distribution in young chétoui olive trees (olea europaea l.) and agronomic applications c. masmoudi-charfi *, m. masmoudi ** n. ben mechlia** * institut de l’olivier, tunisie. **institut national agronomique de tunisie. key words: irrigation, overall root length, root-canopy ratio, root density, root volume, water requirement. abbreviations: etc (mm)= crop evapotranspiration as determined by the fao method (allen et al., 1998). et* (m3)= evapotranspiration volume of an individual tree relative to its root area. kc =crop coefficient. kr = minorative coefficient introduced in the formulae of etc fao to take into account the soil coverage. ksupply = a supply ratio determined in order to link the water supplied to trees to the evaporative demand, it takes into account only the tree-related quantities. eto (mm)= reference evapotranspiration determined following to penman-monteith equation. pe (mm)= effective rainfall determined following the usda-scs method (fao, 1976). i (mm)= irrigation amount supplied during the irrigation period. i*(m3)= irrigation amount supplied by localized system or in small basins around the trunk. p (mm)= total rainfall. p* (m3)= effective rainfall for a single tree received around the trunk. sc (m2)= the maximum projected canopy area determined for each of the six trees assuming a circular shape. sr (m2) = area concerned by tree transpiration i.e. where roots are active. (t)= the number of years from planting. lo, lx= dimension of interest respectively at planting and at maximum growth. α, β = adjustment parameters within the logistic root and canopy growth curve. abstract: the study was carried out to have a comprehensive view of the root system behavior of young olive trees cultivated under field conditions. the experiment involved irrigated trees (olea europaea l., cv., chétoui) cultivated at 6x6 m2 spacing in mornag (36.5°n, 10.2°e), northern tunisia. the way in which roots explore the soil volume during the first years after planting was explored through ‘in situ’ root system drawings and estimation of root densities. the relationship between canopy and root growth parameters was also investigated. the last section of this paper proposes a methodological approach for determining irrigation requirements of young olive trees and how water supply could be linked to the development of canopy and root system during the first years of cultivation when ground cover and the root system are not completely developed. some agronomic applications were then deduced concerning water and fertilizers for such orchards. results show that the main development of the olive root system occurs during the two to four first years of cultivation confining most roots (70%) to the top soil layers (20-40 cm). maximum root densities were observed at this depth at a distance of 0.4 m from trunks. for young trees, water and fertilizers should be supplied at these depths and distances from trunk to allow easy and efficient root absorption. obtained results also show a significant relationship between canopy and root areas which can be approximated by a linear model (r = 0.94). the root-canopy ratio estimated from their areas decreased rapidly beginning from the second year after planting, resulting from the establishment of competition between vegetative growth and fruiting. the optimum ratio root length/leaf canopy area of 2.3 km m-2 was found for the six-year-old tree indicating good equilibrium between the above and underground parts. the mathematical model developed on the basis of canopy cover and root extension allows precise estimation of water needs taking into account the actual root surface. however, while the canopy cover measurement was relatively easy to carry out, it was much more difficult to determine the surface covered by the root system. results obtained in the present work also show an over-estimation of water needs when the fao method is adopted to estimate the evapotranspiration of young trees. 113 1. introduction the primary function of the root system, i.e. water absorption and acquisition of soil nutrients, has a great influence on many of the physiological processes in the tree (doussan et al., 2003). however, despite of this importance, the root system is possibly the least explored area in crop physiology because of the difficulty involved in reaching it, in addition to the highly spatial-temporal variability which can generate many constraints to root extension. amongst the first papers dealing with this area, are those of yankovictch and berthelot (1947), vernet and mousset (1963) and abdel-rahman et al. (1966) which were carried out in north africa, mainly on cultivars chemlali and picholine marocaine. research conducted a few years later in spain and italy investigated the relationship between water and root extension (pisanu and corrias, 1971; bohm, 1979; nunuez-aguilar et al., 1980; martin-aranda et al., 1982; michelakis and vougioucalou 1988; pastor et al., 1998; smit et al., 1999; palease et al., 2000). it was shown that apart from genetics and the origin of the plant (ayachi-mezghani, 2009) root distribution and extension can be markedly influenced by neighboring trees and soil texture and depth (ben rouina et al., 1996). also, roots proliferate within the potential root zone regardless of irrigation application and method (fernandez et al., 1991, 1992, 2003; fernandez and moreno, 1999; connor and fereres, 2005). these last authors noted that localized irrigation increased root length density of manzanilla olive trees but it decreased their spread, largely confining them within the wetted volume and nearby trunks. they reported also that except under the canopy, roots were less frequent in the top layers than in deeper strata. root extension is also dependent on the available carbohydrate resources (dichio et al., 2002) and growth stage (michelakis, 2000). rapid growth is observed in spring and autumn; it depends on water supply. root growth precedes shoot growth and may be drastically limited by the previous year’s fruit load. in fact, when no competition for carbohydrates occurred with other organs, for example for young olive trees or/and for vigorous canopy growth trees, important root extension and greater root densities were reported (palease et al., 2000). in contrast, limited carbohydrate resources led plants to reduce their canopy growth and root length and even could deteriorate the root-canopy ratio as a result of competition between shoots, flowers, fruits and roots (dichio et al., 2002). this relationship between root growth and the aboveground development is complex because it integrates many other factors and physiological processes like temperature, radiation, hormones, variety and alternate bearing. reduction of the root-canopy ratio implies systematic reduction of the capacity of the rooting system to absorb water. in terms of root balance, the importance of the water collecting system resides in its capacity to obtain water to support the transpiring leaf area (connor and fereres, 2005; connor, 2006) and it can be determined via an estimation of total root length through monitoring of root density. these techniques are reported by tennant (1975) and fernandez and moreno (1999). such measurements could provide reliable estimates of comparative activities. for olive trees, connor and fereres (2005) reported root densities ranging between 0.1 and 1.0 cm cm-3. these values are lower than those provided for herbaceous crops and some deciduous orchards, although olive root systems can be extensive and deep. it appears from this short review that fundamental research on this subject is of prime interest: when and where the roots grow is crucial to understanding the functioning of the root system and its relationship with the above-ground organs. in fact, without precise information on root distribution, we cannot expect to efficiently manage the irrigation of the orchard. for these purposes, we have carried out the present study in order to have a comprehensive view of the root system behavior of young olive trees cultivated under field conditions. in this work, we examine how roots explore the soil volume during the first years after plantation. the relationship between root and canopy development was also investigated. the last section of this paper proposes a methodological approach to determine irrigation requirements of young olive trees and considers how water supply could be linked to the development of canopy and root system during the first years of cultivation when ground cover and the root system are not yet completely developed. 2. materials and methods olive orchard the study was carried out during the period 19982003 at the experimental farm of the institut national agronomique de tunisie, located 15 km south of the capital tunis (36.5ºn, 10.2ºe), northern tunisia. in this region, climate is mediterranean with yearly averages of 450 mm rainfall and 1200 mm reference evapotranspiration. it is dry and hot from may to september. the orchard, of 1.6 ha, was planted in 1998 at 6x6 m2 spacing on a textural clay soil (29%c, 49%l, 23%s) of about 2 m depth. the volumetric soil water content was measured in the laboratory at field capacity (50%) and at the wilting point (26%). crop management practices carried out in the orchard, i.e. pruning, fertilizer (masmoudi-charfi and ben mechlia, 2009) and pest management practices, were similar to those applied in intensive orchards (masmoudi-charfi, 2006; masmoudi-charfi et al., 2006). the trial concerned trees of cultivar chétoui, which is the main oil variety of northern tunisia. 114 climatic data and irrigation management daily crop evapotranspiration (etc) was determinedaccording to allen et al. (1998) for the non-standard conditions such as: etc = eto ×kc ×kr, kc rangingbetween 0.3 and 0.5 according to age, while kr valueswere determined experimentally and varied between 0.69 and 0.75. for this purpose, a large white grilled (10 cm/10 cm) sheet was used. it was placed below the tree and the shade squares were counted and compared to the total number of squares (those lighted by sun and those shaded by leaves). this percentage represents the kr value. daily reference evapotranspiration (eto) wascomputed according to the penman-monteith equation, with maximum and minimum yearly values of 1320 mm (1999) and 1212 mm (2003), respectively. data relative to rainfall, eto and temperature are reported intable 1. all climatic data were recorded continuously with an automatic weather station located about 150 m from the young olive orchard. during the six years of the study, rainfall amounts varied from 327 mm (2001) to 790 mm (2003), while effective rainfall amounts ranged between 226 mm and 546 mm. these values were determined according to the usda-scs method (fao, 1976). accounting for these conditions, olive trees were irrigated every year during the spring-summer season. water flows were programmed four times per season regardless of the critical stages and water availability. irrigation was supplied by furrows (basin and drain) during the four first years and then by a drip system (2002 and 2003). two parallel drip lines were fixed on the soil surface at about 0.5 m from trunks. there were four emitters per tree, two at each side of the tree trunk, separated 1 m from each other; each having a 4-l h-1 flow rate. the area wetted by irrigation application varied between 1 m2 (1st year) and 6 m2 (6th year). watering conditions for the whole period are given in table 2. water requirements were covered at levels varying between 0.3 etc and 1.1 etc according to year andwater availability. measurements soil water content. the volumetric water content of the soil was measured with a neutron probe (solo 25) which was previously calibrated for the soil in question (masmoudi-charfi, 2008). twenty-eight access tubes, 1.5 m long, were placed at the corners of a square of 2 m2 below the canopy but also within the tree line and between tree lines. the soil moisture in each of these tubes was recorded frequently during the irrigation period every 0.3 m to 1.2 m depth, and the mean calculated separately for each position: below the canopy, far from the emitters, along and between the lines of tree (unpublished data). for the top 0.2 m soil layer, soil water content was determined by gravimetry. more details are given in masmoudi-charfi (2008). table 1 climatic data recorded during experimentation (1998-2003) annual rainfall (mm) effective annual rainfall (mm) absolute tmax (°c) absolute tmin (°c) average tmax (°c) average tmin (°c) annual eto (mm) 1998 1999 2000 2001 2002 2003 376 260 47.0 3.0 25.0 13.3 1313 440 304 41.0 1.0 23.7 15.0 1320 410 283 44.0 4.0 25.2 14.8 1293 327 226 42.0 3.0 25.8 15.8 1282 345 238 43.0 3.0 25.6 15.5 1231 790 546 46.0 3.0 24.9 14.9 1212 table 2 water requirement and irrigation application for young olive trees of cultivar chétoui during the experimental period irrigation system first irrigation last irrigation dose (m3/tree) irrigation amount (m3/tree/year) i + pe (mm)* etc (mm)* i+pe / etc 1998 1999 2000 2001 2002 2003 basin march august 0.12 0.84 140 243 0.6 basin may september 0.18 0.72 61 241 0.3 drain april september 0.22 0.88 180 291 0.6 drain april september 0.44 1.76 141 287 0.5 drip march august 0.7-1.7 4.98 248 273 0.9 drip may september 0.3-1.0 5.41 389 368 1.1 (*) indicates that values are determined for the irrigation period. pe is the effective rainfall determined according to the usda-scs method (fao, 1976) and i is the irrigation amount. the ratio i+pe / etc was calculated for the irrigation period. the tree downward projection canopy flat area varied between 2% (first year) and 33% (sixth year). 115 root distribution. distribution of the root system was studied during the rest period (november-december) on the same chétoui olive trees by extensive observations of their root system. the trench method was used as described by fernandez et al. (1991). for this purpose, a large pit was opened at 0.4 m from the trunks and roots were counted on the internal trench wall, which was divided into five layers of 0.2 m width each and down to 1.0 1.2 m depth. root diameter was measured by means of a caliper 1/100. maximum distance of roots from trunk was determined at each soil layer in order to estimate lateral root extension. total volume of soil and the area explored by the root system were determined assuming central symmetry to the trunk. root density. root densities were determined on the same chétoui olive trees by using the cylinder method as described by fernandez et al. (1991). soil samples were taken during the rest period by a conventional auger at 0.4 m, 0.8 m and 1.2 m from trunks in order to quantitatively assess the importance of the root system through an estimation of root densities as described by tennant (1975). samples were taken within layers of 0.2 m width, down to 1.0 1.2 m depth, following east and south directions, along the line of drippers (south) as well as perpendicular to this. they were then washed out abundantly and sieved through a 0.5 mm screen. extracted roots were counted by adopting a reference scale (tennant, 1975). root length was then derived from the average root density value for each of the six trees. figure 1 presents details on both protocols. with this scheme, it was possible to obtain information on root distribution in the zones affected and not affected by irrigation. canopy measurements. canopy diameter measurements were monitored at the same time as the study of the root system and on the same experimented trees. the maximum projected canopy area (sc) was determi-ned for each of the six trees assuming a circular shape. these measurements were used to set a typical model of growth and to examine the relationship between root and canopy development. canopy leaf area was determined for the six-yearold olive tree by computing the number of leaves on representative shoots and estimating its specific leaf area. it reached 14 m2 on may 2003. this value was adopted to calculate the root length/leaf canopy ratio. methodological approach to determine irrigation requirements of young olive trees. this section proposes a methodological approach to determine irrigation requirements of young olive trees and how the water supply can be linked to the development of the canopy and root system during the first years of cultivation when ground cover and root system are incompletely developed. determination of water requirements according to the fao method (allen et al., 1998) is adequate for standard conditions, i.e. when soil coverage reaches 60% or more. however, when the coverage area is less, a reductive coefficient kr is introduced (coi,1997; allen et al., 1998). in some cases, particularly for young and new orchards (low tree canopy cover), this coefficient may not be precise enough to allow good estimation of water needs. in addition to problems estimating kr values, the kc is strongly affectedby conditions that influence evaporation from the soil surface (orgaz et al., 2006). recently, testi et al. (2004) proposed a simple linear relationship between the olive ground cover (and leaf area index) and the average kc of the summer months, valid for groundcover fractions up to 0.25, along with its variation when wet surface soil spots are present. these authors indicate that this relationship does not apply outside a rainless summer, and the contribution to soil evaporation from the drip system depends on the surface area and location of the wet spots and is not scalable. thus, we developed the following approach which is designed to determine the consumptive use of olive trees in relation to their canopy growth and root development during the first six years after planting. before full development of the root system, only a fraction of rainfall water is accessible to trees. thus, the water balance equation should consider the area concerned by tree transpiration i.e. where roots are active (sr); sr is assumed to be circular and to increasefollowing a logistic-shaped curve. root extension, as well as canopy increase, seems to coincide with a logistic growth curve as given by the following equation: l(t)=lo+ lx-lo1+exp[α(t-β)] where (t) is the number of years from planting; lo, lx dimensions of interest, respectively, at planting and at maximum growth; α, β are adjustment parameters. in order to link the water supplied to trees to the evaporative demand, a supply ratio (ksupply) that takesinto account only the tree-related quantities is defined by this equation: ksupply = (p* + i*) / et* fig. 1 scheme of sampling to determine root distribution and root densities for young olive trees aged one to six years. root profiles were mode following to nw direction while samples of root density determination were taken at 0.4 m, 0.8 m and 1.2 m from trunks to 1.2 depth following to se direction. : emitter. 116 considering that irrigation (i*, m3) is supplied by a localized system or in small basins around the trunk, only a small surface is wetted and affected by soil evaporation and transpiration. irrigation water is therefore assumed to be fully accessible to the root system of the tree. on the other hand, effective rainfall for a single tree (p*) is taken as the volume of rainfall water available to the root system which could be approximated by the following equation: p* (m3) = p (m) x sr (m2) p is rainfall, considered here as total rainfall. the evapotranspiration volume of an individual tree (et*) can be estimated from the root area of the tree as: et* (m3) = kc x eto (m) x sr (m2). different water supply ratios are determined as kc -fao, i/eto, p*+i*/et*and i*/et*. the ratio i/eto isthe irrigation supply, p*+i*/et* is the volumetric total supply and i*/et* is the volumetric irrigation supply. these ratios are for the period april-august over the first six years of olive tree cultivation. values are represented in the same figure to compare results. 3. results soil water status simultaneous monitoring of soil moisture carried out during the 2003 campaign at the canopy limit and near the emitters showed that soil water contents vary from 15 to 39% according to depth and distance to trunk (fig. 2). low values of soil water content were observed in the upper layers, while minimums were recorded within the superficial strata (0-20 cm) as a result of soil water evaporation and root absorption. this result confirms the concordance between root development and soil water depletion. the results showed large variation between measurements at the limit of the canopy, while low variation of soil moisture was observed near the emitters with values ranging between 32 and 38% according to depth (fig. 2). root system drawings root profiles for the tagged trees show two or three types of roots according to age (fig. 3). during the first years after planting, trees developed fine roots in the upper 0.2 m of the soil layer, which then extended fig. 2 soil water content (%) measured at two sites: on the left at 10 cm from the emitters and on the right at the limit of the canopy during the 2003 campaign. table 3 maximum number of roots and root diameter emerging from the trench face for each soil layer for olive trees aged one to six years 0-20 20-40 40-60 60-80 80-100 total number of roots maximum root diameter (mm) 2 3 4 5 6 6 2 0 0 0 8 2 2 3 8 2 0 15 6 16 6 3 4 0 29 23 10 5 1 4 0 20 32 9 5 3 5 3 25 24 51 91 116 97 81 472 27 1soil layer (cm) age (year) fig. 3 drawings of the root system of young olive trees of cultivar chétoui aged one to six years. roots were counted on the internal trench wall, down to 1.0 1.2 m depth depending on age. rapidly in lateral and vertical directions with inclinations varying from 30° to 60° depending on their size and position. for older plants, larger roots were observed beyond the first 0.3 m and they developed horizontally with numerous fine roots. the number and diameter of roots which emerged from the lateral face of the trench are summarized in table 3. results indicate that most roots (70%) are localized in the first 0.6 m of soil. the maximum number is found in the top layers, with diameters ranging between 2 mm (one-year-old tree) and 32 mm (four-year-old tree). some roots developed in deeper strata, reaching 1.0 m depth. very few roots were found below this depth even for the oldest tree. 117 extension of the root system results presented in table 4 show that the main development of the root system occurred during the first two to four years of cultivation, horizontally and within the top layers (0.2-0.3m). during this period, the soil volume explored by roots increased at a regular rate of about 1.0 m3 yearly. for the three-year-old tree, roots explored a volume of 3.65 m3. the soil volume explored by the root system of the five-year-old-tree represents 47% of that reached by the older tree (sixyear-old tree). root density results relative to root density estimation are reported in figure 4. a noticeable root concentration is observed for both east and south directions and close to trunk in the top layers around each of the six trees. average values varied between 0.001 cm cm-3 and 0.670 cm cm-3 depending on depth, distance to trunk, direction and tree age. greater values, by up to 0.5 cm cm-3, were recorded in the first 60 cm and at 0.4 m from trunk. these values decreased significantly as the distance to trunk increased (except some measurements for twoand threeyear-old plants). roots were less frequent at all depths outside the canopy limit and particularly for the deeper layers. at these depths, however, it should be mentioned that root densities rarely exceed 0.4 cm cm-3 for both directions, while average values ranged between 0.067 cm cm-3 and 0.303 cm cm-3 (table 5). root system length the overall length of the root system varied from 1.0 km to 33.9 km depending on age (table 6). a significant increase of the overall length of the root system was observed for the six-year-old tree. it was 4.8 times greater than that recorded the previous year. the lowest value was recorded for the four-yearold tree. there was no apparent cause which could explain this result. root development and canopy growth results presented in figure 5 showed for tree aged one to four years that roots grew at higher rates than table 4 maximum distance of roots to trunk (m) and volume of soil explored by the root system (m3) for olive trees aged one to six years 0-20 20-40 40-60 60-80 80-100 explored soil volume (m3) 2 3 4 5 6 1.05 1.15 0 0 0 1.45 1.05 1.10 1.00 0.80 0 2.55 1.25 1.30 1.25 1.00 0 3.65 1.45 1.45 1.25 1.25 0 4.60 1.50 1.45 1.25 1.25 1.00 5.30 2.12 1.95 1.80 1.65 1.55 11.2 1 depth (cm) age (year) fig. 4 root densities (cm cm-3) recorded for olive trees of cultivar chétoui aged one to six years based on direction and depth. for each tree, three measurements were carried out for both directions at different distances from trunk; the first observation was made at 0.4 m, the second at 0.8 m and the third at 1.2 m. table 5 average root densities (dr, cm cm-3) determined for trees aged one to six years dr (cm cm-3) 2 3 4 5 6 0.067 0.079 0.196 0.075 0.133 0.303 1 table 6 the overall length of root system (lr, km) for trees aged one to six years lr 2 3 4 5 6 1.005 1.975 7.056 3.450 7.049 33.936 1 fig. 5 maximum root distance from the trunk (m) and maximum canopy radius (m) following to age for olive trees chétoui ages one to six years. di sta nc eo rr ad iu s( m ) 118 canopy radius. then, differences between the canopy radius and the root-to-trunk-distance decreased. roots reached for the six-year-old tree a maximum distance to trunk of 2.10 m, while the canopy limit was observed at 1.95 m. the projected canopy area (sc) increa-sed slowly after planting to reach 0.21 m2 for the oneyear-old tree and 11.94 m2 for the six-year-old-tree (table 7), while the root area progressed at a constant rate of 1.2 m2 per year to reach 13.8 m2 for the six-year-old-tree. a significant relationship was found between canopy (sc, m2) and root (sr, m2) areas, which can beapproximated by a linear model with a correlation coefficient r of 0.94, as illustrated by figure 6, where sc = 1.183 sr 3.602 (r2 = 0.876) the sr/sc ratio derived from both canopy and rootareas decreased significantly from 20 to 0.9 depending on tree age. for the four-, fiveand six-year-old trees, this ratio approximated the unit. a decrease of the sr/sc ratio implies a tendency toequilibrium between the under-ground and aboveground organs beginning from the fourth year after planting, which apparently results from the establishment of competition between shoots, roots and fruits (and explains the decrease of this ratio). in fact, trees began to produce olives within the second year after planting and the first commercial crop arrived in year four (6.5 kg / tree). results indicate also that plants seem to be able to adjust their root systems to the larger above-ground development during the winter rest. this feature is well represented by the root length/leaf canopy area ratio. a value of 2.3 km m-2 of leaves for the six-year-old tree was found in the present study, a value which is considered optimum for such conditions. irrigation supply as a function of canopy and root development in order to link the water supplied to trees to the evaporative demand, a supply ratio (ksupply) that takesinto account only the tree-related quantities is defined as developed in section ‘measurements. methodological approach to determine irrigation requirements of young olive trees’. this ratio could be considered as a crop coefficient for young trees when reference evapotranspiration, rainfall and irrigation amounts are computed according to the previous equations and expressed in m3/tree. adoption of such a ratio allows estimation of irrigation requirements for different rainfall and evapotranspiration regimes. the different water supply ratios, kc fao, i/eto, p*+i*/et* and i*/et*, deter-mined for each of the six olive trees are given in figure 7 for comparative purposes. results show that the ratio of applied irrigation (i, mm) to reference evapotranspiration (eto, mm) duringthe dry season from april to august was very low. it increased from 0.02 to 0.14 when trees grew from one to six years. when using the volume method to calculate the irrigation and precipitation falling on the area covered by roots, ksupply comes very close to the kc-fao. estimation of effective precipitation remains however big challenge for using the proposed method. table 7 canopy and root area estimations (m2) of olive trees aged one to six years root area (sr) canopy area (sc) sr / sc 4.20 0.21 20.00 1 3.80 0.82 4.60 2 5.30 1.86 2.80 3 6.60 3.79 1.70 4 7.10 8.04 0.90 5 13.80 11.94 1.20 6 fig. 6 relationship between canopy and root areas for young olive trees aged one to six years. fig. 7 variation of kc-fao, irrigation supply (i/eto), volumetric total supply (p*+i*/et*) and volumetric irrigation supply (i*/et*) ratios calculated for the period april-august over the first six years of olive tree cultivation, 1998-2003, mornag tunisia. 119 4. discussion and conclusions this study provides preliminary results on root distribution of young olive trees of cultivar chétoui, which could be exploited to manage young olive orchards efficiently. root profiles for trees aged one to six years show rapid extension of the root system during the first two to four years of cultivation following to horizontal direction. most roots (70%) are localized in the first 0.6 m of soil with a maximum number developed in the top layers. some roots developed in deeper strata, reaching 1.0 m depth, but at this age very few roots were found below this depth. the largest roots were observed beyond the first 0.3 m with maximum diameters between 2 mm and 32 mm according to age. results indicate also that lateral fine roots are abundant and give rise to a fibrous root system which represents the main absorbing surface as it was reported by palease et al. (2000). these roots originate from the branching of a parent root and constitute their ramifications, generally at right angles, as indicated by doussan et al. (2003), who classified the roots into three main categories according to their ontogenesis: primary, adventitious and lateral. in our case, the primary root constitutes the main root of the cutting. it was not dominated at the outset by a principal axis as it occurs in trees grown from seedlings. rather, many adventitious roots are produced from the base of the cutting. similar results were found in the literature for young olive trees, although studies were carried out under different conditions. abd-el-rahman et al. (1966) reported for young trees, aged seven years and grown under 150 mm of rainfall, that roots are contained in the shallow tillage (0.15-0.30 m) to approximately 0.3 m from trunk. in sardinia, pisanu and corrias (1971) observed a very shallow root system in the roots of excavated trees. their photographs and drawings clearly illustrate the horizontal development of the roots and the fact that roots of contiguous trees avoid competition by developing outwards from the tree row. in spain, nunuez-aguilar et al. (1980) observed for 12-year-old ‘manzanilla’ olive trees, that most roots are localized in the outer layers at 0.45 m from the trunk with diameter less than 0.5 mm. mickelakis and vougioucalou (1988) observed for five-year-old ‘kalamon’ olive trees cultivated in create, a maximum number of roots at a depth of 0.4 m. later, bongi and palliotti (1994) indicated that the root system of young olive trees is mainly confined to the top meter of soil, growing at depths between 0.15 and 0.40 m at a maximum distance of 0.30-0.40 m from the trunk. results relative to soil volume exploration showed a regular increase of about 1.0 m3 yearly but this rate is apparently lower than that reported in other studies. for three-year-old trees cultivated on loamy soil in southern italy, dichio et al. (2002) found volumes of about 8.6 m3 for the irrigated trees and 5.1 m3 for those cultivated under rain-fed conditions (670 mm/year of rainfall). in our case and for trees of the same age, roots explored a volume of 3.65 m3 only. this extension represents, according to fernandez and moreno (1999), doussan et al. (2003), fernandez et al. (2003) and connor and fereres (2005), the plants’ evolutionary response to the spatiotemporal variability. it explains, in our case, the lateral spread of roots and the depths they achieve; soil characteristics (clay) and its mechanical resistance may adversely affected root exploration. increases in soil strength during the summer months, as a consequence of occasional water shortage (interval between irrigations varying between 20 and 50 days), may have reduced the average number of laterals developed on the primary axes. during the following years (fifth and sixth years) the application of drip irrigation led trees to limit their root development, confining most roots to the upper layers with a noticeable root concentration observed for both east and south directions close to the trunk. an increase of root density is however observed with average values varying between 0.001 and 0.670 cm cm-3 depending on depth, distance to trunk, direction and tree age. similar values ranging between 0.1 and 1.0 cm cm-3 were reported by connor and fereres (2005). greater values of up to 0.5 cm cm-3 were recorded in the first 60 cm and at 0.4 m from the trunk. values of root density then decreased, significantly as distance to trunk increased (except some measurements for twoand three-year-old trees). roots were less frequent at all depths outside the canopy limit and particularly for the deeper layers. nunez-aguilar et al. (1980) observed similar results for 12-year-old ‘manzanilla’ olive trees with highest values of about 0.7 cm cm-3 at 0.45 m from the trunk. for seven-year-old olive trees growing with only 150 mm mean annual rainfall, abd-el-rahman et al. (1966) also found maximum root densities in the top layers at 0.15 0.30 m and up to 0.3 m from the trunk. these results show good concordance between soil profiles made for the six experimental trees and their root density distribution, having agronomic applications, since they could be used to manage more efficiently irrigation and also fertilization. water and fertilizer supplies should be given at these distances from trunks for young trees to guarantee their efficacy. many factors are cited to explain root density distribution (fernandez and moreno, 1999) amongst the cultural practices are reported in most papers. in our case, the six-year-old tree provided the highest values with average density of 0.303 cm cm-3, however the root densities recorded for the three-year-old plant were greater than those observed for the older trees. genetic factors inherent to the potentialities of that tree may be involved (michelakis and vougioucalou, 1988). however, it seems that the most influential factor that affected root density is the heterogeneous distribution of water in the orchard. results showed spatial variability of soil moisture with lower differences between measurements near the emitters (values ranging between 32 120 and 38% depending on depth) and larger variation between measurements at the limit of the canopy. this result was unexpected, but it may indicate lower rates of root absorption around the emitter despite the high densities observed at this distance from trunk (0.4 m). for such a situation, fernandez and moreno (1999) indicated that sites of maximum root density may coincide with low root activity as a compensation mechanism; thus root activity may be higher in zones of low root density than in zones of high density. the influence of soil water content on root distribution is reported by fernandez et al. (1991), who observed that adequate watering makes roots continue to grow during the dry season, thus, increasing the period of their activity and preventing their shrinking during this period. palease et al. (2000) and bongi and palliotti, (1994) indicated that root extension depends largely on the distributed water amounts and the irrigation frequency. larger volumes of water would favor the existence of wider wet bulbs and could increase root length density. in opposite, low water availability can slow down root growth because roots are able to sense the soil dryness and order stomata to close; thereby reducing water losses and preventing excessive water stress. water shortage may also increase mortality of fine roots even in the irrigated orchards; roots developed outside the wetted area during the rainy period may die. root distribution and densities are also highly dependent on leaf area and canopy development. in fact, this trial shows that olive tree establishes equilibrium between root and canopy development rapidly, around the fourth year after planting despite the larger extension of roots observed during the first two years in comparison to canopy growth. such increases in root area could be explained as a need to adequate the root system to a more vigorous canopy development. inversely, greater leaf area could provide greater total carbohydrates reserve for root activity. this relationship between leaf and root is very important to consider because dry soil conditions determine a cumulative effect over the years which indirectly affected root activity through an integrated chemical and hydraulic signaling mechanism controlling leaf water relationships, as stated by fernandez and moreno (1999). it could be represented by the under-/above-ground ratio. our results show high values of this ratio during the first year after planting, indicating a greater availability of water per unit of leaf area. however, beginning from the second year after planting this ratio decreased rapidly to attain a minimum value of 0.9. dichio et al. (2002) explains that a decrease of root-canopy ratio is a consequence of lack of water during the growing phase, which led plants to several physiological modifications; thus it can be used as an indicator of tree adaptation to water shortage. other reasons could be evoked to explain the decrease of this ratio such as the establishment of competition for nutrients between shoots, roots and fruits, which are considered the strongest sinks. the establishment of such competition is important to insure a balanced development of the tree, once it begins to set fruits. during this period of youth and first-fruit-set, the tree re-orientates the mobilization of carbohydrates (proietti and tombesi, 1996; palease et al., 2002) and high amounts of assimilates are drain to growing olives against the competing demand of the growing roots and shoots. as a results, the number of roots and their length could be reduced because their growth remain highly dependent of the available assimilates. under adequate watering conditions, olive trees seem to be able to adjust their root systems to the larger above-ground development during the winter rest essentially when no (or low) competition with other organs occurs. such result was reported by palease et al. (2002) and connor and fereres (2005) who indicate that this feature is well represented by the root length /leaf canopy area ratio. in our experiment we found a value of 2.3 km m-2 of leaves for the six-year-old tree. this ratio is concordant with the optimum values of 2.2 2.9 km m-2 which were determined for intensive plantations (connor and fereres, 2005), and this result is very important for the current work because it indicates that olive trees were adequately irrigated. such management of water ensured good development of the root system and the canopy despite the difficulties involved in fixing the irrigation amounts for such young trees with regard to the incomplete soil coverage and root development. for such young orchards, it is known that only a fraction of rainfall water is accessible to trees. thus, the water balance equation should consider only the area concerned by tree transpiration i.e. where roots are active. this area is assumed to be circular and results show that it increases following a logistic-shaped curve (masmoudi et al., 2007). this factor was taken into account to develop a mathematical model which allows estimation of irrigation needs of young trees, based on the study of the root/canopy over a long period of time. in this model a supply ratio was determined as shown previously in order to link the water supplied to the evaporative demand that takes into account only the tree-related quantities. results show that the ratio of applied irrigation to reference evapotranspiration during the dry season from april to august was very low. it increased from 0.02 to 0.14 when trees grew from one to six years. when using the volume method to calculate the irrigation and precipitation falling on the area covered by roots, the supply ratio comes very close to kc-fao. estimation of effective precipitationremains, however, a big challenge for using the proposed method. the present study provides preliminary results on root distribution of young olive trees of cultivar chétoui, which could give insight into the efficient management of intensive orchards. however, these results 121 should be enhanced by root activity observations. furthermore, development of a more detailed study on young trees would be useful to get more information on the relationship between root activity and root distribution because sites of heavy root density may present lower root activity, even in young trees. in such study, the root system should be viewed as a population of roots with varying, although coordinated, morphological and physiological properties. measurements of carbohydrate status at different stages of development at both root and canopy levels would also improve these results and give us more valuable information on the relationship between the rooting system distribution and canopy development, essential for irrigation requirement estimation as they determine evapotranspiration and water available for the root system. more knowledge is needed on root growth in young trees because they are more vulnerable to water shortages. for such trees, and particularly plants obtained from rooted cuttings, water uptake remains highly dependent on the effective areas of transpiration and water absorption. thus, it is probably more convenient to consider evapotranspiration, rainfall and irrigation in terms of volume of water/tree instead of mm. our progress in the future will be measured by our capacity to integrate knowledge on water supply, evaporative demand and the soil volume explored by the root system for different locations and planting densities. references abd-el-rahmane a.a., el-sharkawi h.m., 1974 response of olive and almond orchards to partial irrigation under dry farming practices in semi-arid regions. water relations in olive during the growing season. plant and soil, 41: 13-31. allen r.g., pereira l.s., raes d., smith m., 1998 crop evapotranspiration. guidelines for computing crop water requirements. fao irrigation 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meteorol., 121(1-2): 1-18 therios i., 2008 irrigation of the olive. in: therios l. (ed.) olives. crop production science in horticulture series. cabi, oxon, wallingston, uk, pp. 166. vernet a., mousset p., 1963 ecologie de l’olivier: alimentation en eaux. rapport de l’ecole nationale supérieure d’agriculture de tunis. yankovictch l., berthelot p., 1947 rapport sur l’enracinement de l’olivier et des autres arbres fruitiers en tunisie. annales du service botanique et agronomique de tunisie. 20: 109-176. 212 1. introduction black cumin, nigella sativa (ranunculaceae), is an annual herbaceous plant. the genus nigella is represented in 20 species of mediterranean-western asian origin (dantuono et al., 2002). only n. sativa, n. damascene and n. arvensis are of interest in jordan; n. sativa is the only species planted by farmers. there is no accurate data about planted area, but the annual production for the year 2005/2006 was 3-5 tons (personal communication). n. sativa is a hermaphroditic species with determined flowering patterns, starting with the flower terminating the main shoot and ending with the flowers on the lowermost branches. in the natural forms, flowers are delicate, and usually colored pale blue and white, with 5-10 petals and characterized by the presence of nectaries. the androecium comprises a large number of stamens, which shed their pollen as the filaments curve outward during the male phase. the gynoecium consists of up to five completely united follicles, each with a long, indehiscent style and composed of a variable number of multi ovule carpels, developing into a follicle after pollination, with single fruit partially connected to form a capsule-like structure. seeds are generally small in size (1-5 mg) dark grey or black (filippo et al., 2002). the fruit is large and its inflated capsule contains numerous seeds. n. sativa is extensively used in traditional medicine for healing various respiratory disorders from morocco to pakistan and in southern europe (filippo et al., 2002). the seeds have been widely added as a spice to a variety of foods such as bread, yoghurt, pickles, sauces, and salads for flavoring. they are also used in jordanian traditional folk medicine for some respiratory, gastrointestinal, rheumatic and inflammatory disorders (nafisy, 1989; zargari, 1990; amin, 1991). n. sativa seeds have been reported to contain essential oil, fixed oil, flavonoids, saponins, alkaloids, and proteins (zargari, 1990; burits and bucar, 2000; al-ghamdi, 2001). pollination studies of n. sativa are very limited in the literature. lloyd (1979) showed that n. sativa is self pollinated without mentioning the mechanism; zohary (1983) showed that n. sativa is capable of setting seed without being cross pollinated. the flowers of n. sativa are visited by honeybees (ricciardelli and oddo, 1981). 2. materials and methods the research considered specific plant species (landraces) of n. sativa, which were planted on-site at different elevations: location a, 150 m below sea level; and location b, 200 m above sea level. n. sativa was obtained from botanical gardens in jordan (ncartt). the seeds were planted in hills 30 cm apart on 5 november 2005. the rows were 20 m long, with 1 m between rows. water was supplied daily by drip irrigation and extra fertilizers (n p k) were applied. black plastic mulch was used. each plant was represented by three rows per pollination of nigella sativa l. (ranunculaceae) in jordan valley to improve seed set k.a. abu-hammour*, d. wittmann** * college of pharmacy, al-isra university, p.o. box 22, 23 isra, university, 11622 amman, jordan. ** institute of natural resource, faculty of agriculture, bonn university, germany. key words: black cumin, jordan, style movement. abstract: in jordan, pollination is one of the problems faced by plants under plastic houses, in open fields and in offseason planting. therefore this study was conducted in jordan to investigate the role of pollinators and to investigate the systems of pollination in nigella sativa species grown at two different altitudes, 150 m under sea level and 200 m above sea level. up to now little attention has been paid to the events associated with pollination such as seed set, and to address this deficit, we examined six pollination treatments of the selected plant species. field work was conducted, repeated and recorded from 2005 to 2007 in jordan. controlled pollinations were carried out in selected individual’s plant at the time of maximum stigma receptivity and anthesis. n. sativa flowers had anthesis intervals which last for five days, then followed by stigmatic receptivity which last for few hours. plants are pollinated trough outcrossing and complete selfing to insure the reproductive assurance. however, self-pollination was occurred due to style movement. the observations confirmed that a mixing mating including a combination of out-crossing and selfing is a better strategy than selfing alone. adv. hort. sci., 2011 25(4): 212-222 received for publication 22 february 2011 accepted for publication 13 november 2011 213 location. missing hills were replanted when necessary. the plants were thinned to two plants per hill when they were at twoto three-leaf stages. the two locations were kept weed-free by cultivation and hand weeding. the time of stigmatic receptivity was determined with the aid of a dissecting microscope. the direct test of receptivity was an assay that detects the presence of stigmatic peroxides. to determine receptivity, the stigmas were treated with hydrogen peroxide 3%: small air bubbles that form by maturation of the stigma indicate that the flower is in the female phase (dafni and maues, 1998). to determine receptive periods, 50 flower buds per plant of each species were marked, 10 flower buds of the same age were bagged a day before the opening of flowers during the anthesis period. on the following day, 10 flower buds were taken to the laboratory in order to check for stigma receptivity. the timing of anthesis was checked in the field using a hand magnifier. after bending, anther capsules were observed with the naked eye. the mechanism of pollen release is described based on direct observations in the field. any rupturing of the capsule causes pollen to release where it is verified by anther dehiscence. in order to observe pollinator visitation tour, the number of visits per bee was estimated by counting the number of visits with anther or stigma contact from the beginning of pollination to fertilization. the counts were conducted every 15 minutes for a period of eight hours on a daily basis during flowering period. controlled pollinations were carried out on selected individual plants at the time of maximum stigma receptivity and anthesis. pollinated flowers were observed periodically for fruit set. the reproductive success of the studied species was assessed by performing a spontaneous self pollination, manual self and cross pollination treatments. following the initiation of the first flower bud, flowers were selected randomly and tagged: 180 flower buds of n. sativa in each location. thirty flower buds were marked for each pollination treatment. pollination treatments were performed from february to march 2006 to determine the best pollination treatment in each locations. in order to conduct geitongamy and xenogamy pollinations, all stamen organs of each flower were removed using special scissors (emasculation). the flowers were pollinated using pollen from freshly dehisced anthers from male flowers (of the same plant) by using a fine brush for geitongamy pollination treatment and from another plant for xenogamy pollination treatment (cross pollination). the flowers were left exposed to any insect as occurs in nature for open pollination treatment. to test the bagged self pollination, flower buds, bagged till the end of pollination stage, were left untreated and uncovered again in order to avoid any negative impact on their germination. in order to check forced self pollination on the same hermaphrodite flower, flowers were bagged till the last day of the male stage. the flowers were pollinated using pollen from freshly dehisced anthers from male to female flowers on the same hermaphrodite flower by using a fine brush. with regard to emasculation, flower buds of nearly the same age were selected in order to remove male flowers to investigate the differences between the role of pollinator and the role of plant, by numbering of fruit set. the anthers were removed with a pair of tweezers and were left to pollinate by pollinator. if an emasculated flower sets fruit, then it must have received pollen from a pollinator. however, if an emasculated flower fails to set fruit, a pollinator will have had no role in fertilization. changes in the relative positions of anthers releasing pollen and the styles was also documented. a total of 30 flower buds were monitored during the study period using a hand magnifier. a single flower from this group was monitored from the morning to the end of the day. each flower was scored for the number of anthers on the flower, the number of anthers dehisced, the position of the dehisced anthers and the positions of anthers relative to the stigma. the length of anther and style were measured using a special caliber. representative photographs were taken of flowers at each stage. thirty marked flower buds were selected to count the number of ovules in order to determine the standard number of ovules in the stigma. the number of ovules per capsule were counted, averaged for both locations and the average number was used as a reference in the calculation. data were analyzed as complete randomized design with three replicates. comparisons between means were made using least significant difference (lsd) at 0.05 probabilities lend (spss). for statistical data, standard descriptive statistics were performed for each of the following quantitative parameters: the number of produced fruits, the number of seed for each stigma, the number of ovules, the number of chambers per capsule, the number of non fecundated seeds and the total number of fecundated seeds. mean number of buds and stigmas of plants, standard deviation, and differences between pollination treatments in terms of seed set per fruit were calculated. the statistical program package spss was used. insect visits were standardized by calculating the number of visits per flower per plant. these data were summarized over the season by taking an average of the observations. minimum and maximum value was observed and graphical analyses were applied. 3. results anthesis and receptivity styles are the first floral organ to emerge and extend, followed by extension of the stamens. when the style has almost straightened, the anthers began to dehisce. after the dehiscence of anthers about half an hour when it is considered as the first day for pollen shedding till fifth day, the male stage activated between 8:30 am to end of the day and anthers were sink down. the male 214 phase is initiated a few days before the stigmas become receptive and male stage lasted for five days. by the fifth day of the male stage, female stage started during this day, stigmatic peroxides tests indicate that receptivity occurred between 8:00-13:00 pm and for one day only. male and female stages synchronized in the last day of the flowering period (fig. 1). the weight of pollen was 0.064 mg/flower, whereas the volume of nectar was 0.13 µl. affluent floral rewards (both nectar and pollen) during the male phase of the flowers. fig. 1 blooming stages of nigella sativa, a) flowers opening, b) anthesis and c) receptivity stage. movements of stigma and anthers the male and female organs at bud stage are presented in (fig. 2 a) at onset of the male stage, all the stamens stand erect (fig. 2 b). they curve outwards one by one, roughly in whorls and strictly reflecting the order of initiation (fig. 2 c). when the anthers reach a horizontal position, the pollen is released (fig. 2 d). then, the stamens sink down. an anther takes 4-7 hours to empty its contents. the stamen movement is not continuous, but it is divided into three phases. in the first phase (12-14 hours) the lower part of the filament inclines slightly, while the upper part curves more strongly, so that the anther is brought into a horizontal position. after reaching this position, movement comes to a standstill. the second phase towards the ends of the male stage, the styles of the five carpels usually curve down (fig. 2 e) and twist (fig. 2 f). this ensures that in the female phase the stigmatic crests, whose bends were making an angle of 45°, continue to make a right angle with ovary to run down nearly the whole length of the style to touch the top of the anther at several points (fig. 2 f). the third stage, in which the stamen sinks down, is much shorter than the previous ones (4-6 hours). finally, the empty anthers curve up. this is a purely passive movement, apparently without any function. after uptaking the pollen, the stigma is pollinated (fig. 2 g), and then the stigma inclined upwardly erect as the order of initiation and makes an angle of 180° with the ovary (fig. 2 h). the maximum style length reached 1.73 cm, whereas the maximum anther length was 1.72 cm. this indicates the equal length of style and anther. pollination season one. location a. n. sativa’ flowers produced a non significant number of ovules under all treatments conditions with an average of 96±0.5, as shown in (table 1). generally, all flowers under the different treatments produced seeds (table 1). open pollinated flowers produced significantly higher seeds as compared with other treatments 74.9±1.4. hand cross, hand geitongamy and hand forced self ranked secondly in seed set and produced a nonsignificant differences between them with a seed set average of 82.9±1.6, 73.5±1.5 and 78.9±1.6 respectively. a non-fecundated seed production is also a common feature of n. sativa’ flowers under the different treatments. in the first location, open pollination occupied the lowest average of non-fecundated seeds all over other treatments (table 1). hand cross, hand geitongamy and hand forced self ranked secondly in producing a non-significant fecundated seeds with an average of 12±1.7, 21.6±1.6 and 18±1.4 respectively. there were significant differences (p≤0.05) in the percentage of seed set between treatments (table 1). seed set percentage after open pollination (86.8% seed) was significantly higher than all other treatments (p≤0.05). non significant differences were found between the average percentage of seed set when hand cross, hand geitongamy and hand forced self was used on flowers (79.8%, 75.4% and 81% respectively). location b. n. sativa’ flowers produced a non significant number of ovules under all treatments conditions with an average of 91.1±0.5, as shown in (table 2). generally, all flowers under the different treatments produced seeds (table 2). open pollinated flowers produced significantly higher seeds as compared with other treatments 82.9±1.5. hand cross, hand geitongamy and hand forced self ranked secondly in seed set and produced a nonsignificant differences between them with a seed set average of 72.4±1.4, 70±1.3, and 77.5±1.1 respectively for the first location. characteristics of producing fecundated seeds in the second location were fairly constant in value and regulated mainly by treatments conditions. a non-fecundated seed production table 1 seeds set after different pollination treatments in nigella sativa, location a. season one treatment of pollination no. of ovules/capsule no. of fecundated seed/capsule no. of non fecundated seed/capsule percentage of seed set/ capsule open 93.3±0.5 a 74.9±1.4 a 19.0±1.5 a 86.8±1.2 a hand cross 95.6±0.7 b 82.9±1.6 b 12.0±1.7 b 79.8±1.3 b hand geitonogamy 95.1±0.6 b 73.5±1.5 b 21.6±1.6 b 75.4±1.2 b hand forced self 96.0±0.5 b 78.9±1.6 b 18.0±1.4 b 81.0±1.4 b a and b are symbols related to difference in comparison. (a) (b) (c) 7:00 8:00 9:00 10:00 11:00 12:00 13:00 14:00 15:00 16:00 215 fig. 2 a) plant at bud stage; b) the stamens stand erect; c) first phase of stames movement: the stamens curve outwardly in whorls; d) pollens releasing; e) first phase of style movement: the styles of the usually five carpels curve down; f) twisting point of style with anther; g) the stigma is pollinated; h) the stigma inclined upwardly erect. a b c d e f g h 216 is also a common feature of n. sativa’ flowers under the different treatments. in the first location, open pollination occupied the lowest average of non-fecundated seeds (table 2) all over other treatments. hand cross, hand geitongamy and hand forced self ranked secondly in producing a non-significant fecundated seeds with an average of 18.7±1.3, 25±1.5 and 18.1±1, respectively. there were significant differences (p≤0.05) in the percentage of seed set between treatments (fig. 3). seed set percentage after open pollination (87% seed) was significantly higher than all other treatments (p≤0.05). non significant differences were found between the average percentage of seed set when cross, hand geitonogamy and hand forced self was used on flowers (79%, 73% and 80% respectively). fig. 3 nigella sativa seed set percentage upon pollination treatments in location a and b season one. season two. location a n. sativa’ flowers produced a non significant number of ovules under all treatments conditions with an average of 92.3±1.42, as shown in (table 3). generally, all flowers under the different treatments produced seeds (table 3). open pollinated flowers produced significantly higher seeds as compared with other treatments in both locations 83.4±0.67. hand cross, hand geitongamy and hand forced self ranked secondly in seed set and produced a non-significant differences between them with a seed set average of 74.6±0.68, 73.6±0.67, and 79.7±0.32 respectively for the first location. bagged self pollinated flowers ranked thirdly and produced 44.1±0.75 seeds. the lowest seed set was recorded in the case of emasculated flowers with an average seed production of 12.4±0.33. hand cross, hand geitongamy and hand forced self ranked second. a nonfecundated seed production is also a common feature of n. sativa’ flowers under the different treatments. in the first location, open pollination produced non-significant fecundated seeds with an average of 12±1.7, 21.6±1.6 and 18 ±1.4, respectively. there were significant differences (p≤ 0.05) in the percentage of seed set between treatments (fig. 4). seed set percentage after open pollination (87% seed) was significantly higher than all other treatments (p≤ 0.05). non significant differences were found between the average percentage of seed set when hand cross, hand geitongamy and hand forced self was used on flowers (79%, 78% and 83%, respectively). nearly half of the produced set seed in bagged flowers with an average of 47%. emasculated flowers (13%) recorded the lowest seed set from other treatments with significant difference. location b. n. sativa’ flowers produced a non significant number of ovules under all treatments conditions with an average of 97.2±1.67, as shown in (table 4). generally, table 2 seeds set after different pollination treatment in nigella sativa location b. season one treatment of pollination no. of ovules/capsule no. of fecundated seed/capsule no. of non fecundated seed/capsule percentage of seed set/capsule open 95.6±0.48 a 82.9±1.5 a 12.8± 1.5 a 87.0±1.3 a hand cross 91.1±0.50 b 72.4±1.3 b 18.7± 1.3 b 79.0±1.2 b hand geitonogamy 95.0±0.55 b 70.0±1.3 b 25.0±1.5 b 73.6±1.0 b hand forced self 96.0±0.60 b 77.5±1.1 b 18.1±1.0 b 80.0±0.99 b a and b are symbols related to difference in comparison. table 3 seeds set after different pollination treatment in nigella sativa location a. season two treatment of pollination no. of ovules/capsule no. of fecundated seed/capsule no. of non fecundated seed/capsule percentage of seed set/capsule open 96.8±2.19 a 83.4±0.67 a 13.4±1.93 d 87±1.67 a hand cross 96.0±2.31 a 74.6±0.68 b 21.4±2.39 c 79±1.98 b hand geitonogamy 93.0±2.81 a 73.6±0.67 b 21.1±1.81 c 78±1.46 b hand forced self 97.2±1.67 a 79.7±0.32 b 17.6±1.77 c 83±1.54 b bagged self 94.2±1.50 a 44.1±0.75 c 51.9±0.73 b 47±1.10 c emasculation 97.6±1.67 a 12.2±0.33 d 85.4±0.33 a 13±0.46 d a, b, c, and d are symbols related to difference in comparison. 217 all flowers under the different treatments produced seeds (table 4). open pollinated flowers produced significantly higher seeds as compared with other treatments in both locations 82.4±0.57. hand cross, hand geitongamy and hand forced self ranked secondly in seed set and produced nonsignificant differences between them with a seed set average of 71.8±0.57, 67.9±0.62, and 78.6±0.5, respectively. bagged self pollinated flowers ranked third and produced 43±0.74 seeds. the lowest seed set was recorded in the case of emasculated flowers with an average seed production of 12.4±0.5. open pollination occupied the lowest average of non-fecundated seeds 13.7±1.76 (table 4) all over other treatments. hand cross, hand geitongamy and hand forced self ranked second, a non fecundated seed production is also a common feature of n. sativa’ flowers under the different treatments in producing a non-significant fecundated seeds with an average of 20.2±1.57, 25.1±1.62 and 14.5±1.6, respectively. there were significant differences (p≤0.05) in the percentage of seed set between treatments (fig. 5). seed set percentage after open pollination (87% seed) was significantly higher than all other treatments (p≤0.05). non significant differences were found between the average percentage of seed set when hand cross, hand geitongamy and hand forced self was used on flowers (78%, 73% and 85% respectively). nearly half of the produced set seed in bagged flowers with an average of 46%. emasculated flowers (13%) recorded the lowest seed set from other treatments with significant difference. behavior of honey bee visitors during our observation, honey bees were the only visitor and pollinator that visited n. sativa in the morning around 7:00 a.m. every flower had one bee at least. each bee spent different time with an average of 12.5 s for nectar collecting, 8 s for pollen collectors. the only diurnal visitor and pollinator were honey bees. honey bees were frequent visitors to n. sativa in the jordan valley. the honey bee had same behavior in the two locations. in the evening, no pollinators were found in the flowers in both sites. the major pollinator was honey bees. n. sativa’ flowers’ mean visit rates for the three replicates in both locations were 14.9 and 14.6 daily visiting tours, respectively. the ultimate activity during the three replicates was approximately from 9:30 to 12:30 in both locations. 33% of the total bees observed were pollen collectors, while the rest 67% were nectar collectors (table 5). honey bees fig. 4 nigella sativa seed set percentage upon pollination treatments in both locations, season two. table 4 seeds set after different pollination treatments in nigella sativa location b. season two treatment of pollination no. of ovules/capsule no. of fecundated seed/capsule no. of non fecundated seed/capsule percentage of seed set/ capsule open 96.1±1.76 a 82.4±0.57 a 13.7±1.76 d 87±1.56 a hand cross 92.3±1.42 a 71.8±0.57 b 20.2±1.57 c 78±1.28 b hand geitonogamy 93.1±1.68 a 67.9±0.62 b 25.1±1.62 c 73±1.22 b hand forced self 93.1±1.53 a 78.6±0.50 b 14.5±1.60 c 85±1.39 b bagged self 93.6±1.50 a 43.0±0.74 c 50.6±1.71 b 46±1.10 c emasculation 94.9±1.9 0a 12.4±0.50 d 82.5±2.01 a 13±0.62 d a, b, c, and d; are symbols related to difference in comparison. table 5 behavior of honey bees and their bearings for nigella sativa behaviors of bees average spending time/flower (second) landing on departure of percent of bees according to their bearingspetals anthers twisting petals anthers twisting nectar collector were observed 12.5 • • 67% pollen collector were observed 8.0 • • 33% fig. 5 nigella sativa seed set percentage upon pollination treatments in both locations, season two. 218 visiting tours were conducted in two stages during five days. anthesis period took place in the first four days, and anthesis and receptivity periods were in the fifth day. visiting tours were for functional nectar collecting. on the first day, honey bees landed on petals and then collected nectar during circular stepping upon petals, without getting directly exposed to anthers. on the second till the fourth day, the same behavior occurred. the pollen grains fell down upon bees back from the horizontal anthers during circular motion. on the fifth day, receptivity period began, in which the styles inclined towards anthers, and then the styles twisted themselves around the anthers. honey bees were landing directly on this synapse (not on petals). after that, they left and flew to another flower. 4. conclusions the male phase is initiated a few days before the stigmas become receptive, where the anthesis duration remains for five days full flowering started with the appearance of bright blue petals. male stage started as the anthers started to shed their pollen, since the first day till fifth day, the male stage activated between 8:30 a.m. to the end of day. the viability of one anther remained during one day then started to sink down. it is interesting to point out that anthers remain active for five days, which leads to synchronize the receptivity period in the fifth day. because the flowering period for n. sativa coincides with good temperature in april in jordan, this may lead to an increase of the interval of anthesis since the pollen responds to temperature. it is surprising for pollen of n. sativa to continue for five days. another reason for this long period of anthesis is the large number of anthers in staminate. climatic factors affect the anthesis intervals in n. sativa, there is evidence that high temperature had a direct effect on pollen performance since the pollen responds to temperature. however, at the same time they are advantageous for the pollen by hastening its tube growth rate. on the other hand, low temperatures may act against the pollen by reducing its germination and growth rate, which could limit the fertilization success (thompson and liu, 1973; jakobsen and martens, 1994) the duration of stigmatic receptivity in nigella sativa was approximately hours in angiosperms, the stigma is the first female structure, the pollen grains and pollen tubes have to face on their way to the female gametophyte. the stigma provides an adequate environment for pollen grain germination (knox, 1984; helsop-harrison and shivanna, 1997). one of the most important features of stigmas is stigmatic receptivity, defined as the ability of the stigma to support pollen germination, which is a decisive stage in fertilization success and has a large variability among plant species (helsopharrison, 2000). at the end of the fifth day on the male stage, the female stage started to be active during 8:00 a.m. to 13:00 p.m. and then ended up. it is interesting to point out that the stigma of n. sativa is receptive throughout anthesis. inspite of the flowering period in april when we don’t have high temperature which may hurt the plant; the stigma receptive only for hours. the explanation for that is that the stigma is exposed in direct way to the sun which may increase the exposed area. in addition, the receptivity of stigma occurred after the stigma lost most of the anthers that surrounded the stigma so that the whole stigma is exposed to the sun which may also increase the exposed area to sun. that means high temperature affects stigma receptivity and reduces receptivity interval. there is evidence that ensures stigma responds to high temperature. high temperatures are detrimental for the female part by reducing the length of stigmatic receptivity and accelerating ovule degeneration (postweiler et al., 1985). it is well documented that the reproductive phase, especially from pollination to fertilization, is highly vulnerable to the prevailing environmental conditions including temperature (hall, 1992; stephenson et al., 1992). the duration of stigmatic receptivity is variable depending on the species, and it is also variable within genus. there is evidence that indicates duration of stigmatic receptivity is variable, that the duration of stigmatic receptivity is variable depending on the species and is usually greater in wind-pollinated than in insect-pollinated species (khadari et al., 1995). thus, the stigma can be receptive for not much more than an hour or so, as in avena or dactylis, to as long as several days, as in other grass species (pennisetum or zea) or eucalyptus in which it can remain receptive for more than a week, particularly in hostile environments (helsop-harrison, 2000). from an agricultural perspective, stigmatic receptivity has also a clear practical implication as it limits floral receptivity, the effective pollination period (guerrero-prieto et al., 1985) and hence fruit set (reviewed in sanzol and herrero, 2001). moreover, in an ecologist context, by altering stigmatic receptivity, flowering plants may influence the likelihood of fertilization by indirectly controlling the number and the quality of mating through the control of the number of pollen grains deposited and the time of germination (cruden et al., 1984; primack, 1985; galen et al., 1986). autonomous pollination first of all, i would like to define the autonomous phrase for the reader to understand. as lloyd, 1992 defines it: prior self pollination within-flower: self-pollination that occurs before the opportunity for outcross-pollen receipt for that flower has occurred, competing self pollination within-flower; self-pollination that occurs during the opportunity for outcross-pollen receipt for that flower has occurred, and delayed selfing pollination within-flower; self-pollination that occurs after the opportunity for outcross-pollen receipt for that flower has occurred. one of these three types of self pollination occurred in our research in n. sativa, which is delayed selfing pollination. automonous delayed selfing late in n. sativa flower’s life 219 is favored when honey bees service and thus outcross-pollen receipt is unpredictable. n. sativa flowers attract honey bees but they can also autonomously perform delayed self pollination, which provides reproductive assurance if pollinators fail to visit. the delayed self pollination occurred in our research because the synchronization between male and female occurred in the end of flowering period. i agreed with darwin (1877), muller (1883), baker (1955, 1965) and lloyd (1979, 1992) that pollinator absence or low pollinator abundance during some periods within or among flowering seasons favor shifts from outcrossing to autonomous self-fertilization because self-pollinated seeds provide reproductive assurance. some authors support the research result that absence of pollinators can shift to delayed self pollination; the extinction of pollinators or range expansion in a plant lineage can favor shifts to biotic modes of pollination, including wind pollination and autonomous self fertilization (baker, 1955; stebbins, 1957; regal, 1982; cox, 1991; weller et al., 1998). the results agreed with barrett and harder (1996), and ramsey and vaughton (1996) that pollinator scarcity and reduced pollinator services may result in high selfing rates. cross pollination and bagged self pollinations occur; approved by seed set achieved by all treatments applied on the research where bagged selfing and outcrossing boosted seed production means of 45% and 77% respectively. the results agreed with zohary (1983) as he found that n. sativa are capable of setting seed without being crosspollinated, but he didn’t mention the mechanism for such a result. the results also agreed with faegri and van der pijl (1971) who reported: there are a few flowers that can selfpollinate by their own, but this limits them to in breeding. the results agreed with goodwillie (1999) in believing the ability of self pollination to provide some insurance against pollination failure. in addition to the reproductive assurance benefits, prior selfing could be favored since it reduces the costs associated with the longer floral maintenance time required for outcrossing, and sets the stage for the evolution of reduced investment in cues for pollinators and the amount of pollen per flower. in contrast with early selfing, later-selfing species will retain floral traits and costs associated with outcrossing (i.e., cues to attract pollinators, pollinator rewards, and prolonging floral maintenance relative to prior selfing species. at one extreme, selfing early in a flower’s life (prior) is favored when a population requires pollinators are chronically absent (lloyd, 1992), or when population size is so low as to be undetectable by pollinators (lloyd, 1992; fausto et al., 2001; goodwillie, 2001), or when a population experiences high levels of interspecific pollen flow (fishman and wyatt, 1999). many authors are interested in common type of pollination as cross, open and self pollination, but through our research i have been devoted all our efforts to point out some thing out of traditional efforts such as delayed self pollination. thus, delayed selfing may be achieved by either a partial overlap in timing of male phase with female phase or changes in the relative position of anther and stigma during development. for example, delayed selfing in hibiscus laevis (klips and snow, 1979) and campanula species (faegri and van der pijl, 1979) is characterized by a progressive downward curling of the stigmatic area towards the style where anthers or pollen are located. conversely, in the protogynous aquilegia canadensis (eckhert and schaeffer, 1998) the stamens progressively elongate towards the exerted stigma. in kalmia latifolia (lyon, 1992), anthers collapse into the stigma on the final day of floral development; thereby achieving self pollination. others have found in self pollination late in floral life without changes in morphology. the breakdown of self incompatibility as the flower ages in both lilium and longifolium (ascher and peloquin, 1966) is attributed to degradation of the proteins that control self incompatibility and can be viewed as another form of delayed selfing. faegri and van der pijl (1971) used the term ‘’self pollination’’ or “autogamy” when pollination takes place within one flower (idiogamy), and “allogamy” or “cross pollination” when pollen from one flower is carried out to the stigma of another one. allogamy may further be divided into “geitonogamy” if the flowers are on the same plant and “xenogamy” if they are from different plants. however, it is that geitonogamy that has the ecological properties of cross-fertilizer but the genetic properties of self fertilization. thus, geitonogamy appears to be equivalent to autogamy (lloyd and schoen, 1992). style movement acts towards promoting self-pollination and leads nigella sativa to delayed self-pollination weber (1995) has produced a presentation film showing the pollination mechanism for nigella arvensis. he concisely presented the mechanism in written steps. the mechanism was demonstrating style movement in n. arvensis which exactly resembled our observations on style movement of n. sativa; through pictures shown above. i used his written description has quotation for its meaningful. i have measured the style and anther length and style twisting angle. hence the equal length of anther and stamen demonstrate the style twisting, whereas weber (1995) did not mention the length. weber (1995) mentioned that insects bear pollen on their thorax after touching the horizontal anthers. our observations showed that honey bees are landing on the horizontal anthers and twisting point of style and anther to bear pollen grains on their legs. so, how could insect carry pollen to another flower if the pollen is on their thorax. the beginning of receptivity caused a strong twist for stamens and style that leads to self pollination. this was observed as the end of male stage and the beginning of the receptivity stage. style movement acts towards promoting self-pollination as in n. sativa. in another plant, style movement leads to avoiding self-pollination and promoting cross-pollination as verma and magotra (2004) reported for eremurus himalaicus where they observed the mechanism of the stigma movement away from the dehiscing anthers, hence, it avoided receiving any left over pollen, and so self pollination is impossible. it is interesting to point out that n. sativa plant relies solely on animal vectors to move pollen among individuals, and if 220 pollinators are absent or in low numbers at certain times or years, individuals of n. sativa, that can self pollinate if not previously out crossed, will be at a selective advantage. this reproductive assurance process has been termed delayed selfing. n. sativa mixed mating is a better strategy than selfing alone mixed mating is a better strategy; that means open pollination system is better to seed setting than other pollination treatment. this open system leaves the plant exposed to biotic and abiotic factor. the plant will be without any restriction which may cause any reduction in seed setting. the open system includes the role of honey bees and role of plant to pollinate itself by delayed self pollinated flowers. the manual pollination, which included: hand cross, hand geitongamy and hand forced self, ranked second after open pollination, and this significant difference is attributed to human performance which is not like natural performance. excluding the biotic and abiotic factor from the plant by bagged self, that means plant will be restricted without honey bees, and the plant depends on itself to develop its style to reach the maximum length to catch the anthers in order to twist. in spite of the style movement towards the anthers, it gained half of the seed setting from open pollination, and this attributed to the fact that the style’s movement occurred once the stigma was receptive and at the final stage of anthesis when there is small number of anthers, and then sink down, this may not be enough to get high percent of seed setting as there isn’t enough quantity of pollen. honey bees are pollinator to n. sativa which is considered unattractive to wild bees the only diurnal visitor and pollinator were honey bees. honey bees frequently visited n. sativa in the jordan valley. the honey bee had similar behavior in the two locations. in the evening no pollinators were found in the flowers in both sites and seasons. flower visitors can only be considered pollinators if four pollination conditions have been met: pollen transfer to the vector is observed; pollen transport by the vector is observed, pollen transfer from vector to stigma is observed; and pollen deposited by the vector is shown to result in fertilization of the value (cox and knox, 1988). the flowers of n. sativa were unattractive to wild bees’ visitors. an important aspect used in many pollination studies is the number of visits made by a pollinator (proctor et al., 1996). apis mellifera engaged in pollen and nectar collection as a pollinator of n. sativa flowers with low frequency. the unattractively of n. sativa flowers to wilds bees may be attributed to several factors such as the presence of other floral resources. during our research, n. sativa flowering coincided with that of other species such as centurea syriaca and s. arevensis which are important for apiculture in jordan due to their abundant nectar and the large floral patches through out the area. the attractiveness of any species is a function such as favor, color, nectar volume, sugar concentration (frisch, 1967), and the bees fly to plant species that yield the greatest nectar and pollen (gary, 1979). the role of honey bees in the pollination of n. sativa is too small honey bees’ role as pollinator in fertilizing n. sativa flower buds was very small compared to the role of plant itself and the role of open natural conditions in pollination. the emasculated buds were let exposed to the pollinators in order to fulfill the pollination where it sets up 12% of seed formation percent, while the natural conditions and self pollination conditions gave 87% and 45%, respectively. it is necessary to ask whether the removal of stamens affected subsequent flower development, e.g. the growth of the perianths, a factor that would make it difficult to distinguish between the costs of stamens or pistils and the costs of structures associated with display and reward (andersson, 2003). such effects seem likely considering the work of andersson (2000), who detected a cost of producing and maintaining sepals and petals in a related species (n. degenii), and plack (1957), who found a negative effect of emasculation on corolla size in hermaphroditic plants of the gynodioecious glechoma hederacea (lamiaceae). in the present study of n. sativa, stamen removal caused significant reduction in the mean of seed set. the results agreed with andersson’s study (2003) where he observed the stamen removal produced reduction in total seed number. as a furthermore for n. sativa, andersson (2003) carried out removal of styles from n. sativa flowers and he found that; style-less plants initiated almost three times more flowers and invested 57% more biomass in stamens, than plants whose flowers were permitted to set fruit. he found also stamen-less plants produced significantly heavier seeds after hand-pollination. these observations indicate that stamens draw upon the same pool of resources as the other floral organs and that the removal of immature stamens therefore influences patterns of resource allocation. furthermore, andersson and jorgensen (2005) carried out removal of perianth from n. sativa flowers and found that; perianth removal produced 12.5% heavier seeds and allocated 15.8% more biomass to seed production than plants on which all perianths were left intact, whereas differences in flower production and total seed number were not significant. perianth removal did not significantly affect the proportion of seeds that germinated, but caused a shift toward earlier germination dates. the ultimate visitation rates for n. sativa flower in both locations was diurnal visitation type especially at early morning the ultimate visitation rates for flower in both locations were during 9:30 a.m. to 12:30 p.m., because the bees’ activity is limited by environmental factors; the radiation rate and the daily temperature. visitation rate was estimated by counting the number of visiting tours, those with anther or stigma contact. counts were made for one hour periods during (8) hours a day, while 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(suppl.), 9: 325-326. weller s.g., sakawi a.k., rankin a.e., golonka a., kutchure b., ashby k.e., 1998 dioecy and the evolution of pollination systems in schiedea and alsinidendron (caryophyllaceae: alsinoideae) in the hawaniian islands. american journal of botany, 85: 1377-1388. zargari a., 1990 medicinal plants. tehran university publications, tehran, fifth edition, vol. 1, pp. 43-44. zohary m., 1983 the genus nigella (ranunculaceae). a taxonomic revision. plant systematics and evoluation, 142: 71-107. impaginato 3 adv. hort. sci., 2023 37(1): 3­13 doi: 10.36253/ahsc­13902 fruit maturity and antioxidant activity affecting superficial scald development in ‘abate fétel’ pears a. bonora 1 (*), a. venturoli 1, 2, m. venturi 1, a. boini 1, l. corelli grappadelli 1 1 department of agricultural and food science, university of bologna, viale giuseppe fanin, 46, 40127 bologna, italy. 2 current affiliation: terremerse soc. coop., via cà del vento, 21, 48012 bagnacavallo (ra), italy. key words: antioxidant capacity, fruit quality, preharvest factors, pyrus commu­ nis, superficial scald, total phenolic content. abstract: superficial scald (ss) is one of the main physiological disorders affect­ ing postharvest of pears. its onset is linked to oxidative processes. antioxidant compounds such as ascorbic acid and phenolics could play a key role in pre­ venting ss. growing environment and fruit quality also have an influence on ss symptoms occurrence. the aim of this project is to understand the relationship between antioxidant activity, phenolic content, and development of ss in ‘abate fétel’ pear. moreover, the effect on ss of fruit maturity at harvest was assessed using multivariate statistical approach. data were collected in thirty orchards in the emilia­romagna region (italy) in three seasons (2018, 2019 and 2020), and the fruit were stored in a regular atmosphere for 120 days. antioxidant capacity was determined by 2,2­diphenyl­1­picrylhydrazy (dpph) method and total phenol content by folin­ciocalteau colorimetric protocol. the results showed that 340 mg of ascorbate/100 g of fw and 300 mg of gallic ac./100 g of fw at least provide good protection against ss. multivariate analy­ sis indicated that pulp firmness and index of absorbance difference (iad) seem to keep low the ss occurrence, when at harvest are higher than 6.3 kg and 1.9, respectively. in conclusion, it would be possible to build a forecasting model to control ss that considers pre­harvest data and content of antioxidants in differ­ ent orchards, to improve the postharvest management of ‘abate fétel’. 1. introduction superficial scald (ss) is one of the main physiological storage disorders of european pears (pyrus communis l.). ss is a skin disorder that appears as brown or black patches on the fruit. ss is considered a chilling injury which induces a damage and death within the surface layers of cells in localized regions (lurie and watkins, 2012). during ss development necrosis of the hypodermal cortical tissue seems to be induced by oxida­ tion products of the sesquiterpene (e, e)­α­farnesene (bain and mercer, 1963; rowan et al., 2001). α­farnesene, accumulates at a relatively high (*) corresponding author: a.bonora@unibo.it citation: bonora a., venturoli a., venturi m. boini a., corelli grappadelli l., 2023 ­ fruit maturity and antioxidant activity affecting superficial scald development of ‘abate fétel’ pears. ­ adv. hort. sci., 37(1): 3­13. copyright: © 2023 bonora a., venturoli a., venturi m. boini a., corelli grappadelli l. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 28 october 2022 accepted for publication 17 november 2022 ahs advances in horticultural science https://doi.org/10.36253/ahsc-13902 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2023 37(1): 3­13 4 level in the fruit peel during low­temperature storage (whitaker et al., 2009; yazdani et al., 2011; lu et al., 2013; calvo et al., 2015). the observation that ss could be inhibited by certain antioxidant treatments and low oxygen in the storage rooms atmosphere has provided evidence that development of the disorder was associated with oxidative processes (huelin and coggiola, 1970; whitaker, 2004; vanoli et al., 2015). thus, the conjugated trienols (ctols) that result from the oxidation of α­farnesene are assumed to play a causal role in the occurrence of ss (whitaker, 2007; giné bordonaba et al., 2013). nevertheless, it is gen­ erally accepted that the accumulation of both α­far­ nesene and ctols may be mediated by ethylene which is effectively correlated with ss development (bai et al., 2009; lu et al., 2013; xie et al., 2014; yazdani et al., 2011). therefore, it has been suggest­ ed that α­farnesene oxidations is a direct conse­ quence of free radical reactions occurring during chilling injury and α­farnesene is not always required for the induction of ss but rather in aggravating the symptoms in fruit already compromised by oxidative stress (rao et al., 1998; rupasinghe et al., 2000). in this context, it has been suggested that superficial scald mainly results from an imbalance between the fruit capacity to generate antioxidants and the reac­ tive oxygen species (ros) produced during cold stress ( a h n e t a l . , 2 0 0 7 ; g u e r r a e t a l . , 2 0 1 2 ; j u a n d bramlage, 2019). nevertheless, the antioxidant sys­ tem in fruit includes an enzymatic and a non­enzy­ matic component that play an important role modu­ lating oxidative damage to cell walls (ahn et al., 2007; lurie and watkins, 2012; li et al., 2016). furthermore, non­enzymatic antioxidants can pre­ vent oxidation­linked damages responsible for super­ ficial scald through biosynthesis of phenolics that are involved in protective redox­linked pathways under cold stress (larrigaudière et al., 2016; sarkar et al., 2018). the nonenzymatic scavengers of reactive oxy­ gen species include low molecular mass antioxidants with high­reducing potentials, such as ascorbic acid (aa) and glutathione (gsh). ascorbic acid acts as an antioxidant compound since it can protect fruit mem­ branes from lipid peroxidation (shewfelt and del rosario, 2000) and acts against reactive o2 species in concert with α­tocopherol (jimenez et al., 1997). nevertheless, aa tends to decrease during storage and processing of fruit and vegetables (haffner et al., 1997). a relationship was found between aa content and the susceptibility to browning during experimen­ tal storage under various brown core­inducing condi­ tions (pintó et al., 2001). in pears the antioxidant capacity is well explained by phenolics content (galvis sánchez et al., 2003). several studies have demonstrated that these compounds are associated with resistance to ss development in apples and pears (ju et al., 1996; zhao et al., 2016). phenolic compounds are particularly sensitive to storage fac­ tors such as controlled atmosphere (amiot et al., 1 9 9 3 ) . v a r i a b i l i t y o f p h e n o l i c s i n p l a n t ti s s u e s depends on many pre­harvest factors, such fruit maturity and environmental conditions, including temperature, uv light, and nutrition (markham et al., 1998; rivero et al., 2001; rühmann et al., 2002). casero et al. (2004) used the partial least squares regressions (pls), a multivariate technique, and found correlations between fruit quality attributes, such as fruit acidity and firmness, and storage disor­ ders with nutrients such as calcium, potassium and phosphorus, both in the leaf and fruit. moreover, pca biplots were helpful in showing the segregation between ss classes and their associations with the various physicochemical attributes (cronje et al., 2015). in pear, pulp firmness is one of the most rele­ vant quality parameters (saquet, 2019). softer fruit had rounder cells separated by larger intercellular spaces than firmer fruit. on the other hand, firmer fruit have smaller cells with less interspace which means denser tissues and longer storage than soft fruit (johnston et al., 2002). moreover, the da­ meter, a handheld device that measures chlorophyll concentration several millimetres into the flesh of fruit providing the index of absorbance difference (iad) (ziosi et al., 2008), can discriminate the ripening stage of climacteric fruit for postharvest tailored cold storage (bonora et al., 2013; gagliardi et al., 2014; sadar and zanella, 2019). fruit ripeness is also well predicted by starch degradation using a multivariate s t a ti s ti c a l a p p r o a c h ( z u d e ­ s a s s e e t a l . , 2 0 0 2 ) . conversely, in ‘abate fétel’ pear fruit the starch index is not always employed even if some studies have reported the use of this procedure to predict pear storability and postharvest issues (kingston, 1992; le lezec and belouin, 1994; agar et al., 1999; calvo et al., 2011). in pears starch pattern degrada­ tion can be influenced by environmental and man­ agement factors such as temperatures, harvest date and deficit irrigation affecting the kinetics of starch accumulation and degradation (watkins et al., 1982; kramer, 1983; lopez et al., 2013; lindo­garcía et al., 2019). total sugar content is an internal fruit quality trait that is crucial for consumer acceptance (osorio bonora et al. ‐ maturity and antioxidants affecting ‘abate fétel’ storage 5 and fernie, 2014). total soluble solids in ‘abate fétel’ and ‘forelle’ pear are mainly fructose, glucose and sucrose (mesa et al., 2016), and they increase in con­ centration after storage since starch is converted via hydrolysis into sugars over time (visser et al., 1968; crouch and huysamer, 2011; rizzolo et al., 2015). additionally, sorbitol accumulates in the fruit still attached to the tree (mesa et al., 2016), acting as cry­ oprotectant in cellular structures during cold storage by preventing dehydration of membranes and pro­ t e i n s t h r o u g h a n o s m o ti c a d j u s t m e n t p r o c e s s (busatto et al., 2018). therefore, the aim of this work was to research relations between antioxidant activi­ ty, phenolic content, and ss development on ‘abate fétel’ pears. furthermore, preharvest maturity and non­destructive postharvest quality parameters, as well as antioxidant activity and phenolic content, influencing the occurrence of superficial scald using multivariate analysis and regression trees were inves­ tigated to develop new reliable hypotheses of their effects in ss development, without compromising consumer acceptance and nutritive value. 2. materials and methods fruit material and superficial scald evaluation fruit were harvested during three consecutive seasons (2018, 2019 and 2020) from different ‘abate fétel’ orchards located in the emilia­romagna region, italy. fruit from 30 and 23 farmers were col­ lected and their maturity assessed in 2018 and in seasons 2019 and 2020, respectively. the farmers were indicated by three digit­numbers. in all seasons, two orchards with historical higher ss and two with lower ss were subjected to biochemical analysis at harvest and during storage. in 2018, eighteen 15 kg boxes for each farm were placed in a regular atmo­ sphere (0.5°c and >90% of relative humidity ­ rh). after 3 (t1), 4 (t2), and 5 months (t3) of storage, the room was opened, following the calendar normally applied by the company. in 2019 and 2020 only six 15 kg boxes per orchard were harvested and placed with a regular atmosphere in a cold room which was opened after 4 months (t2). afterwards, the pres­ ence of superficial scald was assessed in 30 fruits per farm. we defined four classes depending on the severity of symptoms in the skin of pears: class 0 where there was no peel browning, class 1 from 0% to 25% fruit peel showing ss, class 2 from 25% to 50% ss, and class 3 over 50% ss after shelf life. a ss index was computed as follows (bonora et al., 2021): 4 ss index = ∑ (index level) x (fruit at this level) 0 total number of fruit analysis of the physical characteristics in all seasons, 30 fruits per orchard at harvest (t0) were subjected to qualitative analysis such as fruit size, index of absorbance difference (iad), pulp firm­ ness, soluble solid content and starch content. moreover, non­destructive fruit quality such as size and iad after 4 months (t2) of cold storage were con­ sidered. weight and dimensions (diameter and height) of each fruit were measured with an auto­ matic caliper (s_cal work, sylvac, switzerland) and an electronic balance (kb 1200­2n, kern, germany) connected to a notebook. individual fruit ripeness expressed as iad was measured with the da­meter 53500 (sinteleia, bologna, italy) on the fruit side most exposed and less exposed to the sun. individual fruit flesh firmness (fff) was determined by fta (fruit texture analyser, güss instruments, strand, western cape, south africa) fitted with an 8 mm diameter tip, after removing the fruit peel from opposite sides at 180°. the mean value of fruit ripeness and firmness, from the two sides, was calcu­ lated. soluble solid concentration (ssc;°brix) was determined by measuring the refractive index of the juice for each fruit with a digital refractometer (pal­ 1, atago). the stage of starch hydrolysis was deter­ mined by dipping half­cut pears into a lugol solution and scoring the fruit according to the ctifl­eurofru scale (1­10; 1 = minimum, 10 = maximum starch hydrolysis) (planton, 1995). finally, at harvest (t0) and during storage (t1, t2, t3) pieces of the same size with pulp and peel of fruit from all the orchards in 2018 and from four representative farmers in 2019 and 2020 were frozen in liquid nitrogen and stored at ­80°c. these plant materials have been used for quantification of antioxidant activity and total pheno­ lic content. quantification of antioxidant activity to estimate the antioxidant activity, the 2,2­ diphenyl­1­picrylhydrazy (dpph) method was used (adapted from brand­williams et al., 1995). the dpph working solution was prepared in 70% acetone (v/v), with a final concentration of 0.02 mg/ml (w/v) and stored at 4°c until needed. afterwards, antioxi­ dant compounds from 0.5 g of pear (flesh and peel) were extracted in 10 ml of acetone 70%. the frozen adv. hort. sci., 2023 37(1): 3­13 6 material (0.5 g of pear) was homogenised in a ultraturrax (ika t25 digital ultra­turrax) with 10 ml of extraction solution (acetone 70%) for 2 min­ utes on ice. after vortexing, the tubes were sonicated in a bath­type sonicator for 15­20 minutes and the homogenates were centrifuged at 1,500 x g for 20 minutes at 5°c. fruit extracts (0.1 ml) were allowed to react with 3.9 ml of the dpph solution for 30 min­ utes in the dark, and the absorbance at 515 nm by uv­vis spectrophotometer (libra s80pc vbw uv/vis, biochrom), was measured. the dpph working solu­ tion was considered as the blank and the calibration curve was made using ascorbic acid. total phenolic content phenolic compounds quantification was per­ formed using the folin­ciocalteau colorimetric method (adapted from vieira et al., 2009). total phe­ nolics from 0.5 g of pear (flesh and peel) were extracted in 10 ml of 70% acetone. the frozen mate­ rial (0.5 g of pear) was homogenised in a ultraturrax (ika t25 digital ultra­turrax) with 10 ml of extrac­ tion solution (acetone 70%) for 2 minutes on ice. after vortexing, the tubes were sonicated in a bath­ type sonicator for 15­20 minutes. the homogenates were centrifuged at 1,500 x g for 20 minutes at 5°c. 250 μl of supernatant were added to 2 ml of deion­ ized water and 250 μl of folin reagent. after mixing, samples were incubated for 5 min and 5 ml of sodi­ um carbonate (na2co3) and 5 ml of distilled water were added. following 1 h incubation in the dark, absorbance was measured at 750 nm by uv­vis spec­ trophotometer (libra s80pc vbw uv/vis, biochrom). the phenolic concentrations were determined using gallic acid as a standard. data treatment and statistical analysis all the results of antioxidants and phenolics were s t a ti s ti c a l l y e v a l u a t e d b y a n a l y s i s o f v a r i a n c e (anova). furthermore, these data were presented considering four key producers at harvest (t0), after 3 (t1), 4 (t2) and 5 months (t3) of regular air storage. these producers were selected according to the inci­ dence of ss: two had a high incidence of ss (131 and 432) and the others had a low development of ss (272 and 351). moreover, the fruit quality data were subjected to multivariate analysis to highlight which among the factors considered appears to be more related to the onset of superficial scald. multivariate statistical analyses, such as canonical correspon­ dence analysis (cca) and recursive partitioning and regression trees (rpart) analysis, were performed using the statistical software r (r core team, 2020), by addition of packages “vegan” (oksanen et al., 2019) and “rpart” (therneau and atkinson, 2019). cca was used to estimate the interactions between the frequencies of ss classes and the numeric vari­ ables. the blue vector indicates the increase of the factors in a certain direction (ss class). finally, we considered the total variability explained by two components (cca1 and cca2) and how each variable a ff e c t s t h e fi r s t a n d t h e s e c o n d c o m p o n e n t . therefore, maturity data at harvest and ss after 4 months in all seasons were considered to elaborate the overall picture. finally, rpart analysis was applied to detect which factors could contribute more to ss and to understand their thresholds. green and red lights indicate a decrease or an increase in ss index, respectively. 3. results in 2018 antioxidant capacity in fruit during stor­ age decreased significantly (fig. 1 and table 1). regarding phenolic compound content in fruit of dif­ ferent producers, the differences were not statistical­ ly significant at harvest and during conservation (table 1). this can be explained looking at the differ­ ent producers’ behaviour (fig. 2). indeed, two differ­ ent trends can be observed during the first 3 months of storage: in 272 and 351 phenols tend to increase, while in 131 and 432 they decrease. thereafter, phe­ nols in 131, 432 and 351 increase from t1 to t2 fig. 1 ­ evolution of antioxidant capacity in season 2018 (mg ascorbic acid/100 g of fresh fruit) of four farmers (131, 272, 351, 432) and their average trend. bars represent standard error of the mean (±sem). points followed by the same letter in every sampling point are not signifi­ cantly different from each other. mean separation by lsd test (p≤0.05). bonora et al. ‐ maturity and antioxidants affecting ‘abate fétel’ storage 7 before decreasing notably again. on the other hand, in 272 we note only a slightly decrease from t1 to t2. in our study there is a clear distinction between t1, t2 and t3 in terms of ss occurrence in the first season (table 1). in addition, figures 3, 4, and 5 con­ firms the great variability of the incidence of ss among the different producers in t2 in all seasons. the evolution of ss index in 2018 of the 30 producers is also shown in table 1. at t1 the index is low while there is a considerable increase of ss incidence at t2 and at t3, while antioxidants decrease significantly. among the key producers of the first season in figure 3, two farmers had a higher ss index (131, 432), while two producers had a lower ss index (272, 351). in detail, the results show that the producers with the lowest ss (351, 272) are those in which phenols increase during the first three months of storage (fig. 2). therefore, has been hypothesized that fruit were able to initially react and use these substances to p r o t e c t t h e m s e l v e s f r o m o x i d a ti v e s t r e s s . epochs ss index antioxidant capacity (mg ascorbic acid/100 g of fresh fruit) total phenolic content (mg gallic acid/100 g of fresh fruit) t0 mean / 480.92 a 281.99 sem / 19.04 15.91 t1 mean 5.89 b 370.40 b 312.62 sem 0.80 11.62 17.85 t2 mean 35.46 a 300.38 c 299.37 sem 2.52 7.85 12.90 t3 mean 43.08 a 264.41 c 263.16 sd (%) 2.66 13.05 18.95 significance (p<0.05) *** *** ns levene test ns ns ns table 1 ­ mean and standard error of the mean (sem) of ss index, antioxidant capacity, total phenolic content in season 2018 at harvest (t0), after 3 months (t1), 4 months (t2), 5 months (t3) in cold storage data represent the average of fruit quality of 30 producers between epochs for each variable. values followed by the same letter in colu­ mns are not significantly different from each other. means separation by lsd test (p<0.05). *** significant at p≤0.001; ns = not significant. fig. 2 ­ evolution of total phenolic content in season 2018 (mg gallic acid/100 g of fresh fruit) of four selected farms (131, 272, 351, 432) and their average trend. bars repre­ sent standard error of the mean (±sem). values followed by the same letter in every sampling point are not signifi­ cantly different from each other. mean separation by lsd test (p≤0.05). fig. 3 ­ evolution of superficial ss index of four selected farms (131, 272, 351, 432) and their average trend during stor­ age in 2018. bars represent standard error of the mean (±sem). values followed by the same letter in every sam­ pling point after harvest are not significantly different from each other. mean separation by lsd test (p≤0.05). 8 adv. hort. sci., 2023 37(1): 3­13 particularly, 351 accumulated phenols till 4 moths which drop from t2 to t3 even below 431, probably, consuming their reducing power instead of antioxi­ dants avoiding polyphenol oxidase activity and browning. on the other hand, the producers (131, 432) with the greatest ss are those in which the phe­ nols drop during the first three months of storage, even if they rise again in the following months (fig. 2). probably, the damage caused by oxidative stress is already underway. notably, we found a drastic decrease of antioxidants between t1 and t2 in pro­ ducer 272, even if denoted the highest initial antioxi­ dant values at harvest (fig. 1). nevertheless, 272 had a low incidence of ss and this could be explained by the fact that during the first three months the antiox­ idants were high, and phenols increase reaching and keeping a certain threshold value till t3. weather and physiological factors in the second and the third season appear to also influence the average nonenzymatic scavengers’ level and the ss occurrence (fig. 4 and fig. 5). thus, we found a gen­ eral high presence of antioxidants and low ss in 2019, characterized by a rainy and cold season. on the contrary, the protective compounds decreased, and ss increased in all producers in 2020 when the temperatures and yields were higher. moreover, the data shows that antioxidants drop in the first three months of storage in all the four producers consid­ ered (fig. 4 and fig. 5). however, in both seasons the incidence of ss in producers 131 and 432 was higher when the antioxidants decrease drastically after 3 months of cold storage, regardless of the level at har­ vest. in figure 6 and figure 7, cca and rpart analysis are applied to study the effects of maturity of ‘abate fétel’ pear at harvest and during storage against ss development at t2 during three consecutive seasons (2018, 2019 and 2020). the multivariate model fig. 4 ­ evolution of antioxidant capacity (mg ascorbic acid/100 g of fresh fruit) and ss index after 4 months of cold storage (t2) in seasons 2019 of four farmers (131, 272, 351, 432) and their average trend. bars represent standard error of the mean (±sem). values followed by the same letter between four producers are not significantly different from each other considering dpph values at t0 and t1 or ss index during storage. mean separation by lsd test (p ≤ 0.05). fig. 5 ­ evolution of antioxidant capacity (mg ascorbic acid/100 g of fresh fruit) and ss index after 4 months (t2) of cold storage in seasons 2020 of four farmers (131, 272, 351, 432) and their average trend. bars represent standard error of the mean (±sem). values followed by the same letter between four producers are not significantly differ­ ent from each other considering dpph values at t0 and t1 or ss index during storage. mean separation by lsd test (p≤0.05). fig. 6 ­ canonical correlation analysis (cca) of superficial scald classes in ‘abate fétel’ pear after 4 months of cold stor­ age (clas0 0%, clas1 1%­25%, clas2 26­50%, and clas3 51­ 100% of peel symptoms) against qualitative orchard fea­ tures at harvest during three seasons 2018, 2019 and 2020 (blue vectors) and the scores of producers (black circles). total variability explained (53%): cca1 (90%); cca2 (8%). the following abbreviations have been used: weight of the fruit at harvest (sizehrv), weight of the fruit after 4 months of cold storage (sizet2sl), pulp firm­ ness at harvest (firmhrv), soluble solid content at har­ vest (brixhrv), iad­meter values at harvest (iadhrv), iad values after 4 months of cold storage (iadt2sl), starch pattern index at harvest (spihrv). bonora et al. ‐ maturity and antioxidants affecting ‘abate fétel’ storage 9 explains 27% of the observed ss variability (cca1 89% and cca2 7%). in our study we found that flesh firmness at harvest can prevent ss after cold storage considering all seasons and its contribution to com­ ponent 1 is 0,95 against ss (fig. 6). the orchards (23%) with pulp firmness at harvest higher than 6.3 kg developed low ss (7.2 ss index), while the ss index increased three times in the farms (67%) which, at harvest, scored less than 6.1 kg of firmness (fig. 7). however, in figure 6 we noted that bigger fruit at harvest and after storage are more prone to ss (its contribution to principal component is 0.11 at harvest and 0.40 after storage towards ss). in our research starch content at harvest in different pro­ ducers and seasons influences ss during cold storage with an important contribution to component 1 and component 2 (0.43 and 0.51 respectively towards class 3 after 4 months). the non­destructive iad­ meter values also contribute to preventing ss (fig. 6), although its contribution to component 1 is lower than firmness and spi (0.30 and 0.25, at harvest and during storage respectively against ss). furthermore, in figure 7 we found a specific value of iad which con­ tributed to ss occurrence for three consecutive years. among the farms which scored firmness value lower than 6.1 (67%), a fraction (13%) with iad higher than 1.9 developed an average ss index of 17. the 54% with firmness and iad lower than 6.1 and 1.9 respectively denoted a ss index higher than 33. moreover, we found that °brix promotes resistance to ss during storage of ‘abate fétel’ pears in emilia romagna (fig. 6) and its contribution to component 1 is remarkable (0.20 against ss). 4. discussion and conclusions as shown in our research, several studies confirm that antioxidant capacity, in particular ascorbic acid, drops during storage (lee and kader, 2000; franck et al., 2003), promoting a variable ss development in pear between orchards located in different environ­ ment (bonora et al., 2021). indeed, silva et al. (2010) reported that storage reduced differences in antioxi­ dant capacity between producers at harvest. about phenolic content, fruit may react and produce more phenols when stored for few months. this behaviour is reported in apples by leja et al. (2003) who showed that phenolic compounds are synthesised during storage. moreover, calvo et al. (2015) high­ lighted that in addition to the initial value of antioxi­ dants, it is important the level of protective com­ pounds be maintained. regarding quality factors affecting ss, wang and arzani (2019) also reported a good and negative cor­ relation between high flesh firmness at harvest and ss development in ‘d’anjou’ pears. nevertheless, fruit with a high flesh firmness are more unripe (stow, 1988) and more prone to contain less antioxi­ dants (kaur et al., 2021). furthermore, larger fruit generally ripe faster and are characterised by lower firmness and dry matter after storage, by probably increased respiration rate, oxidative stress, and water loss as consequence (gwanpua et al., 2013). accelerated senescence, and increased susceptibility to chilling injury have been reported to result from weight loss (prange and wright, 2023). on the other hand, the higher surface­volume ratio of larger fruit seems to prevent ss by a reduced evapotranspiration and weight loss during storage (pasquariello et al., 2013). although stow (1988) described starch pattern index as an unreliable method to determine opti­ mum harvesting date of pears, szczesniak and ilker (1988) reported that parameters influencing storabil­ ity and fruit textural characteristics of ‘forelle’ pears include the starch content. in contrast with our study, the incidence of superficial scald in apple fig. 7 ­ recursive partitioning and regression tree (rpart) analy­ sis, correlation between quality factor and scald index. t o w a r d s g r e e n p o i n t h y p o t h e s i s ( f i r m h r v ≥ 6 . 1 ; firmhrv≥6.3; iadhrv≥1.9) is confirmed, to red point is not satisfied. numbers in the circle represent scald index and the percentage of producer that are included in that value of scald index. the colour of the boxes represents the severity of ss: low ss (scald index: 0­15; most fruits do not show ss or show slight symptoms), medium to severe ss (scald index: 16­30; occurrence of progressive­ ly more severe symptoms), severe ss (scald index: >30; most fruit show severe symptoms and other very severe symptoms). the following abbreviations have been used: pulp firmness at harvest (firmhrv), index of absorbance difference at harvest (iadhrv). adv. hort. sci., 2023 37(1): 3­13 10 declines when the starch pattern index advances ( w a t k i n s e t a l . , 1 9 8 2 ; m d i t s h w a e t a l . , 2 0 1 5 ) . concerning iad meter values, a three­year study by delong et al. (2014) to develop optimal harvest time for ‘honeycrisp’ in nova scotia (canada) led to fruit with a low incidence of disorders after 3 months of storage. indeed, ‘abate fétel’ pears with higher iad values at harvest ripen less over 6 months of cold air storage (rudell et al., 2017). in fact, the content of primary photoassimilates certainly supports the pro­ duction of secondary metabolites such as antioxi­ dants (mellidou et al., 2021). to conclude, the development of ss seems to be the consequence of the occurrence of many quality and biochemical traits. therefore, it is important to highlight that it is not possible to consider only one variable at a time to find a solution in pears. we explored the possibility to use multivariate analyses to help understand the relationships between all the factors that may influence ss. antioxidant capacity is essential in ‘abate fétel’ pear to prevent ss occur­ rence. moreover, good pulp firmness, increased iad values, high total soluble solids and low starch degra­ dation at harvest seems to have a positive impact on ss development. furthermore, rpart analysis of fruit maturity at harvest confirms the importance of reaching threshold values, as indicators of potential fruit susceptibility to ss during storage, in addition to t h e a b s o l u t e t r e n d s i n m u l ti v a r i a t e a n a l y s i s . therefore, pre­harvest quality and antioxidant values at harvest can be compared with threshold values to discriminate batches of fruit based on their potential to develop ss symptoms. however, it is important to consider that for application purposes it would be necessary to develop faster systems for the quantifi­ cation of fruit maturity and antioxidant capacity at harvest in the orchards or during storage, using reli­ able, non­destructive methods. accordingly, the fruit industry may consider a predictive software to help manage the storage, minimising ss in pears and improving cold room fulfilment and energy efficiency, 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characterize the progression of ripening in peach fruit ‐ postharvest biol. technol., 49: 319­329. z u d e ­ s a s s e m . , t r u p p e l i . , h e r o l d b . , 2 0 0 2 ­ a n approach to non‐destructive apple fruit chlorophyll determination. ‐ postharvest biol. technol., 25(2): 123­ 133. 151 1. introduction summer pruning is a fairly broad term comprising a set of practices performed on the canopy during the growing season with an array of aims, including regulation of size, vigour and crop and reduction of the susceptibility to biotic and abiotic stress. if it is considered that at least two such operations, e.g. selective shoot and cluster thinning, still require manual execution, the total amount of necessary seasonal labour, calculated as man × hr/ha, readily exceeds the demand for winter pruning and becomes a primary determinant of vineyard economics (intrieri and poni, 1995). while it is commonly heard that the ‘perfect’ vineyard needs no summer pruning, perfect in reality has proved to be a very rare occurrence. yet, we should certainly like to see vineyards of the future moving towards a more focused application of summer pruning operations. the major change is that a given summer cut is not solely or exclusively seen as something the grower “has to do”, say, to accommodate adjustments for excessive shoot growth or canopy density. rather it should also be viewed as something that the grower may ‘use’ to head vine and cluster growth towards better grape composition or to specific features consonant with adjustments needed because of climate change. along with traditional summer pruning operations, which define the grapevine canopy management strategy and include cluster and shoot thinning, shoot positioning and hedging, elimination of lateral shoots and late season basal leaf removal, over the last few years innovative summer techniques such as pre-flowering leaf removal (poni et al., 2006; intrieri et al., 2008; poni et al., 2008; diago et al., 2010 a; palliotti et al., 2011 b) or early and late season anti-transpirant sprays (palliotti et al., 2010 and 2011 a) have been introduced. these latter management practices are useful in any situation where the main aims are to reduce the vine yield and improve both technological and phenolic maturation. moreover, global warming is leading to a progressive shift toward sub-tropicalization of several viticulture areas, shorter time intervals between phenological stages (schultz, 2000; jones et al., 2005) as well as increased probability for berry sunburn (spayd et al., traditional and innovative summer pruning techniques for vineyard management a. palliotti*, s. poni** * dipartimento di scienze agrarie e ambientali, università degli studi di perugia, borgo xx giugno, 74, 06121 perugia, italy. ** istituto di frutti-viticoltura, università cattolica del sacro cuore, via emilia parmense, 84, 29100 piacenza, italy. key words: cluster thinning, grape composition, leaf removal, shoot hedging, shoot thinning, vegetative growth. abstract: this review paper highlights physiological and vine performance effects of widely adopted summer pruning operations such as leaf removal, shoot trimming and positioning and cluster thinning. leaf removal is addressed either under its traditional configuration, i.e. removing in dense canopies some or all leaves around clusters usually pre-veraison to improve fruit microclimate and facilitate spraying and early (pre-flowering) defoliation primarily aimed at inducing looser clusters via a concurrent reduction of fruit-set and berry size. time consuming and still non mechanisable cluster thinning is evaluated primarily in terms of response variability vs. season and intensity with emphasis on lack of significant reduction of final yield per vine in thinned treatments when large crop compensation occurs. variability of expected final grape composition improvements in thinned vines is also discussed based on the actual vine balance when the operation is performed. although fully mechanisable, shoot trimming is still a debated choice in terms of timing and severity. while severe (i.e. fewer than six or seven main leaves retained) and late (i.e. several weeks after bloom) cuts should possibly be avoided, the effects of shoot trimming on final grape composition is discussed as a function of seasonal changes in leaf area development, demography, fraction of lateral leaves from the total and leaf to fruit ratio. it is indicated that, for vertically shoot-positioned trellises, if the support trellising is correctly designed and vine vigour is balanced, timing and severity of trimming are dictated by the vine “itself” rather than by grower choices. overall, this review underscores the importance of leading the vineyard to a “natural” control of vegetative growth, which would minimise the need for an extensive use of summer pruning. in other words, such vineyard operations should be viewed not just as something the growers “have to do”, instead as specific tools used to achieve targeted final grape composition. adv. hort. sci., 2011 25(3): 151-163 received for publication 9 may 2011 accepted for publication 6 june 2011 152 2002; tarara and spayd, 2005; greer et al., 2006). finally, clear evidence does exist for faster ripening leading to significant increases in grape sugar concentration at harvest (dokoozlian, 2009). 2. leaf removal this operation has been historically defined as “the removal of some leaves from the fruiting area between fruit set and veraison” (smart, 1973) with the prevailing aim to ameliorate bunch microclimate and reduce rot incidence in canopies that are too dense (gubler et al., 1991). ongoing research has provided knowledge to distinguish two types of leaf removal aimed at quite distinct goals. traditional leaf removal although this practice may have different purposes, it is usually employed from fruit set to veraison on high-density canopies to improve light exposure and air circulation around the clusters, with substantial benefits in terms of pigmentation and tolerance to rot (smart, 1985; bledsoe et al., 1988; gubler et al., 1991; percival et al., 1994; reynolds et al., 1996). this operation can be done manually, requiring up to about 60 hr/ha, although increasing labour costs nowadays strongly advise a mechanical approach which can be easily performed in less than 2 hr/ha. the best timing for machine use is about one to two weeks prior to veraison when berries are still hard while specific bunch weight is already much higher than that of leaves. yield may not change (bledsoe et al., 1988; smith et al., 1988; hunter et al., 1995) or might even occasionally increase as compared with non-defoliated vines (zoecklein et al., 1992). the variability of the impact that leaf removal has on yield and their components is likely dependent upon the negative effects on fruit set and berry growth in the current year and positive effects on bud induction and differentiation for the next year’s crop via an improvement in canopy microclimate. although this type of leaf removal usually leads to undeniable improvements in fruit composition, which more frequently are a slight increase in sugars and ripe fruit characters and a decreased malic acid content and attenuated herbaceous and grassy wine characters (smart, 1985; reynolds et al., 1996; zoechlein et al., 1992; scheiner et al., 2010), its popularity has probably decreased over the last two decades due to either advancement in leaf and whole-canopy physiology and new pressure from global warming. a study from petrie et al. (2003) found that leaf removal from the lower quarter of the canopy during the lag phase of berry growth caused a significant decrease of whole-vine photosynthesis, even on a per-unit leaf area basis, thus suggesting that the lower portion of the canopy contributed more than the upper portion to the whole-vine carbon budget. a possible explanation of this finding is that although basal, and hence older leaves are removed by defoliation, they are also the largest leaves along the shoot and their size can offset lower photosynthetic rates (poni et al., 1994). therefore, lowering shoot photosynthesis might not be negligible especially for leaf removals performed after fruit set. removal of all the leaves from the fruiting area, which thereby exposes the clusters to full sun, might lead in warm climates to compromised fruit composition because of excessive berry temperatures, which can hinder colour formation and cause a sharp drop in malic acid concentrations (spayd et al., 2002; tarara et al., 2008). for such reasons and in association with increasing concern for berry sunburn, criteria for applying leaf removal have become more restrictive and more often conceive retaining some leaf cover around the fruiting area. differentiation in the actual need and/or severity of leaf removal also depends upon specific planting choices. for instance, no or very light defoliation is usually applied on the south facing side of an east-west oriented row, whereas more severe leaf stripping might be required on the north facing row side; basically the same applies for westand east facing sides of north-south oriented rows, respectively. more physiological insights have also been provided about “why” a traditional leaf removal might become mandatory. backward to the still shareable rule indicated from dr. shaulis in that “no leaf removal is needed if while standing in front of a canopy at veraison about 50-60% of the clusters are visible”, other more recent findings have shown that in a significant number of cases, excessive canopy crowding in the bunch zone leading, in turn, to the need of stripping leaves, is caused by other wrong or rushed vineyard management choices (fig. 1). one example is worthwhile above all: spur pruned vertically shoot positioned (vsp) cordontrained canopies are usually prone to leaf removal due to too high shoot density per meter of canopy length. yet, this often happens as vines burst many either secondary or base bud originated shoots casting additional shade in the bunch area. more equilibrated vines would better comply with the shared requirement that, on average, one shoot is expected from each single retained node and, if so, the subsequent leaf removal would become quite likely unnecessary. fig. 1 interrelationships of excessive shoot vigour stimulated by a too narrow within-row vine spacing and related consequences on summer pruning needs (drawn by authors). 153 early leaf removal this practice has mainly been inspired from long-standing knowledge according to which carbohydrate supply at flowering is a primary determinant of fruit set (coombe, 1959; may et al., 1969). the temporary source limitation induced by removing an average of six main basal leaves before flowering has led, under a broad array of genotypes and growing conditions, to a significant decrease in fruit-set, which in turn increases cluster looseness and tolerance to rot (gubler et al., 1991; poni et al., 2006; intrieri et al., 2008; poni et al., 2008; diago et al., 2010 a). yet, the most important outcome is that, irrespective of genotype, this early leaf removal markedly improves grape composition and wine sensory properties as compared to non-defoliated shoots (poni et al., 2006; diago et al., 2010 a; palliotti et al., 2011 b). there are multiple mechanisms involved in such a positive response. defoliated shoots generally have a higher final leaf-to-fruit ratio than control, thus implying that the yield reduction induced by defoliation was more than proportional to the leaf removal constraint due to a fruit-set and berry-size effect (poni et. al., 2006). furthermore, it is known that a precocious source limitation carried out in the form of defoliation or darkening the basal shoot zone hastens translocation of assimilates towards the cluster (quinlan and weaver, 1970). improved grape composition in the defoliated shoots also relates to the ‘quality’ of the source. for example, it is indeed true that removing the main six basal leaves at pre-bloom causes an abrupt and severe decrease in vine photosynthesis [75% less than with not-defoliated (nd) according to poni et al., 2008]. however, removing source leaves around bloom also triggers a series of dynamic changes in canopy growth, age and photosynthesis. defoliated vines have a ‘younger’ canopy at veraison since median and apical shoot leaves at this time are now mature and more lateral leaves may be present as a compensating reaction to early main leaf removal, while some, albeit temporary, photosynthetic compensation usually occurs in both main and lateral leaves of defoliated plants. poni et al. (2008) have recently shown that whole canopy net co2 exchange rates (ncer) monitored uninterruptedly for three months in defoliated (d) vs. non-defoliated sangiovese vines indicated no differences in data expressed on a per-vine basis. yet when the same data were given on a per-unit leaf area basis, defoliated vines showed higher rates than nd vines (4.75 µmol m-2 s-1 vs. 4.16 µmol m-2 s-1) and, most importantly, ncer/ yield increased by 38% in d vines, thus resulting in enhanced carbohydrate supply for ripening (table 1). however, the most intriguing outcome from these early-season defoliation tests is that a significant increase in relative skin mass has consistently been found in separate field studies conducted on a three-year basis in cv. barbera (poni and bernizzoni, 2010), regardless of absolute berry mass (fig. 2). it is reasonable to think that such an early table 1 effects of early defoliation on yield components and whole shoot net co2 exchange rate (ncer)/fresh fruit mass treatment flowers/cluster (no.) fruit set (%) total berries/ cluster (no.) cluster weight (g) berry weight (g) ncer shoot/yield (nmol/s x g) cluster compactness (rating) control 435 38.8 169 334 1.98 2.43 6.60 defoliated 487 21.0 103 207 2.01 3.31 4.25 significance ns ** ** ** ns ** ** **, ns= significant at p ≤ 0.05 or not significant, respectively. fig. 2 correlation between relative skin and berry mass in 2006, 2007 and 2008 for non defoliated and defoliated barbera grapevines (from poni and bernizzoni, 2010). 154 basal leaf removal, besides favouring berry hardening in the long run, would also impose more favourable microclimate conditions for cell division and berry skin deposition, which typically takes place within four to five weeks after flowering. mescalchin et al. (2008) have shown in pinot gris that the earlier the defoliation, the lesser the incidence of skin burning on vsp and pergola-trained varieties due to both more time allowed for cluster cover after treatment and adaptation towards the formation of a thicker skin. mechanization is feasible by preferably using at preflowering (i.e. closed-flower stage) an air pressure blowing machine which can run two passages per row in about 5-7 hr/ha (intrieri et al., 2008). best performance is obtained on canopies characterized by vertical and well positioned shoots and on cultivars having mostly erect inflorescences. it has to be kept in mind that early leaf removal is specifically recommended in highly productive vineyards which often present heavy, thick bunches very susceptible to rot. based on the constancy of the results obtained under the above circumstances, this practice is nowadays an interesting alternative to traditional methods of crop control such as bunch thinning. advantages are feasibility of mechanization, hence cost saving, and different mechanisms by which the crop level on the vine is adjusted. if early leaf removal is chosen, the primary regulation for crop restriction is via a decrease in fruit set with or without a significant reduction in berry size. therefore, cluster number is unchanged, yet each bunch is smaller and looser. conversely, hand bunchthinning, besides being time consuming, drastically lowers bunch number per vine and favours undesirable yield compensation mechanisms such as larger berries and heavier clusters (ough and nagaoka, 1984; keller et al., 2005). anti-transpirant applications a very recent development of the above work investigated whether the precocious, albeit temporary, source limitation sought with early leaf removal can be induced through the non-invasive and easy-to-do application of anti-transpirants (palliotti et al., 2010). their use could sort out the inherent limitations of high labour demand for manual work while eliminating the risks of direct damage to the inflorescences linked to the use of a leaf plucker. results reported for cvs. sangiovese and ciliegiolo subjected to pre-bloom treatment of anti-transpirant vapor gard® (a.i. di-1-p-menthene at 3% concentration, intrachem bio italia, grassobbio, bg, italy) show similar reductions of net photosynthesis (from 30% to 70%) over several weeks after spraying as compared to control vines (fig. 3). the treated sangiovese vines showed reduced yield, berry weight, cluster compactness and, on a two-year basis, lower vigour and unchanged vine capacity per year. at harvest, the treated vines showed higher °brix in all seasons and higher anthocyanin concentration two years out of three. overall, early-season applications of a filmforming anti-transpirant caused a leaf function limitation strong enough to reduce yield and cluster compactness through smaller final berry size. over the last decade, climate change along with improvements in vineyard management and clonal selection have exerted a strong impact on vine yield and grape and wine composition. among the most important effects, the increase in grape sugar concentration at harvest, is to be considered, which resulted in wines with high alcohol config. 3 seasonal trends of air vapour pressure deficit (vpd) and total photosynthetic active radiation (par) (a), assimilation rate (b), transpiration rate (c) and intrinsic water use efficiency (d) recorded on fully expanded, median sangiovese (top image) and ciliegiolo (bottom image) leaves sprayed twice with anti-transpirant vapor gard® at 3% (t) or left unsprayed (c). bold arrows indicate the time of application. data are means ± se (from palliotti et al., 2010). 155 tent (vierra, 2004; duchêne and schneider, 2005; godden and gishen, 2005). there is a surge of interest from the wine industry in tools suitable to lower wine alcohol content such as the de-alcoholisation process which also agrees with the eu legislative measure no 606/2009. conversely, it would thus be helpful to find strategies able to reduce grape sugar concentration in the vineyard, thus limiting the need to operate in the winery without detrimental effects on wine characteristics. in association with traditional management practices which can be used to slow down the accumulation of sugars in the grape berry, interest is growing in late season applications of anti-transpirants. in a recent contribution by palliotti et al. (2011 a), the anti-transpirant vapor gard® sprayed about one month before harvest significantly delayed sugar accumulation in sangiovese, tocai rosso and trebbiano toscano berries which, at harvest, had -1.2 to -2.7 less °brix than the un-sprayed control according to genotype and crop load. the temporary reduction of photosynthesis, due to the film formed by the anti-transpirant, limited the amount of assimilates translocated into the ripening berry, thus lowering must sugar concentration with a potential effect on wine alcohol content. 3. cluster thinning the achievement of an adequate balance between growth and fruiting can be obtained by the regulation of crop level through cluster thinning treatments. despite additional labour costs, cluster thinning might play an important role in all cases where over cropping occurs (e.g. excess of vigour due to cultivar and rootstock, high soil fertility, low planting density, use of drip fertigation, etc.) and in cases where winter pruning severity has not overcome cropping due to high bud fertility. the negative effects of over cropping include delay in grape maturation, worsening of overall grape quality, increased susceptibility to biotic disease and poor wood maturity (winkler et al., 1974). furthermore, different environmental parameters, particularly air temperature, light intensity, photoperiod and soil water content, together with phyto-hormones and the availability of mineral ions are known to influence bud fertility and fruit-set (srinivasan and mullins, 1981). therefore, it is not always possible to regulate the yield level by solely adjusting bud load, especially in vineyards with low planting density and in years and areas characterized by unfavourable environmental conditions. however, the results regarding the effects of high yield levels on fruit composition (sugar, acidity, colour, etc.) and wine quality (taste, flavours, colour and potential for aging) are quite contradictory. for example, some authors found an increase in anthocyanin concentration upon cluster thinning (bravdo et al., 1984 a, reynolds, 1989; guidoni et al., 2002), whereas no improvement in anthocyanin content or wine colour in cluster-thinned vines were found by bravdo et al. (1984 b) and ough and nagaoka (1984). location, application time and intensity of cluster thinning treatment significantly affected the results and can therefore justify, at least in part, the discrepancy of the experimental results in literature. the results of a three-year trial on the effects of three levels of cropping (0%, 20% and 40% cluster thinning treatments) applied just before veraison in sangiovese, merlot and cabernet sauvignon showed that this management practice caused a significant reduction of yield only at the 40% severity and in two out of the three seasons studied (table 2) (palliotti and cartechini, 1988). in each cultivar, in 1995 and 1996, yield was linearly correlated with cluster thinning intensity. cluster thinning treatment at the 40% level caused a reduction of vine yield that ranged from 22% to 47%. the reduction of yield observed was, in general, not proportional to the cluster thinning intensity due to a significant increase of berry and clustable 2 effects of cluster thinning on yield and cluster characteristics in sangiovese, merlot and cabernet sauvignon grapevine cultivars cultivar thinning yield (kg/vine) cluster/vine (n°) cluster weight (g) berry weight (g) 1995 1996 1997 1995 1996 1997 1995 1996 1997 1995 1996 1997 sangiovese 0% 12.4 11.3 10.1 40.6 46.4 39.9 306 245 251 2.30 2.36 2.33 20% 11.8 9.5 10.0 35.1 34.3 32.9 340 271 300 2.47 2.60 2.68 40% 9.5 6.9 9.5 25.2 22.6 24.3 381 308 387 2.70 2.82 3.38 significance ** *** ns ** *** ** *** *** *** *** *** *** r2 0.76 0.92 --0.76 0.94 0.75 0.90 0.89 0.92 0.87 0.94 0.96 merlot 0% 8.1 8.7 8.7 57.8 64.1 60.5 147 137 149 1.70 1.82 2.03 20% 7.7 8.0 8.2 49.5 52.1 50.5 159 154 160 1.76 1.83 2.12 40% 6.1 6.6 7.9 35.4 38.8 37.5 172 170 212 1.92 1.94 2.53 significance * *** ns *** *** *** *** *** *** *** * ** r2 0.47 0.87 --0.84 0.91 0.83 0.89 0.91 0.84 0.85 0.46 0.73 cabernet s. 0% 7.2 7.9 6.2 56.1 58.9 51.6 131 135 123 1.35 1.94 1.39 20% 7.4 7.0 6.1 44.2 47.6 42.2 167 146 146 1.60 2.02 1.57 40% 5.6 4.2 6.0 32.2 27.9 30.5 176 154 198 1.70 2.06 1.89 significance * *** ns *** *** *** *** *** *** ** ** *** r2 0.42 0.84 --0.90 0.93 0.92 0.85 0.87 0.84 0.72 0.70 0.84 *,**,***, ns= linear component significant at p ≤ 0.05, 0.01, 0.001, or not significant, respectively. 156 ter weight. at the 20% intensity of cluster thinning, vine self-regulation warranted full yield compensation through significantly increased berry size and cluster weight. in 1997, due to quite favourable environmental conditions for ripening, +156 and +143 degree-days, base 10°c, as compared to 1995 and 1996, respectively, and lower rainfall during the two months prior to harvest, the impact of the 40% cluster thinning on vine yield was negligible. total soluble solids, anthocyanins and phenolics increased linearly with thinning severity in two out of the three seasons (tables 3 and 4). juice ph and titratable acidity (ta) were rather variable, although cluster thinning tended to reduce ta and increase ph (table 3). in 1995 and 1996, improvements in soluble solids content in cluster-thinned vines were consistent with lower yield levels (table 3) whereas the reduction of titratable acidity and the slight increase of juice ph were probably attributable to an earlier ripening. similar results have also been reported by looney (1981), bravdo et al. (1984 a) and reynolds (1989). table 4 effects of cluster thinning on anthocyanins, polyphenols and total nitrogen content at harvest in sangiovese, merlot and cabernet s. grapevine cultivars cultivar thinning anthocyanins (mg/cm2 berry skin) polyphenols (mg/cm2 berry skin) total nitrogen (% s.s.) 1995 1996 1997 1995 1996 1997 1996 1997 sangiovese 0% 0.412 0.453 0.602 1.42 1.95 1.34 0.35 0.56 20% 0.580 0.596 0.652 1.89 2.37 1.84 0.56 0.56 40% 0.610 0.692 0.639 1.94 2.42 1.87 0.49 0.70 significance *** *** ns ** * ns ns ** r2 0.83 0.96 --0.80 0.57 ----0.65 merlot 0% 0.491 0.487 0.576 1.51 1.63 1.24 0.42 0.49 20% 0.571 0.554 0.641 1.73 2.00 1.46 0.63 0.49 40% 0.824 0.742 0.653 2.10 2.37 1.56 0.49 0.53 significance *** *** * ** ** ns ns ns r2 0.90 0.91 0.49 0.68 0.77 ------cabernet s. 0% 0.652 0.786 0.691 1.80 1.91 2.08 0.38 0.29 20% 0.670 0.772 1.021 2.10 2.60 2.52 0.70 0.42 40% 1.024 0.942 1.073 2.70 2.84 2.55 0.56 0.56 significance ** * *** *** ** ns ns *** r2 0.78 0.62 0.83 0.81 0.79 ----0.95 *,**,***, ns= linear component significant at p ≤ 0.05, 0.01, 0.001, or not significant, respectively. table 3 effects of cluster thinning on soluble solids, titratable acidity and ph at harvest in sangiovese, merlot and cabernet sauvignon grapevine cultivars cultivar thinning soluble solids (°brix) titratable acidity (g/l) juice ph 1995 1996 1997 1995 1996 1997 1995 1996 1997 sangiovese 0% 17.3 17.1 21.4 8.5 8.2 6.3 3.01 3.08 3.26 20% 18.0 18.9 21.8 8.8 7.5 6.1 3.04 3.11 3.22 40% 18.4 21.1 22.0 8.0 7.2 5.9 3.04 3.12 3.21 significance * *** ns ns ** ns ns ns ns r2 0.54 0.94 ----0.71 --------merlot 0% 20.6 21.0 21.4 9.7 6.8 6.5 3.13 3.29 3.32 20% 21.4 21.2 22.8 9.5 6.7 6.3 3.15 3.27 3.28 40% 22.6 22.8 22.6 8.8 6.5 6.4 3.18 3.44 3.36 significance ** ** ns ** ns ns *** ns ns r2 0.79 0.67 --0.64 ----0.85 ----cabernet s. 0% 20.2 21.0 21.6 9.7 8.0 7.6 3.05 3.21 3.22 20% 20.0 21.4 22.2 9.5 7.9 7.1 3.09 3.19 3.23 40% 22.0 23.2 22.0 8.8 7.5 7.2 3.13 3.25 3.27 significance * ** ns * ns ns * ns ns r2 0.55 0.74 --0.55 ----0.46 ----*,**,***, ns= linear component significant at p ≤ 0.05, 0.01, 0.001, or not significant, respectively. 157 data pooled from cultivars and years resulted in negative correlations between total soluble solids and yield level, while positive linear relationships were found between anthocyanins in berry skin and soluble solids in berry juice (fig. 4). overall, regulation of yield through cluster thinning is strictly dependent on year; the grape composition is generally improved and this assumes particular importance in seasons marked by unfavourable environmental conditions or in very productive vineyards due to either high fertility cultivars (i.e. sangiovese) or soils. the increase of polyphenols and anthocyanin content recorded in both 20% and 40% cluster-thinned vines is of great significance for the production of high quality red wine, especially when targeted to aging. since manual cluster thinning is a very expensive operation due to large labour requirements, its mechanization is a very needed, yet largely unresolved issue. in grenache and tempranillo grapevines trained to vertical, shoot-positioned mechanical berry thinning performed with a grape harvester was effective to reduce yield while achieving more ripened grapes and wines with higher alcohol and ph values, more intense colour and increased phenolic compounds (diago et al., 2010 b). 4. shoot hedging practices aimed at manipulating vegetative growth during late-spring and summer, particularly in vigorous vineyards, can substantially influence yield and grape composition (intrieri et al., 1983; kliewer and bledsoe, 1987; reynolds and wardle, 1989). hedging is a common management practice used to maintain canopy shape, reduce vine vigour, improve the microclimate in the fruiting zone, increase the efficiency of disease treatments and facilitate harvest and access of machines to the vineyard rows. compared with other summer management practices used for similar purposes, such as leaf removal and pulling of lateral shoots, hedging is commonly used because it can fig. 4 relationship between yield per vine and total soluble solids (left) and total soluble solids and anthocyanins content in the berry skin at harvest (right). 158 be done completely mechanically and therefore is easy, fast and cheap. the effects of hedging on yield and fruit quality, considering the variables of timing and severity of application, are strictly associated to the ability of the cultivar to develop lateral shoots and their photosynthetic capacity from veraison to harvest (cartechini et al., 1998). the impact of hedging severity on vine performance is well known; severe hedging, i.e. less than six main leaves retained per shoot, generally reduces grape quality (kliewer and bledsoe, 1987; reynolds and wardle, 1989; palliotti, 1992), whereas the time of application is rather controversial because other factors may also influence these effects such as bud load, shoot orientation, training system, environmental conditions, soil characteristics, water availability, and so on (intrieri et al., 1983; reynolds and wardle, 1989). vertical shoot positioned (vsp) training systems are normally trimmed when their shoots exceed the wires placed at the top of the canopy. therefore, the timing is poorly dependent on grower’s decisions and it is instead a function of intrinsic shoot vigour and vine balance. a balanced vineyard would reach the height suitable for trimming around fruit set, whereas an excessively vigorous one would get to the same growth stage much earlier, therefore making shoot trimming more likely to be repeated again later in the season. timing of trimming follows different rules when performed on sprawl canopies (i.e. a single high wire trellis) where an early (pre-flowering) shoot trimming might be made necessary by the need to induce mostly upright shoot growth habits. a two-year trial, aimed at assessing the effect of timing of hedging (one and five weeks after full bloom, afb) on yield and grape composition in different red and white grapevine cultivars grown on fertile clay soil and trained to a single high wire trellis, showed that hedging at the 9-10th node on primary shoots, carried out one week afb, markedly changed canopy characteristics, yield and grape composition (fig. 5 and tables 5, 6, 7 and 8) (cartechini et al., 1998). in untrimmed sangiovese, cabernet sauvignon and verdello vines, leaf area build up progressed rapidly from about 30 to 120 days after bud burst (fig. 5). the development of laterals and relative leaf area occurred from 60 to 110 days after bud burst in sangiovese and from 60 to 140 days after bud burst in cabernet sauvignon and verdello. fig. 5 development of primary and lateral leaves in sangiovese, cabernet sauvignon and verdello grapevine cultivars hedged one and five weeks after full bloom (afb) as compared to the untrimmed control (n = 3 ± se). 159 in all the cultivars, from flowering to veraison, the total leaf area increased more than three-fold. at the end of canopy growth, the sangiovese had less total leaf area than cabernet sauvignon and verdello (-1.5 and -2.0 m2/vine, respectively) and the laterals represented 18, 32 and 22% of the total leaf area in sangiovese, cabernet sauvignon and verdello, respectively. up to the end of canopy growth, sangiovese, cabernet sauvignon and verdello hedging one and five weeks afb produced about 1.1, 3.9 and 3.5 and 0.9, 3.4 and 3.1 m2 of new leaves per vine, respectively, derived mainly from lateral development. in all cultivars, early-hedging, one week afb, generally increased the contents of soluble solids, total nitrogen and total polyphenols (tables 6, 7 and 8) as well as anthocyanins content in the red cultivars (table 8). early-hedging significantly reduced the titratable acidity and juice ph in all the cultivars (table 6 and 7). late-hedging, five weeks afb, instead significantly reduced yield in sangiovese and, except for sauvignon blanc, the soluble solid content was significantly reduced as well as anthocyanins content in both red cultivars. the positive outcomes of the early-hedging were likely dependent upon a cultivar’s ability to develop lateral shoots after trimming (fig. 5). all the cultivars with a good capacity to produce laterals, such as cabernet sauvignon, verdello, drupeggio and sauvignon blanc, responded better to early summer pruning as shown by the increased cluster weight and yield and improved contents of soluble solids, total polyphenols and nitrogen content. trimming vines increased lateral growth and table 5 yield and average cluster weight at harvest in vines of different grapevine cultivars hedged one and five weeks after full bloom (afb) and control (n= 60) cultivar yield (kg/vine) cluster weight (g) control hedged 1 week afb hedged 5 weeks afb control hedged 1 week afb hedged 5 weeks afb sangiovese 7.4 b 7.3 b 6.0 a 279.8 b 292.4 b 253.5 a cabernet s. 6.0 a 7.8 b 5.5 a 122.9 a 143.7 b 110.5 a verdello 7.0 a 8.2 b 6.9 a 215.6 a 276.6 b 218.7 a drupeggio 7.4 a 9.1 b 7.2 a 238.7 a 275.7 b 235.4 a sauvignon b. 4.0 a 5.2 b 3.9 a 106.5 a 129.3 b 103.8 a for each grapevine cultivar, the means followed by different letters are significantly different at p ≤ 0.05. table 6 soluble solids content and titratable acidity at harvest in different grapevine cultivars hedged one and five weeks after full bloom (afb) and control cultivar soluble solids (°brix) titratable acidity (g/l) control hedged 1 week afb hedged 5 weeks afb control hedged 1 week afb hedged 5 weeks afb sangiovese 23.2 b 23.9 b 21.8 a 6.6 b 6.1 a 6.8 b cabernet s. 23.4 b 23.7 b 22.9 a 7.1 b 6.6 a 7.3 b verdello 19.4 b 21.0 c 17.8 a 8.5 b 8.0 a 8.6 b drupeggio 20.3 b 21.9 c 18.1 a 8.4 b 7.8 a 8.3 b sauvignon b. 20.5 a 23.1 b 20.4 a 8.8 b 8.2 a 9.0 b for each grapevine cultivar, the means followed by different letters are significantly different at p ≤ 0.05. table 7 juice ph and berry nitrogen content at harvest in vines of different grapevine cultivars hedged one and five weeks after full bloom (afb) and control cultivar juice ph total nitrogen (% d.w.) control hedged 1 week afb hedged 5 weeks afb control hedged 1 week afb hedged 5 weeks afb sangiovese 3.42 b 3.35 a 3.36 a 0.48 a 0.63 b 0.45 a cabernet s. 3.40 b 3.22 a 3.29 a 0.63 a 0.98 b 0.55 a verdello 3.06 b 3.00 a 2.99 a 0.42 a 0.59 b 0.41 a drupeggio 3.08 b 3.03 a 3.04 a 0.44 a 0.68 b 0.38 a sauvignon b. 3.07 b 3.01 a 3.02 a 0.51 a 0.66 b 0.45 a for each grapevine cultivar, the means followed by different letters are significantly different at p ≤ 0.05. 160 the total final leaf area was always less than that recorded in control vines (from 15 to 49% less). at harvest, in all the grapevines tested, early-hedging reduced the leaf/ fruit ratio from 33 to 45% in comparison to the control vines and improved the soluble solids content (from 0.3 to 1.6°brix), whereas late-hedging caused a reduction of both leaf/fruit ratio and soluble solid accumulation in the berries (fig. 6). the rejuvenation of leaf area in the canopy following early-hedging and their high photosynthetic efficiency from veraison to harvest of the newly formed lateral leaves (fig. 7) likely reduced the leaf area per gram of fruit required to achieve adequate ripeness. these laterals also translocate assimilates to the subtending clusters very efficiently (candolfi-vasconcelos and koblet, 1990). negative results found on late-hedged vines, also reported by other authors (intrieri et al., 1983; palliotti, 1992), are probably linked to the fact that lateral shoots compete with the developing grapes for carbohydrates, causing delayed berry growth and sugar accumulation. early-trimming reduced titratable acidity as compared to control vines due to greater cluster exposure to sunlight table 8 anthocyanins and total polyphenol content at harvest in the berry skin of different grapevine cultivars hedged one and five weeks after full bloom (afb) and control cultivar anthocyanins (mg/cm2 berry skin) polyphenols (mg/cm2 berry skin) control hedged 1 week afb hedged 5 weeks afb control hedged 1 week afb hedged 5 weeks afb sangiovese 0.754 b 0.958 c 0.412 a 1.65 b 2.24 c 1.09 a cabernet s. 1.095 b 0.998 b 0.773 a 2.07 a 2.96 b 1.90 a verdello ------0.88 a 1.25 b 0.80 a drupeggio ------0.91 a 1.19 b 0.81 a sauvignon b. ------0.82 a 1.12 b 0.75 a for each grapevine cultivar, the means followed by different letters are significantly different at p ≤ 0.05. fig. 6 relationship between must total soluble solids and leaf/fruit ratio at harvest in vines of sangiovese, cabernet sauvignon and verdello either untrimmed or trimmed one (a) and five (b) weeks after full bloom (afb). fig. 7 evolution of net photosynthesis of primary and lateral leaves from veraison to leaf fall in sangiovese, cabernet sauvignon and verdello grapevine cultivars (n = 8 ±se). 161 and a consequent decrease of malic acid content due to respiration activity. in addition, the reduced must ph with early-hedging is probably linked to the reduction of both the malic acid and potassium contents in the must in association with lower total leaf area. bledsoe et al. (1988) found a significant positive correlation between these two parameters and juice ph. in all the grapevine cultivars that develop many laterals after hedging, the greater transpiration rate (from +15 to 35%, data not shown) assessed in these leaves, compared with primary ones, particularly in august and september, may aggravate susceptibility to vine water stress especially in hot environments and in particularly dry years. during the first two weeks of november, the laterals on the vines had net photosynthesis values that ranged from 0.7 to 1.6 µmol co2 m -2 s-1 (fig. 6), in a period when all the carbohydrates fixed are very useful for the reserve accumulation, and therefore for increased cold hardiness (wample and bary, 1992) and even for budbreak and initial shoot growth the following season. thus, at the end of the season care must be taken to maintain the integrity of these leaves until total abscission occurs. early winter pruning, practiced in some viticulture areas, should be avoided. 5. shoot positioning in vsp canopy trellis systems, shoot positioning is performed to maintain canopy form and shoot separation, to create a uniform distribution of leaves that minimizes cluster shading as well as to optimize canopy light interception and allowing the transit of mechanical equipment between rows. shoot positioning also exerts a positive effect on disease incidence and severity; usually disease pressure is lessened due to increased air flow and sunlight penetration inside the vine canopy. another important effect of this canopy management technique is that it has a positive impact on the development of fruitful buds and therefore for the vine yield in the following year. the way shoot positioning is performed depends mainly on the training systems. in a vsp system the process consists of directing the shoots growing up between a set of catch wires as they develop. the vertically positioning of shoots can be done manually or using movable wires and done several times during the growing season. mechanical shoot positioning on vsp trellis systems with specialized equipment has undergone a notable increase in recent years. on geneva double curtain (gdc) training system the shoots are positioned downward and separated out from the permanent cordon in order to reduce the vigour of shoots and attain optimal canopy density. in the gdc trellis, shoot positioning is performed on the interior part of the canopy to maintain two distinct canopies avoiding excessive shading in the central part of the canopy. usually, in most training systems, shoot positioning is performed one or two weeks after bloom, before tendrils have become firmly attached. for best results, however, two or three shoot positioning runs during the season are needed. 6. conclusions vineyard management should aim to achieve and maintain high efficiency over time, which is closely dependent on the ability to control the competition both between-and intra-vine. this approach would warrant a fair and fruitful balance between vegetative and productive activity of the vines and the best expression of grape quality (smart and robinson, 1991) without costly additional inputs. since the “perfect” vineyard able to reach and maintain this equilibrium in a natural way during the season is generally utopia, summer pruning often plays a crucial role. in light of the climate change in progress, an important challenge for old and new vineyards will be the matching of tradition and innovation. this raises the question of new techniques of canopy management, availability of rootstocks of low-to-moderate vigour, new cultivars better adapted to higher temperatures and water shortage and more intense mechanization. the latter assumes particular importance especially when the wines produced must be sold in un-bottled form or within large organized distribution (lod) chains, like supermarkets, hypermarkets and discount markets. currently, at least in italy, lod commercialize about 70% of the entire italian wine production (which corresponds to about 48-50 million hl per year) (ismea, 2007), where the binomial “adequate quality”“moderate selling price” is still dominant. global warming requires rapid adaptation and poses the crucial question of ripening modulation. in white grape varieties, the major challenge is the preservation of organic acids and primary grape flavours; whereas in black-berried cultivars the priority is producing wines with moderate alcohol content without modifying colour intensity and wine sensory. some traditional and innovative canopy management practices can help to achieve these results, such as light pruning (petrie et al., 2003), early leaf removal (poni et al., 2006; palliotti et al., 2011 b), late defoliation and severe summer pruning (stoll et al., 2010), use of anti-transpirants (palliotti et al., 2010, 2011 a), canopy treatment of exogenous auxins (böttcher et al., 2010) and brassinosteroid and brassinazole steroidal 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kliewer w.m., lider l.a., 1974 general viticulture. univ. calif. press. berkeley, usa. zoecklein p.w., wolf t.k., duncan n.w., judge j.m., cooke m.k., 1992 effects of fruit zone leaf removal on yield, fruit composition and fruit rot incidence of chardonnay and white riesling (vitis vinifera l.) grapes. am. j. enol. vitic., 43: 139-148. 51 adv. hort. sci., 2011 25(1): 51-63 received for publication 10 may 2010. accepted for publication 4 march 2011. review paper crop physiology of elephant foot yam [amorphophallus paeoniifolius (dennst. nicolson)] v. ravi, c.s. ravindran, g. suja, james george, m. nedunchezhiyan, g. byju, s.k. naskar central tuber crops research institute, sreekariyam, 659 017 thiruvananthapuram, india. key words: corm, dormancy, sprouting, elephant foot yam. abstract: amorphophallus paeoniifolius (dennst. nicolson), syn. a. campanulatus (roxb.) bl. exdence (also elephant foot yam) is largely cultivated in the philippines, java, indonesia, sumatra, malaysia, bangladesh, india and china. in india, it is cultivated in the states of andhra pradesh, west bengal, gujarat, kerala, tamil nadu, maharashtra, uttar pradesh and jarkhand. sree padma, gajendra, sree athira (a hybrid), bidhan kusum and nda-9 are some of the high yielding amorphophallus varieties released for cultivation. the corm production potential of this crop is 5080 t ha-1 and net economic return is about 2000 – 3000 us$ per ha. plant growth and corm yield is influenced by the size of planting material (corms/cormels/corm pieces), plant spacing, nutrient management and water availability. nevertheless, the production aspect of this crop is less understood as scanty research has been conducted in this crop. the available literature on growth and productivity of elephant foot yam is briefly described in this article. 1. introduction amorphophallus paeoniifolius (dennst.), syn. a. campanulatus (roxb.) bl. exdence (also elephant foot yam) is an herbaceous, perennial c3 crop. it is basically a cropof southeastern asian origin. it serves as a source of protein as well as starch. it has long been used as a local staple food in many countries such as the philippines, java, indonesia, sumatra, malaysia, bangladesh, india, china and southeastern asian countries (chandra, 1984; sugiyama and santosa, 2008). it is commercially cultivated due to its production potential and popularity as a vegetable in various indian cuisines. in india, it is cultivated in andhra pradesh, west bengal, gujarat, kerala, tamil nadu, maharashtra, uttar pradesh, and jarkhand states whereas in northern and eastern states, the wild, local cultivars grown are generally used for making vegetable pickles and medicine preparations for various aliments. the crop is also cultivated as an intercrop along with turmeric (fig. 1) and under coconut (fig. 2) or banana. in recent years, farmers in bihar and uttar pradesh have also begun cultivation. under improved cultural practices and high yielding varieties the production potential of this crop varies between 30 and 100 t ha1 and the net profit (economic return) is about 2000 – 3000 us$ per ha (aicrp, 2004, 2005, 2006 a, b, 2007, 2008, 2009). this crop also offers export potential in india since it is not commercially cultivated in other fig. 1 elephant foot yam as an intercrop with turmeric. arrows indicate elephant foot yam plants. fig. 2 elephant foot yam under coconut. 52 countries (misra and shivalingaswamy, 1999; misra, 2000; misra et al., 2001). in india, ‘sree padma’, ‘gajendra’, ‘sree athira’ (a hybrid), ‘bidhan kusum’ and ‘nda-9’ are some of the high yielding amorphophallus varieties released for cultivation (aicrp, 2006 a). the corms are usually eaten as a vegetable after boiling or baking and are rich in calcium, (50 mg g-1), phosphorus (34 mg g-1) and vitamin a (260 iu g-1). the leaves are used as a vegetable by local tribes in india because they contain a high concentration of vitamin a (rajalakshmi et al., 2001). elephant foot yam plants grow well in medium to light soils (coarse-textured sandy soils) with adequate amounts of organic matter because they prefer well-aerated soils. the crop can tolerate temporary flooding, but anaerobic water logging causes corm rot. in kerala, elephant foot yam is planted in february and harvested during november-december under rainfed conditions. in andhra pradesh, the crop is planted during september-october and harvested in june (winter season crop) or planted in june and harvested in january (rainy season crop) under irrigated conditions. in west bengal, the crop is planted in october and harvested in june under irrigated conditions. this review summarizes the available literature on growth and productivity of elephant foot yam. 2. shoot characteristics the new shoot (leaf) sprout emerges from the cut corm pieces or full corm used as planting material (plate 1 a and b). the time of emergence (sprouting) of new shoots depends on the dormancy status of the planting material. if the planting material has completed its dormancy before planting, then the new shoot sprout will emerge as soon as it is planted. leaf emergence is delayed when the apical buds of seed corms are damaged or cut pieces of corm are planted. leaves were found to emerge earlier when whole corms were planted than when cut corms were planted, irrespective of corm size (sen et al., 1996). when whole corms, bud portions or upper half sections were planted, buds sprouted 2-3 weeks after planting. however, buds started to sprout 47 weeks after planting when vertical 1/2, 1/4 and 1/8 corm sections and lower half corm sections were planted (sugiyama and santosa, 2008). once the sprout is initiated, further development of new shoots may be completed within 30 days (plate 2 a to f). leaves are basal, compound, pinnate, solitary and erect. leaves are medium to very large in size. the plant develops leaves by using preserved carbohydrates in seed corms (planting material) and then daughter corms (new corms) enlarge by using plate 1 new sprout emerging from cut corm pieces (a) and full corm (b) of elephant foot yam before planting. emerging sprout from cut pieces of corm emerging sprout from the apical bud meristem zone 53 assimilates synthesized by the leaves. in general, amorphophallus corms have one apical bud, which exists inside the cavity in the head part of seed corms. three or four small cataphylls existing in the head part of corms cover the apical bud in which the first leaf primordium has already differentiated at planting. the cataphylls elongate concomitantly with leaf development. possibly, they protect a leaf from damage by soil impedance during development. furthermore, subepidermal cells of cataphylls may contain needle-like crystals of calcium oxalate which presumably offer protection to a young leaf from damage by pests. cataphyll size depends on corm size and plant age. the cataphylls wither after leaves become mature. leaves are composed of a petiole (pseudostem) and three rachises with many leaflets. the number of leaves which develop during the growing season is dependent on corm age. during a growing season up to 12 leaves may be produced successively. as such, more than two leaves may coexist at the same time. the number of leaves is plate 2 different stages (a-f) of leaf development from the sprout in elephant foot yam. canopy pseudostem newly developing corm mother corm c early stage of leaf emerging out from the cataphyll d leaf emerging out from the cataphyll e spreading canopy b cataphyll enclosing the leaf and the corn showing profuse root development a sprouting corm showing the newly forming corm f full plant. 54 also determined by the size of planting materials. plants originating from small corms (10 g) produce three to eight leaves, while large corms (500 g) usually produce one or two leaves during a growing season. under field conditions, weeds grow much before shoot development from planted corms because of corm dormancy and delay in sprouting. under weedy conditions, leaves are submerged under weeds (fig. 3) and the number of leaves, total leaf area, leaf thickness and fresh masses of corms decreases markedly (santosa et al., 2006 c). when preflowering and post flowering corms with similar fresh masses were planted both types of corms sprouted at about the same time; however, leaf sizes (length of petioles and rachis) were larger in preflowering corms than in postflowering corms (sugiyama and santosa, 2008). up to 150-250 leaflets may be produced per leaf and this may vary among accessions. the leaf area of any one of the three lobes of a. campanulatus leaves showed a highly significant correlation (r = 0.93 to 0.97) with total leaf area (patel and mehta, 1987). the number of stomata in the lower epifig. 3 a heavily weed-infested elephant foot yam field. plate 3 a and b stomata in elephant foot yam leaf. a) stomata on the abaxial leaf surface (10x10). b) single stoma with subsidiary cells (40x10). dermis increased from 10.22 per unit area at 50 days after planting (dap) to 17.78 per unit area at 150 dap (gopi et al., 2008). a stoma has two adjacent cells surrounded by four subsidiary cells (plate 3 a and b). the leaf area index increased with time and reached a maximum (6.1) at 120 dap at a planting density of 140 x 103 plants ha-1 (das et al., 1997). on the other hand, the lai reached 4.4 and 5.4 at a planting density of 100 x 103 and 120 x 103 plants ha-1 respectively. petioles (pseudostem) look like the stems of normal plants and are cylindrical in morphology. in general, large petioles indicate that the corm is also large. depending upon the variety, plant spacing or size of planting material used, the mean shoot length varied between 47.3 and 122.5 cm (mukhopadhyay and sen, 1986; ravindran and kabeerathumma, 1991; sen and das, 1991; goswami and sen, 1992; james george and nair 1993; geetha, 2001; suja et al., 2005, 2006; aicrp, 2004, 2005, 2006, 2007, 2008, 2009; saraswati et al., 2008). increases in n application from 50 to 150 kg ha-1 increased shoot length by 11%, (mukhopadhayay and sen, 1986) or did not increase shoot length and girth (geetha, 2001), while increases in k application from 50 to 150 kg ha-1 did not have any significant effect on shoot growth (mukhopadhayay and sen, 1986; geetha 2001). regardless of plant spacing, an increase in size of planting material increased plant (pseudostem) height; plant height was maximum (84.6 cm) when 1 kg cut corm piece was used as planting material. closer plant spacing (60 x 45 cm) increased plant height (53.8 cm) more than wider plant spacing (90 x 90 cm) (james and nair, 1993). plants produced from whole seed corms were taller than those produced from cut pieces of corm of the same size. this may be due to early sprouting and better root ramification (sen and das, 1991). canopy spread was found to vary between 70.2 and 143.8 cm (ravindran and kabeerathumma, 1991; sen and das, 1991; goswami and sen, 1992; james and nair, 1993; aicrp, 2004, 2005, 2006 a, b, 2007, 2008, 2009). regardless of plant spacing, increases in the 55 size of planting material increased canopy spread and canopy spread was maximum (132.7 cm) when 1 kg cut corm piece was used as planting material at wider plant spacing (90 x 90 cm) (sen and das, 1991). canopy spread was greater in plants raised by planting whole seed corms than in plants produced from cut pieces of corms of the same size. this was presumably due to early sprouting and better root ramification (sen and das, 1991). biomass production of shoots (leaf and pseudostem/petiole) increased up to 120 and 150 dap respectively and declined thereafter, whereas corm dry weight and total dry matter production (tdmp) showed a steady increase up to maturity. the corm drymatter production (cdmp) per ha increased with increases in planting material size or plant density and the highest cdmp (25.6 t ha-1 and 19.4 t ha-1 respectively) was observed at six months after planting (map) by using 250 g cut corm pieces as planting material or with high plant density (14 plants m-2) (das et al., 1997). crop growth rate (cgr) increased gradually up to 120-150 dap and sharply declined at maturity as crop growth ceased. however, the relative growth rate (rgr) continued to decrease with crop age and was the highest at the early growth stage (das et al., 1997). the leaf area increased with increases in planting material size or plant density and the highest leaf-area index (5.4) was observed between 4 and 5 map by using 250 g cut corm pieces as planting material or with high plant density (14 plants m-2) (das et al., 1997). similary cgr increased with increase in planting material size or plant density and highest cgr (25.3-32.2 g m-2day-1) was observed at 5 months by using 250 g cut corm piece as planting material. the cgr was 22. 4 g m-2 day-1 at a plant density of 14 plants m-2 (das et al., 1997). treating corm pieces from the bottom portion of corm with growth regulators thiourea, kno3 and ga3 effectively influenced thegrowth characters and ga3 gave the maximum cormyield (das et al., 1997). 3. plant growth regulators application of triazole compounds (systemic fungicides) triadimefon (tdm), paclobutrazole (pbz) and propiconazole (pcz) through soil drenching increased total root length (by 8.85-75.92%), dry weight of whole plant (by 71.44-84.91%), intercellular co2 concentra-tion (by 25.12-27.91%), leaf thickness, number of spongy and palisade cells, number of chloroplasts per cell, net photosynthetic rate (pn) (by 15.7-28.92%) andwater use efficiency (wue) (by 56.81-87.9%) as compared to untreated control plants. in contrast, total leaf area, transpiration rate (tr) and stomatal conductancedecreased (gopi et al., 2005, 2008, 2009). 4. root characteristics roots grow out from the surface of newly developing daughter corms at the base of the pseudostem through the remnants of the cataphylls concomitantly with leaf emergence. these roots extend horizontally and are densely distributed at a shallow depth of the top 15-30 cm soil depth. the roots are cylindrical and 2 to 5 mm thick. roots grow more than 1 m in length under adequate soil moisture conditions or under adequate rain and are known as “rain roots”. under dry soil conditions, the root length decreases to less than 30 cm length. the transverse section (t.s.) of root shows about 25 layers of thin walled parenchymatous cortex cells surrounding a central stellar portion with eight protoxylem points (plate 4 a and b). plate 4 a and b root anatomy of elephant foot yam (4x10). a) t.s. of root showing stele. b) t.s. of root showing cortex. 56 5. corm development and yield a new daughter corm is formed at the region between the petiole (pseudostem) and seed corm when a sprout grows out from the corm (fig. 4). then, roots appear from the surface of new corm and attain a maximum dry mass at 90 dap. the daughter corm begins to enlarge after a leaf has fully expanded (one to two months) and remarkable enlargement occurs later. the dry mass of seed corms (planting material) decreases gradually, finally decomposing within three months after the new shoot sprouts and develops. after the corm has been initiated, it continuously grows and bulks as long as there is adequate moisture in the soil. morphologically, the corm is a shortened stem with compressed nodes and internodes. there are many small lateral buds (about 1 mm in height) and one large lateral bud (5-15 mm in height) arranged in a definite pattern in concentric nodes of corms. the number of lateral buds per node ranges from 13.0 to 43.3. the number of lateral buds is larger in the middle region of corms than in the head and bottom regions. about 20% of visible lateral buds develop into cormels in the head and middle regions of corms, while about 8% of visible buds develop into cormels in the bottom region. therefore, the middle region of corms produces a larger number of cormels than other regions (sugiyama and santosa, 2008). corm growth rate (corm bulking rate) increased steadily between 1 and 5-6 map. maximum corm bulking rate (7.2-8.2 g plant-1 day-1) was observed dur-ing the fifth or sixth map (mukhopadhayay and sen, 1986; nair et al., 1991). corm bulking efficiency (final corm weight or size as compared to that planted) follows four rules of thumb: 1) at identical plant spacing, corm bulking efficiency decreases with increases in planting material size in both cut pieces and whole corm (table 1); 2) at a given constant planting material size, corm bulking efficiency increases with increases in plant spacing (table 2); 3) corm bulking efficiency is greater in the case of whole corm than cut pieces of corm used as planting material (table 3); and 4) under rainfed conditions, using constant size of planting material, corms harvested from a particular field or a plot show gradient sizes (table 4). nevertheless, the proportion of gradient sizes may narrow under the best management and soil conditions. thus, corms of desired size can be produced by using appropriate sized planting material and plant spacing. production of full corms of 1 kg size is suitable for home consumption because cut corms may perish rapidly. increasing the level of n from 100 to 200 kg ha-1 or k2o from 75 to 150 kg ha-1 increased the plant height fig. 4 new corm formed at the base of the apical sprout before planting. cut pieces 100 100 200 250 250 300 500 500 500 750 750 full corm 250 500 750 1000 400-500 g (z) 1 kg (z) 5 kg (z) rajib et al., 2007 nedunchezhiyan, 2006, james and nair, 1993 rajib et al., 2007 james and nair, 1993 sen and das, 1991 ghosh et al., 2008 james and nair, 1990 sen and das, 1991 ghosh et al., 2008 james and nair, 1990 sen and das, 1991 sen and das, 1991 nedunchezhiyan, 2006 ravindran (pers. comm.) nedunchezhiyan (pers. comm.) size of corm planted (g) plant spacing size of corm harvested (g) corm bulking efficiency reference 50 x 50 cm 60 x 20 cm 50 x 50 cm 60 x 45 cm 50 x 50 cm 55 x 50 cm 60 x 45 cm 50 x 50 cm 55 x 50 cm 60 x 45 cm 50 x 50 cm 50 x 50 cm 50 x 50 cm 50 x 50 cm 50 x 50 cm 90 x 90 cm 1.2 x 1.2 m 1.5 x 1.5 m 513 500-700/1000 659 740 840 810 1063 950 1320 1017 1230 1360 1480 1800 2530 3-4 kg 5 kg 15 kg 5.1 5-7/10 3.3 3.0 3.4 2.7 2.1 1.5 2.6 1.4 1.6 5.44 3.0 2.4 2.5 6-10 5 3 table 1 at identical plant spacings, corm bulking efficiency decreases with increase in planting material (cut pieces or full corm) size (z) corm bulking efficiency in farmer’s field under the best management practices. new corm showing newly emerging roots from the surface sprout 57 100 100 100 200 200 200 250 250 250 300 300 300 300 300 500 500 500 500 500 500 500 500 750 750 750 rajib et al., 2007 james and nair, 1993 ghosh et al., 2008 ghosh et al., 2008 james and nair, 1993 james and nair, 1993 size of corm planted (g) plant spacing (cm) size of corm harvested (g) corm bulking efficiency reference 50 x 30 50 x 40 50 x 50 50 x 30 50 x 40 50 x 50 60 x 45 90 x 45 90 x 90 40 x 40 55 x 50 65 x 60 70 x 70 90 x 85 40 x 40 55 x 50 65 x 60 70 x 70 90 x 85 60 x 45 90 x 45 90 x 90 60 x 45 90 x 45 90 x 90 279 373 513 389 510 656 740 927 1143 810 1005 1265 1450 1585 1170 1320 1610 1800 1970 1063 967 1533 1017 1290 1880 2.8 3.7 5.1 1.9 2.6 3.3 3.0 3.7 4.6 2.7 3.4 4.2 4.8 5.3 2.3 2.6 3.2 3.6 3.9 2.1 1.9 3.1 1.4 1.7 2.5 table 2 at a given constant planting material size, corm bulking efficiency increases with increase in plant spacing 250 500 750 1000 size of corm planted (cut pieces) (g) size of corm harvested (g) multiplication ratio size of whole corm planted (g) 840 950 1230 1740 3.4 1.5 1.6 1.7 250 500 750 1000 1360 1480 1800 2530 5.4 3.0 2.4 2.5 size of corm harvested (g) corm bulking efficiency table 3 corm bulking efficiency is greater in the case of whole corm than the cut pieces of corm used as a planting material (sen and das, 1191) and corm bulking rate (sen et al., 1996). increases in n application from 50 to 150 kg ha-1 increased corm growth (corm bulking rate) by 10.6-27.6% during the six month growth period (mukhopadhayay and sen, 1986). the effect of n was more pronounced during the initial growth period than during the later growth period. the increase in corm bulking rate due to increase in n application from 50 to 150 kg ha-1 was highest (27.6%) during the four month growth period but declined to 15.3% and 10.6% during the fifth and sixth map respectively. the increase in n application from 50 to 150 kg ha-1 increased the mean corm weight per plant by 21.3%. the corm yield per ha increased by 20% with an increase in n application. the corm yield was 84.6 and 102.3 t ha-1 with n at 50 and 150 kg ha-1 application respectively. increases in k application did not significantly increase corm growth, mean corm weight per plant and corm yield per ha. however, n and k had significant interactive effects on corm growth (corm bulking rate), mean corm weight per plant and corm yield per ha and this appears to be mainly due to n (mukhopadhayay and sen, 1986). shoot height, basal shoot (pseudo-stem) girth, and dry matter accumulation in shoot increased and reached a peak at 120 dap. corm and total (shoot and corm) dry matter increased up to 150 days and declined thereafter. maximum shoot height (85.2 cm), shoot girth (16.4 cm), shoot dry matter (6.63 t ha-1) and corm yield (67.83 t ha-1) were obtained with the application of 150 kg ha-1 n and k in two splits (verma et al., 1995). treating planting material (corms) with 2% azotobacter solution at the time of planting and application of 9.0 kg ha-1 of culture mixed with 40 kg of soil at the root zone of the crop along with 150 kg n ha-1 resulted in high corm yield (64.9 and 62.2 t ha-1 respectively) (mukhopadhayay and sen, 1999). size of harvested corms proportion (%) table 4 under rainfed conditions, with use of constant size of planting material, corms harvested from a particular field or a plot shows gradient sizes 2-2.5 kg 1.5-2.0 kg 1.0-1.5 kg 900 g 700 g 600 g 500 g 400 g 300 g 200 g 100 g 50 g 1-2 5 5 5-6 6-7 7-8 15 12 10 10 10 10 58 corm yield varied between 30.9 and 85.4 t ha-1 depending upon the variety, cultural practices (particularly plant spacing) and manurial practices (mukhopadhayay and sen, 1986; nair et al., 1991; ravindran and kabeerathumma, 1991; goswami and sen, 1992; james and nair, 1993; kundu et al., 1998; geetha, 2001; suja et al., 2005, 2006, 2007, suja and sundaresan, 2008 a, b). corm yields between 39.6 and 98.9 t ha-1 were obtained due to application of 100-200 kg n and 100-150 kg k2o5 each per ha (nair et al.,1991; sen and das, 1991; kundu et al., 1998). application of farmyard manure at a rate of 30 t ha-1 increased the fresh mass or corms by 15 %, while application of n at 150 kg ha-1 increased yield by 6.5% (patel and mehta, 1984). kabeerathumma et al. (1987) reported that 100 kg ha-1 n, 38 kg ha-1 of p2o5 and 267 kg ha -1 of k2o were removed from the field every yearwhen 33 t ha-1 of corms were produced. organic farming (fym at 35 t ha-1 + green manuring with cowpea to generate 2 0-2 5 t ha-1 of green matter + neem cake at 1 t ha-1and ash at 3 t ha-1) increased corm yield by 25.37% (62.67 t ha-1) as compared to traditional method (farmer’s practice, fym 25-30 t ha-1 + and ash at 3 t ha-1) (49.99 t ha-1) and by 19.21 % as compared to conventional method (fym 25 t ha-1 + npk @ 100: 50: 150 kg ha-1) of cultivation (52.57 t ha-1) (suja et al., 2005, 2006, 2007, suja and sundaresan, 2008 a, b, 2009). the corm yield was significantly influenced by the size of seed corm and higher yields were recorded from planting materials of 1 kg size (sen et al., 1984; asokan, 1984; sen and das, 1991). increasing the size of planting material from 2 50 g to 1 kg increased mean corm weight per plant from 0.75 to 1.74 kg whereas the corm yield per ha increased from 21.6 to 77.34 t (sen et al., 1984; asokan, 1984; sen and das, 1991; james and nair, 1993; das et al., 1995). comparatively more corm yield was obtained by planting whole seed corms: about 45% greater than the corm yield obtained from cut pieces of corms of the same size (table 5 and 6). this was presumably due to early sprouting and better root ramification (sen and das, 1991). nevertheless, a seed corm size of 400 500 g at 90 x 90 cm spacing would be ideal for economic cultivation of elephant foot yam (james and nair, 1993; yadav et al., 2008). for production of small size (< 1 kg) corms for home use, planting materials of 100-300 g may be used (das et al., 1995; mondal and sen 2004; rajib et al., 2007). to prevent decay after planting due to the presence of several soil borne pathogens, cut corm pieces are dipped in cow dung slurry mixed with mancozeb (0.2%) + monocrotophos (0.05%) for 10 min and surface dried under shade for 24 hr before planting. biofertilizers and other beneficial microorganisms may be added to the cow dung slurry for high productivity (nedunchezhiyan et al. 2006). it was found that planting depth affected plant growth and yield (santosa et al. 2004 a). deeper planting of seed corms led to deformation in daughter corms. at a depth of 30 cm, most corms were elongated or became pyriform. therefore it is desirable to have corms at a depth of 10 cm below the soil surface (sugiyama and santosa, 2008). the multiplication ratio in amorphophallus could be enhanced to 1:15, from the conventional 1:4, by adoptcut corm piece (g) 250 500 750 1000 whole corm (g) 250 500 750 1000 cd (0.05) 37.4 42.2 54.7 77.3 60.5 65.8 80.0 112.4 seed corm size (g) shoot length(cm) canopy spread (cm) mean corm weight (kg) corm yield (t ha-1) 62.6 72.3 81.0 84.6 69.1 75.4 88.8 96.8 0.8 85.8 89.0 114.8 132.7 88.6 99.7 117.9 134.9 0.8 0.84 0.95 1.23 1.74 1.36 1.48 1.8 2.53 0.06 250 500 750 14.1 18.9 23.2 seed corm size (g) shoot length(cm) canopy spread (cm) mean corm weight (kg) corm yield (t ha-1) 36.5 40.4 48.7 99.8 97.1 114.4 1.14 1.53 1.88 table 5 effect of seed corm size on shoot length, canopy spread, mean corm weight and corm yield source: sen and das, 1991. table 6 effect of seed corm size on shoot length, canopy spread, mean corm weight and corm yield source: james and nair, 1993. 59 ing the minisett technique developed in ctcri (james et al., 2004). minisetts produced corms in the range 600 g to 1.5 kg. treating setts of corm pieces from the bottom portion of corm with ga3 (200 ppm) resulted in maxi-mum corm yield (das et al., 1997). various integrated nutrient management practices (combination of inorganic fertilizers, organic manures and biofertilizers) and weed management practices enhanced plant height, canopy spread, corm size, and corm yield per ha (aicrp, 2004, 2005, 2006 a, b, 2007, 2008, 2009). the mean starch content of amorphophallus corm varied between 9.2 and 23.8% and the increase in n or k application did not have a significant effect on starch content (mukhopadhayay and sen, 1986; geetha, 2001). organic practices favoured starch content of elephant foot yam corm (suja et al., 2005, 2006, 2007; suja and sundaresan, 2008 a, b). the starch content was found to range from 3.6 to 11.5% on a fresh mass basis in indonesian accessions (santosa et al., 2002), and from 7.0 to 14.3% in indian accessions (moorthy et al., 1994). little variation was noted in the average size of starch granules (9-13 µm) and amylase content (22-24%) among different accessions (moorthy, 2002). 6. corm dormancy amorphophallus corms exhibit dormancy for about three to five months after harvest. as a result, planting and harvesting are done at a particular time of the year. amorphophallus is propagated by corms as such or by cut corm pieces having a part of apical meristem. sprouting percentage was greater (98%) with top cut portion of corm than the cut corms from the lower half of the mother corm (dhua et al., 1988; nedunzhyan and mohankumar, 1997; mondal and sen, 2004; santosa et al., 2006 b). the bottom portion of the corm is not generally used as planting material due to its lower sprouting efficiency (dhua et al., 1988; nedunzhian and mohankumar, 1997; mohankumar and ravi, 2001). therefore, a greater portion (about 25%) of the harvested produce is again lost as source of planting materials. also, the apical bud from the corm can be excised and used as planting material. removal of the apical bud results in development of one or two adjacent buds within two weeks which also can be excised and used as planting material (fig. 5). ethrel or ethephon was reported to induce early sprouting in amorphophallus corm (dhua et al., 1988; bala and indira, 1992). treating cut pieces of corms from the lower half with chemicals significantly improved sprouting, subsequent growth and yield. among the different chemicals used, thiourea, potassium nitrate and ccc were effective in promoting sprouting. thiourea (200 ppm) and kno3 (1000 ppm) and kinetin (5 ppm) increasedcorm sprouting by 24.3-92.0, 17.8 and 13.4% respectively as compared to control (table 7) (dhua et al., 1988; kumar et al., 1998). however, mean corm weight was greater in plants from corms treated with thiourea (100 ppm), potassium nitrate (kno3) (500 ppm) and ccc(0.02 ml l-1) yielding 722, 821 and 806 g per plant respectively (dhua et al., 1988). however, corm yield per ha did not increase significantly in plants from corms treated with chemicals, as compared to plants from untreated corms. exposing the whole corms to smoke for 6 h per day for six weeks increased sprouting by 58.3% as compared to untreated corms presumably due to ethrel in smoke. similarly exposing the corms to high temperature (32-45oc) increased sprouting by 83.3% as compared to untreated corms (mohankumar and ravi, 2001; archana et al., 2009). pre-harvest, foliar application of potassium nitrate (2%) and thiourea (1%) had greater influence on breaking dormancy and inducing early sprouting (bhagavan et al., 2008). this may be due to an increase in the availability of sugars as a result of an increase in respiration at higher temperature. compared to smoke and heat treatments, soaking corms in different chemicals [kno3, thiourea, ammonium sulphate (nh 4so4)] for a short period (20-30 min) for 1-2 hr had nosignificant effect on inducing early sprouting (mohankumar and ravi, 2001). however, treating the apical portion of corm (after removing the apical bud) with thiourea and subsequently wetting the apical portion for a period of 10 days induced early sprouting with more sprouts (archana et al., 2009). darkness had an adverse fig. 5 emerging sprout from the lateral bud after the apical bud is scooped. emerging sprout from the lateral bud after the apical bud is scooped new daughter corm scar of the scooped apical bud 60 effect on sprouting (kumar et al., 1998). in a. konjac, abscisic acid (aba) and ferulic acid were extracted from the dormant corms and exogenous application of aba (10 mg l-1) and ferulic acid (400 mg l-1) inhibited sprouting and growth of the terminal buds of non-dormant corms, suggesting that aba and ferulic acid are inhibitors of sprouting of dormant corms (sun et al., 1996). corms are acrid before dormancy, but it decreases after dormancy (santosa et al., 2003). 7. ecological requirements elephant foot yam grows well under tropical, warm, humid conditions with maximum day-time temperature ranging between 25 and 35oc, minimum night-time temperature ranging between 20 and 25oc and annual rainfall ranging between 1000 and 3000 mm spread over a period of about six to eight months. it grows well in sandy loam or sandy clay loam soil with good drainage and ph of 6.0 to 7.0. it can also be grown in laterite soil (with about 40-50% gravel) but heavy clay soil is not suitable for this crop. soil with high organic matter favours good crop growth and corm yield. planting material (whole or cut pieces of corm) is planted shallow in pits of 60 x 60 x 45 cm size dug out in well-ploughed soil. the top soil dug out is then mixed with farm yard manure or compost (2.0-2.5 kg per pit) and the mixture is put back into the pit prior to placing the planting material over it. the planting material is placed vertically in the pits and is then covered with soil and compacted lightly. 8. response to shade elephant foot yam tolerates shade conditions. therefore, it can be intercropped between young trees. corm yield decreased by 66 % when light intensity was reduced to 2 5% of full sunlight (pushpakumari and sasidhar, 1992). on the contrary, santosa et al. (2006 a) reported that the fresh biomass of corms increased with a decrease in light intensity; 75 % shading produced the largest corms and 0% shading produced the smallest. under full sunlight necrosis and curling at either the edge or the tips of leaflets occurred causing 25 % loss of the crop. no damage was observed in the 25, 50 and 75 % shading. however, shading treatments significantly decreased the leaf number. the short life span of leaves might enhance the production of new leaves resulting in a larger number of leaves under full sunlight. shading treatments significantly affect the length of petioles and rachis. plants developed the shortest petioles under full sunlight but the longest under 75 % shading. 9. effect of water deficit stress little research work has been done on the response of amorphophallus to water deficit stress. soil moisture status does not influence sprouting but further development of new shoot depends on adequate soil moisture. elephant foot yam plants produce large corms and yield more when the water supply is adequate (aicrp, 2008). about 1000-1500 mm of rainfall per year is optimum for the crop. many plants enter dormancy earlier than usual when the rainy season is shorter than four months and supplementary irrigation is necessary for high productivity under the same conditions. plants produced a larger number of leaves under frequent watering (one-, threeand five-day intervals) than under sevenand 15-day intervals; the third leaves were produced in treatments up to seven-day intervals, but neither the second nor the third leaves were produced with 15-day intervals. furthermore, frequent watering produced large leaves and extended their life span compared to less frequent waterthiourea (100 ppm) thiourea (200 ppm) kno3 (500 ppm) kno3 (1000 ppm) ethrel (0.025 ml l-1) ethrel (0.125 ml l-1) kinetin (5 ppm) kinetin (10 ppm) ccc (0.02 ml l-1) ccc (0.1 ml l-1) control (soaked in water) control (unsoaked) top cut portion cd (0.05) 28.5 31.3 30.9 28.9 18.9 29.3 24.5 16.7 32.4 19.7 30.9 22.2 36.7 11.6 treatments sprouting(%) mean corm weight (g) total corm yield (t ha-1) 73.3 91.1 82.2 86.6 75.5 73.3 82.2 75.5 84.4 68.8 68.8 66.6 97.7 16.7 721.6 488.0 820.6 528.6 434.3 679.6 390.0 410.6 806.3 563.6 587.3 540.6 722.6 218.3 table 7 effect of chemicals on sprouting of corm, mean corm weight and corm yield (source: dhua et al., 1988) 61 ing (santosa et al., 2004 b). a decrease in the dry mass of seed corms was more evident with frequent watering, suggesting that reserved carbohydrates in seed corms are not easily metabolized under a limited water supply. the ratios of dry mass of daughter corms to that of seed corms are 6.1, 1.1, 0.6, 0.4 and 0.2 at one-, three-, five-, seven-, and 15-day intervals, respectively. the high ratios under frequent watering treatments could be ascribed to the fact that the soil water availability affects not only the utilization of dry matter in seed corms but also the production and translocation of photoassimilates into daughter corms (sugiyama and santosa, 2008). the roots dried earlier than usual when the soil water content decreased to less than 40 % of field capacity (santosa et al., 2004 b) and the crop tolerates water deficit stress conditions for about 30-60 days but prolonged stress may affect corm yield (santosa et al., 2004 b). in green-house conditions, plant growth was not affected when plants were watered at one-, threeor five-ay intervals. nevertheless, infrequent watering (watering at sevenor 15-day intervals) reduced corm yield and forced the corms to enter into dormancy. soil moisture conservation methods like mulching induced a higher percentage of early sprouting, greater canopy spread, plant height, greater mean corm weight and corm yield (mohankumar et al., 1973). in india, mulching the field with paddy straw resulted in maximum plant height (78.2 and 88.2 cm respectively), girth (14.1 and 14.4 cm respectively) and corm yield (47.44 and 56.74 t ha-1 respectively) as compared to control (aicrp, 2004, 2006 a, b). also cowpea live mulch produced greater yield (41.72 t ha-1) than control (aicrp, 2006 a, b). maximum corm yield was also obtained by black polythene mulching (82.48 t ha-1) and straw mulch (64.82 t ha-1) ranked second (aicrp, 2004). although the corm yield and net return in the straw mulch treatment was lower than polythene mulch, the cost:benefit ratio of straw mulch (1:3.18) was greater than polythene mulch and other treatments (aicrp, 2004). mulching with sesame leaves also resulted in better corm yield (41.8 t ha-1) than straw and black polythene mulch (aicrp, 2004, 2006 a, b). paddy straw mulch also resulted in greater corm yield (13.8 t ha-1) than control (aicrp, 2004, 2006 a, b). when considering mulching with straw, black polythene or cowpea, corm yield was significantly greater only in the first two cases (11.69-14.12 t ha-1) whereas live cowpea mulching significantly reduced corm yield (5.68 t ha-1) compared to control (7.98 t ha-1) (aicrp, 2004, 2006 a, b). maximum corm yield (44.3 t ha-1) was recorded with the application of 100% recommended dose of fertilizer (rdf) along with flood irrigation and the yield (43.5 t ha-1) was on par with the application of 100% rdf plus irrigation at 100% cpe (aicrp, 2009). corm yield was significantly reduced when irrigation was less than 100% cpe (aicrp, 2009). finally, the corm yield of elephant foot yam was greater (37.3 t ha-1) under micro-irrigation (drip-irrigation) at 60% cpe daily for the first 15 days and then on alternate days for the next 15 days, at 80% cpe between two and six months and then at 60% cpe between seven and eight months, than under surface irrigation (26.4t ha-1) (nedunchezhiyan et al., 2008). 10. seed dormancy successful seed production has been reported in amorphophallus (arakeri, 1950). a seed dormancy of five to six months has been reported in this crop (arakeri, 1956). exposing seeds to running water for six days resulted in greater sprouting (55.5%) than in control (2.7%). however, exposing seeds to water for more than six days led to a lower percentage of sprouting (rajendran and hrishi, 1976). 11. future thrust since the whole corm and cut corm pieces are used as planting material, a large portion of harvested produce is used for propagation. therefore, the development of plantlets through in vitro culture of apical/lateral buds (irawati et al., 1986; archana et al., 2009; unnikrishnan and mohan, 2009) should be further refined and exploited for planting material production. furthermore, more detailed investigation of the physiological aspects of growth and productivity of amorphophallus needs to be developed in the following areas: effect of photoperiod and temperature on leaf area development, crop growth rate, stomatal characteristics, photosynthetic rate, root development and rooting pattern, corm development and bulking rate, light interception, dry matter production and partitioning (harvest index), varietal variation in these aspects and physiological factors limiting corm yield. effect of exogenous application of growth regulators such as benzyl adenine and other such growth promoters on maximizing corm yield. since amorphophallus needs a long duration (8 months growing period) for maximum corm yield, studies on factors controlling corm bulking could reveal the physiological basis for developing rapid bulking, short duration varieties. studies to determine water, light and thermal degreeday requirements and the effect of water deficit stress, high temperature (>35oc) (heat stress), salinity and shade on growth and productivity. factors controlling corm dormancy, breaking of dormancy and sprouting and related gene expression. references aicrp, 2004 elephant foot yam. all india coordinated research project on tuber crops, annual report 2003-2004, pp. 54-62. aicrp, 2005 elephant foot yam. all india coordinated research project on tuber crops, annual report 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n., panda p.k., 1995 growth, corm development and uptake of n and k as influenced by doses and methods of n and k application in elephant foot yam (amorphophallus campanulatus). j. potassium res., 11: 68-74. yadav r.k., sanwal s.k., deka b.c., buragohain j., 2008 effect of seed size on tuber yield of elephant foot yam (amorphophallus paeniifolius) under meghalaya condition. nat. sem. amorphophallus: innovative technologies, abst., pp. 111. 129 1. introduction yield and quality performance of fruit crops results from the co-ordinated and integrated functioning of several components which constitute the “orchard system”. this system must be carefully assembled in all its components in order to respond to the diverse environmental, social and economic constraints. designing the system means to define the physical arrangement of the trees, their shape and all the agronomic practices to apply during the entire life span of the orchards. all the system components must be implemented at the time of the orchard design determining its basic traits such as planting density, training system, and scion and rootstock combination. in addition to these physical traits it is necessary to define the proper pruning strategy to be adjusted year by year to the chosen training system taking into account also the agronomic operations direct to the management of the soil root relationship. pruning is the basic tool to manipulate fruit tree architecture and behaviour in order to achieve economically sound crop yield and fruit quality. “pruning” includes a very large and diversified number of operations involving the aerial organs and the root system, performed both during rest and vegetative season (for a glossary of pruning terms see: baldini and scaramuzzi, 1962; liebster and pesserl, 1982; ingels et al., 2002). once the major traits of the orchard have been defined, the pruning strategy may then enter into discussion. the main objective in training fruit trees is the modulation of competition among the vegetative and reproductive parts of the tree, which essentially means the manipulation of the source-and-sink relationships occurring in the plant taking into account other basic objectives such as light interception and distribution in the canopy, and biomass partition properly directed toward the fruits. the relationships between pruning and processes of growth, fruit bearing and senescence assume a strategic relevance as almost every pruning operation affects the growth of a tree or its organs on either a shortor longterm scale. pruning is a very powerful tool but it cannot overcome the effects of basic mistakes in design and management of the orchard. the significance of the architecture and growth traits of trees and their relationships with reproductive activities are essential to understanding how pruning may affect the functioning of the orchard system. in this paper the principal aspects of plant architecture and growth relevant to pruning are examined; since “pruning” includes many diverse operations, a large range of combinations of winter and summer pruning manipulations is possible and therefore their potential effects on tree behaviour are considered and explained in terms of the sourceand-sink relationship and competition among sinks. a number of the references cited in this paper may seem dated, but it must be said that studies and experiments on pruning have recently become rare whereas during the 1980s and ‘90s scientists, researchers and field experts paid great attention to this inalienable orchard management practice. italian contributions to the knowledge of the physiological and practical basis of pruning have been particularly important, as shown by fundamental papers and reference books on this topic (dotti, 1949; morettini, 1963; bargioni, 1992; sansavini and errani, 1998; branzanti and ricci, 2001). it seems, however, useful to reasbasic considerations about pruning deciduous fruit trees c. giulivo dipartimento agronomia ambientale e produzioni vegetali agripolis, università degli studi di padova, via romea, 16, 35020 legnaro (pd), italy. key words: architecture, correlate function, fruit trees, growth, pruning. abstract: crop yield and fruit quality performance determine the economic value of the orchard system. once the basic traits of the orchard design are defined, pruning may represent a powerful tool to modulate the vegetative and productive behaviour of the trees. the various pruning manipulations involve variations in growth, size and geometry of the plant and their effects are explained in terms of correlate functions among the tree organs. in spite of its potential, pruning can not overcome the effects of some basic mistakes made in design and management of the orchard. basic concepts about pruning developed in the past are reassessed in light of recent knowledge on architecture and growth of the tree. adv. hort. sci., 2011 25(3): 129-142 received for publication 13 may 2011 accepted for publication 27 july 2011 130 sess these contributions in light of recent knowledge on the architecture and growth of trees. 2. tree architecture the identification of tree architecture traits is relevant to the pruning strategy. according to costes et al. (2006) plant architecture includes two independent notions: branching and connection between plant units (topology), and the spatial location, orientation, size and shape of the vegetation elements (geometry). tree architecture includes physical structures and physiological functions of fruit trees, and is therefore related to space and time aspects. the multilevel approach of the tree (whole tree, branch, leaf and fruit) should be stated and tree architecture should be described by canopy height, width and shape, by fruiting and vegetative shoot types and their relative proportions, spatial distribution, branching, and growth dynamics. canopy porosity or density should also be considered as light interception and distribution, and pest occurrence are concerned (simon et al., 2007). the orchard structure, function and production may be understood if, in addition to tree architecture, phenotypic plasticity and phenology are considered. trees are modular organisms which develop by reiteration of elementary botanical elements whose anatomical, morphological, dimensional and functional traits change during ontogeny and accordingly to various life stages (bathélémy and caraglio, 2007). during ontogeny plant appearance varies with form and/or structure and with temporal and/or topological changes and, therefore, it is of high practical relevance to orchard design and management (fig. 1). tree architecture depends on the nature and relative arrangement of each of the tree’s parts. at any given time, the architecture is a result of equilibrium between internal growth events and external environmental constraints. pruning must take into account this natural fact and hence it must follow the natural behaviour of the fruit tree and direct it toward the desired economic goals. the main morphological traits used in describing tree architecture are the growth processes, branching modalities, morphological differentiation of axes and location of the reproductive structures (barthélémy and caraglio, 2007). growth processes. organogenesis and extension are two distinct but co-ordinated morphogenetic events that result in the primary growth of a plant. these events basically determine the stem of a plant, which can be considered a succession of internodes and nodes, on which a leaf (or leaves) and axillary bud (or buds) are located; the node and the subtending internode represent the basic structural unit of the plant body (metamer or phytomer). a tree, whatever its final size, is initially formed by the activity of at least two primary meristems, one to develop the aerial part and one the root system, but the subsequent increment in diameter of woody axes (aerial and subterranean) is determined by the activity of secondary meristems. in many species, the apex of the aerial axes may abscise or abort after a time of functioning or it may shift into reproductive structure or other organs: this behaviour is defined as determinate growth. on the other hand, when the apical meristem of the axes maintains indefinitely its growth potential, an indeterminate growth (or extension of the axis) occurs. continuous growth occurs when a shoot does not show a marked endogenous cessation of extension, a case usually occurring in uniform equatorial climates or environments. when a shoot shows marked endogenous extension periodicity and cessation, rhythmic growth occurs. rhythmic extension of leafy shoots is the typical pattern of deciduous fruit trees in which meristem activity shows an alternation of periods of rest and of active extension. the rest period marks on an axis portion a zone of short internodes and/or cataphylls corresponding to the protective organs of the bud from which the axis derives. rhythmic growth of the stem may combined with continuous or more frequently with a rhythmic organogenesis pattern. preformation and neoformation. when metamers and organs of a shoot are already present in the bud before the elongation of the axis deriving from it, the shoot is called preformed. in other cases, more metamers and organs than those included in the bud appear on the shoot and are neoformed by the apical meristem. fruit trees show a strong polymorphism in axis development (costes et al., 2006) (fig. 2). preformed shoots are usually short axes (brachyblast) which after bud burst elongate slightly due to the extension of the preformed metamers. in horticulture short shoots are referred to different names according to the species and to their reproductive or vegetative structures. in stone fruit fig. 1 evolution of the tree crown during plant life (from hilkenbäumer, 1953). 131 species, they are called ‘leaf rosettes’ or ‘clusters’ if they bear, respectively, only leaves or an apical vegetative bud and a variable number of lateral flower buds. in pome fruit species, the short preformed shoots are called ‘dards’ when they are vegetative or ‘spurs’ when they bear also flowers (fig. 2). in many fruit species (cherry, apple and pear trees in particular) the spurs can be one-year-old brachyblasts or consist of a perennial set of branched shoots, which have all remained brachyblasts. longer shoots may be also preformed (mesoblasts) and in this case their limited length derives only from the elongation of internodes; mesoblasts can carry only vegetative buds or carry some lateral flower buds (stone fruits) or an apical fruit bud (pome fruits) and, in any case, are called ‘brindles’(fig. 2). preformation and neoformation can also be combined to produce much longer shoots (auxiblasts) in fruit trees; they are usually vegetative axes in pome fruits (fig. 2), whereas in stone fruits they can bear numerous lateral flower buds as in peach tree. branching process. the complex architecture of a tree consists of several axes, one derived from another by repetitive processes (fig. 3). the branching process is based on axillary meristems located just beside the initiated leaf at a node. more than one axillary bud may be found at the axil of a leaf as in the case of mixed shoots of peach trees or other stone fruit species. the branching pattern of an axis can be monopodial or sympodial according to its indeterminate or determinate growth pattern, respectively. in the case of a sympodial pattern, one, two or more branches may develop after the death, abscission or transformation of the apex. in fruit trees one or two branches arise more commonly. rectilinear stems may be composed of a succession of metamers or growth units or annual shoots, all produced by a single meristem or by a linear succession of sympodial modules (barthélémy and caraglio, 2007). continuous branching occurs when all the axillary meristems of a stem develop into lateral shoots; rhythmic branching occurs when lateral axes are grouped as distinct tiers with an obvious regular alternation of unbranched and branched nodes on the parent stem. branching may be diffuse if only some nodes of the parent axis bear a lateral axis or if a regular distribution of branches in tiers is no evident. acrotony, basitony and mesotomy. the positional preferential development of lateral branches on a vertical parent axis may be classified into three categories. acrotony is the prevalent development of lateral axes in the distal part of the parent axis or shoot, whereas basitony consists in the prevalent development of branches on the proximal part; mesotony is used to denote a privileged development of branches in the middle part of the axis. the topological arrangement of lateral branches along a parent axis is often associated with an increasing or decreasing gradient in length and/or vigour of the branches. in fruit trees, all three categories of lateral axis development are present with some variations even in the same species. the topological arrangement of branches in the tree crown can be strongly modified by pruning. hypotomy, epitomy and amphitomy. considering that an axis may diverge from the vertical, the privileged arrangement of lateral axes on a parental axis is very different if this is horizontal, curved or slanted. the privileged zone may be the upper, lateral or basal position of the parent axis and the terms are respectively epitony, amphitony and hypotomy (fig. 4). in many fruit trees epitony is fig. 2 polymorphism of the axes on an apple branch. left: s (brachyblast) and bs (brachyblast on a bourse) and br (mesoblast) are preformed shoots, ls (auxiblast) are preformed and neoformed shoots. right: long shoot, brindles and spurs on a bourse. fig. 3 evolution of a one-year axis (a) into two-year (b) and threeyear (c) branches. fig. 4 development of lateral axes on an axis. slanted parent axis of peach tree (left) and curved parent axis of apple tree (right). 132 a very common feature (costes et al., 2006), it is often associated with the survival of old branches in old trees (fig. 5). hypotomy is characterized by a privileged development of lateral axes in the curvature zone of a branch. amphitony occurs frequently on rectilinear horizontal or slightly slanted branches. the latter two features may be combined in slanted and curved branches and their incidence in the expansion of lateral branch complexes is of the outmost importance in the aerial architecture of many woody plants. amphitony is a frequent behaviour in rectilinear branches while epitony and hypotony are characterized by the predominant development of lateral axes on the convex side of the curved, downwardlyor upwardlyoriented branches. as they are highly influenced by axis orientation, these branching features are frequently combined with topological arrangement along axes (acrotony, basitony, mesotony) and these combinations can strongly influence the bud fate according to their topological position and space orientation within the tree canopy. polymorphism of axes. the orientation of an axis and the spatial disposition of its leaves are of major importance in the growth strategy of a tree. within a single tree, some of these axes are essential in plant skeleton edification; some are involved in space exploration whereas others are more directed toward reproductive function or light interception and photosynthesis. the differentiation of axes and bud fate may be highly specialized and very different structures (i.e. flowers, inflorescences, spine, shoot, etc.) may be found in a single leaf axil and in a precise position, but the differentiation of an axis may not be an irreversible process. depending on modifications of internal or external conditions or after architectural traumatism or manipulation (pruning), reversion of axis differentiation may frequently occur, indicating that shoot differentiation and bud fate are controlled by a whole plant network of correlated and environmental conditions. the polymorphism of axes is common in fruit trees and represents a morphological differentiation determined by meristem expression and activity. it is common that several types of axis coexist on the same individual tree. however axis specialization is very significant when a tree is trained and pruned for fruit production since in this tree a balanced distribution among skeleton axes, reproductive axes and vegetative axes used for renewal fruiting shoots (fig. 6) must be found. orthotropy, plagiotropy, and mixed axes. orthotropic axes are generally erect to vertical with a radial symmetry, bear leaves in spiral, opposite or verticillate disposition, and lateral axes in all spatial directions; orthotropy is generally associated with plant skeleton edification and the colonization or exploration of vertical space. by contrast, horizontal to slanted oriented axes (plagiotropy) show a bilateral symmetry with distichous phyllotaxis and lateral axes arranged in one plane; plagiotropy is generally concerned with exploration and exploitation of the horizontal space and reproductive functions. in many trees an axis may present an orthotropic proximal portion and a plagiotropic distal end or vice versa. the superimposition of such “mixed axes” is a distinctive feature of trunk edification in trees, but it can be strongly manipulated by pruning (fig. 7). the position of sexuality and reproductive organs can be terminal or lateral and the onset of these organs involves dramatic changes in the architecture of the trees because its impact on plant growth and branching. the lateral or terminal position of reproductive organs is of great relevance for the orientation of the axis that supports the organs (fig. 8). fig. 5 evident epitomic behaviour in an old apple tree (from liebster and pesserl, 1982). fig. 6 in the tree canopy the polymorphims of axes is differently located. in some parts vegetative structures prevail and in others reproductive structures are predominant (redrawn from hilkenbäumer, 1953). 133 architectural model. the growth pattern of a fruit tree species which determines the successive architectural phases must be taken into account if a proper and specific training and pruning is to apply to the trees. the architectural model derives from an inherent strategy that defines both the manner, in which the plant elaborates its form, and the resulting structure. the model results from the nature and the sequence of activity of endogenous morphogenetic processes as determined by the basic growth program on which the entire architecture is established and realized under the environmental and cultivation constraints. a particular combination of simple morphological features may identify typical architectural models: a) the growth pattern (determinate vs. indeterminate growth; rhythmic vs. continuous growth); b) the branching pattern (terminal vs. lateral branching vs. no branching; monopodial vs. sympodial branching; rhythmic vs. continuous vs. diffuse branching; immediate vs. delayed branching; c) the morphological differentiation of axes (orthotropic vs. plagiotropic vs. axes with orthotropic and plagiotropic portions); d) the position of reproductive structures (lateral vs. terminal fruiting) (barthélémy and caraglio, 2007). the tree architecture can be considered a hierarchical branched system in which the axes are grouped into categories according to their morphological, anatomical or distinctive functional features. this branch system, even if very complex, is composed of a simple sequence of axes characterizing its basic architecture. the simple architectural unit lasts during the whole life span of some tree species while in most trees it is possible to recognize repeated architectural units during their development, late in ontogeny, or under particular conditions. this process is called “reiteration”; more precisely it is a morphogenetic process through which the tree duplicates its own elementary architecture. this process may be demonstrated in several structures such as water shoots, root suckers, etc. these reiterated structures may derive from dormant meristems and in this case are called proleptic or delayed. in other cases, reiteration may be a consequence of a shift in the functioning of the apical meristem of a growing shoot that will finally produce a less differentiated structure; in this case, the reiteration is described as sylleptic or better immediate. the trees develop by the repetition of elementary construction units conforming to their model and a differentiation sequence in the activity of their whole set of meristems. the specific and exact structure of a particular organ in a given location within the architecture of the tree may be considered as the result of a complex of several ontogenetic and morphogenetic factors that influences all plant organization levels, at each stage of plant development and during its whole life span. although environmental factors may modulate these sequences of differentiation, they almost never modify the inherent morphogenetic and ontogenetic constructional project of the plant organization. this is also true for horticultural manipulations such as training and pruning, confirming that it is advisable to follow the fruit tree’s development without radically changing its vegetative and reproductive behaviour. in other words, the branching order of an axis must be respected since the higher the order, the higher its degree of differentiation. when the architecture of a branched tree must be manipulated, the modification must be carefully considered according to the specific branching pattern, gradients (acrotony, basitony, mesotony) and the axis spatial orientation and/or geometry (hypotony, epitony, amphitony). in a given plant species, or even in a fig. 7 the relative location of vegetative and reproductive activities may be changed by variations of growth direction of axes imposed by pruning. fig. 8 natural variation in growth direction of a pear brindle with an apical fruit bud (from grisvard, 1957). 134 single tree, very different situations may be observed: at a given stage of development, homologous botanical structures with different features coexist on the same individual (e.g. short vs. long shoots or reproductive vs. vegetative shoots) whereas, by contrast, similar elementary structures with the same morphological features (short shoots with vegetative or reproductive features) may be observed in very different plant ages or stages of development. this may be defined as the physiological age of the meristem, which may generally be characterized by a particular combination of several morphological, anatomical and/or functional attributes of a given plant structure derived from this meristem. the physiological age of the meristem depends on its location in the plant architecture and on the stage of development of the plant; its expression may be modulated by environmental factors and obviously by cultural practices, particularly by pruning (fig. 9). the physiological age of a meristem relates to the degree of differentiation of the structure it produces. typical features of the physiologically aged structures are, for example, the short axes of many trees: growth units are short, bear flowers and may have a short lifetime. these highly differentiated axes may be considered physiologically old whatever their moment of appearance. by contrast, main axes consisting of vigorous growth units and/or annual shoots may be considered physiologically young products and generally appear only in young trees or in strongly pruned fruit trees. identification of meristem age of is very important in order to understand the comprehensive architecture of a plant or even its plasticity, i.e. the effects of the environment and/or of cultivation practices on its development and structure (phenotypic plasticity). 3. tree growth the growth of a living being means the irreversible increase of dry matter, and more currently the term is used for any positive variation in shape, size or fresh weight of an individual or a part of it. trees are characterized by secondary growth processes and by a continuous formation of woody tissue, part of which is non-living biomass. the woody part has the essential functions of tree support, sap transport, and storage of water, nutrients and carbohydrates. from the stand point of modern horticulture, trees of large size are not desirable because they invest a large part of energy and resources in building up and maintain their woody frame. the growth rate of the whole tree is of great economic meaning since it determines, in addition to dry matter accumulation, other important events such as the overcoming of juvenility (if the tree develops from a seed), the duration of the initial low productivity stage, and the acquisition of high fruiting and competing capability. practically speaking, in a grafted fruit tree a fast initial growth rate is an economic advantage for rapidly attaining its definitive size, which in modern fruit trees should be as small as possible in order to realize a high ratio between fruit load and woody frame (bargioni, 1988). tree growth (i.e. permanent increase of dry matter) is balanced between the development of new leaves and new roots. the new leaves exert a positive feedback on the assimilate production and similarly the new roots a positive feedback on absorption of more water and nutrients. at the beginning of a tree’s life, the major part of assimilates produced by the leaves is invested in the construction of the root system. as long as the root system does not achieve an adequate size to supply a sufficient amount of water and nutrients, the reproductive potential of the tree remains zero or extremely low. during maturity the canopy-root ratio is relatively stable and fruiting is kept more or less constant. when the tree attains a given size, the costs of maintaining the structure become very high and fewer and fewer resources are available to sustain root system growth, since its competition potential is weak in relation to other plant organs, particularly fruits. less water and nutrients are then available to the crown, reducing net photosynthesis and assimilate potential and, as a consequence, senescence of the tree is triggered. the net investment of the various resources which enter in and exit out of the tree and partition of the biosynthesis products are two important determinants of the balanced growth of a tree. water, nutrients, carbohydrates, organic acids, lipids, proteins, hormones etc. may be utilized in different fig. 9 formation of lateral axes of different types and ages on an apple tree branch pruned according to the various pruning criteria used in four training systems, which diverge from the natural behaviour very little (a) to very much (d) (redrawn from hilkenbäumer, 1953). 135 ways and times in relation to the physiological balance of the tree. the amount and partition of photosyntates gives a good indication of the growth potential of the tree or of its organs because they are the source of energy which can be used at the time or stored. source-sink interactions, that is the transitory destination of assimilates and their final partition, determine the rate and expression of the relative growth of the plant organs and therefore the architecture and size of the tree. the aim of tree and orchard management is to channel the major part of assimilates toward the reproductive structures, limiting that directed toward other plant organs without affecting the functioning of the whole system. individual tree architecture can vary greatly based on the growth strategy of the plant but can be modified by the environment and cultivation conditions (phenotypic plasticity). an example related to orchard design is the competitive stress induced by planting density (fig. 10). in comparison to a widely spaced population, closely spaced tree populations have a more limited volume of soil available for the individual root systems and thus fewer resources are supplied to the tree canopy. with close spacing, individual trees are smaller and less branched, but the ratio between fruits and woody structure (or volume of the crown) is higher than in individuals in a widely spaced population. not all plant characters are affected equally by the competitive stress, and some effects tend to be speciesand environmentspecific (cannel, 1983). light interception and distribution in the canopy may play a concomitant role in plant growth; in the tree canopy the behaviour at high incident irradiance depends on the degree of light saturation of the leaves, which in turns depends on their geometrical arrangement and the geometry of the tree, which determine the ratio between direct and diffuse radiation (connor, 1983). from outside to inside the canopy the quantity of light decreases sharply, as does the light quality (proctor, 1978). taking into account the variation of light quality inside the canopy (visible light, red/far red, uv), plant form and reproductive performance may be different in dense or open canopies because of the photomorphogenic and photoperiodic effects (connor, 1983). in stands composed of genetically identical trees, like orchards, the effects of competitive stress can be even more emphasized if appropriate pruning and management practices are not applied. the contribution of pruning to the shape, growth and functioning of the tree can be relevant but it does not introduce dramatic variations in the natural vegetative growth habit of the fruit tree. 4. correlative functions the correlative functions within an individual tree (i.e. competition and cooperation among the plant organs) deserve particular attention because they offer some basic concepts for pruning, which l.h. bailey illustrated very well a long time ago in “the pruning manual” published as first edition in 1898. it is worthwhile to report exactly what he wrote. “a tree is essentially a collection or a colony of individual parts. every branch, even every joint of the branches, may do what another branch does it may bear leaves, flowers and seed. every branch competes with other branches; and there are more germs of branches that is, more buds than there can be branches on any tree, or in any other plant that by its nature produces many branches. no two branches of a tree are exactly alike, but are what their position or condition or heredity makes them to be. some are strong and some are weak. that is, there is no definite or proper size or shape for any branch, as there is for different members of an animal or of a flower. the limbs and organs of an animal are not competitors but copartners, each performing some functions or office, that another does not, and they all attain a definite maturity of size and shape. but a branch in a tree-top never attains its full size until it ceases to grow and thereby begins to die. branches are not so much organs as competing individuals. if all these statements are true, then three conclusions follow: there is a contest among the branches of a plant, and some of the contestants perish; the destruction of these branches may conduce to the betterment of those remaining; all the branches of a tree are not necessary to it, but some of them may be superfluous or detrimental to it. in other words, pruning may follow as natural course.” in this context the manipulation of trees by pruning is amply justified in an orchard. citing again bailey, “of course there is a kind of partnership between the branches of a tree, for we assume that each strong branch makes a contribution to the development of the root-system and trunk-system, and there is not the same separateness as between wholly different plants; yet the contest between these branches is apparent, and it has special significance to the present discussion.” of course these considerations may be extended to the other organs of the tree: buds, twigs, shoots, flowers, fruits and roots. fig. 10 effect of planting density on tree structure. closely planted trees bear more fruits per unit canopy volume than widely spaced trees because of phenotypic plasticity (cannel, 1983). 136 taking into account the functional equilibrium between the size and activity of the shoots (carbon fixation) and size and activity of the fine roots (absorption of water and nutrients), that in a constant environment, favouring continuous growth, tends to maintain a constant ratio of root/shoot relative growth rates (cannel, 1985), it is evident that removing a part of a tree by pruning represents a loss of a given amount of tissue and in replacing the lost part the tree expends a certain amount of assimilates. this may seem nonsense, but it is necessary to keep in mind the goals of the orchard, which are the economics and adjustment of treeenvironment interactions. tree vitality is not impaired by removing a part of it unless the removal is so great that it interferes with nutrition (growth) of the remaining parts, for example a great removal of photosynthetic leaf surface or of adsorbing roots. taking into account plant homeostasis, it is obvious that if part of the shoots or branches are removed, assimilates are devoted to rebuilding the lost part and fewer are directed toward the root system, and vice versa. if part of the roots are suppressed, the shoots resume their growth only when the pruned part of the root system is rebuilt and the root/shoot equilibrium is re-established, other aspects remaining constant (richards and rowe, 1971). therefore, shoot pruning temporarily checks root growth, while root pruning temporarily checks shoot growth: the more pruned off, the greater the check in growth, and the longer it takes for the plant to recover the root/shoot relationship that existed before pruning. thus, shoot pruning is a means of promoting new shoot growth, and root pruning is a means of promoting new root growth (cannel, 1985). therefore when part of the treetop is cut away, if not too severely, the tree resources are directed toward the remaining growing points and the tree develops, for example, more vigorous shoots. the consequence is that a correctly pruned tree appears more vigorous and also, as reported by dotti (1949), more productive and longliving than an unpruned tree. for the purpose of this paper, the genetic and metabolic processes causing and governing these correlative functions will not be considered but instead some of the most relevant organ and function competitions for the tree resources are addressed from a phenotypic point of view. hierarchic but not univocal relationships are often established among tree organs and they contribute to the correlative functions that may involve organs of the same or different type. 5. correlative relationships between organs of the same type in this section relationships between meristems, buds, shoots, branches, flowers and fruits are considered. the most important correlative functions are those among the meristems present on a shoot since they originate all the organ typologies. the role of apical dominance, that varies amply according to genotype, development stage and environmental conditions, is known. apical dominance also affects the functional relationships of the buds, determining different vegetative gradients along the shoot. these phenomena offer an important basis for pruning practices. in peach the number of sylleptic shoots appeared to be related to the growth rate displayed by the parent shoot during the early part of the growing season (four to five weeks after bud break). parent shoots with high growth rate formed sylleptic shoots in a greater number than the pinched back ones in which apical dominance was completely abolished. therefore growth rate in the early growing season may play a stronger role than pinching in feather formation. apex removal by pinching (summer pinching) changes the physiological status of a growing shoot, whereas bending affects only the shoot growth rate, thus its feathering (giulivo and ramina, 1974). an interesting case, reported by hilkenbäumer (1953), regards the number and distribution of buds on a tree. three cases are considered and are shown in figure 11: a) when a tree carries a very high number of buds, on each axis a large number of weak shoots are produced and a part of buds remain silent; b) if all the axes are heavily and evenly pruned back, the number of buds is reduced and fewer but vigorous shoots originate in the same manner on each branch; c) if one axis is heavily pruned back and the other two lightly, weak shoots are produced by the former and strong shoots by the latter axes in contrast with case b. cases a and b may be explained by the assimilate partition among many or few buds (hilkenbäumer, 1953). for case c, which is more difficult to explain, it is suggested that the less pruned axes dominate the heavy pruned axis because they bear more buds and sprout earlier, becoming thus stronger sinks. this may be considered an example of the independence-competition among axes of a tree. the fewer the shoots are allowed to grow with a severe pruning, the longer they are (fig. 12). when only very few shoots are present on the tree, not only are they thicker with larger leaves and have longer internodes, but some of the lateral buds grow out to form short shoots or feathers (abbot, 1984). the leading shoot (dominant position) is however always longer than those below it (fig. 13). whichever parent axis is manipulated (fig. 14), there is fig. 11 vegetative responses of branches headed back with different intensities (a and b) or uneven intensity (c) (redrawn from hilkenbäumer, 1953). 137 always one lateral axis that assumes dominance over the other axes. these factors clearly indicate the competition between homologous vegetative organs. a typical case of competition among homologous organs is the high abscission potential or reduced development and growth of fruits that are located, respectively, below or above the fruit that sets first in the corymbs of apple and pear trees (fig. 15). the effect of fruit set priority on competition was also reported in peach tree where the fruitlets which set first and start to grow rapidly have the lowest probability of abscission (ramina 1981; giulivo et al., 1981 b). 6. correlative relationships between organs of different types the strength of relationships among organs are dynamic and change over the growing season and year. usually the organs that have the larger mass (size) and grow more actively dominate the other organs of the plant. the relationship between vegetative and reproductive structures is a typical case. when shoot growth is very intense, fruit growth is limited and vice versa (fig. 16) (giulivo et al., 1981 b; pitacco and giulivo, 1992). in an unpruned tree parts of canopy exist where vegetation dominates and parts where fruiting is prevalent. the position of a vegetative axis in space determines the vegetative-reproductive relationship (fig. 6); in a vertical axis the vegetative activity is strongly favoured and fruiting is repressed. if an axis occupies a lower position in fig. 13 elongation in apical (a), sub-apical (b, i) long shoots and in brindle (br) shoots of three cultivars of peach tree with different ripening time (giulivo et al., 1981 a). fig. 14 position of the dominant shoot in headed-back vertical (a), curved (b) and slanted (c) parent axes (da grisward, 1953). fig. 12 effects of light, medium and severe pruning (one, two, three shoots on a headed-back one-year-old apple tree) (abbott, 1984). 138 the crown and tends toward a plagiotropic position, the relationship is reverted. this phenomenon obviously offers a great opportunity to manipulate a tree with winter and summer pruning. it can be assumed that every manipulation limiting growth can increase the reproductive activity, i.e. fruit bud differentiation and fruit development and growth. two typical cases may be reported: a) by bending, the elongation rate of a growing shoot is decreased but bud differentiation is favoured; b) with limited nitrogen availability the vigour of the tree is depressed but the fruiting capability is increased (hilkenbäumer, 1953). the relationship between fruitlets and shoot carried by a bourse of apple tree can be an interesting case of transient competition between a leafy shoot and a reproductive organ carried by the same plant structure (brachyblast). usually the fruitlets carried by the bourse corymb present a steady abscission for some weeks after full bloom but, if the shoot of the bourse is removed early, fruitlet abscission is delayed and reduced; the fruits on bourse deprived of the shoot growth less and at their final size results much smaller than that of fruits on a normal bourse carrying a shoot (abbot, 1984). this is a consequence of fruit abscission in the corymb, but it may also be an effect of the transport of assimilates to the fruit which takes place when the leaves of the bourse shoot become active exporting organs. as the season progresses, the developing fruits have an increasing demand for assimilates: after some weeks from fruit set they are diverted from shoot and root growth, bud differentiation and later from reserve storage. increasing the fruit load has a dramatic effect: fruit bud differentiation is progressively reduced to a certain threshold, beyond which it is completely abolished (fig. 17). the presence of fruits on the tree slows down the formation of fruit buds as long as the fruits remain on the tree (fig. 18); the later the harvest, the stronger the effect (giulivo et al., 1981 a). a heavy crop load, induced by a very low fruit load during the previous year, strongly reduces shoot growth, flower differentiation for the next year and storage of reserve fig. 16 seasonal growth rate of shoots and fruit of peach tree, cv. andross. when the former is high, the latter is low, and vice versa (pitacco and giulivo, 1992). fig. 17 effects of increasing fruit load of apple tree on shoot and fruit bud formation and on elongation of the longest shoot. (abbott, 1984) fig. 15 competition of the first setting fruitlet over the other fruitlets of the same corymb of pear (left) and apple tree (right). 139 materials (giulivo, 1990). this can explain the succession of onand off-years. a large number of developing fruits requires a great amount of assimilates which are diverted from vegetative growth and bud differentiation and this is a clear example of competition between different plant organs or functions. flower or fruit thinning in the on-year is a powerful tool for modulation of the competition exerted by the fruits (ramina, 1981; abbot, 1984). over-cropping and undercropping, subsequent to offand on-years, can be to some extent overcome by winter pruning, decreasing or increasing the number of fruiting structures of the tree. the onset of fruiting is a dramatic event in the behaviour of a tree and the relative growth of plant parts is radically changed (chalmers and van den ende, 1975): as the crop potential of the tree increases with time (fig. 19), fewer and fewer resources are allocated in the permanent structures of the tree (trunk, branches and root system). this means that cropping of the tree is over time an aging factor and its regulation is a way to delay senescence of the plant. the regulation of cropping by summer and/or winter pruning represents a powerful tool for the maintenance of long-lasting economic performance of fruit trees. during the growing season a heavy fruit load reduces root growth; very strong vegetation acts in a similar way, limiting fine root formation (willianson and coston, 1989). the partitioning of assimilates between shoots and roots is strongly affected by the water status of the tree (schultze, 1982). in many cases and in some stages of development the competition between tree organs is often connected with cooperation. the dynamic relationship between shoots and fruit within the whole tree, and also within a single leafy shoot, is particularly relevant. in fact, in an early stage of fruit growth (fig. 20) the relationship shoot/fruit depends on the number of sinks and their relative position along the mixed shoot, as shown by an experiment on one-year-old peach shoots (giulivo and ramina, 1975). complex relationships among sinks occur, even within a single node of a shoot as observed in peach tree. it is fig. 18 fruit growth and percent of fruit bud formed on shoots of three cultivars of peach tree with different ripening times (giulivo et al., 1981 a). fig. 19 dry matter partition among fruits (points), tree crown (crosses) and root system (asterisks). at increasing tree age represented by the trunk circumference less and less dry matter is allocated in the permanent structure of the tree (redrawn from chalmers and van den ende, 1975). fig. 20 fruitshoot correlative relationships on peach one-year-old mixed shoots 0.2 m long. (circles = fruit; arrows = shoots; square = basal leaf of mixed shoot treated with 14c, all other organs were removed). the organ (or organs) at the higher position was always the strongest sink. the fruit was a very strong sink (situation a); when the fruit was in between two shoots its strength was strongly reduced (situation b); when the fruit was above two shoots it was dominant (situation c) and therefore the relative organ position changed the sink relationship; the number of shoots above and below the fruit change the relationship between the organs (situations b and d). (giulivo and ramina, 1975). 140 known that different combinations of vegetative and flower buds occur at the nodes of peach mixed shoots; if a single fruit originates at a node initially it grows faster than the one or two fruits which are associated with a node with a growing shoot, but after some time, when the shoot rate decreases, these fruits attain a larger size (casella, 1949; giulivo, data not published). this may be explained by the contribution of the shoot leaves to the assimilate requirement by the fruits, in agreement with the results reported by abbot (1984) on the shoot/fruit relationship carried by an apple bourse. it would be possible to mention many other correlative functions, but the cases mentioned here should be adequate to stress the significance of these relationships in pruning management. 7. conclusions the efficiency of the orchard system is a primary requirement for economic success in fruit growing and therefore the architecture of the stand, with an appropriated use of the space available, can permit an efficient use of light, water and nutrients to sustain the basic physical and physiological processes involved in the functioning of the system. sometimes space utilization (planting density) is too low or too high; in the former case light interception by the canopies becomes too scarce and soil evaporation too high; in the later, reciprocal shading of the trees assumes considerable weight. sometimes the structure of the canopy is not optimal, as it may be too dense or too sparse and consequently the conditions inside the canopy are such that leaf and fruit functioning may be modified, pest proliferation may be promoted or light interception may be too scarce to maintain the various functions and processes of the canopy. in designing tree architecture, three basic indexes must be considered: the ratio between the tree parts above and below the ground, the ratio between leaf area surface and canopy volume, and the leaf area surface per unit of fruit weight. these indexes are satisfied differently in the various orchard models in relation to the applied training system and pruning criteria. different training systems with a similar pruning criterion tend to have more or less the same production behaviour while different pruning criteria applied to the same training system tend to induce different crop performance (sansavini and musacchi, 1994). pruning practices are thus a powerful means to modify tree functioning: pruning generally reduces the primary production of the orchard but may induce a great improvement of the performance of the system. the details of pruning for maximum fruit bud production and adequate fruit size and ripening differ for different species but it is possible to identify some general principles. the various pruning techniques have very different effects on the growth and fruiting of a tree; heading-back or removal of organs, variations of spatial position and/or orientation of vegetative axes and transient modification of sap transport induce very different reactions. interventions directed to shortening or removing an axis generally involve vegetative responses which are more or less strong in relation to location, intensity and when they are performed. particular attention is required because strong negative or positive effects on the vegetative and reproductive equilibrium of the tree may arise. the negative effects regard mainly an excessive vegetative reaction but, if the pruning is carefully performed, the tree develops a species-specific fruiting structure more adequate to the economic goal of the orchard. the detachment of fruits (thinning) performs a particular role in realizing a correct fruit-to-leaf ratio in the tree where the crop load is too heavy in relation to the leaves of the canopy. changing the position or the growth direction of the axes in the space (rectilinear, slanted, curved) generally decreases the growth rate and vigour of shoots and branches, improving the formation of reproductive structures. therefore this type of manipulation assume a great importance in training the fruit trees. some pruning techniques, which tend to modify sap transport (incision above or below a bud, bark ringing, strangulation or girdling, shoot twisting and crashing, etc.), influence the relationships among sinks and the source-sink and thus they may stimulate or limit the development of an organ. whatever the manipulations of the above-ground part of the tree, root functioning is modified, but taking into account tree homeostasis, any modification to the root system affects the performance of the canopy. therefore root pruning may be very useful for controlling tree vigour or improving root growth in adult trees. all pruning manipulations mentioned involve variations in plant growth, size and geometry, thus modifying the architecture and functioning of the tree and affecting its productive life. the timing of pruning manipulations is of great importance since the effects on the tree may be very different. in recent years summer pruning tends to prevail over winter pruning in most fruit tree species (bargioni, 1988; sansavini and corelli, 1990; costa, 1997; sansavini et al., 1999; neri and sansavini, 2004). this tendency is mainly based on two reasons: to form small-sized trees and to follow the natural species-specific behaviour of the tree. under the same cultivation conditions winter pruning tends to stimulate the vegetative reaction, whereas summer pruning generally has positive effects on reproductive activity. this may be due to the more powerful action of summer manipulations on source-sink relationships, and among sinks. summer pruning, however, may reduce leaf area surface or the ratio between young, mature and old leaves and this may have some consequence on the photosynthetic performance of the canopy, which can be of some importance when light availability is limited. the timing of summer manipulation of the canopy is critical: if performed when vegetation is growing very fast. in this case the responses of tree may be too strong and consequently a consistent amount of resources are invested in the edification of new 141 vegetative structures or the density of the canopy may be increased. the timing of root pruning is as important as the time of canopy pruning since it has positive effects in the spring when it controls tree vigour, whereas it acts negatively if performed in late summer when storage of reserve materials is needed for the next year. in the last few decades summer pruning (mainly pinching) has been extended to nurseries to train trees of some fruit species (apple, peach, etc.) carrying some feathers which speed up the construction of the tree crown in the orchard. in this way is possible the shortening of the unproductive period or to have young trees more suited for high density orchards or some particular training system (sansavini and corelli, 1990; vigl, 1999; neri and sansavini, 2004). in the 1990s some attempts were made to reduce production costs by omitting pruning during the training of trees and thereafter applying very simplified and superficial pruning, relying on thinning to equilibrate fruit load and leaf area surface. the performance of this technique, called ‘no-pruning’, was quite disappointing because of the serious drawbacks on fruit quality, such as variability in size and ripening of the fruits. even in high density planting, without an appropriate adaptation of the training system, pruning was not able to overcome the excessive tree competition that determines premature aging of the fruiting structures, excessive shading, crop alternation and poor fruit quality (sansavini, 1999). therefore it may be concluded that pruning will always be an unavoidable operation, taking into account the economical goal of the orchards. in conclusion, the best pruning derives from a clear production target and from precise planning while considering tree general physiology, the peculiar characteristics of the fruit species, cultivars, clones, scion-rootstock combinations, and the local environmental and cultivation conditions. finally, it is important to remember that pruning represents only one of the many manipulations which can be performed in the orchard yet it cannot overcome basic mistakes made in designing the system. references abbot d.l., 1984 the apple tree physiology and management. grower books, london, uk, pp. 90. bailey l.h., 1916 the pruning manual. being the eighteenth edition, revised and reset, of the pruning-book, which was first published in 1898 macmillian company, new york, usa, pp. 407. baldini e., scaramuzzi f., 1962 glossario dei termini usati nella potatura degli alberi da frutto a foglia caduca. tipocolor, firenze, italy, pp. 24. bargioni g., 1988 forme di allevamento e tecniche di potatura: come cambieranno? frutticoltura, l(1-2): 97-103. bargioni g., 1992 potatura degli alberi da frutto, pp. 393-494. in: baldoni l., g. bargioni, m. devreux, o. failla, c. fideghelli, g. fontanzza, f. gorini, r. jona, f. lalatta, g. maracchi, r. paglietta, g. rapparini, c. xiloyannis, and a. zocca. frutticoltura generale. reda, roma, italy, pp. 644. barthélémy d., caraglio y., 2007 plant architecture: a dynamic, multilevel and comprehensive approach to plant form, structure and ontogeny. ann. bot., 99: 375-407. branzanti c.e., ricci a., 2001 manuale di frutticoltura. edagricole, bologna, pp. 103-141. cannel m.g.r., 1983 plant management in agroforestry: manipulation of tree, population densities and mixture of trees and herbaceous crops, pp. 455. in: huxley p.a. (ed.) plant research and agroforestry. international council for research in agroforestry, nairobi, kenya, pp. 617. cannel m.g.r., 1985 dry matter partitioning in tree crops, pp. 160. in: cannel m.g.r., and j.e. jackson (eds.) attributes of trees as crop plants. institute of terrestrial ecology publisher, pp. 592. casella d., 1949 mezzi per attenuare la cascola delle gemme, dei fiori e dei frutticini nelle specie legnose fruttifere. atti iii conv. naz. di frutticoltura, ferrara, italy, pp. 200-212. chalmers d.j., van den ende b., 1975 productivity of peach trees: factors affecting dry-weight distribution during tree growth. ann. bot., 39: 423-432. connor d.j., 1983 plant stress factors and their influence on production of agroforestry plant associations, pp. 401. in: huxley p.a. (ed.) plant research and agroforestry. international council for research in agroforestry, nairobi, kenya, pp. 617. costa g., 1997 potatura di produzione e diradamento dei frutti negli impianti di pianura. proceeding symposium melicoltura di pianura: quale futuro, verona, italy, 12 april. costes e., lauri p.e., regnard j.l., 2006 analyzing fruit tree architecture: implications for tree management and fruit production. horticultural reviews, 32: 1-61. dotti f., 1949 recenti vedute sulla potatura dei fruttiferi. atti iii conv. naz. di frutticoltura, ferrara, pp. 213-230. giulivo c., 1990, basi fisiologiche della potatura degli alberi da frutto. proceedings “la potatura degli alberi da frutto negli anni 90”, verona, italy, 27 april. giulivo c., ramina a., 1974 osservazioni sulla formazione dei germogli anticipati nel pesco. ii contributo. riv. ortoflorofrutt. it., 3: 170-176. giulivo c., ramina a., 1975 competizione tra germogli e giovani frutti di pesco. riv. ortoflorofrutt. it., 4: 273-280. giulivo c., ramina a., costa g., 1981 b il contributo del sottoprogetto 5 alle conoscenze dei processi di abscissione e maturazione dei frutti proceedings i fitoregolatori nel controllo chimico della produzione degli alberi da frutto, ferrara, italy, 26-27 march. giulivo c., ramina a., scrittore g., 1981 a accrescimento e sviluppo riproduttivo in cultivar di pesco con diversa epoca di maturazione dei frutti. proceedings i fitoregolatori nel controllo chimico della produzione degli alberi da frutto, ferrara, italy, 26-27 march. grisvard p., 1957 la taille des arbres fruitiers. la maison rustique, paris, frances, pp. 160. hilkenbäumer f., 1953 obstbau. paul parey, berlin und hamburg. 142 ingles c., geisel p.m., unruh c.l., 2002 fruit trees: training and pruning deciduous trees. anr, publication 8057, ucd, davis, ca, usa, pp. 7. liebster g., pesserl g., 1982 fachausdrücke des obstbaumschnittes lexikon in deutscher, wörterbuch in englischer, französicher, italienischer und niederländischer sprache. mitteilungen klosterneuburg, 32: 50-100. morettini a., 1963 frutticoltura generale e speciale. reda, roma, italy, pp. 612. neri d., sansavini s., 2004 attualità della potatura nella frutticoltura intensiva. frutticoltura, lxvi(1): 14-23. pitacco a., giulivo c., 1992 dinamica della crescita dei germogli e dei frutti in cultivar di pesco a diversa epoca di maturazione. giornate scientifiche s.o.i., ravello, italy, 8-10 april. proctor j.t.a., 1978 apple photosynthesis: microclimate of the tree and orchard. hort. sci., 6: 641-643. ramina a., 1981 la dinamica della cascola ed alcuni aspetti fisiologici dell’abscissione nel diradamento chimico dei frutti di pesco (prunus persica, l. batsch). conv. i fitoregolatori nel controllo chimico della produzione degli alberi da frutto, ferrara, 26-27 march. richards d., rowe r.n., 1971 effects of root restriction, root pruning and 6-benzilaminopurine on the growth of peach seedlings. ann. bot., 41: 729-740. sansavini s., 1999 potatura in libertà. frutticoltura, lxi(3). 5-6. sansavini s., corelli grappadelli l., costa g., lugli s., marangoni b., tagliavini m., ventura m., 1999 ricostituzione degli impianti e revisione degli indirizzi produttivi della peschicoltura romagnola. frutticoltura, lxi(3): 8-20. sansavini s., corelli l., 1990 la potatura e le forme di allevamento del melo. proceedings la potatura degli alberi da frutto negli anni ’90. verona, italy, 27 april, pp. 65-99. sansavini s., errani a., 1998 frutticoltura ad alta densità, impianti, forme di allevamento e tecniche di potatura. edagricole, bologna, italy, pp. 593. sansavini s., musacchi s., 1992 modelli di impianto, architettura e potatura nei moderni pereti. frutticoltura, lvi(2): 7-18. schultze e.d., 1982 plant life forms and their carbon, water and nutrient relations, pp. 615-676. in: lange o.l., p.s. nobel, c.b. osmond, and h. ziegler (eds.) physiological plant ecology, ii. encyclopedia of plant physiology. springer verlag, berlin, germany. simon s., sauphanor b., lauri p.e., 2007 control of fruit tree pests through manipulation of tree architecture. pest technology, 1(1): 33-37. vigl j., 1999 l’allevamento del melo a “spindle”. frutticoltura, lxi(3): 29-32. williamson j.g., coston d.c., 1989 the relationship among root growth, shoot growth, and fruit growth of peach. j. amer. soc. hort. sci., 2: 180-183. 3 1. introduction world production of pineapple (ananas comosus l.) reached 19 million tonnes in 2008 with the industry dominated by brazil followed by thailand, the philippines and indonesia. in australia, pineapple, as an exotic species, is grown almost exclusively in queensland, producing 104,000 tonnes annually with an industry average yield of 37.9 t ha-1 in ~2700 ha (dhungel et al., 2009) contributing an annual farm gate value of au$50 million (fao, 2011). thanks to crassulacean acid metabolism (cam), pineapple is adapted to dry environments. however, pineapple is sensitive to water-logging and therefore requires a well-drained soil with good aeration when grown with irrigation. pineapple response to irrigation is generally high for yield and quality. industry interest in developing irrigation for pineapple has been triggered by recurring episodes of drought brought about by climate change, and a major shift in the historical rainfall pattern, and under such circumstances strategic and supplementary drip irrigation are imperative for a sustainable industry (camp et al., 1993). increased cost of water, reduced ground water reserves and vocal public pressure have forced growers to look for more effective alternatives to the traditional surface flood and furrow irrigation. in response, increasing adoption of localized micro irrigation such as sprinkler, drip irrigation (di) or subsurface drip irrigation (sdi) is taking place to improve water use efficiency (wue) and to minimize environmental impacts by reducing runoff and deep drainage (thompson et al., 2002). the irrigation efficiency of sprinkler systems is less than for drip systems due to evaporative losses and the tendency to promote foliar diseases in the former. although far more efficient, di and sdi can induce temporal hypoxic conditions in the rhizosphere due to their sustained wetting fronts, particularly in fine textured soil (machado et al., 2003) where oxygen may not be sufficiently available for root respiration. localized water-logging purges the soil pores of air and causes hypoxia, reducing root metabolic activity and function (goorahoo et al., 2002; bhattarai and midmore, 2009). alleviation of hypoxic rhizosphere conditions can be achieved through the use of aerated water for irrigation, increasing oxygen availability in the root zone (su and midmore 2006; dogan et al., 2008). oxygation, the term we use for aerated irrigation water with sdi, has been shown to benefit growth of a range of crops, particularly in heavier soils. this is because even under normal irrigations, roots can suffer due to a lack of soil oxygen. an increase in crop yields and wue has been aerated water irrigation (oxygation) benefits to pineapple yield, water use efficiency and crop health j. dhungel, s.p. bhattarai(1), d.j. midmore centre for plant and water science, faculty of science engineering and health, central queensland university, rockhampton, qld 4702, australia. key words: ananas comosus, cam photosynthesis, ‘d’ leaf, phytophthora, root respiration. abstract: pineapple roots need adequate oxygen to function, sustaining growth and yield. the crop is susceptible to soil saturation caused by natural rainfall or irrigation, or even with drip irrigation that creates sustained wetting fronts. drip and subsurface drip irrigation can develop sustained wetting fronts, particularly in low permeability soils, predisposing plant roots to a low oxygen environment. we evaluated the use of aerated irrigation water “oxygation”, employing mazzei air injectors which mix air with irrigation (12% air by volume of water) in-line, increasing oxygen concentration in the irrigation water stream. the effect of this treatment was evident in growth, development, and leaf gas exchange parameters. total fruit yield increased by 44 and 26% whereas industry yield increased by 11 and 6% due to oxygation compared to the control and no irrigation, respectively. high yield was associated with an increase in fruit size and not the number of fruits produced. phytophthora infestation in the oxygation (3% of plants) was significantly reduced compared to the control (4.9%), and without irrigation treatment (10.5%) suggesting that reasonable management of phytophthora, which is one of the major pathological problems for pineapple production in australia and elsewhere, can be addressed through aerated water irrigation. oxygation responses were mediated through root and soil processes involving greater root biomass, root respiration, increased microbial diversity and enhanced soil aeration status. adv. hort. sci., 2012 26(1): 3-16 (1) corresponding author: s.bhattarai@cqu.edu.au received for publication 8 april 2011 accepted for publication 18 december 2011 4 confirmed by bhattarai et al. (2004) with the use of aerated water irrigation in a number of annual crops with c 3 metabolism. other studies on other crops in a range of soil types with drip and sdi have also been implicated in creating a wetting front that can induce the possibility of hypoxia in the rhizosphere (goorahoo et al., 2002; midmore et al., 2006). drip irrigation which involves point source water application in the rhizosphere could lead to constantly poor aeration around the root zone and predispose pineapple plants to a number of physiological disorders and susceptibility to phytophthora root rot. oxygation, using aerated water that has potential to ameliorate the hypoxic/ anoxic conditions, may be of benefit not only to improve the wue, yield and quality but also to minimize disease caused by phytophthora, with symptoms of root and fruit rot in pineapple. previous research in annual crops has demonstrated the potential of oxygation to improve yield and wue (bhattarai and midmore, 2009). most of the effects of water-logging in pineapple are mediated through the roots, including predisposition of the root system to a number of diseases including root and fruit rot. generally field grown pineapples tend to develop roots in the upper soil layer, where they are confined and follow the wetting area. as the root activities are bound to the wetting fronts, the susceptibility to phytophthora is generally high. recent adoption of drip irrigation for pineapple in australia and elsewhere is encouraging. introduction of aerated drip systems can be pivotal in improving irrigation water use efficiency and minimising the infestation by phytophthora in pineapple. the main objective of this trial was to determine under what conditions there is a measurable positive effect of aeration and how that relates to impact upon incidence of phytophthora and on general crop growth and development. the study was designed to evaluate the effectiveness of oxygation in a perennial crop pineapple with the cam pathway for carbon fixation, evaluating the benefits on fruit yield, quality, and water use efficiency. we report the results of field research carried out in collaboration with valley syndicate pineapple farm in yeppoon, qld australia from 2007 to 2011 in order to evaluate the above mentioned effects of aerated water for drip irrigation. 2. materials and methods trial site and soil the field experiment was conducted at valley syndicate pineapple farm, yeppoon, central queensland, australia (23o9’31.12os, 150o42’51.36oe). the crop was grown over the period 2007-2011 from which two harvests were taken as main crop and first ratoon crop. total area of the experimental site was 2.15 ha on a calcareous sandy loam soil, with organic carbon 0.68-1.2%, total nitrogen 0.06-0.09%, potassium (colwell) 25-139 mg/kg, and phosphorus (colwell) content of 18-39 mg/kg. the crop seasons were relatively wet compared to long-term averages. the region is described as a semiarid tropical environment, with summer-dominant rainfall (fig. 1). fig. 1 total monthly rainfall (mm), monthly average maximum temperature (°c), and monthly minimum temperature (°c) over the experimental period at yeppoon. experimental design the experiment was a randomised block design, with two sdi irrigation treatments, with and without oxygation. air injection into the irrigation water for oxygation was supplied by a 1583™ air injector venturi (mazzei corp, usa) installed in-line immediately before the field plot, regulated to ingress 12% air by volume of water following bhattarai et al. (2006). the experiment was replicated seven times within 14 field plots (average dimensions 16 m x 70 m) where seven plots received aerated water by sdi and seven plots were irrigated without aerated water. an adjacent block, comprising three plots, received no irrigation (although all other inputs such as fertilizer, flowering regulation chemicals, fungicides and insecticides were identical) and was included as a third treatment (noirrigation) for comparison purposes. planting materials and crop management crowns of pineapple variety gc1 were planted on 24 october 2007, in a double row, raised bed (10 cm high) system to accommodate up to 53,333 plants/ha. inter row space was 50 cm and between plants was 25 cm, whereas centre to centre between the bed was maintained at 1.5 m. the oxygation treatment commenced 14 march 2008 (139 days after planting). ethylene (ethephon) was applied twice as a saturated solution in water and activated charcoal to enhance absorption to induce flowering, as pressurized spray late in the evening or at night for enhanced uptake through the stomata. plants were of an optimum size (~2 kg) for forcing in order to obtain even flowering. irrigation design and scheduling irrigation was blocked in four units. a mazzei air injector was installed near the block (3 m from the first plot), whereas the pump was installed near to reservoir about 1 km distance from the plot. the inlet pressure of 45 psi was achieved at the point of the mazzei air injector installation. a pressure differential across the air injector was maintained at 45 and 15 psi for the inlet and outlet, respectively, to maintain air injection at 12% by volume 5 of water (fig. 2). the drip tube (manufactured by plastro australia) consisted of pressure compensated emitters at 30 cm intervals emitting at the rate of 1.2 l/hr, buried at 150 mm from the soil surface, one drip tube per bed running between two 50-80 m length crop rows. fig. 2 mazzei air injector installed in a pressurised irrigation line close to the plot for air injection into irrigation water for oxygation (top), and the first year crop at fruit development stage (b) being marked for destructive sampling. irrigation and soil water monitoring soil water content was monitored at depths of 10, 20, 30 and 40 cm, logged at 15 min intervals but averaged daily, using a calibrated odyssey greenlight-redlighttm (glrl) sensor (capacitance probes manufactured by data flow system pvt ltd, nz). there was one monitoring site in each plot. soil moisture was also measured (at 10 cm intervals from 10-60 cm depths) during the diurnal measurement of photosynthesis over a period of five days in january 2010; a calibrated odyssey micro-gopher system, the probe of which consists of a capacitance sensor (soil moisture technology, australia) was used. scheduling of irrigation was based on the averaged readings from the glrl sensors at 20 cm depth; the amount applied was calculated to take soil moisture content when at c. <50% of the field capacity (fc 36 mm h 2 o per 100 mm soil depth) to refill the soil water reservoir by irrigation to reach field capacity. this required different durations to bring the soil to fc. water applied to individual blocks (oxygation and control) was measured with two calibrated water meters installed near the pumping station and the rainfall data were accessed from a nearby weather station (<1 km aerial distance). nutrients to the crop were supplied through an industry standard rate of basal application of macro and micro nutrients and top dressing by spreading in the non-irrigated plot and by fertigation on drip irrigated plots supplied at the equivalent dose for all treatments. the industry strategies for increasing pineapple yield and fruit size included side dressing of nitrogen (160 k/ha), phosphorus (60 kg/ ha) and potassium (193 kg/ha) over five split applications in a year. an additional dose of magnesium (12 kg/ha) was also applied once. soil oxygen monitoring the o 2 concentration in the soil was measured at 15 cm depth between two emitters and offset 5 cm from the drip tube using pst3 o 2 sensitive fibre-optic minisensors with fibox-3 oxygen meters (presens gmbh, germany) as described by klimant et al. (1995). sensors were installed in the soil for five days prior to data collection and soil oxygen monitoring took place for two days before oxygation, during oxygation and two days post oxygation event in the oxygation and control plots following the procedure of chen et al. (2010). soil oxygen monitoring was also performed in the ratoon crop over the period of four days before (-48 hr), during (0 h) and post (+48 hr) irrigation. destructive plant sampling for dry matter partitioning destructive plant sampling for dry matter accumulation and partitioning was carried out on 22 nov 2008 (392 dat) and 15 january 2010 (713 dat) to evaluate the treatment effects in the main and ratoon crops. plant samples collected from two whole 2 m linear lengths per bed were separated into leaf, stem, fruits and roots, oven-dried at 70°c, and the fresh and dry weights of each component were recorded. the ‘d’ leaf is always easy to pull from the plant and has leaf margins that are more-or-less parallel all the way to the leaf base (bartholomew, 2008). the ‘d’ leaf is defined as the youngest physiologically mature leaf on the plant and also is the tallest leaf on the plant. plant height and sla were measured on the ‘d’leaf. diurnal changes in gas exchange and plant parameters light interception by the canopy was measured using an accupar ceptometer (decagon, usa) and canopy temperature was recorded using an everest ag multimeter. leaf photosynthesis (a), transpiration (e) and stomatal conductance (sc) were measured at 6 hr intervals using an infrared gas analyser (irga) lca-4 (adc, uk) on two fully-expanded topmost sunlit leaves per plot on each occasion at early morning (dawn), midday, dusk and at night following adams et al. (2002). soil respiration was 6 measured in the soil 3-5 cm from the plant using irga principle with an egm-3 (pp systems (uk) following hanson et al. (2000) on 392 dat for the main crop and 713 dat for the ratoon crop. data collection was also carried out to measure the gas exchange responses of the crop before, during and after oxygation events in the aerated sdi, the control sdi and no irrigation treatments. pre-irrigation data were collected over a 24 hr period, on 14-15 january 2010 at 6 hr intervals to include dawn, midday, dusk/early evening and night to determine diurnal patterns of leaf gas exchange parameters during and post irrigation, and data on soil moisture and soil respiration were also collected using microgoopher and egm-3 soil respiration systems, respectively. harvesting and yield determination harvesting was performed at two scales, i.e. sample plot harvesting for total yield and industry harvest for marketable fruits yield. for sample plot harvests, all fruits were hand-picked at maturity from two rows of 2 m linear lengths (16 plants) selected from each of the seven replicated bordered plots, and from three plots in the non-irrigated area. the fruits were counted, weighted and processed for quality parameters for both main crop and ratoon crop. change in fruit colour, particularly to the eye, from green to yellowish was considered an index for maturity and harvesting. fruits without crown and peduncle were weighed. the industry harvest represents only marketable fruit yield harvested by the crew of pickers in a mobile harvester. commercially-harvested fruits were weighted in the load cells with wooden crates containing ~500 kg. commercial harvest was carried out for each plot separately at approximately weekly intervals. the main crop was harvested from january to april 2009 and it was left for ratoon which recommenced harvesting in 2010. the ratoon crop was harvested 28 june to 11 october 2010, and the whole crop was then uprooted for planting of a new crop, hence the crop spanned a period of 39 months from planting. fruit quality determination mature fruits harvested from sample areas were used for quality determination. fruit quality parameters measured included °brix, fruit size, volume, density, fruit height and width, flesh colour, skin colour, dry matter, translucency and flavour, following the standard analytical method described by bartolome et al. (1995). fruit quality was assessed on fruits from different harvests in the main and also in the ratoon crop. in total, 20-50 randomly selected mature fruits were assessed for quality in the main and ratoon crops. the flavor score was determined based on smell by a panel group following industry standard; 1= no flavor, 2= little flavor, 3= good flavor. water use efficiency crop water use efficiency was calculated to represent irrigation water use efficiency (iwue) and gross water use efficiency (gwue). iwue is calculated as fruit yield (t) per megaliter of irrigation input, whereas gwue is the fruit yield (t) per megaliter of crop water input, comprising both the inputs from irrigation and rainfall. instantaneous water use efficiency (wue i ) was calculated from the irga data which represent µmol co 2 assimilated for each mmol of h 2 o transpired during measurement of the photosynthesis process. soil physical and chemical parameters changes in soil physical and chemical properties were assessed by measuring soil compaction, bulk density and air filled porosity (following the method of usda, 2010) and soil organic carbon (kuhlbusch, 1995) and nitrogen (iso 13878 soil quality elemental analysis). soil compaction was determined with remek cp4011 soil cone penetrometers (ict international, australia) during the destructive plant sampling period to a depth of 35 cm from the soil surface. soil water samples were collected at 50 cm depth for sub-surface leaching and nutrient analysis particularly nitrate signature using wetting front detectors (csiro, australia). subsurface solution samples were also collected from ceramic solusamplers placed at depths of 20 cm and 50 cm to determine nutrient transfer through the soil profile. at the end of the crop season, soil cores (20 cm deep and 8.6 cm diameter) were collected to determine bulk density, root density, air filled porosity at field saturation, field capacity and in dry soil following the method by peverill et al. (2002). soil microbial and phytophthora determinations fluorescein diacetate hydrolysis activity (fda) analysis provides a surrogate measure of the soil microbial load. soil samples were collected during harvest at the depth 10 cm and 10 cm distance from emitter, and were used for fda analysis following the method described by adam and duncan (2001). the incidence of phytophthora infestation was assessed in the field crop. in each plot, a double row comprising ~500 plants was examined for infestation based on the visual symptoms following pegg (1977). plants with characteristic symptoms of phytophthora on fruit and crown rot were counted and percentage infestation calculated. data analysis data were analysed following the procedures for analysis of variance (anova) for randomised block design in genstat version 11 (vsn international, uk) as the no irrigation plots were contiguous to sdi plots. for most of the crop, only the main effects of soil and water parameters are presented, whereas for all other data collected during the diurnal events (leaf and soil gas exchange parameter) the effects of the treatment on the diurnal course were also analysed. hence some significant interactions between the treatments and diurnal effects have been analysed and presented. means were separated by the least significant difference (lsd) at p≤ 0.05. 3. results and discussion weather conditions rainfall was recorded as 917 mm, 1294 mm, 875 mm, and 1850 mm for 2007, 2008, 2009 and 2010, respective7 ly (fig. 1). 2010 had the highest rainfall, more than four times that of the driest years in the past 20 years. total rainfall input during the entire crop period was 4250 mm. irrigation input irrigation scheduling was based on measurement of the soil moisture deficit. the same delivery system through mazzei injector was utilized for air injection and water application (fig. 2). irrigation commenced when soil moisture reached the refill point. irrigation input during the crop period for the oxygation and control treatments was 252.4 and 240.5 mm per hectare respectively (fig. 3). rain contribution during the same period was 4250 mm per hectare (fig. 1). therefore, the proportion of irrigation to total crop water input was only 5.5%. all irrigation events were scheduled for supplementary and strategic applications to the crop. soil moisture in the profile soil moisture at 40 cm was consistently higher than at 20 cm, irrespective of the treatment (fig. 4). oxygation maintained somewhat less soil moisture compared to the control at both depths. on a number of occasions, the soil moisture content was well above the field capacity (36 mm for 100 mm soil depth) at greater depths, particularly in the control treatment. the results suggested that soil moisture increased with increasing depths (0-60 cm), irrespective of the treatment (fig. 5). the effect of irrigation treatments on soil moisture was evident, as soil moisture always remained lower with oxygation compared to the control for all the depths in spite of slightly higher irrigation input associated with oxygation, suggesting that water loss from the rhizosphere was greater through transpiration for plants with the oxygation treatment. soil oxygen dynamics soil oxygen concentration in the oxygated rhizosphere remained higher (7.5 vs 4.4 ppm) than the control. the highest oxygen concentrations (10.72 and 7.08 ppm) were noted during irrigation for the oxygation and control groups, respectively. the lowest concentrations for oxygation and control were 5.48 and 3.09 ppm, respectively fig. 4 change in soil moisture (mm water 100 mm-1 of soil depth) at two different depths (400 and 200 mm) over the period of two months (march-april 2009) in the oxygation and control treatments measured by red light green light soil moisture sensors. fig. 3 cumulative irrigation input (ml ha-1) over the crop period for oxygation and control treatments. fig. 5 change in soil moisture (mm water 100 mm-1 of soil depth) over a period of four days [before (2 days), during (upon completion of 2 hr irrigation) and after (2 days) irrigation] in the oxygation and control irrigation. 8 (fig. 6). a higher oxygen concentration in the rhizosphere during oxygation events compared to non-aerated water irrigation was also reported by chen et al. (2010) in cotton and wheat crops in both vertisol and ferrosol under similar climatic conditions. changes in soil physical properties soil compaction. there was less soil compaction in the oxygation treatment compared to the control, and it was higher in the non-irrigated treatment, particularly at depths below the position of emitters (fig. 5). soil resistance increased down the soil profile to 37.5 cm depth, and the treatment effect was consistent over this depth. the data suggest that the lower soil resistance down the profile was consistently maintained throughout the profile in the oxygation treatment (fig. 7) compared to the control and no-irrigation treatments, particularly in the positions below the emitter depths. this observation is in contrast to the report presented by bhattarai and midmore (2010 unpublished data) on cotton in vertisol where they showed a greater soil resistance in the wetting fronts of cotton under oxygation compared to the control. such a response was linked with rapid water uptake and transpiration by oxygated plants and, therefore, a drier soil in the wetting front region that contributed to greater soil resistance. soil bulk density and air filled porosity. a tendency for lower air filled porosity was recorded in the no-irrigation treatment compared to irrigated oxgation and control treatments. air-filled porosity at field capacity did not differ significantly between the treatments (table 1). however, the air field porosity at near saturation was significantly greater in the irrigated control and higher in oxygation compared to the no-irrigation treatment. while no significant difference between the treatments was noted for the soil bulk density, root density was significantly lower (reduced by one half) for the no-irrigation treatment compared to both oxygation and control treatment (table 1). non irrigated plants showed shallower root systems, and were easier to pull by hand. instead of putting down deep rooting, they tended to produce adventitious root in the soil surface. consistent with these results, schneider et al. table 1 soil physical and biological properties assessed at the end of the ratoon crop for oxygation, control and no-irrigation treatments treatments air filled porosity (%) bulk density (g/cm3) root density (kg/m3) fluorescence release (µg /g dwt soil/ hr) phytophthora (% plants)near saturation field capacity control 2.6 20.7 1.68 6.32 2.2 4.9 oxyation 2.3 20.6 1.61 7.50 2.2 3.0 no-irrigation 2.0 18.3 1.61 3.56 1.8 10.5 p value 0.021 0.102 0.243 0.047 0.890 <0.001 lsd (p≤0.05) 0.537 ns ns 1.811 ns 1.379 mean separated by lsd. ns= not significant. fig. 6 change in soil dissolved oxygen concentration (ppm) over the period of four days [before (2 days), during (upon completion of 2 hr irrigation cycle) and after irrigation (2 days)] in the oxygation and the control treatment measured at the wetting front. fig. 7 soil compaction measured in the soil at field capacity at the end of the ratoon crop as influenced by soil depth for oxygation (open circle), control (closed circle), and non-irrigated (closed diamond) treatments. 9 (1992) reported deep penetration of roots and greater root biomass near the drip line and major concentration of root mass at 30-40 cm depth in drip irrigated pineapple on a silty clay soil in hawaii usa. soil respiration in the ratoon crop, the rate of soil respiration did not differ significantly between the oxygation and control treatments three days after irrigation, however the rate was greater for the irrigated treatments compared to noirrigation treatment. the diurnal pattern of soil respiration showed a greater soil respiration rate during the day and during the night compared to the early morning and evening (table 2). in the fast growing pineapple crop, i.e. before the first crop harvest, soil respiration was significantly greater in the oxygation (2.2 g co 2 m-2 hr-1) compared to the control (1.4 g co 2 m-2 hr-1) treatment, an increase of 64%, at 394 days after planting (chen et al., 2010), and following 6 hr of irrigation. root respiration has been shown to be enhanced by aeration in a number of previous studies on oxygation. these findings are in accord with those of bhattarai et al. (2005) who showed that oxygation increases the amount of oxygen in the irrigation water and ultimately in the root zone, which drives greater root respiration, and therefore ameliorates the temporal hypoxia associated with wetting fronts. soil biological properties and phytophthora low fda values were noted in the no-irrigation treatment compared to irrigation treatments without significant differences. a marked effect of irrigation treatments was noted on the development of phytophthora symptoms in the field crop (table 2). the oxygation treatment showed significantly lower infection (3%) compared to the no oxygation sdi treatment (4.9%), whereas the highest phytophthora infestation (10.5%) was recorded in the noirrigation treatment. in spite of the lower water application rate and reasonably dry soil surface in the no-irrigation plot, development of phytophthora was more severe in this treatment. exposure of the roots to the soil surface provides poor anchorage to the plant. when the plant was loaded with fruit, the top-heavy weight of the plant resulted in crop lodging and damage to the roots. this may have predisposed the plants to phytophthora contamination particularly when the plot was wet due to rainfall. severe crop lodging was noted in the non-irrigated treatment in this trial site. a study by stirling (2004) also suggested that the pineapple crop in qld in a ferrosol treated with cane trash mulch and under minimum tillage, both of which contribute to maintaining greater soil aeration status, recorded a high fda level and a positive correlation with greater nematode suppression in the field compared to non-mulched traditional tillage treatments. soil chemical properties (nutrients) soil organic carbon, colwell k and colwell p contents were higher (the latter two not reaching significance at p<0.05) in the no-irrigation treatment compared to oxygation and control treatments when analysed at the end of the crop period. in contrast, soil ph (cacl 2 ) was significantly lower, and exchangable k was signifcantly higher in the no-irrigation treaments compared to irrigated control or oxygation treatments (table 3). no significant effects of irrigation treatments were detected for total n, electrical conductivity, and exchangeable calcium and magnesium concentration in the soil. table 3 macro nutrients, organic carbon, soil ph, conductivity (cond) and exchangeable calcium (exc. ca), exchangeable magnesium (exc. mg), exchangeable potassium (exc. k) in the soil sampled after the ratoon crop in oxygation, control and no-irrigation treatments treatments organic carbon (%) n total (%) colwell p (mg/kg) colwell k (mg/kg) cond (ds/m) ph (cacl 2 ) exc. ca (meq/100g) exc. mg (meq/100g) exc. k (meq/100g) control 0.900 0.06 19.00 61.50 0.04 3.45 0.22 0.06 0.04 oxygation 0.890 0.07 17.50 56.50 0.05 3.50 0.18 0.05 0.05 no-irrigation 1.210 0.08 45.00 107.00 0.05 3.30 0.31 0.08 0.12 p value 0.064 0.14 0.009 0.086 0.572 0.033 0.547 0.485 0.008 lsd ns ns ns ns ns 0.129 ns ns 0.031 mean separated by lsd. ns= not significant. table 2 soil respiration, soil temperature, and soil resistance for different irrigation treatments and diurnal sampling treatments soil respiration (g co 2 /m2) soil temperature (°c) soil resistance (kpa) (2.5-30 cm) control 0.82 26.8 1285.2 oxygation 0.82 26.5 1012.8 no-irrigation 0.37 27.1 2067.5 p value (aeration) 0.018 0.030 <0.001 lsd (p≤0.05) (aeration) 0.329 0.423 434.1 day (1300 hrs) 0.89 29.5 evening (1900 hrs) 0.51 26.2 night (2300 hrs) 0.99 26.1 morning (0500 hrs) 0.57 25.2 p value (diurnal) 0.008 <0.001 p value (a x d) 0.287 0.726 lsd (p≤0.05) (diurnal) 0.327 0.420 lsd (a x d) ns ns mean separated by lsd. ns= not significant. 10 plant growth and development dry matter partitioning during growth of the main crop the effect of oxygation was assessed on vegetative and reproductive biomass of the main crop during the early fruit growth stage prior to maturity (392 dat). dry weight in the root, leaf, fruits and total above-ground dry biomass increased significantly due to oxygation compared to the control. however, the stem dry weight was not affected by the treatments. the size of the immature fruits (measured as weight of individual fruit) was significantly larger with oxygation compared to the control harvested at the same time (table 4). the fruit dry weight was greater by 14% with oxygation compared to the control and, dry biomass was 13% greater in the oxygation compared to the control treatment (table 4). these results are consistent with yield increases in previous trials on other crops such as tomato, zucchini and cotton representing the c 3 pathway for co 2 assimilation (bhattarai et al., 2005). dry matter partitioning during the growth of the ratoon crop dry matter partitioning was carried out for the ratoon crop during the growth phase, at the same time as the diurnal measurements of gas exchange (713 dat). above-ground and total dry matter biomass increased with oxygation compared to the control, but without significant differences. however, the increase in leaf dry weight associated with oxygation, compared to the control, was significant (table 5). the leaf weight with oxygation at 22.9 t/ha was 15% greater than that for the control. this result in the ratoon crop was similar to that in the main pineapple crop, where leaf weight increased by 14%. the total biomass was 35.4 t/ha with oxygation, which was 7% more than in the irrigated control (31.2 t/ha). this result is also consistent with, but somewhat less than, the result in the main pineapple crop, where the biomass increase was 14%. the oxygation treatment improved root biomass in the main crop (79%) and much less so in the ratoon crop compared to the non-oxygated control. the result in the main crop was consistent with the hypothesis that oxygation improves oxygen availability in the rhizosphere, which positively influences the availability and uptake of water and nutrients favorable for increased root growth and enhanced soil microbial functions (goorahoo et al., 2002). the lower soil compaction in oxygation treatment plots could also have favoured root growth. crop physiological performance leaf chlorophyll leaf chlorophyll content was estimated using a spad meter. a standard calibration for spad was also made with acetone chlorophyll extraction method (arnon, 1954) and a close agreement was achieved between these two methods (fig. 8) as reflected by the coefficient of determination (r2=0.807). the chlorophyll content in the d leaf of the ratoon crop was recorded as higher with oxygation compared to the control and no-irrigation treatments. the increase in leaf chlorophyll content was to the order of 1117% with oxygation compared to that of the control and no-irrigation treatments (table 6). table 4 effect of oxygation on plant dry weight and its components at the sample harvest of the main crop (392 dat) from irrigated control and oxygation treatments (no irrigation treatments were not sample harvested in main crop) treatments stem (g/m2) root (g/m2 ) leaf (g/m2 ) agdb (z) (g/m2 ) fruit (g/fruit) total (g/m2 ) root/shoot control 585.9 582.3 1760.0 2345.9 833 2928.2 0.248 oxygation 671.8 1056.1 2232.0 2903.8 1002 3959.9 0.363  p value 0.633 0.004 0.025 0.001 0.016 0.004 0.071 lsd (p≤0.05) ns 343.5 555.8 323.7 124.7 367.5 0.109 (z) above-ground dry weight. mean separated by lsd. ns= not significant. table 5 effect of oxygation on dry plant weight and its components at harvest of the ratoon crop (713 dat) treatments stem (g/m2 ) root (g/m2 ) leaf (g/m2 ) crown (g/m2 ) fruit (g/m2 ) agdb (g/m2 ) total (g/m2 ) root/shoot control 549 528 1942 9.9 85 2587 3115 0.204 oxygation 598 533 2289 14.7 105 3007 3541 0.177 no-irrigation 623 409 2136 0.0 0.0 2760 3169 0.148 p value 0.76 0.32 0.08 0.62 0.53 0.20 0.22 0.34 lsd (p≤0.05) ns ns 150 ns ns ns ns ns mean separated by lsd. ns= not significant. 11 ‘d’ leaf characters a significant increase in plant height was recorded in oxygation and control treatments compared to no-irrigation plants. although in the first crop there was no effect of oxygation on the number of leaves per plant, nor on ‘d’ leaf area and the specific leaf area (sla) of the ‘d’leaf (nor on plant height, data not presented), a significant increase in ‘d’ leaf area due to sdi with or without oxygation compared to the non-irrigated control was evident in the ratoon crop (table 6). a larger ‘d’ leaf area has been linked with higher yield of pineapple fruits in a number of previous studies (e.g., fournier et al., 2007). canopy light interception light interception by the canopy increased significantly with oxygation compared to the control and no-irrigation treatments (table 6). light interception by the canopy was highest (92%) in the oxygation treatment and increased by 5% and 7% compared to the no-irrigation and control treatment respectively (table 6). leaf photosynthesis there was a distinct diurnal pattern for leaf gas exchange. no gas exchange activity was recorded during the daytime, while the carbon dioxide exchange rate ranged between 2.4-3.5 µmol m2 s-1 over the early morning, evening and night (table 7). a higher co 2 exchange rate was noted in the oxygation treatment compared to the control table 6 pineapple leaf characteristics of the ratoon crop as affected by irrigation treatments when harvested at the fruit developing stage treatments plant height (cm) leaf (no./plant) ‘d’leaf area (cm2) ‘d’leaf weight (g) sla(z) (cm2/g) chlorophyll (spad units) li (y) (%) control 98.0 9.6 263.23 5.81 46.8 44 87 oxygation 102.0 8.9 280.45 6.15 48.2 49 92 no-irrigation 93.3 7.9 215.31 5.03 45.4 42 83 p value 0.06 0.82 0.01 0.31 0.63 0.08 0.09 lsd (p≤0.05) 5.8 ns 33.11 ns ns 5.2 5.6 mean separated by lsd. ns= not significant. (z) sla = specific leaf area. (y) li = light interception by the canopy. table 7 diurnal variation for leaf co 2 exchange rate, stomatal conductance, transpiration rate, and leaf temperature for different treatments treatments leaf co 2 exchange rate (µmol/m2/s) stomatal conductance (mmol/m2/s) transpiration rate (mmol/m2/s) leaf temperature (°c) control 2.038 0.312 2.75 30.7 oxygation 2.198 0.376 2.80 30.6 no-irrigation 1.850 0.150 2.71 31.1 p value (aeration) 0.39 0.428 0.062 0.033 lsd (p≤0.05) ns ns 0.09 0.396 day (1300 hrs) -0.14 0.018 1.27 38.5 evening (1900 hrs) 3.53 0.196 3.21 27.7 night (2300 hrs) 2.39 0.353 3.69 27.6 morning (0500 hrs) 2.93 1.324 5.30 27.5 p value (diurnal) <0.001 <0.001 <0.001 <0.001 lsd (p≤0.05) (diurnal) 0.585 0.463 1.034 0.451 p value (a x d) 0.791 0.135 0.082 <0.001 lsd (p≤0.05) (a x d) ns ns ns 0.966 mean separated by lsd. ns= not significant. a x d = interactions between aeration and diurnal measurement for the given parameters. fig. 8 relationship between the spad unit and leaf chlorophyll concentration by acetone extraction method. (c h lo ro p h yl l μ /c m 2 ) spad unit 12 and no-irrigation. the diurnal pattern of leaf gas exchange in photosynthesis in pineapple is characteristic of the crassulacean acid metabolism (cam), in which carbon dioxide is temporarily fixed during the night and in conditions of very low light intensity as in other succulent plants (cushman, 2001). pineapple crops are able to cope with seasonal variations in weather such as rainfall, dry atmosphere and drought, all of which reduce productivity, due to their ability to assimilate co 2 via the cam pathway (san-josé et al., 2007). during the day stomata are closed and leaf surface transpiration is at its lowest (zhu et al., 1999). due to this unique cam physiology, pineapple exhibits high wue, several times higher than c 3 and c 4 plants (cushman, 2001). our leaf gas exchange data were collected as point source data, from small portions of the leaf and were instantaneous measurements, hence raising questions as to whether the observations made in a single leaf in instantaneous time frames can reflect the response of the whole plant over an integrated time scale. leaf transpiration, stomatal conductance and temperature leaf transpiration was low during the day and higher in early morning, evening and night (fig. 9), and was lower for control and no-irrigation treatments compared to oxygation (table 7). the stomatal conductance was somewhat higher for the oxygation compared to the control and noirrigation treatments; however, the difference in stomatal conductance between the treatments was not statistically significant (fig. 9). in contrast, the leaf temperature measured during gas exchange decreased significantly with irrigation compared to the no-irrigation treatment (table 7). higher leaf transpiration rate is associated with evaporative cooling of the leaf surface that reduces leaf temperature in relation to the ambient temperature of the leaf environment. a significant interaction between irrigation method and diurnal time scale was due to significantly higher predawn leaf temperature in no-irrigation treatment compared to aerated sdi and sdi control (table 7). this is due to low transpiration, and slow evaporative cooling of leaf in this treatment compared to other irrigated treatments in the experiment (table 7). fruit yield the harvest from sub-sample areas at maturity was comprised of all fruits irrespective of their size and marketability. the total pineapple fruit yield (consisting of main crop and ratoon) was significantly greater with oxygation (133.7 t/ha) compared to that of the irrigation control (106.4 t/ha), and least in no-irrigation treatment (90.4 t/ha). the total yield increase due to oxygation was 48% compared to no-irrigation, and 26% compared to the control (table 8). the harvest yield was greater in the main crop compared to the ratoon crop. the total harvest of the ratoon crop was only 51% that of the main crop, averaged over all three treatments. however, oxygation still maintained a higher yield compared to the control and no irrigation in the ratoon crop. table 8 pineapple fruit yield from the sample area (harvested yield) and industry marketable harvest (industry yield) for the main and ratoon crops treatments harvested yield (t/ha) (z) industry yield (t/ha) (y) main crop ratoon total main crop ratoon total control 68.20 38.17 106.37 50.92 18.25 69.17 oxygation 79.60 54.11 133.71 53.08 20.18 73.26 no-irrigation 71.30 19.07 90.37 49.50 16.42 65.92 p value 0.005 0.001 0.032 0.295 0.051 0.076 lsd (p≤0.05) 6.43 10.76 12.36 ns 3.17 7.39 mean separated by lsd. ns= not significant. (z) harvested yield is all fruits harvested from the sample area. (y) industry yield refers to only marketable yield harvested on whole plot basis by the industry picking process. fig. 9 leaf stomatal conductance (a), transpiration rate (b), photosynthetically active radiation (c), co 2 exchange rate (d), soil temperature (e), soil respiration (f), leaf temperature (g) and instantaneous water use efficiency (h) in oxygation, control and no-irrigation treatment over the time period of 24 hr (bar shows mean, line shows standard error of the means) in a ratoon pineapple crop. morning day evening night morning day evening night 13 total industry fruit yield was highest in the oxygation treatment (73.3 t/ha), followed by control (69.2 t/ha) and no-irrigation (65.9 t/ha). the marketable fruit yield in the industry harvest was greater by 11% due to oxygation compared to the no-irrigation treatment, and 6% compared to the control treatment (table 8). the industry yield as a proportion of the sampled yield was also greater in the main crop compared to the ratoon crop. the total industry harvest in the ratoon crop was only 36% that of the main crop, averaged over all three treatments. the sample plot yields were considerably higher than the commercially harvested yields due to the fact that commercial yield only considered fruit >1.5 kg; the sample plot yields included fruits which were smaller but mature. the benefits of oxygation for pineapple yields are in agreement with data from oxygation field trials on a vertisol, where lint yield of cotton increased consistently over a number of years and the benefit averaged >10% per annum (bhattarai and midmore, 2009). fruit quality the individual fruit weight increased significantly due to oxygation, particularly for the main crop, compared to the control and no-irrigation treatment. the fruits in the oxygation treatment were 230 g and 228 g larger than the control and no-irrigation treatments respectively in the main season crop. in the ratoon crop, the effect of treatments on mean fruit size was not as notable, and the mean fruit size in the no-irrigation treatment had improved considerably. greater annual rainfall (~1900 mm) that was more evenly distributed compared to the previous year (fig. 1) minimized crop water stress, and imparted a positive effect on the fruit size and quality in the no-irrigation treatment in the ratoon crop. other parameters of the fruit size such as fruit height and width were also significantly greater with oxygation compared to the control in the main crop. such increase in fruit size due to oxygation has also been reported for other crops such as tomato (bhattarai et al., 2006). the total soluble solid content measured as °brix and dry matter content remained quite consistent across seasons and treatments. the brix readings were much higher than the minimum brix standard set for golden circle (12° brix) and fresh market consumption. a number of other fruit quality parameters such as fruit translucency, flesh and skin color, flavor and fruit shape were also measured at harvest. fruit translucency at harvest in the main crop was lower with oxygation compared to that of the control treatment (table 9), whereas in the ratoon crop the effect of oxygation was significant in lowering the translucency. low translucency in pineapple at harvest is considered an indicator of better quality fruit. fruit quality measured by ranking of flesh colour was better under the oxygation: the score for flesh colour ranking was 11% higher (3.29) under oxygation than under the control (2.95). fruit quality measured by ranking of skin colour was also better in the oxygation treatment. the score for skin colour was 5% higher with oxygation than in the control. flavor quality of pineapple was also improved with oxygation. the flavor quality score for the sample from the oxygation treatment was 12% higher than the control sample. although a positive effect of oxygation was recorded on these quality parameters, the differences were not statistically significant in either crop, except for flavor in the main crop and translucency in the ratoon (table 10). water use efficiency season-long water use efficiency for the total harvested yield component, the irrigation water use efficiency (iwue), which includes only the irrigation component as the water input, increased by 20% due to oxygation (52.98 t/ml) compared to the control (44.23 t/ml). the gross water use efficiency (gwue), which includes both irrigation and rainfall inputs, increased by 39% due to oxygation (2.97 t/ml), and by table 9 fruit characteristics and quality parameters of pineapple as affected by irrigation treatments in the main crop treatments fruit weight (g/fruit) fruit height (cm) fruit width (cm) brix (º) density (g/cm3) dry matter (%) translucency (1-5) (z) flavor (1-3) (y) flesh colour (1-5) (x) skin colour (1-5) (w) control 832.0 12.71 10.32 16.41 0.89 17.51 1.28 2.45 2.95 2.93 oxygation 1061.8 13.77 10.85 16.55 0.91 17.76 1.10 2.75 3.29 3.07 no-irrigation 834.0 14.50 10.63 15.83 0.97 17.57 2.17 2.83 3.67 3.50 p value 0.045 0.014 0.084 0.719 0.185 0.547 0.11 0.005 0.197 0.564 lsd (p≤0.05) 209.5 0.748 ns ns ns ns ns 0.167 ns ns mean separated by lsd. ns= not significant. (z) translucency rating: 1 = 0% translucency, 2 = 25% translucency, 3 = 50% translucency, 4 = 75% translucency, 5 = 100% translucency. (y) flavor rating: 1 = no flavor, 2 = little flavor, 3 = good flavor. (x) ,flesh colour rating: 1 = 100% white, 2 = 25% yellow, 3 = 50% yellow, 4 = 75% yellow, 5 = 100% yellow. (w) skin colour rating: 1 = 100% green, 2 = 25% yellow, 3 = 50% yellow, 4 = 75% yellow, 5 = 100% yellow. 14 9% due to the control (2.37 t/ml) compared to the noirrigation treatment (2.20 t/ml) (table 11). for the marketable yield component (i.e. the industry harvest), iwue increased only marginally due to oxygation, while gwue increased by 6 % due to oxygation (1.63 t/ml), and by 5% due to the control (1.54 t/ml) compared to the no-irrigation treatment (1.55 t/ml) (table 11). table 11 water use efficiency (irrigation water use efficiency and total water use efficiency) of the harvested sample yield and industry yield in different irrigation treatments for the total yield averaged over the main and ratoon crop treatments wue harvested yield (tonnes/ml) wue industry yield (tonnes/ml) iwue (z) gwue (y) gwue gwue control 44.23 2.37 28.76 1.54 oxygation 52.98 2.97 29.02 1.63 no-irrigation na 2.13 na 1.55 mean 48.60 2.49 28.89 1.57 (z) iwue= irrigation water use efficiency presents tonnes of total harvested fruits per mega liter of applied irrigation. (y) gwue= gross water use efficiency presents tonnes of total harvested fruits per mega liter of applied irrigation + rain contribution in the crop for the entire crop duration. the wue has been presented for harvested yield (total harvest from the sample area), and industry yield harvested by the industry harvesting crew by the machine (represent total marketable fruits). these observations are consistent with, but much smaller than, the findings of bhattarai et al. (2005) where greater wue due to oxygation using sdi tomato was reported, and for cotton and vegetable soybean where season-long wue for fruit and biomass yield and instantaneous leaf transpiration rate were greater with oxygation (bhattarai and midmare, 2009). cost benefit analysis/decision support the additional cost for installing an oxygation unit in an already established sub-surface drip irrigation system involves the purchase of a mazzei air injector model mi 1583 (au$ 365), plus fittings and pressure gauges for an existing 3” irrigation pipe (au$ 135), totaling au$ 500/ ha. the installation cost per unit area can decrease with an increase in the size of the air injector. the estimated yield increment of 7.5 ton/ha/crop with oxygation over the average industry yield of 65.9 ton/ha without irrigation brings an additional return of au$ 3750/ha in the first crop at a sale value of au$ 500/ton of fruits for the investment of au$ 500 for oxygation. for a new sdi installation, however, the cost with oxygation for pineapple is au$ 6000. hence, the repayment period for the investment to oxygated sdi is two crop cycles (six years). sdi infrastructure lasts 15 years, covering five cycles of the crop (three years/crop cycle) with potential additional returns of $18,750/ha over the 15-year period. these comparative estimates have been based on a crop with no-irrigation, particularly in high rainfall years (4500 mm over three years). crop performance under drier years without irrigation is expected to be much less and under such circumstances sdi offers greater opportunity to deliver strategic irrigation. we conclude that oxygation can improve both yield and quality of ratoon pineapple for an industry scale of operation. 4. conclusions the total and marketable fruit yield increased with oxygation and the irrigation control compared to no-irrigation. aerated water increased the marketable fruit yield (73.25 t/ha) by 11% whereas control treatment increased yield (69.2 t/ha) by 6% compared to no-irrigation (65.9 t/ha). total yield (both marketable and unmarketable) was greater, significantly so, due to oxygation (133.7 t/ha), compared to the control (106.4 t/ha), and no-irrigation (90.4 t/ ha). yield gain with oxygation was attributed to the larger area and weight per leaf, greater plant height, higher specific leaf area, chlorophyll content and light interception by the canopy compared to the control and no-irrigation. greater co 2 exchange rates and instantaneous water use efficiency were recorded for the oxygation compared to table 10 fruit characteristics and quality parameters of pineapple as affected by irrigation treatments in the ratoon crop treatments fruit weight (g/fruit) fruit height (cm) fruit width (cm) brix (º) density (g/cm3) dry matter (%) translucency (1-5) (z) flavor (1-3) (y) flesh colour (1-5) (x) skin colour (1-5) (w) control 793.0 12.40 10.17 16.46 0.90 16.37 1.30 2.41 2.74 2.76 oxygation 961.0 13.43 10.62 13.80 0.92 16.61 0.92 2.29 2.74 3.22 no-irrigation 988.0 14.68 10.70 15.90 0.97 16.81 2.40 2.80 3.80 3.60 p value 0.061 0.007 0.138 0.09 0.003 0.674 0.003 0.117 0.077 0.473 lsd (p≤0.05) 203.0 1.16 ns ns 0.03 ns 0.638 ns ns ns mean separated by lsd. ns= not significant. (z) translucency rating: 1 = 0% translucency, 2 = 25% translucency, 3 = 50% translucency, 4 = 75% translucency, 5 = 100% translucency. (y) flavor rating: 1 = no flavor, 2 = little flavor, 3 = good flavor. (x) flesh colour rating: 1 = 100% white, 2 = 25% yellow, 3 = 50% yellow, 4 = 75% yellow, 5 = 100% yellow. (w) skin colour rating: 1 = 100% green, 2 = 25% yellow, 3 = 50% yellow, 4 = 75% yellow, 5 = 100% yellow. 15 the control and no-irrigation treatments. carbon dioxide exchange was not measurable during the day, only in the early morning, evening and night. aerated irrigation water also reduced phytophthora infestation in the field from 11% in the non-irrigated control to 3%, whereas 5% infestation was noted for control drip irrigation. hence, the use of aerated drip irrigation demonstrated multiple benefits for yield, quality and phytophthora disease management. the trial seasons were rather wetter than average years, and the in crop total rainfall was 4250 mm (42.5 ml), requiring only small amounts of irrigation (2.405 and 2.524 ml for control and oxygation respectively). supplementary and strategic irrigation contributed only 5.5% to total crop water input. nevertheless, this strategic use of irrigation and oxygation led to marked benefits for the pineapple crop yield and quality. 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j.g.f. fumes**, r.l. vieites***, n.c. cabia**, r.s.d. castro**** * department of horticulture, faculty of agronomic sciences, unesp, c.p. 237, 18610307 botucatu, são paulo, brazil. ** agronomic engineering, unesp, c.p. 237, 18610307 botucatu, são paulo, brazil. *** department of agribusiness management and technology, faculty of agronomic sciences, unesp, c.p. 237, 18610307 botucatu, são paulo, brazil. **** food engineer, unesp, c.p. 237, 18610307 botocatu, são paulo, brazil. key words: antioxidant capacity, bioactive compounds, persea americana mill., refrigeration. abstract: avocados possess high nutritional value with proven effectiveness in preventing cardiovascular diseases, attributed primarily to their unsaturated fatty acids content. this fruit is also rich in carotenoids and vitamins, particularly vitamin e. this work evaluates the antioxidant capacity and total phenolic content of hydrothermally-treated fuerte avocado. fruits were selected and hydrothermally treated at 45oc for 5, 10, 15 and 20 min. they were then stored in a refrigerator (10 ± 1oc and 90±5% relative humidity) and evaluated over a 15-day period. the total phenolic content increased up to the sixth day of storage, and decreased thereafter, without differences between the treatments. the percentage of antioxidant capacity of the control and the hydrothermally-treated samples for 5 and 10 min increased during storage. untreated fruits showed the highest percentage of antioxidant capacity. however, the antioxidant capacity of avocado fruits subjected to these treatments declined starting on the twelfth day of storage, possibly due to the fruits’ senescence. hydrothermal treatments for 15 and 20 min delayed fruit senescence while the antioxidant capacity continued to increase up to the fifteenth day of storage. no significant correlation was found between antioxidant capacity and total phenolic content. the antioxidant capacity of ripe fuerte avocado was higher than that of unripe or overripe avocado. 1. introduction avocado (persea americana mill.) has considerable nutritional quality, with a high content of fibers, proteins and mineral salts, particularly potassium and vitamins, especially vitamin e (usda, 2007). it also contains significant amounts of unsaturated fatty acids, which are beneficial for the prevention of cardiovascular diseases (tango et al., 2004). previous studies have also shown that this fruit contains anticarcinogenic lipophilic compounds such as carotenoids (ding et al., 2007). wang et al. (2010) pointed out the scantiness of studies on the phytochemical composition of avocados and the lack of knowledge about the total phenolic content and antioxidant capacity of different avocado varieties, or cultivars. the aforementioned authors conducted studies to determine the antioxidant capacity of the pulp, seed and peel of different avocado varieties, but not the fuerte variety. ‘fuerte’ avocados are small and are highly valued in european and american markets. antioxidants, which are compounds that inhibit and/or reduce the effects of free radicals (soares et al., 2005), can be defined as compounds that protect the cells against the harmful effects of oxygen and nitrogen free radicals that are formed in oxidative processes. high free radical levels generate an imbalance, triggering oxidative stress, the metabolic process responsible for the onset of several types of chronic degenerative diseases. antioxidants can be obtained by eating food containing vitamins e and c, carotenoids, phenolic compounds, and other compounds (ali et al., 2008). phenolic compounds are responsible for most of the antioxidant activity in fruits, making them a natural source of antioxidants (heim et al., 2002). the phenolic content in food and plants depends on a number of intrinsic factors such as the genus, species and cultivar, and on extrinsic factors such as agronomic and environmental factors, handling and storage (thomas-barberán and espín, 2001). avocado is a climacteric fruit which ripens a few days after harvest (hardenburg et al., 1986; seymour and tucker, 1993) and whose postharvest behavior can be influenced by temperature and storage time. the literature contains several studies about the increase in 76 the conservation period of avocado, involving the evaluation of storage temperature, the use of modified atmosphere with the application of wax, gamma irradiation and thermal treatment to prevent chilling injury (zauberman et al., 1973; castro and bleinroth, 1982; seymour and tucker, 1993; germano et al., 1996; de oliveira et al., 2000; sanches, 2006; morgado, 2007; donadon, 2009). thermal treatment has been applied postharvest to solve the problem of contamination by fungal diseases and insect infestation in fruit (fawcett, 1922 apud couey, 1989) or to reduce problems caused by low storage temperatures (kluge et al., 2006). to this end, thermal treatments are performed prior to refrigeration, in the form of conditioning, or during refrigerated storage, in the form of intermittent warming. thermal conditioning consists of exposing fruits briefly to moderate (15 to 25oc) or high temperatures (37 to 53oc) before putting them in refrigerated storage (kluge et al., 2006). daiuto and vieites (2008) conducted a study on hass avocado to evaluate the polyphenol oxidase (ppo) and peroxidase (pod) content in unripe and ripe fruits hydrothermally treated at 45oc for 10 min and stored at 9oc (±1). the enzyme inactivation in ripe fruits subjected to the treatment was 78 to 94% compared to untreated fruits. daiuto et al. (2010) evaluated the weight loss and respiratory rate of ‘hass’ avocado by subjecting it to different physical treatments (thermal, uv and gamma radiation) and reported a decrease in the intensity of the fruit’s respiratory peaks. the evaluation of antioxidant capacity has become increasingly important to determine the effectiveness of natural antioxidants in protecting vegetable products against oxidative damage and loss of their commercial and nutritional value. therefore, the present research focused on an evaluation of the antioxidant capacity and total phenolic content of ‘fuerte’ avocado subjected to hydrothermal treatment. 2. materials and methods ‘fuerte’ avocados were harvested carefully at the point of physiological maturation and according to their oil content. the fruits, which were selected with a view to uniform size, color and absence of injuries and defects, were hydrothermally treated in a water bath at 45°c for 5, 10, 15 and 20 min (four treatments), after which they were stored under refrigeration (10±1°c and 90±5% relative humidity). fruits not subjected to the hydrothermal treatment were used as control. the fruits of these five treatments were evaluated at threeday intervals for two weeks. fruit extraction the extraction process was performed with a solvent mixture of ethanol:water (80:20 v/v). fruit extracts were obtained in triplicate. aliquots of 3.0 g of pulp were weighed and placed in falcon tubes, to which were added 30 ml of an ethanol:water mixture (80:20 v/v). the tubes containing pulp and solvent were then processed at room temperature in a turrax crushing disperser for several minutes, and then centrifuged at 5000 x g for 15 min. the extracts were filtered and stored in dark vials at 8ºc for no longer than a week prior to analysis. total polyphenol analysis the total phenolic content was determined by the folin-ciocalteu spectrophotometric method, as described by singleton et al. (1999), using gallic acid as standard. an aliquot of 0.5 ml of the resulting extracts was then transferred to a test tube and 2.5 ml folin-ciocalteu reagent diluted in water 1:10 was added. the mixture was allowed to rest for 5 min, after which 2 ml of sodium carbonate 4% was added and the tubes were left to stand for 2 hr in the dark. the absorbance was measured in a spectrophotometer operating at a wavelength of 740 nm. a blank sample was subjected to the same procedure and conditions. the results are expressed in µg gae/100 g-1 of dry weight. dpph radical scavenging activity the radical scavenging activity was determined by dpph method (mensor et al., 2001). tocopherol and bht at a concentration of 90 µg ml-1 were used as standards. the reaction mixture consisted of 500 µl of fruit extract, 3.0 ml of ethanol 99%, and 300 µl of the dpph radical in a solution of ethanol 0.5 mm, which was incubated for 45 min at room temperature in the dark. the negative control was prepared by replacing the volume of extract for an equal volume of the extraction solvent. a processing time of 45 min was defined after determining the half maximal effective concentration, ec50. to determine the stabilization time, readings of the antioxidant in five concentrations (1, 2, 3, 4 and 5 g) were taken at 15-min intervals (sanchesmoreno et al., 1998). according to do rufino et al. (2007), in subsequent experiments with the same fruit, readings can be limited to the previously established time (ec50 time), accompanied by the initial reading of the control. the blank was prepared by substituting the volume of the dpph solution for an equal volume of solvent. the free radical scavenging activity was determined in the form of antioxidant activity (aa), using the equation: aa (%) = 100[(aa – ab) x 100] / ac, where: aa = absorbance of the sample; ab = absorbance of the blank; and ac = absorbance of the negative control. all the analyses were performed in triplicate and accompanied by a control. a variance analysis was performed using tukey’s test for multiple comparisons of the averages, at a significance level of 5%. the data were then subjected to a regression analysis and to pearson’s correlation for the two parameters evaluated, using the sas version 77 9.2 software program. 3. results and discussion table 1 presents the average and standard deviation of total polyphenols identified in ‘fuerte’ avocado. although the four treatments produced similar results, a difference was detected as a function of storage time (p=0.007). total polyphenol content was higher in the control treatment. mean values of 45.7, 47.0, 47.1 and 47.2 µg gae/100 g-1, respectively, were obtained in 5, 10, 15 and 20 min hydrothermal treatment, while the control showed 49.8 gae/100g-1. the lowest value obtained was 42.7 µg on the first day of analysis and the highest was 62.1 µg for the control treatment on the sixth day of analysis. the composition of phenolic compounds in fruit may be modified as a function of the environment and postharvest factors, including processing and storage. processing and storage can induce prolonged enzymatic and chemical oxidation of phenolic compounds, contributing to their reduction (kaur and kapoor, 2002). many studies have shown that phenolic compounds generally decrease in climacteric fruit such as tomatoes, bananas, mangos and guavas during ripening (haard and chism, 1996; lakshimnarayana et al., 1970; mitra and baldwin, 1997; selvaraj and kumar, 1989). the total phenolic content increased up to the sixth day of storage, decreasing thereafter due to the onset of senescence (fig. 1). daiuto et al. (2010) found that the average respiratory peak of ‘hass’ avocado subjected to different physical treatments occurred on the ninth day of storage, after which senescence set in. this decrease can be attributed to a series of chemical and enzyme amendments that occur during the accelerated process of maturation of this fruit. these changes may include glycoside hydrolysis by glycosidases, phenol oxidation by phenoloxidases, and polymerization of free phenolic content (robards et al., 1999). the average antioxidant capacity measured in the treatments varied from 21.1% (20 min treatment) to 28.5% (control). the lowest value obtained was 17.6% on the first day of analysis and the highest was 67.6% on the twelfth day of analysis in the control treatment (table 2). the overall average, taking into account the days of storage time, indicated an increase in antioxidant capacity. table 1 average and standard deviation of the total polyphenol content in hydrothermally-treated ‘fuerte’ avocado as a function of treatment and storage day treatments storage days 0 3 6 9 12 15 overall average per treatment control 5 min 10 min 15 min 20 min overall average per storage day 42.7±2.7 42.7±2.7 42.7±2.7 42.7±2.7 42.7±2.7 42.7b±2.7 41.8±2.7 51.8±12.8 58.5±2.7 51.6±5.1 49.3±1.7 50.6ab±8.8 62.1±20.5 42.3±13.5 48.0±4.6 58.4±9.1 54.6±10.1 53.1a±13.0 54.2±1.3 52.9±15.4 48.8±6.5 41.4±15.8 45.3±6.8 48.5ab±10.3 36.5±9.3 42.7±3.7 44.1±7.5 42.8±4.5 45.6±2.9 42.4b±6.1 61.7±3.5 42.2±6.0 39.8±15.1 45.6±6.0 45.4±7.9 46.9ab±10.8 49.85±13.2 45.75±9.9 47.05±9.5 47.15±9.4 47.25±6.5 upper case letters compare overall averages on each storage day table 2 average and standard deviation of antioxidant capacity of hydrothermally-treated ‘fuerte’ avocado as a function of hydrothermal treatment and storage day treatments storage days 0 3 6 9 12 15 overall average per treatment control 5 min 10 min 15 min 20 min overall average per storage day 17.6ab±1.1 17.6ab±1.1 17.6ab±1.1 17.6ab±1.1 17.6ab±1.1 17.6±1.1 9.3ab±2.0 24.7aab±1.0 15.4aba±4.0 13.2aab±4.3 17.7aab±2.5 16.1±5.9 20.6abb±7.2 10.8bb±6.4 15.7aba±7.4 38.4aa±5.4 32.6ab±13.3 23.6±12.9 26.6ab±6.8 25.1aab±9.7 34.3aa±16.9 9.3ab±4.5 10.81b±3.8 21.2±12.9 67.6aa±1.4 43.2aa±33.6 16.9ba±0.7 13.2bab±0.7 7.2bb±4.8 29.6±17.5 29.4aab±3.6 29.6aab±12.6 31.7aa±12.2 33.4aab±8.4 40.9aa±34.6 33.0±15.7 28.5±19.5 25.1±16.6 21.9±11.2 20.9±13.4 21.1±17.8 lower case letters compare averages per treatment per day. upper case letters compare averages of each treatment on each storage day. fig. 1 total phenolic content (µg gae/100g-1) of hydrothermallytreated ‘fuerte’ avocado (overall average of storage days). 78 the highest percentages of antioxidant capacity were obtained in the fruit without hydrothermal treatment. the percentage of antioxidant capacity declined in thermally-treated fruits starting on the twelfth day of storage, possibly due to senescence. the fruits thermally treated for 15 and 20 min showed values of 33.4 and 40.9%, respectively, after 15 days of storage. however, the values declined between the sixth and twelfth day of storage. this tendency for the percentage of antioxidant capacity to decrease may be a result of the thermal treatment, but the profile presented here with low values at nine and 12 days may be a consequence of the heterogeneity of fruit samples. this may explain the results of this research, which indicated that the antioxidant capacity of pulp thermally treated for 15 and 20 min increased up to the fifteenth day of storage. with a less intense respiratory peak, the degradation reactions were also diminished. arancibia-avila et al. (2008) reported that total polyphenols, flavonoids and anthocyanins were significantly higher (p < 0.05) in ripe durian fruit than in unripe or overripe fruit (durio zibethinus murr., cv. mon thong). the overall average antioxidant capacity during the storage period was 17.6, 16.1, 23.6, 21.2, 29.6, and 33.0% for the different treatments, indicating the tendency for antioxidant capacity to increase as the fruits ripened. the total phenolic content is not necessarily involved in the quantification of antioxidant activity (jacóbo-velasquéz and cisneros-zevallos, 2009). the correlation analysis of antioxidant capacity and phenolic compounds in fuerte avocado did not reveal significant results (p=0.992 and r=0.001). arancibia-avila et al. (2008) found a correlation of 0.98 between the total phenolic content and antioxidant capacity of durian fruit. these authors concluded that the high polyphenol content was the main factor responsible for the fruit’s antioxidant capacity. wang et al. (2010) found a significant correlation between the total phenolic content and antioxidant capacity (≥ = 0.79) of avocados of different cultivars. the two parameters evaluated by these authors showed no correlation with the chlorophyll and carotenoids content (r<0.1). furthermore, for these authors the high correlation found between procyanidins and the polyphenol content and antioxidant capacity suggests that this compound is the main polyphenol contributing to the antioxidant capacity of avocado. in the present research, the low correlation found for the evaluated parameters may indicate that another food metabolite is responsible for the antioxidant activity of avocado. it should be noted that vitamin e is a powerful antioxidant which may also contribute to the antioxidant capacity of avocado fruits. in a study of the effect of heat treatment on the antioxidant capacity of vegetables, melo et al. (2009) found that several events that occur during this treatment explain changes in the antioxidant activity of foods, which may be increased, reduced or unaltered. in situations in which the antioxidant activity of food increases, heat treatments favor the partial oxidation of the bioactive compound with the highest ability to donate a hydrogen atom to a radical starting from the hydroxyl group, and/or the aromatic structure of the polyphenol is more able to withstand the displacement of the unpaired electron around the ring. moreover, heat treatments may favor the formation of new compounds such as maillard reaction products (reductones), which exhibit antioxidant activity (nicoli et al., 1999). because refrigeration is the most efficient method for controlling fruit maturation, it may have contributed to maintaining the antioxidant capacity of the fruits during the storage period. the heat treatment had a negative effect on the maintenance of the fruit’s antioxidant capacity compared to that of the control. the longer the fruit is exposed to a hydrothermal treatment, the higher the loss of its antioxidant capacity. 4. conclusions the total phenolic content increased up to the sixth day of storage, decreasing thereafter, without differences between the treatments. the antioxidant capacity of the control fruit and the fruit hydrothermally treated for 5 and 10 min increased throughout the storage period. the highest percentages of antioxidant capacity were obtained for the fruit without heat treatment. the percentage of antioxidant capacity of these treatments declined starting on day 12 of storage, possibly due to senescence. hydrothermal treatments of 15 and 20 min delayed senescence, with antioxidant capacity continufig. 2 dpph antioxidant activity of hydrothermally-treated fuerte avocado. legend: t = thermal treatment considering all the treatments, the highest antioxidant capacity was found in the control treatment, followed by the 5-min thermal treatment. figure 2 shows increasing values of antioxidant capacity over storage time in the control and the 5and 10-min hydrothermal treatments. 79 ing to increase up to the fifth day of storage. no significant correlation was found between the antioxidant capacity and the content of phenolic compounds. the antioxidant capacity of ripe ‘fuerte’ avocado was higher than that of unripe or senescent fruit. acknowledgements the authors gratefully acknowledge the company jaguacy (bauru, sp, brazil) for its support of this study, and the brazilian research funding agencies fapesp (são paulo research foundation) and capes (federal agency for the support and evaluation of postgraduate education) for their financial support. references ali s.s., kasoju n., luthra a., singh a., sharanabasava h., sahua a., bora u., 2008 indian medicinal herbs as sources of antioxidants. food research international, 41(1): 1-15. arancibia-avila p., toledo f., park y.-s., jung s.-t., kang s.-g., heod b.g., lee s.-h., sajewicz m., kowalska t., gorinstein s., 2008 antioxidant properties of durian fruit as influenced by ripening. food science and technology , 41(10): 2118-2125. castro j.v., bleinroth e.w., 1982 conservação do abacate em atmosfera controlada e à temperatura ambiente. boletim do instituto de tecnologia de alimentos, 19(2): 165182. couey h.m., 1989 heat treatment for control of postharvest diseases and insect pests of fruits. hort science, 24(2): 198202. daiuto e.r, vieites r.l., 2008 atividade da peroxidase e polifenoloxidase em abacate da variedade hass, submetidos ao tratamento térmico. revista iberoamericana de tecnologia postcosecha, 9(2): 106-112. daiuto e.r., vieites r.l., trecomoldi m.a., citadini russo v., 2010 taxa respiratória de abacate ‘hass’ submetido a diferentes tratamentos físicos. rev. iber. tecnología postcosecha, 10(2): 101-109. de oliveira m.a., dos santos c.h., henrique c.m., rodrigues j.d., 2000 ceras para conservação pós colheita de frutos de abacateiro fuerte, armazenados em temperatura ambiente. scientia agricola, 57(4): 777-780. ding h., chin y.w., kinghorn a.d., d’ambrosio s.m., 2007 chemopreventive characteristics of avocado fruit. seminars in cancer biology, 17(5), 386-394. do rufino m.s., alves r.e., de brito e.s., de morais s.m., de sampaio c.g.; pérez-jiménez j., sauracalixto f.d., 2007 metodologia científica:determinação da atividade antioxidante total em frutaspela captura do radical livre dpph. comunicado técnico on line. 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occupies an area of 19,700 ha with a total production of 460,000 t, but average yield is only 23 t ha-1, which is much below the crop’s potential productivity. such a low yield seems to be due to the imbalance in nutrients applied for the agricultural production of this crop. owing to the current trend of intensive cropping in lebanon, soils have developed multi-nutrient deficiencies. farmers usually diagnose and correct the deficiencies of nitrogen (n) and phosphorus (p), but often neglect the effect of deficiencies of other essential macronutrients such as potassium (k). in addition to n and p, potato is a heavy remover of soil potassium and its response to potassium varies with variety, source and method of potassium fertilizer application (sharma and sud, 2001; kumar et al., 2007; abd el-latif et al., 2011). since biomass and bulking rate of potato tubers are positively affected by synthesis and accumulation of starch, k plays a key role in this regard as it is the most efficient monovalant cation that stimulates the activity of the starch synthase enzyme, catalyzing the incorporation of simple glucose molecules into complex molecules of starch (moinuddin et al., 2004). starch accumulation is coupled with cell and tissue growth of the tubers as k enhances the overall growth of the plants (singh and singh, 1996), and facilitates the translocation of assimilates to the sinks/tubers (moinuddin et al., 2005), which could ultimately increase the tuber bulking capacity and, thereby, its biomass and yield. thus, potato removes large quantities of k and other soil nutrients, particularly n and p, in a short period coupled with a high rate of dry matter production (perrenoud, 1993; singh and trehan, 1998). an optimum k level, along with optimum levels of n and p would, therefore, be required to exploit the full genetic potential of the crop and achieve an improved level of tuber yield and quality. in spite the efforts aimed at optimizing potato response to k fertilization, little has been done on the time of k application for potato farming. this study, conducted in the central bekaa valley of lebanon, aims at assessing potato response to increasing in-season potassium rates (four progressive rates of k) applied at different times of tuber potato response to potassium application rates and timing under semi-arid conditions f. karam*, r. massaad**, s. skaf**, j. breidy**, y. rouphael*** * international center for agricultural research in dry areas, po box 5466, aleppo, syria. ** lebanese agricultural research institute, po box 287, zahleh, lebanon. *** department of crop production, faculty of agricultural engineering and veterinary medicine, lebanese university, dekwaneh-el maten, beirut, lebanon. key words: aggregate tuber yield, potassium application rates, potassium application timing, solanum tuberosum l., specific gravity, tuber dry matter. abstract: a two-year experiment (2004-2005) was conducted at tal amara research station in the bekaa valley of lebanon to evaluate the influence of progressive application of k rates and application timing on yield, yield components and tuber quality of potato (solanum tuberosum l. cv. agria). four levels of potassium (0 (k0), 75 (k75), 150 (k150), and 225 (k225) kg k2o ha -1) and two application timings (tuber initiation and tuber bulking stages) were used in a split-plot design. the progressive application of potassium fertilizer from 0 to 225 kg k2o ha -1 significantly affected the yield and yield components of potato. in both years, small grade tubers and aggregate tuber yield increased quadratically with increasing k application rates up to 150 kg k2o ha -1, reaching a plateau thereafter, showing luxury consumption of the nutrient at 225 kg k2o ha -1. in 2004 when averaged over k application rates, large and medium grade tubers and aggregated tuber yield were 120%, 22%, and 12% greater, respectively, with k application at tuber bulking than at tuber initiation. a similar trend was also observed in 2005, when the small grade tubers and aggregate tuber yield were 20% and 12% higher, respectively, with k application at tuber bulking than at tuber initiation stage. finally, no significant difference among treatments was observed for tuber dry matter (avg. 19.8%) and specific gravity (1.08 g cm-3). adv. hort. sci., 2011 25(4): 265-268 received for publication 2 august 2011 accepted for publication 20 october 2011 short note 266 growth (tuber initiation and tuber bulking stages) and to depict the optimal rate to achieve target yield. 2. materials and methods experimental site field experiments were conducted from april to august during the 2004 and 2005 growing years at tal amara research station in the central bekaa valley of lebanon (33º 51’ 44’’ n lat., 35º 59’ 32’’ n long., 905 m a.s.l). the details of the experimental site have been described elsewhere (karam et al., 2003, 2005, 2006, 2007, 2009 a,b, 2011). tal amara has a well-defined hot and dry season from may to october and very cold conditions for the remainder of the year. average seasonal rainfall is 592 mm, with 95% of the rain occurring between november and march, and a maximum of 145 mm in january. historical data indicate no rain occurrence at tal amara from june to september. rainfall amounts during the growing period were 35 and 25 mm during 2004 and 2005, respectively. soils of the experimental site were deep, non-calcareous, clay eutric cambisols with an average bulk density of 1.2 g cm-3. soil chemical and physical properties were: available n content 45.5 g kg-1, available p content 17.0 g kg-1, and available k content 11.5 g kg-1, organic matter content 1.2% and ph 7.9. crop management, k-treatments and experimental design potato (solanum tuberosum, l.) seeds of cultivar agria were sown under field conditions on 5 april 2004 and 11 april 2005. the soil was plowed and disked each year in anticipation for bed preparation. in both years, seeds were planted in a conventional “hill” system, where single soil beds were separated by relatively deep furrows spaced 70 cm apart, giving a theoretical plant density of 70000 plants ha-1. the experiments were conducted under optimum irrigation conditions in both years. at planting, the soil surface was thoroughly moistened using a sprinkler irrigation system at the application rate of 4.5 mm h-1. when plants reached 8 to 10 cm in height (two weeks after emergence), a drip irrigation system was installed along the furrows. the drip system consisted in polyethylene (pe) distribution lines, 16 mm in diameter, 40 cm spaced drippers, delivering each 4 l h-1 at 1 bar of head pressure. experiments were set up in a split plot design (main plot: potassium application rate; sub-plot: application timing). the trial covered four levels of potassium (0 (k 0 ), 75 (k 75 ), 150 (k 150 ), and 225 (k 225 ) kg k 2 o ha-1) and two application times (tuber initiation and tuber bulking stages), with five replications. each experimental unit consisted of six rows, 5 m in length. in both years, preplant fertilizer was broadcast (150 kg·ha-1; 17n 17p -17k) and incorporated into the soil. moreover, a fertilizer dose of 144 kg n and 96 kg p 2 o 5 ha-1 was applied in two splits after planting (35 days after planting, dap) and at tuber initiation (60 dap) uniformly to all the plots to assure rigorous shoot development. potassium was applied as k 2 o (0-0-46) in one split at tuber initiation (60 dap) and tuber bulking (80 dap) stages in four application rates with irrigation water. the experiments were concluded on 3 august 2004 (120 days after planting) and on 8 august 2005 (119 days after planting). data collection after harvest, the tuber yield was grouped into three grades, grade 1 (200-400 g), grade 2 (85-200 g) and grade 3 < 85 g. the grade-wise and aggregate tuber yields were recorded. tubers were dried in a forced-air oven at 80°c for 72 h and weighed to determine the tuber dry matter (dm). tuber specific gravity (tuber weight in air/tuber weight in water) (dunn and nylund, 1945) was determined on subsamples of acceptable tubers. statistical analysis all data were statistically analyzed by anova using the spss software package (spss 10 for windows, 2001). duncan’s multiple range test was performed at p=0.05 on each of the significant variables measured. 3. results and discussion in experiment 1 (2004), small grade tubers and aggregate tuber yield were significantly affected by k application rates, whereas large and medium grade tubers and aggregate tuber yield were highly influenced by k application timing, with no ‘k rates x k timing interaction’ (table 1). while in experiment 2 (2005), small grade tubers and aggregated tuber yield were significantly influenced by k application rates, k application timing, with no significant ‘k rates x k timing interaction’ (table1). in both years, no significant difference among treatments was observed for tuber dry matter (avg. 19.8%) and specific gravity (1.08 g cm-3) (table 1). these results on tuber quality (i.e. dry matter and specific gravity) are consistent with the findings of davenport and bentley (2001) who observed no response in tuber quality, mainly specific gravity, in response to increasing k rates. in contrast, others have reported that excess k fertilizer reduces dry matter content and specific gravity of tubers (westermann et al., 1994 a, b). explanations for this disagreement could be the different environments in which the plants were grown, and variations between potato genotypes in response to potassium application rates. in 2004 when averaged over k application rates, large and medium grade tubers and aggregated tuber yield were 120%, 22%, and 12% greater, respectively, with k application at tuber bulking than at tuber initiation. a similar trend was also observed in 2005, when the small grade tubers and aggregate tuber yield were 20% and 12% higher, respectively, with k application at tuber bulking than at tuber initiation stage (table 1). in both years, irrespective of k application timing, the highest small grade tubers yield was recorded with k application rates of 150 kg k 2 o ha-1 (avg. 30 and 33 t ha-1, in 2004 and 2005 respectively), 267 whereas the highest aggregate tuber yield was observed at both k 150 and k 225 with no significant difference observed between the two k application rates followed by k 75 and finally k 0 treatment. in both experiments, aggregate tuber yield increased quadratically with increasing k application rates up to 150 kg k 2 o ha-1, reaching a plateau thereafter, indicating the luxury consumption of the nutrient at 225 kg k 2 o ha-1 (table 1). significant increase in tuber yield of potato as a result of k application is well documented (cordova and valverede, 2001; singh et al., 2001; tawfik, 2001; umar and moinuddin, 2001; moinuddin et al., 2004, 2005). in fact, potato has a higher potassium requirement for optimum production compared to cereals, pulses, oilseeds, and other commercial crops and produces much more dry matter in short growth duration. it produces large amounts of starch due to k-mediated carbohydrate metabolism (perrenoud, 1993; singh and trehan, 1998). in addition, it helps in efficient translocation of photoassimilates to the developing sinks/tubers (beringer, 1978) and enabling the plants to fully utilize applied n and p fertilizers (mengel and kirkby, 1987). thus, k helps the potato tubers to attaine large size and heavier weight. this was evident in the current study, as we observed a progressive increase in aggregate tuber yield. these results are consistent with the findings of moinuddin et al. (2004, 2005) and abd ellatif et al. (2011) who showed an increase in tuber yield with a progressive application of k fertilizer from 0 to 225 kg k 2 o ha-1 (moinuddin et al., 2004, 2005) and from 72 to 120 kg k 2 o fed.-1 (abd el-latif et al. 2011). moreover, in line with our results, singh et al. (1997) reported that an increase in k application rates resulted in an increase in the yield of small-grade tubers. to summarize, we can conclude that the progressive application of potassium fertilizer from 0 to 225 kg k 2 o ha-1 significantly affected the yield and yield components of potato. in both experimentation years, small grade tubers and aggregate tuber yield increased quadratically with intable 1 effects of potassium application rates and k application timing on grade-wise and aggregate tuber yield, tuber dry matter and specific gravity of potato plants grown in 2004 and 2005 year k timing k rate grade-wise tuber yield (t ha-1) aggregate yield tuber dry matter specific gravity kg k2o ha -1 grade 1 grade 2 grade 3 t ha-1 % g cm-3 2004 tuber initiation stage 0 0.6 30.7 19.9 51.2 20.0 1.078 75 0.3 23.4 28.1 51.8 19.8 1.079 150 0.9 24.2 28.9 54.0 19.5 1.077 225 0.2 26.0 29.4 55.6 20.2 1.081 tuber bulking stage 0 1.0 27.4 25.5 53.9 19.5 1.077 75 1.7 30.8 24.3 56.8 19.2 1.075 150 0.4 33.0 31.1 a 64.5 20.0 1.079 225 1.3 36.5 25.2 63.0 20.0 1.080 significance (z) k rate ns ns ** ** ns ns k timing * * ns * ns ns k rate x k timing ns ns ns ns ns ns 2005 tuber initiation stage 0 0.8 29.1 22.7 52.6 19.8 1.078 75 1 27.1 26.2 54.3 19.5 1.077 150 0.6 28.0 30 58.6 19.8 1.078 225 0.8 31.2 27.3 59.3 20.1 1.081 tuber bulking stage 0 0.9 28.2 27.3 56.4 19.7 1.077 75 1.3 29 31.4 61.7 19.3 1.076 150 0.5 31.0 36 66.5 19.9 1.079 225 1 33.9 32.8 67.7 20.0 1.080 significance (z) k rate ns ns * ** ns ns k timing ns ns * * ns ns k rate x k timing ns ns ns ns ns ns (z) ns * **. ***non significant or significant at p < 0.05, or 0.01 respectively. creasing k application rates up to 150 kg k 2 o ha-1, reaching a plateau thereafter, showing luxury consumption of the nutrient at 225 kg k 2 o ha-1, indicating the detrimental effect of over fertilization. the results also demonstrated that k application during tuber bulking stage was more effective in terms of yield and yield components, and not in terms of quality, than at tuber initiation stage. references abd el-latif k.m., osman e.a.m., abdullah r., abd el kader n., 2011 response of potato plants to potassium fertilizer rates and soil moisture deficit. advances in applied science research, 2: 388-397. beringer h., 1978 functions of potassium in plant metabolism with particular reference to yield, pp. 185-202. in: sekhon g.s. (ed.) potassium in soils and crops. potash research institute of india, new delhi, india. cordova j., valverede f., 2001 potato response to potassium application in volcanic soil. better crops, 15: 1617. davenport j.r., bently e.m., 2001 does potassium fertilizer form, source, and time of application influence potato yield and quality in columbia basin? american journal of potato research, 78: 311-318. dunn l.e., nylund r.e., 1945 the influence of fertilizers on the specific gravity of potatoes grown in minnesota. american potato journal, 22: 275-288. eppendorfer w.h., eggum b.o., 1994 dietary fiber, starch, amino acids and nutritional value of potato as affected by sulphur, nitrogen, phosphorus, calcium and water stress. acta agric. scand. section b, soil plant sci., 44: 107-115. karam f., breidy j., stephan c., rouphael y., 2003 evapotranspiration, yield and water use efficiency of drip irrigated corn in the bekaa valley of lebanon. agricultural water management, 63: 125-137. karam f., kabalan r., breidi j., rouphael y., oweis t., 2009 a yield and water-production functions of two durum wheat cultivars grown under different irrigation and nitrogen regimes. agricultural water management, 96: 603-615. karam f., lahoud r., masaad r., daccache a., mounzer o., rouphael y., 2006 water use and lint yield response of drip irrigated cotton to the length of irrigation season. agricultural water management, 85: 287-295. karam f., lahoud r., masaad r., kabalan r., breidi j., chalita c., rouphael y., 2007 evapotranspiration, seed yield and water use efficiency of drip irrigated sunflower under full and deficit irrigation conditions. agricultural water management, 90: 213-223. karam f., masaad r., bachour r., rhayem c., rouphael y., 2009 b water and radiation use efficiencies in drip-irrigated pepper (capsicum annuum l.): response to full and deficit irrigation regimes. european journal of horticultural science, 74: 79-85. karam f., masaad r., sfeir th., mounzer o., rouphael y., 2005 evapotranspiration and seed yield of field grown soybean under deficit irrigation conditions. agricultural water management, 75: 226-244. karam f., saliba r., skaf s., breidy j., rouphael y., balendonck j., 2011 yield and water use of eggplants (solanum melongena l.) under full and deficit irrigation regimes. agricultural water management, 98: 1307-1316. kumar p., pandey s.k., singh b.p., singh s.v., kumar d., 2007 influence of source and time of potassium application on potato growth, yield, economics and crisp quality. potato research, 50: 1-13. mengel k., kirkby e.a., 1987 principles of plant nutrition. international, potash institute, 4th ed., bern, switzerland. moinuddin, singh k., bansal s.k., 2005 growth, yield, and economics of potato in relation to progressive application of potassium fertilizer. journal of plant nutrition, 28: 183-200. moinuddin, singh k., bansal s.k., pasricha n.s., 2004 influence of graded levels of potassium fertilizer on growth, yield and economic parameters of potato. journal of plant nutrition, 27: 239-259. perrenoud s., 1993 fertilizing for higher yield potato. international potash institute, 2nd ed., ipi bull. 8. bern, switzerland. sharma r.c., sud k.c., 2001 potassium and management for yield and quality of potato. central potato research institute, pp. 363-381. singh d., singh d., marwaha s.k., 1997 response of potato to nitrogen and potassium application under faizabad condition. journal indian potato association, 24: 40-43. singh j.p., trehan s.p., 1998 balanced fertilization to increase the yield of potato, pp. 129-139. in: barar m.s. and s.k. bansal (eds.) proceedings of the ipi-prii-pau workshop on balanced fertilization in punjab agriculture. international potash institute, bern, switzerland. singh m.v., jaiswal r.c., singh a.p., 2001 effect of phosphorus and potash on yield of potato crop raised from t.p.s. seedlings. journal indian potato association, 28: 267-269. singh v.n., singh s.p., 1996 influence of split application of potato. journal indian potato association, 23: 72-74. tawfik a.a., 2001 potassium and calcium nutrition improves potato production in drip-irrigated sandy soil. african crop science journal, 9: 147-155. umar s., moinuddin, 2001 effect of sources and rates of potassium application on potato yield and economic returns. better crops, 15: 13-15. westermann d.t., james d.w., tindall t.a., hurst r.l., 1994 a nitrogen and potassium fertilization of potatoes: sugars and starch. american potato journal, 71: 433-452. westermann d.t., tindall t.a., james d.w., hurst r.l., 1994 b nitrogen and potassium fertilization of potatoes: yield and specific gravity. american potato journal, 71: 417-432. 268 88 1. introduction strawberry (fragaria x annanasa dutch) has been grown commercially in various parts of the world for many years but in india it was only introduced in the early 1960’s (sharma and sharma, 2004) and it has now acclimatized well in different parts of india. this is essentially a temperate fruit crop hence its expansion in the kashmir valley has been gaining popularity in the last decade. it is not only consumed as fresh fruit but is also used in processed foods such as jam, ice cream, biscuits and so on. the demand for strawberry fruits for domestic as well as export markets has been increasing steadily. the standard planting time in the valley ranges from late october to the first fortnight of november, and harvesting from 15 march to late april in controlled conditions and from the second fortnight of april to late may in open conditions. climatic conditions under various production methods during the growing season may affect fruit quality. soluble solids concentration, acidity and colour of strawberry fruit have all been reported to be affected by environmental factors (sacks and shaw, 1994; vlachonasios et al., 1995), as well as harvest date (shaw, 1988). temperature is an important factor for floral initiation under short day conditions. the optimum temperature for short day floral initiation is 1518oc, while below 10°c and above 25°c short day induction is rather ineffective (manakasem and goodwin, 2001; sonsteby and heide, 2006; verheul et al., 2007). although both diurnal and nocturnal temperatures are important, strawberry requires an optimum daytime temperature of 22°c and nighttime temperature of 13°c for maximum growth and yield (shoemaker, 1977). growing strawberries under polyhouse decreases the dependence of fruit quality on climate and soil conditions. such cultivation system also enables better water, light and temperature control to a certain extent. in kashmir valley, cultivation is generally carried out under open field conditions and takes advantage of the local climatic conditions, but due to the long duration of winters the availability of fruit is very late. there is no information regarding the protected cultivation and management practices on the performance of commercialized cultivars of strawberry in the kashmir valley. hence, the present study was conducted to study the feasibility of growing strawberry under polyhouse to obtain an early crop and to evaluate the cultivars which may be suitable for commercial exploitation. 2. materials and methods experimental site and material the experiment was laid out under polyhouse during 2008-09 and 2009-10 at krishi vigyan kendra, pulwama, jammu and kashmir. the kvk is located at 33° north and 74° east at an altitude of 1601 m amsl. the mean annual rainfall ranges from 500 to 850 mm. the minimum and maximum temperatures of the station during summers range between 10 and 30oc and between -4 and 10°c durgrowth, yield and fruit quality of strawberry under protected cultivation in south kashmir a. kumar, i. ahad krishi vigyan kendra, extension training centre, sher-e-kashmir university of agricultural sciences and technology kashmir, malangpora (pulwama) jammu & kashmir, india. key words: fruit quality, growth, protected cultivation, strawberry, yield. abstract: field experiments were conducted at krishi vigyan kendra, pulwama, jammu and kashmir at an altitude of 1601 m amsl to identify the suitable strawberry cultivars for higher production of good quality fruits. eight strawberry cultivars were evaluated for two consecutive years (2008-09 and 2009-10) under polyhouse conditions. maximum plant spread (27.43 cm) along with maximum number of runners (8.54) was produced by ‘chandler’. ‘tioga’ produced first flower in 97 days after planting, while runners of ‘chandler’ (56.79) flowered for maximum number of days. ‘chandler’ produced maximum number of flowers per plant (27.23) and set maximum berries (86.01%), however final yield of berries was more in ‘tioga’ which recorded maximum yield per plot (2.26 kg), closely followed by ‘chandler’ (2.19 kg). berry weight (12.24 g) and berry size (5.10 cm length and 4.73 cm) was maximum in ‘tioga’. ‘catskill’ registered maximum for all the biochemical characters, closely followed by ‘tioga’. overall, ‘chandler’, ‘catskill’ and ‘tioga’ performed well under polyhouse conditions in kashmir valley. adv. hort. sci., 2012 26(2): 88-91 received for publication 17 october 2011. accepted for publication 21 may 2012. 89 ing winter under open conditions. a polyhouse with steel pipe framework clad with twin layer uv stabilized 200 µm plastic sheet of was used to create a modified environment. the polyhouse was additionally fitted with a high pressure fan on each west side. under the polyhouse the temperature was maintained up to 25°c. the soil of the location is silty clay, loam neutral in reaction (ph 7.07) having organic c 10.02 g/kg, available n 248.6 kg/ha, available p 14.7 kg/ha and available k 250.3 kg/ha. the experimental materials were comprised of eight commercial cultivars (‘catskill’, ‘chandler’, ‘confutura’, ‘gorella’, ‘pajaro’, ‘selva’, ‘tioga’ and ‘fern’) collected from skuast-k and the department of horticulture, ramban, jammu and kashmir. the experiment was laid out in a completely randomized block design (crbd) with three replications. the spacing between the runners was 30 x 30 cm on 1 x 1 m2 raised beds of 15 cm height with 55 cm spacing between the beds. uniform runners were planted in the first week of november 2008 in three rows on each bed accommodating nine runners. for the second year crop, the emerged runners were removed in the last week of october 2009 in order to maintain the proper spacing for the next year’s crop. usual irrigations, manures and fertilizers, weeding and hoeing were applied equally to the experimental plots during the study years. observations recorded data were recorded for different growth, flowering and fruiting characters for three years. plant spread (cm) and length of the runners (cm) was measured with the help of a measuring tape. number of runners per plant, number of flowers per plant and number of berries per plant were counted from five randomly selected plants. days to first flower was recorded from the date of planting of runners to initiation of first flower. flower duration was counted by subtracting the date of initiation of first flower from the date of last flowering. percentage of berry set was calculated by dividing the number of berries by the number of flowers. yield per plant (g) was calculated by weighing whole fruits from a single plant. ten fruits were randomly selected for all the physio-chemical characters. berry weight was determined with the help of a weighing scale; berry length and width were determined using a vernier calliper. tss, acidity and tss/acid ratio were estimated using standard procedures. total sugar and reducing sugar were determined by shaffer somogy, micro method (ranganna, 1991). data on temperature and humidity under polyhouse were recorded with a portable thermohygrometer. data analysis the pooled data of two years were statistically analyzed following panse and sukhatme (1985). the mean of attributes was compared by paired ‘t’ test and the least significant difference was calculated at 5% level. 3. results and discussion the average monthly data on minimum and maximum temperature and relative humidity inside the polyhouse from transplanting to harvesting are presented in figure 1. the pooled data of two consecutive years shown in table 1 reveal that ‘chandler’ had maximum plant spread (27.43 cm) which was statistically at par with ‘catskill’ (25.49 table 1 growth and flowering characters of strawberry cultivars under polyhouse cultivar plant spread (cm) no. of runners/ plant runners length (cm) days taken first flower to produce duration of flowering number of flower/ plant catskill 25.49 ef 7.09 ef 81.94 f 106 def 50.86 ab 26.92 e chandler 27.43 f 8.54 h 82.59 fg 101 bc 56.79 d 27.23 f confutura 24.18 cde 6.11 d 89.78 h 99 ab 53.92 bcd 20.93 ab gorella 25.44 def 5.67 bc 75.40 de 103 cd 51.12 abc 21.90 bc pajaro 21.75 ab 4.89 a 67.58 b 105 de 49.39 a 20.48 a selva 23.08 bcd 5.44 b 73.95 cd 116 h 47.82 a 21.84 bc tioga 20.19 a 7.35 fg 70.42 bc 97 a 55.49 cd 25.64 d fern 22.35 abc 6.93 e 60.92 a 110 g 48.91 a 21.45 bc mean 23.74 6.50 75.32 104.6 51.79 23.31 cd 0.05 2.40 0.29 3.70 3.84 4.43 0.99 fig. 1 average maximum, minimum temperature (oc) and relative humidity (%) month wise from transplanting to harvesting time. 90 cm) and ‘gorella’ (25.44 cm), while minimum plant spread was recorded for ‘tioga’ (20.19 cm). maximum number of runners per plant in pooled data was found in ‘chandler’ (8.54) which differed significantly from all other cultivars, whereas minimum number of runners per plant was recorded in ‘pajaro’ (4.89). the pooled data of two years shows that ‘confutura’ (89.78 cm) produced maximum runner length which differed significantly from the other cultivars; minimum length of runners was observed in ‘selva’ (67.58 cm). two years of data relative to runner length shows that in the first year runner length was greater than in the second year: the material may have degenerated with the passage of time (childers, 1975). ‘tioga’ (97) produced flowers earlier among the considered cultivars and was closely followed by ‘confutura’ (99) and ‘chandler’ (101); ‘selva’ ranked last and took 116 days to produce first flower (table 1). runners of ‘chandler’ (56.79) flowered for the maximum number of days followed by ‘tioga’ (55.49) and ‘confutura’ (53.92) which was statistically at par with both the cultivars, whereas ‘selva’ (47.82) produced flowers for the fewest number of days. kaska et al. (1997) cultivate nine cultivars of strawberry under high tunnels in adana (turkey) observed that ‘selva’ took maximum days to produce first flower and flowered for least number of days while ‘chandler’ flowered for maximum number of days. maximum number of flowers per plant (27.73) (table 1) and maximum number of berries per plant (23.42) (table 2) were both produced by ‘chandler’ which was statistically at par with ‘catskill’ (26.92) with respect to the number of flowers per plant, while for the number of berries per plant the former was significantly higher than all the cultivars. this indicates that the number of flowers per plant certainly has a bearing on the number of fruits per plant to be harvested, i.e. greater the number of flowers/plant, greater the number of fruits to be harvested but the total yield per plant may vary due to berry weight. ‘pajaro’ (20.48) produced the fewest flowers per plant, which was statistically at par with ‘fern’ (21.45), however the lowest number of berries per plant was produced by the latter (14.49) which was statistically at par with ‘pajaro’ (14.74). paraskevopoulou-paroussi et al. (1990) also recorded a minimum number of flowers per plant and number of berries per plant in ‘pajaro’ and ‘fern’ while growing these cultivars under greenhouse in northern greece. the data in table 2 reveal that ‘chandler’ (86.01%) gave the maximum berry set, which was statistically at par with ‘tioga’ (83.48 %); the minimum berry set (71.84%) was recorded for ‘pajaro’. maximum yield per plot of berries was recorded in ‘tioga’ (2.26 kg) which was statistically at par with ‘chandler’ (2.19 kg) and ‘catskill’ (2.17 kg), and minimum yield per plot was recorded in ‘selva’ (1.08 kg). paraskevopoulou-paroussi et al. (1990) also noted that ‘pajaro’ (24%) and ‘fern’ (38%) produced marketable yield under greenhouse conditions in northern greece, however, kaska et al. (1997) reported that ‘chandler’ and ‘selva’ produced the highest and lowest yield, respectively under high tunnel conditions in adana (turkey). observations from two years of data on yield per plant shows that yield was greater in the second year than the first which might be due to substantial annual variation in fruit set in strawberry (smolarz et al., 1968; misic et al., 1976). ‘tioga’ scored as having maximum berry weight (12.24 g) along with maximum berry length (5.10 cm) and berry breadth (4.73 cm), closely followed by ‘chandler’. ‘selva’ had minimum berry weight (8.71 g), however minimum size [i.e. berry length (3.70 cm) and berry breadth (3.62 cm)] was recorded for ‘pajaro’. pathak et al. (2006) observed similar results with respect to weight, length and breadth of berries while growing strawberry cultivars under cover. maximum tss was scored by ‘catskill’ (9.85%), followed by ‘tioga’ (9.32%), however both these cultivars significantly differed from each other (table 3); ‘selva’ had minimum tss (6.72%). minimum acidity was observed in ‘catskill’ (0.88%), closely followed by ‘tioga’ (0.89%), yet the pooled data of two years showed non significant results. ‘catskill’ (10.51) showed maximum tss/ acid ratio which was statistically at par with tioga (10.47) while minimum tss/acid ratio was recorded for ‘selva’ (6.25). maximum reducing sugar (6.27 %) and total sugar (8.09%) were observed in ‘catskill’ which was the highest table 2 yield and fruiting characters of strawberry cultivars under polyhouse cultivar number of berries/ plant berry set (%) yield/plot (kg) berry weight (g) berry length (cm) berry breadth (cm) catskill 22.32 e 82.90 e 2.17 d 11.11 e 4.98 de 4.55 de chandler 23.42 f 86.01 f 2.19 d 12.11 f 4.79 d 4.31 c confutura 16.93 c 80.86 d 1.44 d 10.54 d 4.78 d 4.48 cd gorella 16.62 c 75.81 c 1.28 c 9.76 c 4.25 bc 3.76 a pajaro 14.74 a 71.84 b 1.18 b 9.03 b 3.70 a 3.62 a selva 15.94 b 72.73 b 1.08 a 8.71 a 4.39 c 3.72 a tioga 21.32 d 83.48 ef 2.26 d 12.24 f 5.10 e 4.73 e fern 14.49 a 66.95 a 1.13 ab 9.22 b 4.03 b 4.01 b mean 18.22 77.57 1.59 10.34 4.50 4.14 cd 0.05 0.64 2.73 0.09 0.27 0.22 0.23 91 among all the studied cultivars, and ‘pajaro’ scored minimum reducing sugar (3.71%) and total sugar (5.55%). our investigation showed much variation in the various cultivars for all the characters and this could be attributed to the genetic make up of the cultivars (dhaliwal and singh, 1983; chandel and badiyala, 1996). factors which may significantly influence strawberry composition include mineral and organic fertilization but weather conditions and variety are also important. it is concluded from the present study that ‘chandler’ for growth and yield characteristics, ‘catskill’ for biochemical characters and ‘tioga’ for yield and physical characters of strawberry fruits are profitable for cultivation under polyhouse conditions in the kashmir valley. references chandel j.s., badiyala s.d., 1996 performance of some strawberry cultivars in foothills of himachal pradesh. annals of agricultural research, 17(4): 375-378. childers n.f., 1975 modern fruit science. horticulture publication, rutgers university nichol avenue, new brunswick, n.j., usa, pp. 976. dhaliwal g.s., singh k., 1983 evaluation of strawberry cultivars under ludhiana conditions. haryana journal of horticultural science, 12(1/2): 36-40. kaska n., turemis n., kafkas s., comlekcioglu n., 1997 the performance of some strawberry cultivars grown under high tunnel in the climatic condition of adana (turkey). acta horticulturae, 439: 297-300. manakasem y., goodwin p.b., 2001 responses of day neutral and june bearing strawberries to temperature and day length. j. of hortic. sci. and biotech., 76: 629-635. misic p.d., tdodrovic r.r., lakie n.k., 1976 self fertility in strawberry. jugoslovensko vocarstovo, 10(37/38): 355-360. panse v.g., sukhatme p.v., 1985 statistical methods for agricultural workers. indian council of agricultural research (icar), new delhi, india. paraskevopoulou-paroussi g., vassilakakis m., dogras c., 1991 performance of five strawberry cultivars under plastic greenhouse or field conditions in northern greece. acta horticulturae, 287: 273-280. pathak p.k., gurung m.r., mitra s.k., 2006 performance of strawberry cultivars under cover in the plains of west bengal. the orissa journal of horticulture, 34(2): 35-37. ranganna s., 1991 manual of analysis of fruits and vegetable products. tata mcgraw hill publishing co., new delhi, india, pp. 77. sacks e.j., shaw d.v., 1994 optimum allocation of objective colour measurements for evaluating fresh strawberries. j. of americ. soc. for hortic. sc., 119: 330-334. sharma v.p., sharma r.r., 2004 the strawberry. indian council of agricultural research (icar), new delhi, india. shaw d.v., 1988 genotypic variation and genotypic correlations for sugars and organic acids of strawberries. j. of americ. soc. for hortic.l sc., 113: 770-774. shoemaker j.s., 1977 small fruitculture. the avi publishing company, westport, connecticut, usa. smolarz k.d., sobezy k.w., kostrzena z., michalski w., bustydzienski w., 1968 yield of four strawberry cultivars in different regions of the country. prace. instytutu sadownictwa, 12: 21-26. sonsteby a., heide o.m., 2006 dormancy relations and flowering of the strawberry cultivars korona and elsanta as influenced by photoperiod and temperature. scientia horticulturae, 110: 57-67. verheul m.j., sonsteby a., grimstad s.o., 2007 influences of day and night temperatures on flowering of fragaria x ananassa duch., cvs. korona and elsanta. scientia horticulturae, 112: 200-206. vlachonasios c., vasilakakis m., dogras c., mastrokostas m., 1995 out of season glasshouse strawberry production in north greece. acta horticulturae, 379: 305-312. table 3 biochemical characters of strawberry cultivars under polyhouse cultivar tss (%) acidity (%) tss/acid ratio reducing sugar (%) total sugar (%) catskill 9.85 h 0.88 10.51 g 6.27 f 8.09 e chandler 9.06 ef 0.94 9.64 d 5.45 d 7.18 d confutura 9.24 fg 0.97 10.11 e 5.85 e 7.14 d gorella 8.03 d 1.02 7.85 c 4.63 c 6.65 c pajaro 7.81 b 1.00 7.78 c 3.65 a 5.55 a selva 6.72 a 1.04 6.25 a 4.04 b 5.86 b tioga 9.32 g 0.89 10.47 fg 5.88 e 7.19 d fern 7.83 cd 1.02 7.64 bc 3.76 a 5.70 a mean 8.48 0.97 8.78 4.94 6.67 cd 0.05 0.20 ns 0.35 0.19 0.22 193 1. introduction in western europe plums are grafted on myrobalan b, st. julien a, marianna and other vegetatively propagated rootstocks (nicotra and moser, 1995). in eastern europe it is difficult to propagate dwarfing and semi dwarfing rootstocks for plums and prunes because of long and hard winters. stool beds are often damaged by winter frost and hard-wood cuttings are not properly rooted because of low temperatures in autumn and spring. mother plants in stool beds are also threatened by plum pox. very few nurseries produce trees grafted on st. julien a and gf 655/2. most plum and prune trees are grafted on seedlings of selected types of prunus divaricata. such trees are planted at 4x3 to 5x5 m and trained to open centre form. in poland about 20% of trees are grafted on seedlings of ‘prune wangenheim’ (prunus domestica). this cultivar is self-pollinated, so seed trees grown in isolation produce seedlings with uniform grow habit (sitarek et al., 2001). ‘prune wangenheim’ is compatible with all european plums and prunes. cultivars grafted on ‘prune wangenheim’ are semi dwarf, tolerant to arid soils common in poland and very productive (sitarek et al., 2001). such trees should be suitable for dense plantings in intensive orchards. in germany, zahn (1986, 1994) and brunner (1990) introduced central leader spindle and free spindle (without shoot bending) for dwarf and semi dwarf, densely planted plums and prunes. these systems were tested in belgium (wustenberghs and keulemans, 1996) with 825 and 1250 trees per ha and appeared to be very economical in yielding and harvesting. the goal of the present work was to elaborate an intensive plum orchard suitable for both hand and mechanical fruit harvesting with combined self propelled straddle harvester working in continuous motion, as is used in poland to harvest sour cherries. the harvester requires densely planted trees with central leader up to 3 m high and young flexible shoots coming out of the leader. for this purpose new methods of summer training and pruning were introduced to plum trees. studies were also undertaken on the intensity of tree growth of six cultivars grafted on ‘prune wangenheim’, their growth habit, canopy structure, fruit bud formation in relation to wood age, quality of flower clusters, fruit set, sun irradiation and distribution and yield. these studies enabled to precise the pruning method of trees in full bearing age. 2. materials and methods one-year-old feathered trees of ‘cacanska rana’, ‘cacanska lepotica’, ‘cacanska najbolja’, ‘diana’, ‘katinka’, and ‘silvia’ grafted on semi dwarf seedling rootstock ‘prune wangenheim’ were planted in autumn 2004 on a 0.5 ha plot, on sandy-loam soil at the research institute of pomology, skierniewice, poland. to estimate optimum planting density, trees were spaced 4 m between rows and at various densities in the row: 1.5; 2.0 and 2.5 m (1666; 1250 and 1000 trees/ha). each cultivar was planted in two rows (each consisting of 36 trees): one for hand harvesting, the other for mechanical harvesting. in each row the trees were arranged in three randomized blocks with four trees per plot. in the second year after planting the interrows were grassed down with frequent grass mowing in intensive plum orchard with summer training and pruning a. mika, z. buler research institute of pomology and floriculture, 96-100 skierniewice, poland. key words: canopy structure, fruiting biology, mechanical harvesting, plums, pruning, prunus domestica, training. abstract: an intensive plum orchard model was created for two types of fruit harvesting: by hand and with a self-propelled straddle harvester working in continuous motion. six plum cultivars grafted on semi dwarfing rootstock ‘prune wangenheim’ (prunus domestica) were planted at three densities (1000, 1250 and 1666 trees/ha). a new training system central leader spindle was applied. the leader was not headed after planting and summer training treatments were performed. from the third year onward renewal pruning was done after fruit harvesting. the new training and pruning system resulted in very rapid tree growth, much young wood, fruit bud formation on young wood and early bearing. trees appeared to be suitable for hand and mechanical harvesting within four years from planting. adv. hort. sci., 2011 25(3): 193-198 received for publication 13 april 2011 accepted for publication 1 august 2011 194 conjunction with the maintenance of 1.5-m-wide herbicide strips along the row. a drip irrigation system was installed from the first year. this was necessary because yearly precipitation at skierniewice is around 500 mm whereas plums grown in central poland require 700 mm of rainfall. fertilizers were applied according to the standard recommendation for commercial plum orchards. eight to ten sprayings were essential to control pests and diseases. a new training system with summer pruning was introduced to obtain central leader trees suitable for mechanical harvesting and hand picking (fig. 1). trees having central leaders 1.7 m high at planting time were not headed after planting and side shoots were shortened lightly. subordination of the side branches to the central leader was obtained by summer pruning. at the end of may or beginning of june (depending on the growing season) new shoots that appeared at the top of the central leader were thinned leaving only one to extend the leader. this treatment resulted in numerous short side shoots along the leader most of them setting flower buds in the first growing season. any side shoots growing upright were bent to horizontal position by fixing (clips) pinches to the leader above shoots. these treatments were repeated in the second year. in spring of the third year, trees were nearly 3 m high with at least 1.5 m of canopy diameter and they were able to give the first crop. further training was not necessary. from the fourth year onward, renewal pruning introduced in poland by czynczyk et al. (1976) was performed after fruit harvesting. each branch over three years of age was cut off near the central leader leaving a 10-30 cm stub to ensure new shoot growth (fig. 2). light shoot thinning was carried out as necessary. effect of cultivars and planting distance on tree growth, canopy structure, relation between shoot age and fruit bud formation, and fruit setting was studfig. 1 plum tree training with summer pruning: a) planted feathered tree is left with unheaded central leader and lightly headed side shoots; b) in may side shoots appearing at top of the leader are cut off except for one left for leader prolongation, some lower shoots are bent with clips; c) treatments at top of the leader are repeated in may of the second year; d) tree with fruiting ability in spring of the third year; e) shoot bending with a clip. fig. 2 plum tree pruning by renewal method done after harvesting. old branches are removed to stumps and mainly one-, and twoyear-old twigs are left. 195 ied. to estimate light interception, the irradiation intensity was measured at ground level in rows and interrows with a delta t tube solarimeter tsl and with a light sensor mounted above trees (anderson, 1964). light interception was calculated by subtracting the light intensity at ground level from light intensity above trees and was expressed as percentage of light intercepted by the canopy. light distribution was measured across tree rows on three levels: 0.8; 1.6 and 2.4 m with sun scan probe type ss -1 (delta-t devices ltd, burwell, cambridge, england). harvesting efficiency with a combine harvester was recorded. fruit quality of hand-picked fruit against mechanically harvested fruit was compared. 3. results and discussion in the sixth year after planting (2010) the growth of trees, expressed by trunk cross sectional area (table 1), showed significant differences between cultivars and also significant differences between the most dense tree spacing in the row compared to the two larger spacing treatments. the large differences in growth intensity among cultivars indicate that this factor should be considered in designing intensive plum orchards. treatment in the most densely spaced rows resulted in a dwarfing effect on tree growth. when the orchard is managed over a long period, such dwarfing effect leads to a smaller crop from smaller trees, as is often observed in intensive apple orchards (mika and piskor, 1996). the new applied methods of tree training resulted in rapid vertical growth. all the trees (except for cv. ‘katinka’) reached the required height (3.0-3.5 m) in the fourth year after planting (table 2). in the subsequent years tree height had to be restricted by pruning to coincide with gap required by the harvester. for this reason strong shoots appearing on the top of trees were removed from the fourth year on trees for both machine and hand harvesting. canopy spread (table 3) increased until the sixth year from planting. trees of cv. ‘silvia’ created the most spread whereas ‘katinka’ formed compact trees which were significantly smaller than the other cultivars. some influence of planting density on canopy spread was evident but the differences were not significant. in renewal pruning method, old branches are cut out and replaced by young shoots. due to that, trees are kept in a constant spread. canopy structure is favourable for fruiting because most of new shoots are short, in range 5-10 cm. (table 4). such shoots are able to form fruit buds on one-yearold wood (table 4). there were no significant differences between cultivars and planting density treatments. spur number per tree was also estimated in the fourth year after planting. in spite of renewal pruning, which forced young wood to grow, numerous spurs were present in the tree table 1 influence of cultivars and spacing on tree growth expressed by trunk cross sectional area in the sixth year from planting (2010) influence of cultivars trunk cross sectional area (tcsa) (cm2) ‘cacanska rana’ 46.6 bc ‘cacanska najbolja’ 52.9 d ‘cacanska lepotica’ 35.0 a ‘diana’ 42.8 b ‘katinka’ 33.3 a ‘silvia’ 51.7 cd influence of spacing (m) 4 x 1.5 40.7 a 4 x 2.0 45.6 b 4 x 2.5 44.8 b different letters indicate significant differences separately for cultivars and spacing at p=0.05. table 2 influence of cultivars and spacing on tree growth expressed by tree height in the fourth year from planting (2008) influence of cultivars tree height (m) ‘cacanska rana’ 3.08 b ‘cacanska najbolja’ 3.26 b ‘cacanska lepotica’ 3.20 b ‘diana’ 3.44 b ‘katinka’ 2.50 a ‘silvia’ 3.62 c influence of spacing (m) 4 x 1.5 3.20 a 4 x 2.0 3.17 a 4 x 2.5 3.20 a different letters indicate significant differences separately for cultivars and spacing at p=0.05. table 3 influence of cultivars and spacing on tree growth expressed by tree spread in the fourth year from planting (2008) influence of cultivars tree spread (m) ‘cacanska rana’ 2.47 b ‘cacanska najbolja’ 2.33 b ‘cacanska lepotica’ 2.50 b ‘diana’ 2.44 b ‘katinka’ 1.74 a ‘silvia’ 2.67 c influence of spacing (m) 4 x 1.5 2.49 a 4 x 2.0 2.38 a 4 x 2.5 2.40 a different letters indicate significant differences separately for cultivars and spacing at p=0.05. 196 canopy (table 5). significant differences among cultivars suggests that some variation in pruning methods may be needed in future for cultivars producing a small number of spurs. bare wood, typical for plums under traditional pruning, did not appear. the significant differences between cultivars show the variation in the growth habit of the chosen cultivars. in the fifth year from planting, trees of three cultivars planted at distances of 1.5 and 2.0 m created dense structures in the bottom part of the canopy; for this reason leaf area index (lai), light interception and light distribution at three planting distances were estimated. this very laborious study was performed only on vigorously growing silvia cultivar having the most regular canopy structure (table 6). the results reveal that lai, expressed as ratio of total canopy leaf area to ground area under tree (m2/m2), achieved a value of 2.5 at planting distance 4 x 1.5 m. this was close to the optimum value (2-3) suggested by jackson (1996) for intensive apple orchards. trees spaced 4 x 2.5 m were far from that value. light interception (table 7) was at an acceptable level at planting distance 4 x 1.5 and 4 x 2.0 m but still insufficient as required for an intensively planted orchard; according to jackson (1996) the value should be 60-70%. light interception at planting distance 4 x 2.5 m was very poor. in a number of studies it has been found that light interception is correlated with fruit production per ha (jackson, 1980). in apple orchards, production increases with light interception up to about 70% available light. light distribution within the fruit tree canopy was acceptable in the upper and middle parts of the trees. in the bottom part (0.7 m above ground) it was critical at the 1.5 x 4 m planting distance, low at 4 x 2 m and sufficient at 4 x 2.5 m planting distances. these results show that the most densely planted trees (1.5 m) require more heavy pruning in the fifth year from planting than trees spaced 2 and 2.5 m in the row. the relationship between the age of wood and percent of cluster fruit bud setting was studied in three years table 4 influence of cultivars and spacing on canopy structure (% of shoots in three classes of length) in the fourth year from planting (2008) influence of cultivars class 5-50 cm class 51-90 cm class 91-150 cm ‘cacanska rana’ 73.5 ij 18.7 cde 6.6 ab ‘cacanska najbolja’ 62.1 hi 30.2 ef 4.8 a ‘cacanska lepotica’ 60.4 gh 13.8 bcd 24.7 def ‘diana’ 48.3 g 34.2 f 14.0 bcd ‘katinka’ 74.6 j 14.6 bcd 10.2 abc ‘silvia’ 54.1 gh 22.7 def 21.7 def influence of spacing (m) 4 x 1.5 61.1 c 21.2 b 15.0 ab 4 x 2.0 62.1 c 22.5 b 12.4 a 4 x 2.5 64.1 c 22.1 b 11.0 a different letters indicate significant differences separately for cultivars and spacing at p=0.05. table 5 influence of cultivars and spacing on fruiting spurs per tree in the fourth year from planting (2008) influence of cultivars number of fruiting spurs ‘cacanska rana’ 238.8 b ‘cacanska najbolja’ 260.8 b ‘cacanska lepotica’ 136.6 a ‘diana’ 285.7 c ‘katinka’ 146.1 a ‘silvia’ 265.8 b influence of spacing (m) 4 x 1.5 216.8 a 4 x 2.0 4 x 2.5 219.6 a different letters indicate significant differences separately for cultivars and spacing at p=0.05. table 6 influence of spacing on leaf area, leaf area index (lai), and light interception of ‘silvia’ cultivar in the fifth year from planting (2009) spacing (m) leaf area (m2) lai insolation on ground level (watt/m2)* % of light interception 4 x 1.5 15.16 a 2.52 c 168.0 44.6 b 4 x 2.0 14.20 a 1.78 b 176.3 46.8 b 4 x 2.5 14.75 a 1.48 a 105.5 28.0 a mean insolation above trees 376.7 watt. table 7 influence of spacing on % light distribution in three canopy levels of ‘silvia’ cultivar in the fifth year from planting (2009) spacing (m) canopy level from the ground 0.8 m 1.6 m 2.4 m 4 x 1.5 7.7 a 18.8 bc 48.0 d 4 x 2.0 19.5 bc 29.2 c 48.1 d 4 x 2.5 10.5 ab 32.4 c 49.5 d 197 (2008-2010). there were no significant differences between years. table 8 shows the results in the sixth year from planting on fully-grown trees. many differences were found in the ability of the cultivars to set fruit buds on young wood. in spite of this, most cultivars were able to set 60 to 80% of cluster fruit buds on one-year-old and two-year-old wood. the rest (20-40%) was set on threeyear-old wood. these results indicate that trees treated with renewal pruning produce enough fruiting wood and the pruning method does not have adverse effects on tree yielding. bare wood observed on trees with traditional regulated pruning was not observed in this experiment. detailed studies on flower bud formation revealed that cluster fruit buds on one-year-old wood consist of, on average, fewer flowers than clusters on older wood (table 9). for this reason one can expect that young wood may set less fruit than older wood. however this was not proven. most of the cultivars set 5 to 10% fruit out of 100 flowers (table 10). as the result, the percent of fruit on oneand two-year-old wood was similar to the percent of cluster flower buds (60-80). trees came to blossom in the second year after planting and produced about 0.3 kg of plums per tree. in the third year, blooming was very abundant but spring frost in the first days of may killed all the flowers. for this reason the first yield was obtained in the fourth year after planting. most of the trees produced from 8 to 30 kg per tree. the most productive was ‘cacanska najbolja’, the poorest was ‘katinka’. there were many differences within cultivar and little differences within planting distances. only ‘cacanska najbolja’ and ‘diana’ gave a greater crop when planted at the greater distance. in the fifth year again spring frost in may reduced the crop to less than one kg per tree. acceptable yield was obtained in the sixth year after planting (2010) (table 11) when most trees yielded 15-18 kg per tree. there were again very large differences in tree productivity among cultivars but none in terms of planting distance. this suggests that when renewal pruning is performed trees can be spaced at even 1.5 m in the row. the good yield, calculated per hectare, varied from 13.9 tons at the largest planting distance to 23 tons at the closest spacing. in the fourth and sixth years after planting fruits were harvested by hand and by self-propelled straddle harvester. harvesting was begun when the forces between fruit and stem were 6-8 n, fruit firmness 5 kg/cm, and tss 12-14%. table 8 influence of cultivars and spacing on % of spur fruit buds on young wood in the fourth year from planting (2008) influence of cultivars 1-year-old 2-year-old 3-year-old ‘cacanska rana’ 34.4 efg 42.4 ghi 21.4 bcd ‘cacanska najbolja’ 50.0 i 30.1 def 19.5 bc ‘cacanska lepotica’ 37.5 fgh 25.9 cde 39.4 fghi ‘diana’ 11.9 a 48.2 hi 39.2 fghi ‘katinka’ 40.8 fghi 37.5 fgh 20.6 k ‘silvia’ 16.5 ab 44.5 ghi 38.2 fgh influence of spacing (m) 4 x 1.5 28.2 ab 40.4 e 31.2 abcd 4 x 2.0 29.0 abc 36.7 de 32.0 bcd 4 x 2.5 35.6 cde 36.7 de 24.8 a different letters indicate significant differences separately for cultivars and spacing at p=0.05. table 9 influence of cultivars and shoot age on number of flowers in one spur fruit bud in the fourth year from planting (2008) influence of cultivars number of flowers in one spur fruit bud ‘cacanska rana’ 4.3 d ‘cacanska najbolja’ 1.9 b ‘cacanska lepotica’ 1.9 b ‘diana’ 2.9 c ‘katinka’ 1.3 a ‘silvia’ 1.3 a influence of shoot age 1-year-old 1.9 a 2-year-old 2.1 b 3-year-old 2.5 c different letters indicate significant differences separately for cultivars and shoot age at p=0.05. table 10 influence of cultivars and spacing on yield (kg/tree) in the fourth and sixth years from planting (2008 and 2010 z) influence of cultivars 2008 kg/tree 2010 kg/tree ‘cacanska rana’ 9.7 a 16.5 c ‘cacanska najbolja’ 28.6 c 4.9 a ‘cacanska lepotica’ 11.4 ab 16.1 c ‘diana’ 14.5 b 17.1 c ‘katinka’ 15.0 b 11.7 b ‘silvia’ 9.0 a 11.1 b influence of spacing (m) 4 x 1.5 13.8 a 12.7 a 4 x 2.0 14.3 b 12.2 a 4 x 2.5 16.0 c 13.9 a different letters indicate significant differences separately for cultivars and spacing at p=0.05. (z) yields in 2007 and 2009 were affected by spring frosts. table 11 calculated yield in 2008 and 2010 (t/ha) influence of spacing (m) 2008 2010 4 x 1.5 23.0 21.2 4 x 2.0 17.9 15.3 4 x 2.5 16.0 13.9 198 the effectiveness of mechanical harvesting was about 95%, with 5% fruit left on the tree or lost on the ground. labour efficiency was 150 kg/8-hour-day at hand picking and 15 ton/day with mechanical harvesting. after grading fruit harvested by machine did not differ visually from that picked by hand. when put in cold storage at temperatures close to 0°c the fruit remained in good condition for five to seven days. the fruits were excellent for processing, but rather poor quality as dessert fruit. references anderson m.c., 1964 light relations of terrestrial plant communities and their measurement. biol. rev., 39: 425486. brunner t., 1990 pruning with bending effect on the basis of inducing hypotonic ramification. physiological fruit tree training for intensive culture. akademiai kiado, budapest, pp. 81-97. czynczyk a., mika a., chlebowska d., 1976 progress report on 12 years of experimentation with established spur, renewal and regulated pruning of apple trees. fruit sci. rep., 3(2): 25-28. jackson j.e., 1980 light interception and utilization by orchard systems. hortic. rev., 2: 208-267. jackson j.e., 1996 light interception and canopy characteristics at low latitudes in relation to orchard system design. acta horticulturae, 451: 417-425. mika a., piskor e., 1996 growth and cropping of dwarf ‘jonagold’ (‘jonica’) apple trees planted at the density ranged from 2000 to 10000 per ha and trained as slender spindle, super spindle and v system. acta horticulturae, 451: 473-477. nicotra a., moser l., 1995 orientamenti per la scelta dei portinnesti del susino. l’informatore agrario, 32: 43-45. sitarek m., grzyb z.s., kołodziejczak p., 2001 effect of rootstocks on growth and yield of plum trees. j. of fruit and ornam. plant res., ix(1-4): 19-24. wustenberghs h., keulemans j., 1996 a comparison of 3 plum tree training systems during the first five years of growth. acta horticulturae, 451: 625-631. zahn f.g., 1986 intensivierung von steinobstanlagen durch stärkenbezogene schnittbehandlung. erwerbsobstbau, 28: 124-140. zahn f.g., 1994 hohengerechter pflanzabstand durch stärkenbezogene baumbehandlung. erwerbsobstbau, 36: 213-220. 207 1. introduction coriander (coriandrum sativum) is a culinary and medicinal plant of the umbelliferae, and is an annual herb originally from the mediterranean area; several authors have indicated coriandrum sativum as a wild plant. linnaeus (1780) reported that c. sativum also occurs as a weed in cereals. alefeld (1866) mentioned that c. sativum was a common weed spread from southeastern europe to southern russia. stoletova (1930) reported on wild c. sativum from armenia. all parts of the plant have a strong odor, from which the plant takes its name. cultivation of c. sativum is widespread, but it is planted on a small scale only; it is cultivated as a summer or winter crop. in jordan, it is found in gardens rather than in large fields, while in germany there are many landraces of coriandrum sativum (diederichsen, 1996). description of the plant the plant can reach heights of 20 to 80 cm. the stem is more or less erect, branched sometimes with several side branches at the basal node. each branch finishes with an inflorescence. the color of the more or less ribbed stem is green and sometimes turns to red or violet during the flowering period. the leaves alternate, and the first ones are often gathered in a rosette. the leaves are of two types: lower ones with leaflets and upper ones divided into narrow linear segments (diederichsen, 1996). the coriandrum sativum flower has five irregular-shaped petals, five stamens, five sepals, and two styles. flowering starts with the primary umbel. the first umbels to bloom have hermaphrodite flowers, with possibly a few staminate ones (diederichsen, 1996). the inner flowers of umbellets are staminate. the central flowers are circular, with small inflexed petals. the color of the petals is pale pink or sometimes white. the umbels of higher order usually contain more staminate flowers than the first ones, and their flowering period is shorter (diederichsen, 1996). in a single flower, the five filaments of the staminate are located between the five petals. after the flower opens, the white filaments are visible between the petals. under optimum conditions, many different insect species are pollinators or visitors of c. sativum umbels (diederichsen, 1996), and the species that pollinate the plants depend on the area of cultivation. flowering and pollination biology of c. sativum is typical of that for umbelliferous plants, according to bell (1971). depending on the weather conditions, two to three days after opening of the first flower, the pollen sacs open and spread the pollen. mcgregor (1976) showed that selfing of c. sativum is impossible but glukhov (1955) showed that it is partially self-fertile. he suggested that geitongamy is common and cross is possible. bees are beneficial to c. sativum: glukhov (1955) reported that when they were excluded only 49.4% of the seeds set, but when they were present 68.3% of the seeds set. bogoyavlenseii and akimenko (1966) associated seed yields with greater insect visitation. use of coriandrum sativum this plant is of economic importance since it has been used as a flavoring agent in food products, perfumes and seed contents of coriandrum sativum in jordan valley k.a. abu-hammour*, d. wittmann** * college of pharmacy, al-isra university, p.o. box 22, 23 isra, 11622 amman, jordan. ** institute of crop science and resource conservation (inres), department of ecology of the cultural landscape-animal ecology, university of bonn, d-53127 bonn, germany. key words: coriandrum sativum, fertilized seeds and global warming, petroselinic acid. abstract: the aim of this research was to determine seed contents of coriander (coriandrum sativum). research was conducted in the jordan valley at 150 m below sea level and in northern jordan at 200 m above. analyses of fertilized seeds showed that they contain 14.9% protein while non fertilized seeds had a significantly lower content (4.7%). the seeds contain 7.4% oil, which can vary according to strain. the fatty acid composition varied significantly among the seeds from the selected locations. petroselinic acid was significantly the most concentrated fatty acid (80.10%). this acid (c18:1) can be split to produce c6 (adipic acid) and c12:0 (lauric acid) molecules. adipic acid is used for the manufacture of a wide range of polymers including high grade engineering plastics; at present it is derived from mineral oil by a process that damages the ozone layer and contributes to the releases of gasses such as n2, which affect global warming. petroselinic acid is abundant in c. sativum and could be an alternative, more environmentally friendly raw material for use in industry. adv. hort. sci., 2011 25(4): 207-211 received for publication 18 february 2011 accepted for publication 18 july 2011 208 cosmetics. moreover, the essential oils of the fruits and various extracts from c. sativum have been shown to possess antibacterial (burt, 2004; cantore et al., 2004; kubo et al., 2004), anticancerous and antimtagenic (chithra and leelamma, 2000) properties and the plant has been used in medicine for thousands of years. in jordan, the primary product is the fresh green herb of c. sativum used for its specific flavor, which is completely different from that of the ripe fruits. in other countries the fruits are used as a spice and vegetable. oleum (1993) stated that russia produces high quality c. sativum oil, with a linalool content of 55%. bauer (1942) found that coriandrum sativum attains its greatest yield of volatile oil (0.9%). the fatty oil of coriandrum sativum is of interest because of the high level of petroselinic acid. 2. materials and methods research sites the research was conducted in jordan at two different locations. the first is located 150 m below sea level. this area is humid with warm temperatures in winter, and dry and hot in summer. the other location is 200 m above sea level. it is characterized by rainy, cold winters and dry, mild summers (fig. 1). both locations have produced relatively high biodiversity in wild plants and bees. the jordan valley, latitude 22° 40’ 0’’ and longitude of 35° 30’ 0’’, is located in jordan, a part of the middle east (fig. 2) and extends down the entire flank of jordan 50 km from amman; it is the country’s most distinctive natural feature. the northern segment of the jordan valley, known in arabic as the ghor, is the nation’s most fertile region. it contains the jordan river and extends from the northern border down to the dead sea. several degrees warmer than the rest of the country, its year-round agricultural climate, fertile soils, high winter rainfall and extensive summer irrigation have made the ghor the food bowl of jordan. according to md (2002), the mean maximum and minimum temperatures are 29.9°c and 16.98°c, respectively, with rainfall of around 77-392 mm over 44.84 rainy days yearly. over the last 30 years, mean relative humidity has been 72.45% in winter and 48% in summer. the jordan valley is subjected to ground frost nearly 2.5 days yearly. coriandrum sativum plantation coriandrum sativum seeds, obtained from local markets (landraces), were planted at locations a and b on 5 november 2007. the rows were 20 m long with 1 m between rows. water was supplied daily by drip irrigation, and extra fertilizers (n p k) were applied. both locations were kept weed-free by cultivation and hand weeding. determination of seed content to determine the seed content of c. sativum, 400 g of seeds were collected. oil extraction and preparation of seeds involved the following: drying of seeds, crushing, and extraction. solvents such as carbon dioxide and propane were used to facilitate oil extraction. gas liquid chromatography was used to determine the seed content. seed content analyses were performed at the national center for agricultural research and technology transfer (ncartt) chemist’s lab, amman, jordan. the protein content of c. sativum was analyzed in fecundated and nonfecundated seeds using the international standard method (sop: 130m01-006) (table 1). table 1 analyzed constituents for coriandrum sativum standard method used seed constituent ash sop: 130m01-009 moisture sop: 130m01-010 oil sop: 130m01-001 proteins sop: 130m01-006 trace elements mg mn cu ca k sop no :131m02-005 sop no :131m02-002 oil constituent fatty acids coi/t.20/doc. no. 24 (2001) – aocs ch 2-91 (1997) the seed contains the essential oils based on the analysis applied (table 2). 3. results seed content seeds for testing (for fatty acid composition, essential oils and mineral contents) were chosen from both locations. moisture content was not significantly different among treatments. protein values were significantly different among pollination treatments and ranged from 13.01 to 15.78%. oil content was low in all seeds and varied from 5.61 to 7.40%. ash values ranged between 6.26 and 6.51% (table 2). oleic acid was significantly the most concentrated 80.10% (table 3). all mineral contents fig. 1 jordan valley overview of vegetation covers. 209 location b: 200 m (above sea level).location a: 150 m (below sea level). fig. 2 research sites (53, 15, 30, 30, are the main international highway). table 2 essential oil component and percentage in seed of c. sativum main component total essential oil (%) 1. linalool 66.7 2. alpha-pinene 9.8 3. gamma-terpinene 8.3 4. geranylacetate 3.3 5. camphor 3.0 6. geraniol 1.9 table 3 main components and mineral contents in seed of c. sativum seed content moisture 8.30 % ash 6.31 % protein 15.78 % oil 7.40 % magnesium 0.34 % calcium 0.40 % potassium 1.46 % manganese 18.80 mg/kg cupper 11.60 mg/kg 210 varied significantly among seed samples; manganese and copper were the most prevalent minerals. linalool acid is the common essential oil in coriander and was determined to be 66.7%. fatty acid composition varied significantly among the seeds from the selected locations. (table 4) the fatty oil of coriander is of considerable interest because of the high level of petroselinic acid, which has potential non-food applications in oleo chemistry. protein percentage in coriandrum sativum seeds the protein percent in treated fecundated seeds in coriandrum sativum was 14.9% of the total dry mass, whereas in non-fecundated seeds it was 4.7%. 4. conclusions coriander is an annual herb and is common in the middle eastern and mediterranean cuisine, in which the fresh leaves and dried seeds are the most commonly used parts of the plant. chemicals extracted from coriander have also been used as a traditional treatment for diabetes and hyperlipidemia (chithra and leelamma, 1997; gary and flat, 1999). little is known about the metabolic origin of petroselinic acid (18:1), which is an unusual fatty acid that occurs primarily in seeds of the umbelliferae plant families (tsevegsuren et al., 2004). petroselinic acid is of potential industrial significance because of unsaturation at c-6. through chemical cleavage at its double bond, petroselinic acid can be used as a precursor of both lauric acid, which is a component of detergents and surfactants, and adipic acid, which is the monomeric component of nylon (avato et al., 2001). adipic acid is used for the manufacture of a wide range of polymers including high grade engineering plastics and it has a global market in excess of 2.5 million tons worth over £1 billion. many previous studies have shown that petroselinic acid is the major constituent of the seed oils of many species of umbelliferae such as parsley or coriander and ranges from 15% up to 85% (cahoon et al., 1992; tsevegsuren et al., 2004). keeping with this, the present study illustrated that the major component of the seed oil of coriander tested was petroselinic acid (18:1) and it represented to 80% of the total fatty acid content. recently, derivatives or polymers from under-utilized fatty acids such as petroselinic acid have been regarded as a new raw material, representing an important oleochemical material for the food, cosmetics, chemistry and pharmaceutical industries (avato et al., 2001). additional recent uses include the use as a green vegetable by some ethnic groups and flavoring for dishes and foods such as pickles and sauces. although many studies have focused on the seed content in coriandrum sativum, the percent of protein of the total dry mass in fecundated and non fecundated seeds has not been pointed out. in the present study, the protein percent in treated fecundated and non fecundated seeds for coriandrum sativum was low. protein content in fecundated seeds is more than in non-fecundated seeds, which can be explained by the enzymatic activity of protein synthesis in complete fertilized ovary (embryo) as compared to empty ovary (gamete). in fact, cell division in the fecundated seeds needs more protein in order to complete division. finally, further studies are needed to fully determine the seed contents of coriandrum sativum, and to explore the feasibility of growing coriander on a large scale in jordan. we also believe that the application of advanced plant breeding, together with extensive biochemical studies, could result in more environmentally friendly, high oil, and high petroselinic acid coriander varieties. references alefeld f., 1866 landwirthschaftliche flora. wiegandt and hempel, berline, germany, pp. 165. avato p., fanizzi f.p., rosito i., 2001 the genus thapsia as a source of petroselinic acid. lipids, 36(8), 845-850. table 4 fatty acid content in seed of c. sativum fatty acid trivial name systematic name percentage c 14:0 myristic acid tetradecanoic acid 0.10 c 16:0 palmitic acid hexadecanoic acid 3.33 c 16:1 palmitoleic acid cis-9-hexadecenoic acid 0.42 c 17:0 hexadecanoic acid 0.03 c 17:1 desaturation of cis-9-hexadecenoic acid 0.04 c 18:0 stearic acid octadecanoic acid 0.88 c 18:1, n-7 vaccenic acid cis-11-octadecenoic acid 80.10 c 18:2 linoleic acid cis-9, 12-octadecadienoic acid 14.63 c 18:3 α –linolenic acid cis-9, 12, 15-octadecatrienoic acid 0.29 c 20:0 arachidic acid eicosanoic acid 0.09 c 22:0 behenic acid docosanoic acid 0.03 c 20:1 gadoleic acid cis-9-eicosenoic acid 0.04 c 24:0 lignoceric acid tetracosanoic acid 0.02 211 bauer 1942 landw. jahresber. 92. in: guenther (ed.) the essential oil. vol. 3. robert e. krieger publishing company, huntington, new york, usa. bell c.r., 1971 breeding systems and floral biology of the umbelliferae or evidence for specialization in unspecialized flowers, pp. 93-107. in: heywood v.h. (ed.) the biology and chemistry of the umbelliferae. academic press, new york, pp. 438. bogoyavlenseii s.h., akimenko a.l., 1966 coriander as nectiferous and entomophillous culture. achievements of science and advanced experiment in beekeeping, pp. 119-125. burt s., 2004 essential oils. their antibacterial properties and potential applications in foods a review. int. j. of food microbiol., 94: 223-253. cahoon e.b., shanklintt j., ohlrogge j.b., 1992 expression of a coriander desaturase results in petroselinic acid production in transgenic tobacco. proc. natl. acad. sci. usa, 89: 11184-11188. cantore p.l., iacobellis n.s., de marco a., capasso f., senatore f., 2004 antibacterial activity of coriandrum sativum l. and foeniculum vulgar miller var. vulgare (miller) essential oils. j. of agric. and food chem., 52: 7862-7866. chithra v., leelamma s., 1997 hypolipidemic effect of coriander seeds (coriandrum sativum): mechanism of action. plant foods for human nutriton, 51(2): 167-172. chithra v., leelamma s., 2000 coriandrum sativum effect on lipid metabolism in 1, 2,-dimethyl hydrazine induced colon cancer. j. of ethnophamacology, 71: 457-463. diederichsen a., 1996 coriandrum sativum l. promoting the conservation and use of underutilized neglected crops. 3. institute of plant genetics and crop plant research, gatersleben, germany. gary m.a., flat p.r., 1999 insulin-releasing and insulin-like activity of the traditional anti-diabetic plant coriandrum sativum (coriander). british journal of nutrition, 81: 203-209. glukhov m.m., 1955 honey plants. izd. 6, perer. i dop. moskva, gos. izd-vo selkhov litry, pp. 512. in russian. kubo i., fujita k.i., kubo a., nihei k.i., ogura t., 2004 antibacterial activity of coriandrum sativum volatile compounds against salmonella choleraesuis. j. of agric. and food chem., 52: 3329-3332. linnaeus c., 1780 ded ritters carl von linnaeus, koniglich schwedischen leibarztes u.u. vollstandiges pflanzensystem nach der dreyzehnten lateinischen ausgabe und nach anleitung des hollanddischen houttuynischen werks ubersetzt und mit einer ausfuhrlichen erklarung ausgefertiget. von den krautern, gabriel nicolaus raspe, nurnberg 6: 152-154. mcgregor s.f., 1976 insect pollination of cultivated crops plants. usda agricultural handbook, no. 496, pp. 411. md, 2002 metrological report of jordan. metrological departement of jordan. oleum, 1993 the essential oil catalogue. stroud, glouchestershire, uk. stoletova e.a., 1930 polevye i ogorodnye kul’tury armenni. tr. po prikl. bot., gen. i sel. 23: 290-291. tsevegsuren n., aitzetmuller k., vosmann k., 2004 geranium sanguineum (geraniaceae) seed oil: a new source of petroselinic and vernolic acid. lipids, 39(6): 571-576. 39 1. introduction supply-chain traceability systems are suitable tools for controlling plant disease diffusion and they can be implemented by means of integrated computer-based information systems (icbiss) which incorporate data from different production centers (porto et al., 2011). with regard to propagative material for grapevine in the european union (2005/43/ce), there are four main categories, labeled with a color coding system. the primary source, derived from selected grapevine by a conservative breeder and is grown under his/her responsibility: this source produces pre-basic material, as well as the derived basic material. this material is intended for delivery only to nurseries having the necessary qualification; certified grapevines are derived from this source. italian regulations (dm 8 february 2005, dm 7 july 2006 and dm 24 june 2008) define which propagative material must be periodically assayed for pests: each material stage is intrinsically characterized by increasing risks of re-infection, and rapid and safe retrieval of mother plant data can be useful for prompt intervention when it is necessary to limit spread of the pathogen. surely an efficient and durable traceability system can provide for information retrieval regarding propagated material, particularly if it is supported by an information technology (it) solution: radiofrequency (rfid)-based technologies can be implemented in platforms to share and manage data in agriculture, providing a safe and durable link between items such as plants and information (sørensen et al., 2010), with positive effects on traceability. sørensen et al. (2011) stated that communication and automated processing of data require a digital form and a machine-readable format which can be interpreted unambiguously by all entities involved in the information flows. increasingly, common data sources and sensor systems in agriculture produce digital, machine-readable data which can be used for decision making. with regard to grapevine propagative material, rfid technology has been successfully used to identify all plants during propagative material of grapevine: rfid technology for supporting traceability of “basic” and “certified” material along the wine production chain a. luvisi*, a. panattoni*, r. bandinelli**, e. rinaldelli***, m. pagano***, e. triolo* * dipartimento di coltivazione e difesa specie legnose, sezione di patologia vegetale, università degli studi di pisa, via del borghetto, 80, 56124 pisa, italy. ** associazione toscana costitutori viticoli, tos.co.vit, via vecchia di marina, 6, 56010 san piero a grado (pi), italy. *** dipartimento di scienze delle produzioni vegetali, del suolo e dell’ambiente agroforestale, sezione di coltivazioni arboree, università degli studi di firenze, viale delle idee, 30, 50019 sesto fiorentino (fi), italy. key words: conservative breeder, foundation block, electronic identification. abstract: four main categories of propagative material in the european union (eu) have been indentified for grapevine: the primary source, pre-basic, basic and certified material. each type of material has to be periodically assayed for pathogens and each material stage is intrinsically characterized by increasing risks. radiofrequency (rfid) can be considered an efficient and durable traceability system to provide retrieval of propagated material or check producer identity. rfid tagging of grapevines of different categories along the production line could establish a durable link between stakeholder and products. to evaluate this approach, histological observations and growth parameters of basic or certified rfidtagged grapevine were performed, as well as requirement analysis for management of sanitary checks and for traceability of the wine production chain. basic material can be safety tagged with rfid tags to establish mother plant vineyards; derived certified material can also safely be tagged. no detrimental performance in terms of vegetative growth and bud production were reported for mother plant vineyards from the first year of production life. requirement analysis made it possible to individuate traceability objectives, materials and stakeholders involved, as well as the rfid-tagging steps and methods to collect sanitary and production data that are useful for traceability purposes. adv. hort. sci., 2012 26(1): 39-43 received for publication 2 february 2012 accepted for publication 5 march 2012 40 sanitary, ampelographic and genetic checks during selection, with no losses in retrieving information from plants (pagano et al., 2010). grapevine tagging with a rfid microchip (tag) can be performed internally (bandinelli et al., 2009) in order to guarantee a durable identification of plants: a microchip identification code can also be associated to an electronic identification datasheet (eid) to store virtually unlimited data for each microchip/plant (luvisi et al., 2010 a). in this perspective, rfid tagging of grapevines of different categories belonging to a production line could establish a durable link between stakeholder and products: interestingly, this application could be combined with corks containing a rfid inlay for highvalue wines (collins, 2005; launois, 2008). histological observations and growth parameters were performed in four-year-old rfid-tagged basic plants to evaluate plant response to implanting method at production stage: tests were also performed on one-year-old derived plants, the information from which was linked to mother plants. requirement analyses for plant management were also performed, while the wine production chain was described in order to define a traceability scheme based on rfid microchips linked to each production stage, from primary source registration to bottle tagging. 2. materials and methods plant materials in order to evaluate the traceability procedures from foundation block to grower, pre-basic vitis vinifera cv. sangiovese were externally tagged with a rfid wristband. then, derivate v. vinifera cv. sangiovese cuttings was grafted on rootstock 1103 paulsen (vitis berlandieri x vitis rupestris) by a foundation block in 2007, in order to obtain two rows of basic material. hardwood cuttings were obtained from these plants in 2009 and 2010, and used by a nursery to graft on rootstock 1103 paulsen in 2010 and 2011 respectively, in order to obtain two rows of plants of certified material. plants belonging to basic or certified categories were tagged with rfid tags, following the luvisi et al. (2010 b) procedure: microchips were inserted inside pith of rootstock after 4-cm depth direct drilling of pith from a distal cut of rootstock just before omega grafting. the microchip was positioned 3 cm below grafting point. untagged plants were used as control in order to evaluate production performances. in order to evaluate the tagging system along the entire grapevine production chain, conservative breeder activities were included in the trial. to minimize experimental times, v. vinifera cv. sangiovese plants involved in a clonal selection procedure started in 2007 were used as primary source of plant used for the foundation block. similarly, barrels and wine bottles were marked with external tagging to simulate data matching with plants, thus completing the traceability system of the wine production chain using rfid. electronic materials and requirement analysis transponder glass rfid tags were used (diameter 2.1 mm, length 12 mm), working at a frequency of 125 khz (intermedia sas, forlì, italy, www.rfid360.net). they were used for internal implanting or attached to plastic wristbands for the external tagging procedure. tags were electronically read using a card flash reader connected by sd slot to a palm-pc (dell axim x51) able to identify the microchips from a distance of 100 mm. software for managing data was java™ and adobe® flex®. tag reliability, as number of readable microchips out of the total, and accuracy, as number of readable microchips within 30 sec, were evaluated in implanted basic (four-year-old) or certified (one-year-old) grapevine plants. requirement analysis was performed by stakeholder identification, interviews, collecting data relative to regulations, farm rules and existing software for managing rfid-tagged plants. according to porto et al. (2011), the requirement analysis step regarded the a) identification of traceability objectives; b) analysis of the regulatory requirements concerning grapevine production, c) identification of plant propagating materials produced along the grapevine production chain; d) definition of rfid-tagged materials; e) analysis of the material flows within the plant supply chain; and f) analysis of the information to be managed in the traceability system. plant assay in order to evaluate the productivity of rfid-tagged basic plants, growth of branches expressed mean relative growth rate (mrgr, mg d-1) with one sampling period of 45 or 90 days was calculated from when shoots started growing in 2009 and 2010. in addition, mean number of buds recovered for grafting and mean diameter of buds were evaluated at the same time. considering these parameters, mean data from 20 plants per treatment are reported. to estimate long-term damage or losses in productivity, effects of implanting procedures on functional vascular tissue area (%) were measured at three heights in rfid-tagged basic (four-year-old) or certified (one-yearold) grapevine. measurements were performed on fresh trunk sections in proximity of the microchip location, at approximately mid-length of the microchip (“height 0”), 3 mm higher (“height 3”) and 3 mm lower (“height -3”). in unmarked plants, sections were taken at the same height as in marked plants. vascular tissue area was calculated using software for image analysis (cerri et al., 1993), measuring total vascular tissue area and non-necrotic vascular tissue area. for histological observations, fresh transversal sections (20 μm thick) were made with a rotary microtome (reichert-jung, autocut 2040, austria) and stained with toluidine blue o (sigma-aldrich corporation, usa); sections were immediately observed with a light microscope (leica, wetzlar, germany). data analysis the effects of treatments were compared by analysis of variance in a random design. duncan’s multiple range test 41 at 5% level was calculated in order to compare treatments for functional vascular area, characterized by undamaged vessels, and in which xylem rays are developed as control, and for growth parameters as well. data in percentage were normalized by arc sin square root transformation. 3. results requirement analysis and microchip tests the objectives for traceability of requirement analysis were: to record genetic links among plant categories; to record mandatory or voluntary assays; to identify structures or people involved in plant production and resulting wine; and to use an identification system which relies on rfid codes associated with electronic identity cards (luvisi et al., 2010 a). regulations concerning non standard grapevine production require mandatory assay for conservative breeders or foundation stock (2005/43/ce). data relative to assays, in particular periodical health assays, can be associated to tags through the use of a database. in any case, other checks, for example those performed by phytosanitary services or those for voluntary certification, can be implemented within the system. identification of plant propagation materials produced along the grapevine production chain led to the creation of six plant categories to be included in the plant management system (fig. 1): plants belonging to these categories can be tagged diffig. 1 data flow within rfid-tagged grapevine production chain. 42 ferently in relation to their morphological characteristics. external tagging is for grapevines selected by conservative breeders, primary sources and non grafted pre-basic material: these plants cannot be tagged using methods proposed thus far. taking into account trunk dimension (which in the case of older plants may be adequate), transversal drilling of trunk and microchip implanting may be possible. on the other hand, tags can be implanted in basic and certified material by foundation blocks and nurseries, respectively. furthermore, to establish a link with the final product, barrels and bottles can be externally tagged. figure 1 offers a scheme to analyze the material flows within the plant supply chain. analysis of the information to be managed in the traceability system identifies the information as: information from grapevine cultivar registries (i.e. the italian registro nazionale delle varietà di vite); information useful for grapevine purchasers, such as botanical name and cultivar, plant category, clone and rootstock specifications, name of producer, rfid-related codes; product history, such as the conservative breeder, mandatory or voluntary assays, information about stakeholders; a digital map accessible via mobile or desktop systems, providing a “virtual vineyard” in which grapevine plants marked by rfid can be viewed (luvisi et al., 2011); and details of wine production, wine producer and cellar. with regard to tests of microchip reliability, tags were fully functional in both plant categories and the implanting procedures did not compromise readability. tags were readable within 30 s in more than 98% of the reading tests, without significant differences between plant categories (table 1). table 1 microchip reliability (as number of readable microchips out of the total) and accuracy (as number of readable microchips within 30 s), implanted in basic (four-year-old) or certified (one-year-old) grapevine plants grapevine category microchip reliability microchip accuracy basic 100.0 a (z) 98.8 a certified 100.0 a 98.1 a (z) within each parameter, values in the same column followed by the same letter do not differ significantly according to duncan’s multiple range test (p=0.05). plant assay rfid-tagged grapevines belonging to the basic category did not show detrimental effects for parameters affecting their productivities such as growth and bud production. in fact, after two years from tagging, no differences in mrgr at 45 or 90 days were measured compared to control (table 2). rfid-tagged plants develop a comparable number of buds with adequate diameters compared to control (table 3). the absence of detrimental effects of rfid implanting in both categories was confirmed by measurement of the functional vascular tissue area, not affected by tag presence (table 4). table 2 growth of branches of rfid-tagged basic grapevine expressed as mean relative growth rate (mrgr, mg d-1) with one sampling period of 45 or 90 days, calculated from when shoots started growing year mrgr (mg d-1) 45 days mrgr (mg d-1) 90 days control rfid-tagged control rfid-tagged 2009 0.095 a (z) 0.100 a 0.121 a 0.125 a 2010 0.0101 a 0.099 a 0.128 a 0.126 a (z) within each parameter, values in the same line followed by the same letter do not differ significantly according to duncan’s multiple range test (p=0.05). mean data from 20 plants per treatment are reported. table 3 mean number of buds for rfid-tagged basic grapevine recovered for grafting and mean diameter of buds year mean no. of buds/plant mean diameter of buds (mm) control rfid-tagged control rfid-tagged 2009 27.3 a (z) 25.1 a 8.5 a 9.1 a 2010 29.4 a 32.5 a 10.8 a 10.0 a (z) within each parameter, values in the same line followed by the same letter do not differ significantly according to duncan’s multiple range test (p=0.05). mean data from 20 plants per treatment are reported. table 4 effects of implantation procedures on functional vascular tissue area (%), measured at three heights, in rfid-tagged basic (four-year-old) or certified (one-year-old) grapevine procedure functional vascular tissue area (%) height -3 height 0 height 3 basic certified basic certified basic certified control 99.1 a (z) 98.7 a 99.9 a 99.4 a 99.8 a 99.0 a rfidtagged 99.2 a 99.1 a 99.8 a 98.7 a 99.9 a 98.8 a (z) values in the same column followed by the same letter do not differ significantly according to duncan’s multiple range test (p=0.05). mean data from 20 plants per treatment are reported. 4. discussion and conclusions plant labeling represents a key step in the certification scheme, with labels supplied by certifying authorities such as a government agency or an officially recognized private organization. for various woody plants, labeling is strictly regulated by law, and currently electronic identification is not an viable option for substituting plastic labels. however, interest in these tools by organizations such as the european commission cannot be ignored, as reported for bovines, already in use in several eu member states on a private basis mainly for farm management purposes. this approach seems to be crucial for localizing and tracing individual animals for veterinary purposes as a tool to control infectious diseases (european commission, 2011). 43 pets can also be legally subjected to microchip implanting, as reported for dogs in italy (ordinanza 6 agosto 2008). even if plants cause less concern about human health when compared to the meat production chain, implications do exist in terms of worldwide spread of plant pathogens (in particular viruses) and chemical residuals. plant traceability by rfid can be a useful tool when it comes to risk management. considering the grapevine production line, basic material can be safely tagged with rfid tags to establish mother plant vineyards, the same for derived certified material. no detrimental performance in terms of vegetative growth and bud production were reported for mother plant vineyards from the first phase of production life. tags were readable to check identities after four years from implanting. data associated to basic material can be linked to previous categories such as pre-basic and primary sources that can be externally tagged, completing the traceability of the grapevine production chain. requirement analysis made it possible to individuate traceability objectives, materials and stakeholders involved, as well as rfid-tagging steps and methods to collect information and match data, from the plant to the wine bottle. even if the availability of user-friendly details about wine production may seem sufficient for the final user (i.e. details about the cellar or viewing of virtual vineyards), the system guarantees retrieval of detailed, exhaustive data for inside users, thus supporting a virtuous circle of trust. references bandinelli r., triolo e., luvisi a., pagano m., gini b., rinaldelli e., 2009 employment of radiofrequency technology (rfid) in grapevine nursery traceability. adv. hort. sci., 23(2): 75-80. cerri s., panattoni a., triolo e. 1993 studio, progetto e sperimentazione di una procedura semiautomatica per l’analisi di alterazioni del legno in barbatelle di vite. consiglio nazionale ricerche, nota interna, b4-25: 1-24. collins j., 2005 wine bottles get corked with rfid. http://www.rfidjournal.com/article/articleview/2117/1/1/ european commission, 2011 electronic identification of bovines to further strengthen food safety and animal health in the eu. http://europa.eu/rapid/pressreleasesaction.do?reference=ip/11/991&format=html&aged=0&lan guage=en launois a., 2008 rfid tracking system stores wine bottle data. http://www.foodproductiondaily.com/packaging/rfidtracking-system-stores-wine-bottle-data luvisi a., pagano m., bandinelli r., rinaldelli e., gini b., scartòn m., manzoni g., triolo e., 2011 virtual vineyard for grapevine management purposes: a rfid/gps application computers and electronics in agriculture, 75: 368-371. luvisi a., panattoni a., bandinelli r., rinaldelli e., pagano m., gini b., triolo e., 2010 b rfid microchip internal implants: effects on grapevine histology. scientia horticulturae, 124: 349-353. luvisi a., triolo e., rinaldelli e., bandinelli r., pagano m., gini b., 2010 a radiofrequency applications in grapevine: from vineyard to web. computers and electronics in agriculture, 70: 256-259. pagano m., bandinelli r., rinaldelli e., panattoni a., triolo e., luvisi a., 2010 rfid technology for clonal selection purposes. adv. hort. sci., 24(4): 282-284. porto s.m.c., arcidiacono c., cascone g., 2011 developing integrated computer-based information systems for certified plant traceability: case study of italian citrus-plant nursery chain. biosystem engineering, 109(2): 120-129. sørensen c.g., fountas s., nash e., pesonen l., bochtis d., pedersen s.m., basso b., blackmore s.b., 2010 conceptual model of a future farm management information system. computers and electronics in agriculture, 72: 37-47. sørensen c.g., pesonen l., bochtis d.d., vougioukas s.g., suomi p., 2011 functional requirements for a future farm management information system. computers and electronics in agriculture, 76: 266-276. impaginato 32 adv. hort. sci., 2011 25(1): 32-36 received for publication 24 august 2010. accepted for publication 21 february 2011. fibrous root distribution in pineapple orange trees under semi-arid irrigated ecosystem r.p.s. dalal, a. thakur punjab agricultural university, regional station, dabwali road, bathinda, punjab, 151001 india. key words: cleopatra (citrus reshni), depth, radial distance, root excavation, root length density, rootstocks, rough lemon (citrus jambhiri lush), troyer citrange (poncirous trifoliate x citrus sinensis osbeck). abstract: the root distribution pattern of 17-year-old pineapple orange trees budded on rough lemon, cleopatra and troyer citrange rootstocks were studied by root excavation method at four radial distances, 0-75, 75-150, 150-225 and 225-300 cm from tree trunk, and at three depths, 0-15, 15-30 and 30-60 cm. fibrous root length density (frld) and fibrous root length percentage differed significantly at various depths and radial distances among rootstocks. frld was closer to tree trunk on both horizontal and vertical planes. root density decreased from 0.183 to 0.084, 1.051 to 0.238 and 0.238 to 0.095 cm.cm-3 from 0-15 cm to 30-60 cm depth within 0-75 cm radial distances from tree trunk in trees on rough lemon, cleopatra and troyer citrange, respectively. cleopatra contains the highest 0.231 cm.cm-3 frld as compared to 0.051 cm.cm-3 in rough lemon and troyer citrange. troyer citrange has intensive lateral root development with 84% fibrous roots (fr) within 75 cm radial distance, whereas rough lemon and troyer has an appreciable amount up to 225 cm distance (extensive lateral). cleopatra contained 57% fr in upper soil layer (0-15 cm) (intensive vertical). in rough lemon and troyer 54% fr are confined to lower depth 15-60 cm (extensive vertical root development). troyer and rough lemon had the same vertical, whereas rough lemon and cleopatra showed the same horizontal rooting pattern under arid irrigated ecosystem. thus, irrigation depth and fertilizer placement should be critically rootstock specific. 1. introduction citrus production depends not only upon soil, climate and high density planting but also rootstocks play an important role as different rootstocks have different intensities of root proliferation and penetration (castle and krezdorn, 1975; neves et al., 2004; morgan et al., 2007). moreover, roots are the principal organ for absorption of nutrients and water from soil. root system structure determines the volume of the soil accessible to the crop plant and it is important to maintain sufficient water and nutrient concentration within the soil occupied by the crop root system for optimal nutrient and water uptake (kramer and boyer, 1995; scholberg et al., 2002). increasing the density of fibrous root within a crop root system increases the amount of water and nutrients available to the crop (eissenstat et al., 1999; tinker and nye, 2000). the rootstock in turn can be influenced by the scion and soil environment. performance of rootstock in a certain environment is related to total volume, configuration, lateral distribution and depth of the root system (cintra et al., 1999). the root distribution pattern of a tree varies from region to region and from one rootstock-scion combination to another. even a single rootstock-scion combination may differ in root distribution with a change in climatic condition. mikhail and elzefhoui (1979) found that 79% of the total fibrous root of valencia orange occurred in the first 60 cm of soil depth on sandy soil, whereas clay soil contained 94% in the same depth. boman et al. (1999) reported that citrus production in deep sandy soils with a high volume irrigation system tends to cause the upper soil layer to dry out between long irrigation intervals and this condition favours deep rooting. hipondoka et al. (2003) reported that most of the root activities in trees with regard to water uptake are performed near the soil surface in arid ecosystems of africa. these differences in rooting pattern among rootstocks and soil environments are more likely to reflect the adaptation of plants to a given environment. since citrus growth and root distribution system is rootstock-dependent and may be modified as a result of changes in the root environment, a clear understanding of the root system is important to best deal with management practices such as irrigation and nutrient application and fixing the geometry in a particular ecosystem. keeping in mind the above facts, the present investigation was carried out with the objectives to 33 determine the rooting pattern of 17-year-old pineapple orange budded on three rootstocks under an arid ecosystem of punjab (india). 2. materials and methods study sites the trial was conducted at the experimental orchard of the punjab agricultural university, regional station, bathinda located at 211 m above mean sea level, latitude 74° 58’ e and longitude 30o 17’, and average rainfall 400 mm/year. however 80% of the rainfall is received during the southwest monsoon season (first week of july to mid september). the mean maximum temperature is 40-45°c in june with hot winds and minimum temperature is 4-5°c in january. soil characteristics the soil samples collected from the experimental orchard at a depth of 0-30 cm were analysed for their physical and chemical properties. the soil type was loamy sand with clay content 13%, bulk density 1.5g/cc with moisture holding capacity of 40-45%, moisture at field capacity 25-28%. the ph of the site was 8.32 with electrical conductivity (ec) 0.2 dsm-1 and calcium carbonate 5-12%. the available n, p, k contents were 160-182, 13-17 and 320-346 kg/ha, respectively. treatments mature pineapple orange trees budded on three rootstocks, i.e. rough lemon (citrus jambhiri lush), cleopatra (citrus reshni) and troyer citrange (poncirous trifoliate x citrus sinensis osbeck), at a spacing of 6x6 m planted in 1990 were selected for the study. all three sets of five mature 17-year-old trees were grown under uniform cultural practices (i.e. irrigation with flooding); fertilizer application at 880 g nitrogen and 440 g. phosphorus/plant/year and mechanical weeding/hoeing were selected randomly in a randomized block design and examined for the root distribution system. sample collection for each plant a circle with a radius of 3 m from the tree trunk was marked. this radius was further divided into four segments with 0-75, 75-150,150-225 and 225300 cm radius. the circle circumference was divided into eight parts and one-eighth sections were excavated at three depths, viz. 0-15, 15-30 and 30-60 cm (fig. 1). the roots of 15 plants were excavated with a jet of water at a pressure of 10-15 psi. the plants were exposed to a radial distance of 3 m from the trunk and down to a depth of 15 cm from the ground surface; exposed roots were painted red. the roots were then excavated to a depth of 30 cm (i.e. between 15-30 cm) and exposed roots were painted yellow. the roots were further excavated to a depth of 60 cm (i.e. between 3060 cm) and these roots were kept as such to distinguish them from other roots. after the entire root system was exposed, the roots were collected from each segment of depth and radial distance separately and washed. the root diameter was measured with the aid of a vernier caliper and those having diameter < 0.2 cm were categorized as fibrous roots. the fibrous root length of each segment was measured using a meter scale separately. fig. 1 scheme of the root sampling areas around the trunk (radial distances: a = 0-75 cm; b = 75-150 cm; c = 150-225 cm; d = 225-300 cm). data collection we estimated root length per soil volume represented by the volume of soil calculated as the radial distance from the trunk by the depth increment (0-15; 1530; 30-60 cm) for each of the 15 orange trees. the surface area of the ring of radial distance 0-75 cm (a) was determined by calculating the area of a circle with radius of 75 cm. the area of ring between radial distances 75-150 cm (b) was equal to the area of a circle with a radius of 150 cm minus the area of ring a. similarly the areas of the other rings (c and d) were determined by subtracting consecutively the area of the adjacent smaller circle from the larger one. the volume of soil used to determine the estimated root length at each sampling location was the product of the area of each ring determined by the sampling distance and soil depth (0-15, 15-30 and 30-60 cm) then divided by 8 because only one-eighth of each ring was excavated. fibrous root length density (frld) was determined by dividing the sample fibrous root length for each sampling location by their respective sample soil volume and expressed as cm cm-3. the root length percentage at various depth zones and radial distances from tree trunk was determined on the basis of total root length, irrespective of radial distance and depth respectively. th 34 statistical analysis the experiment was set up in randomized block design with three sets and five replications. the frld and root length percentage of the three rootstocks at various depths and radial distances from the trunk were analysed by one-way anova using duncan’s multiple range test (p< 0.05). 3. results fibrous root length density (frld) was significantly different among the rootstocks. therefore the frlds were pooled and analysed for interaction among rootstocks, soil depths and distances from tree trunk. although the average frld to a 60-cm depth was statistically significant for cleopatra (0.231 cm.cm-3) in respect to rough lemon (0.048 cm.cm-3) and troyer citrange (0.051 cm.cm-3) which were otherwise at par (table 1). a significant interaction of rootstock and depth suggests distinctly different root distribution patterns among the three rootstocks. trees on cleopatra had significantly greater frld than trees on rough lemon and troyer citrange, whereas the frld was not statistically significant between rough lemon and troyer citrange at all soil depths. frlds decreased significantly with every increase in soil depth in rough lemon and cleopatra, whereas with troyer citrange frld was at par between 0-15 and 15-30 cm depth. the maximum frlds (0.067, 0.390 and 0.070 cm.cm-3) were observed in the top 15-cm soil layer in rough lemon, cleopatra and troyer citrange, respectively. a high proportion of fibrous root length (frl) was found in the upper 0-15 cm soil containing 46, 57 and 45% of rough lemon, cleopatra and troyer citrange, respectively, which also did not differ significantly. the proportion of frl differed significantly with every increase in soil depth in rough lemon and cleopatra and at par in troyer citrange at 0-15 and 15-30 cm depth. however trees grown on rough lemon and troyer citrange have more frl (57%) deeper than 15 cm compared with trees grown on cleopatra (43%), resulting in only 45% of rough lemon and troyer citrange root length at more than 15 cm depth. frld and percentage root length of cleopatra differed significantly compared to rough lemon and troyer citrange at all depth zones, whereas rough lemon and troyer citrange were not significantly different. unlike soil depth, distance from trunk had more effect on distribution of fibrous roots among rootstocks (table 2). cleopatra had significantly greater frld at all 75-cm increments in radial distances from trunk in respect to rough lemon and troyer citrange rootstocks. troyer citrange showed significantly more frld (0.173 cm.cm-3) compared to rough lemon (0.129 cm.cm-3) at the 0-75 cm radial distance, whereas for greater radial distances, rough lemon contained significantly more frld compared to troyer citrange. frlds differed significantly with every increase in radial distance in rough lemon and cleopatra while in troyer frlds at 75-150 and 150-225 cm radial distances did not differ significantly. the highest proportion of frl was observed close to the trunk (i.e. 0-75 cm radial distance from trunk) in all the rootstocks. however, troyer citrange showed maximum frl (84%) within 75 cm radial distance whereas, trees table 1 pineapple orange tree mean fibrous root length density (frld) and percentage of root length in the radial distance up to 300 cm of the soil for rootstock and soil depth soil depths (cm) rough lemon frld (cm.cm-3) root length 0-60cm (%) cleopatra frld (cm.cm-3) root length 0-60cm (%) troyer citrange frld (cm.cm-3) root length 0-60 cm (%) 0-15 15-30 30-60 average 0.067 a 0.048 b 0.030 c 0.048 46.19 a 33.24 b 20.56 c 0.390 a 0.210 b 0.093 c 0.231 57.02 a 29.39 b 13.59 c 0.070 a 0.057 a 0.028 b 0.052 45.11 a 36.74 a 17.88 b fibrous root length density (frld) and root length (%) separation by duncan’s multiple range tests. values followed by different letter within a column are significantly different (< 0.05) from other values in the same column. mean (n=5). table 2 pineapple orange tree mean fibrous root length density (frld) and percentage of root length in the upper 60 cm of soil for rootstock and distance from the tree trunk radial distances (cm) rough lemon frld (cm.cm-3) root length 0-300 cm (%) cleopatra frld (cm.cm-3) root length 0-300 cm (%) troyer citrange frld (cm.cm-3) root length 0-300 cm (%) 0-75 75-150 150-225 225-300 0.129 a 0.031 b 0.022 c 0.012 d 66.54 a 15.87 b 11.39 c 6.19 d 0.642 a 0.139 b 0.097 c 0.035 d 70.31 a 15.23 b 10.58 c 3.87 d 0.173 a 0.017 b 0.011 bc 0.004 c 84.20 a 8.26 b 5.37 bc 2.15 c fibrous root length density (frld) and root length (%) separation by duncan’s multiple range tests. values followed by different letter within a column are significantly different (< 0.05) from other values in the same column. mean (n=5). 35 grown on rough lemon and cleopatra, showed 82-85% of frl within 150 cm radial distance. the proportion of frl beyond 75 cm radial distance was for rough lemon and cleopatra at par and significantly more than troyer citrange. all rootstocks differed significantly for frld with every increment in radial distance, but cleopatra and rough lemon did not significantly differ in root length percentage and differ significantly compared to troyer citrange. however, the greatest frld in the top 15-cm depth ranged from 0.08 to 1.05 cm.cm-3 soil at a distance of 300 cm or less for trees on cleopatra, whereas frlds ranged from 0.016 to 0.183 and 0.006 to 0.238 cm.cm3 at the same depth and distance from trees on rough lemon and troyer citrange, respectively (fig. 2). the figure illustrates that the fibrous roots are concentrated closer to the tree trunk (i.e. up to 75 cm radial distance and 0-15 cm depth). beyond the radial distance of 75 cm there was a very sharp decrease in frlds in all rootstocks. the effect is more pronounced in troyer citrange at 0-60 cm depth, followed by cleopatra (1560 cm) and rough lemon (30-60 cm). 4. discussions and conclusions fibrous root density was influenced by depth and distance from trunk and rootstock. however, fibrous root length observed was lower than earlier reports (kaufman et al., 1972; castle, 1980; morgan et al., 2007) which may be due to sampling time in late spring because in citrus root growth is periodic; root activity declines during fall/winter with unfavourable environment and moisture stress condition then a spring growth flush takes place. root activity then increases immediately after the cessation of shoot elongation in summer months. there is a gradual decrease in frld with depth and distance from tree trunk. however the frld’s were highest near the surface and closer to trunk in all rootstocks (castle, 1980; kurien et al., 1991; swietlik, 1992; zhang et al., 1996). cleopatra had more overall fibrous root length compared to rough lemon and troyer citrange, the latter which showed the same intensity. this may be due to differences in their rooting pattern or genetic make-up. cleopatra had more roots (57%) in the upper layer (0-15 cm) compared to rough lemon and troyer citrange (45% each), however cleopatra rootstock showed only 42% fibrous roots between 15-60 cm while rough lemon and troyer citrange contained 54%. hence cleopatra may be classified as shallow rooted. thakur et al. (1981) concluded that citrus is basically a surface feeder. similarly, avilan et al. (1985) reported that most cleopatra roots (80%) were located in the top 30 cm of soil under the canopy of the tree. similar results were reported previously by zhang et al. (1996): root density was greater (75%) at 0-15 cm depth when field is flooded and nitrogen is spread and less than 10% at 30-60 cm depth in grapefruit on sour orange. neves et al. (2004) found that 80% of the roots grow under 31 cm for african rough lemon and more root area was observed at lower horizon of the soil in p. trifoliata and c13 citrange as compared to rough lemon and sunki mandarin for tahiti lime. sharma and chauhan (2005) found in apple nearly all fibrous roots above the 50 cm depth with very few roots between 75100 cm. troyer citrange showed 84% fibrous root closer to tree trunk (0-75 cm) and at higher distance there was a very sharp decrease showing less than 10% at 75-150 cm distance, whereas, rough lemon and cleopatra has 82-85% fr within 150 cm radial distance with less than 10% fibrous root length beyond 225 cm. thus troyer citrange has an intensive lateral root development and cleopatra and rough lemon showed an extensive lateral root system. the maximum root growth in citrus takes place during summer months following rainy season, hence this may cause more lateral and less vertical root development due to the availability of water in the upper layer during active root growth period. in arid climates, higher root density are in irrigated compared to non-irrigated zones and effect fig. 2 changes in fibrous root length density as a function of soil depth from the surface and radial distance from the tree trunk for pineapple orange trees on (a) rough lemon (n=5); (b) cleopatra (n=5) and (c) troyer citrange (n=5) rootstock. r o o t le n g th d e n s it y (c m .c m -3 ) r o o t le n g th d e n s it y (c m .c m -3 ) r o o t le n g th d e n s it y (c m .c m -3 ) 36 of irrigation is closer to tree trunk due to shading effect or lower evaporation under the canopy (bielorai, 1985; roth and gardner, 1985; morshet et al., 1989). furthermore, rough lemon and cleopatra have a dense and large canopy in comparison to troyer citrange, hence the dense and large canopy reduced soil water losses by evaporation forming a favourable environment for root development in the upper layer. secondly, the largest part of roots are formed within the 0-15 cm depth, the most important layer for plant nutrient supply specially, phosphorus that stimulates root growth in layer fertilized with nutrients. troyer citrange showed a reduced and somewhat upright growth of canopy hence more moisture loss under the canopy took place which make roots to divert to lower horizon for water uptake. these results are in accordance with those of misra et al. (2003) in grape fruit budded on trifoliate orange. carrizo citrange rootstock has intensive type root system and less lateral development in hamlin rootstock (castle and krezdorn, 1975; morgan et al., 2007). similarly castle (1980) and cintra et al. (1999, 2000) found that rough lemon and cleopatra have large root system and rough lemon extensive lateral and vertical development. kurien et al. (1991) reported that most root activity (75-80%) was confined within a radius of 80 cm and 24 cm in depth in acid lime on karna khatta (citrus karna) in this study, we have observed that frld distribution of pineapple orange trees grown on rough lemon, cleopatra and troyer citrange rootstocks decreased with soil depths and lateral distances. the overall maximum frld was recorded in cleopatra at all the depths and radial distances. the density of feeder roots was concentrated at a depth of 0-15 cm within 75 cm radial distance. trees grown on troyer citrange and rough lemon showed an appreciable amount of fr up to 60 cm in depth and may be classified as plants with an extensive vertical root development, whereas, in cleopatra and rough lemon a noticeable amount of fr is confined up to 225 cm radial distance and hence can be considered as extensive lateral development. trees on troyer citrange have fr very closer to tree trunk (0-75 cm) i.e. intensive lateral roots. cleopatra showed more roots in the upper soil layer (0-15 cm) and it can be considered as upper intensive root development. therefore depth of irrigation and placement of fertilizer based on root distribution should be rootstock specific and deep ploughing should be avoided. references avilan r.l., menesses l., sucre r., 1985 root distribution system in fine textured soil on cleopatra rootstock budded with valencia orange. agronomia tropical, 33: 509-534. bielorai h., 1985 moisture, salinity and root distribution of drip irrigated grapefruit. third intl. drip/trickle irr. congr., 2: 567573. boman b., levy y., parsons l., 1999 water management, pp. 72-81. in: timmer l.w. and l.w. duncan (eds.) citrus health management. aps press, st. paul, mn, usa. castle w.s., 1980 fibrous root distribution of ‘pineapple’ orange trees on rough lemon rootstock at three tree spacing. j. amer. soc. hort. sci., 105: 478-480. castle w.s., krezdorn a.h., 1975 effects of citrus rootstocks on root distribution and leaf mineral contents of ‘orlando’ tangelo tree. j. amer. soc. hort. sci., 100: 1-4 cintra f.l.d., libardi p.l., jorge l.a.c., 1999 distribuicao do sistema radicular de porta-enxertos de ecossistema de tabuleiro costeiro. revista brasileira de fruticulture, 21: 313-317. cintra f.l.d., libardi p.l., saad a.m., 2000 balanco hidrico no solo para porta-enxertos de citros em ecossistema de tabuleiro costeiro. revista brasileira de engenharia agricolae ambiental, 4: 23-28. eissenstat d.m., whaley e.l., volder a., wells c.e., 1999 recovery of citrus surface roots following prolonged exposure to dry soil. j. exp. botany, 50: 1845-1854. hipondoka m.h.t., aranibar j.n., chirara c., lihavha m., macko s.a., 2003 vertical distribution of grass and tree roots in arid ecosystem of southern africa: niche differentiation or competition? j. arid environ., 54: 319-325. kaufmann m.r., boswell s.b., lewis l.n., 1972 effect of tree spacing on root distribution of 9-years old ‘washington’ navel oranges. j. amer. soc. hort. sci., 97: 204-206. kramer p.j., boyer j.s., 1995 water relations of plants and soils. academic press, ny, usa. kurien s., goswami a.m., deb d.l., 1991 the distribution of root activity in acid lime (citrus aurantifolia). exp. agri., 27: 431-434. mikhail e.h., el-zefhoui b.m., 1979 effect of soil types and rootstocks on root distribution, chemical composition of leaves and yield of valencia oranges. australian j. soil res., 17: 335342. misra k.k., singh n., jaiswal h.r., 2003 studies on root distribution pattern of grapefruit (citrus paradisi macf) budded on trifoliate orange rootstock. indian j. hort., 60: 49-52. morgan k.t., obreza t.a., scholberg j.m.s., 2007 orange tree fibrous root length distribution in space and time. j. amer. soc. hort. sci., 132: 262-269. morshet s., cohan y., fuchs m., 1989 water use and yield of a mature ‘shamouti’ orange orchard submitted to root volume restriction and intensive canopy pruning. proceeding of international society of citriculture, 2: 739-746. neves c.s.v.j., murata i.m., stenzel n.m.c., medina c.de c., borges a.v., okumoto s.h., lee r.h.c., kanai h.t., 2004 root distribution of rootstocks for ‘tahiti’ lime. scientia agricola, 61: 94-99. roth r.l., gardner b.r., 1985 root distribution of mature orange trees irrigated by pressurized systems. proceeding of third international drip/trickle irrigation congress, 2: 579-586. scholberg j.m.s., parsons l.r., wheaton t.a., mcneal b.l., morgan k.t., 2002 soil temperature, nitrogen concentration and residence time affect nitrogen uptake efficiency of citrus. j. environ. quality, 31: 759-768. sharma d.d., chauhan j.s., 2005 effect of different rootstocks on root distribution of apple. acta horticulturae, 696: 167-171. swietlik d., 1992 yield, growth and mineral nutrition of young ‘ray ruby’ grapefruit trees under trickle or flood irrigation and various nitrogen rates. j. amer. soc. hort. sci., 117: 22-27. thakur r.s., rajput m.s., srivastava k.k., 1981 root distribution studies in some fruit crops with special reference to tracer technique. a review. haryana j. hort. sci., 10: 45-53. tinker p.b., nye p.h., 2000 solution movement in the rhizosphere. oxford university press, ny, usa. zhang m., alva a.k., li l.c., calvert d.v., 1996 root distribution of grapefruit trees under dry granular broadcast vs. fertigation method. plant and soil, 183: 79-84. impaginato 21 adv. hort. sci., 2011 25(1): 21-25 received for publication 30 november 2010. accepted for publication 3 march 2011. growing spinach in a floating system with different volumes of aerated or non aerated nutrient solution a. lenzi, a. baldi, r. tesi dipartimento di scienze delle produzioni vegetali, del suolo e dell’ambiente agroforestale, università degli studi di firenze, piazzale delle cascine, 18, 50144 firenze, italy. key words: hydroponics, hypoxia, leafy vegetables, nitrate accumulation, oxygen, spinacia oleracea l. abstract: vegetables grown in a floating system may encounter problems of hypoxia at root level, especially in the summer when temperature is high. depending on the species, oxygen deficiency may cause a lower yield due to a reduction in water and mineral uptake by the plants. on the other hand, plants under oxygen stress may reduce nitrate accumulation, thus ameliorating produce quality. in the present work spinach was grown in summer and autumn in a floating system in different volumes (252, 126 and 60 l per m2 of cultivated area) of aerated or non aerated nutrient solution. aeration kept oxygen concentration at 7-8 mg l-1 while in the non aerated solution oxygen decreased gradually reaching at harvest, on average, values of 1.92 mg l-1 and 2.83 mg l 1 in summer and autumn respectively. such levels of hypoxia did not affect yield and did not reduce nitrate accumulation either. on the contrary, in the summer cycle leaf nitrate content was significantly lower when the nutrient solution was aerated. reduction of the volume of the solution to 60 l m-2 of cultivated area induced a decrease in nitrate accumulation without negative effect on yield. no significant aeration x volume interaction was observed. 1. introduction a floating system is a soilless cultivation technique where plants are grown on alveolate or fissured polystyrene panels floating in tanks containing the nutrient solution (lazzarin et al., 2001; tesi, 2002). the system is particularly suitable for growing small-size, shortcycle species like leafy vegetables, especially if aimed at producing ready-to-use salads (nicola et al., 2007). as in other hydroponic systems, plants grown in a floating system may encounter problems of oxygen deficiency (hypoxia) at root level, as roots themselves gradually consume the oxygen dissolved in the nutrient solution. oxygen deficiency may cause a reduction in water and mineral uptake by the plants, with consequences on the development of their aerial part and therefore on yield (morard and silvestre, 1996). the problem is surely more urgent in the summer, since with higher temperatures the quantity of oxygen dissolved in a solution decreases and root respiration rate increases (boisseau et al., 1988). in a floating system the stillness of the solution, that may favour the occurrence of hypoxia, is compensated for by the large volumes normally adopted (150-250 l per m2 of cultivated area) and the shortness of the growing cycles. in any case, in order to avoid any risk, growers aerate the nutrient solution to enrich it with oxygen, thus incurring additional costs. on the other hand, plants under oxygen stress are known to increase the activity of nitrate reductase (garcia-novo and crawford, 1973; lambers et al., 1978; veen, 1988), the key enzyme for nitrate utilization by plants (campbell, 1988). therefore, the reduction of oxygen concentration in the nutrient solution could actually control nitrate accumulation in hydroponically-grown vegetables, as suggested by ferrante et al. (2003). in the present work oxygen trend during summer and autumn cultivation of spinach in a floating system equipped or not with a device to aerate the nutrient solution was studied and the possible repercussions on spinach yield and quality (namely nitrate content) were investigated. the aim of the work was to verify if, when the nutrient solution was not aerated (thus achieving a cost reduction), oxygen level was kept high enough to ensure spinach yield but at the same time decreased enough to control its nitrate accumulation. furthermore, different volumes of solution were tested in order to verify if a cost saving may be achieved in the floating system by reducing water and nutrient consumption without negative effects on yield and produce quality, at least in spinach, and if such eventuality may be limited by oxygen concentration when the nutrient solution is not aerated. 22 2. materials and methods growing conditions spinach (spinacia oleracea l.) cv. seven r was cultivated in a floating system in a glasshouse in sesto fiorentino (fi, italy). two cultivation cycles were carried out, one in the summer (sowing on 1 july 2008) and the other in autumn (sowing on 30 september 2008). plants were grown in polystyrene 32.5x51.4x5 cm 160-alveoli trays (three seeds per alveolus). trays floated in polypropylene 36x56x29 cm tanks, lined with a black (inside)/white (outside) polyethylene sheet, on 42, 21 or 10 l of continuously aerated or non aerated nutrient solution (252, 126 or 60 l of nutrient solution per m2 of cultivated area, respectively). the bottom of the tanks containing 10 and 21 l of nutrient solution was raised in order to bring the cultivated panels at the same height as panels floating on 252 l m-2 of nutrient solution. aeration was provided by aquarium aerators (air flux: about 2 l hr-1 per l of nutrient solution). the nutrient solution was composed as follows (macronutrients in mmol l-1, micronutrients in µmol l1): 12 n-no3, 3.8 n-nh4, 2.8 p, 8.4 k, 3.5 ca, 1.4 mg,40 fe, 10 mn, 40 b, 5 zn, 1 cu, 1 mo. during the cultivation cycles the nutrient solution consumed by the plants was restored once (after 10 days and 14 days of cultivation in the floating system for the summer and autumn cycle, respectively) by adding tap water. tap water was composed as follows (macronutrients in mmol l-1, micronutrients in µmol l1): 0.08 n-no3, <0.0027 n-nh4, traces of p, 0.9 k, 1.8ca, 0.57 mg, 0.14 fe, <0.04 mn, 10.17 b, 0.09 zn, 0.36 cu, 0 mo. at harvest, the overall consumption was on average 79.9 l m-2 in summer and 74.0 l m-2 in autumn, without differences due to the imposed treatments (aeration/volume). plants were harvested on 23 july and 30 october 2008 for the summer and autumn cycle, respectively, when their developmental stage was suitable for ready-to-use salads (about 12 cm height). during the cultivation period, maximum, minimum and mean air temperature were 38.3, 19.9 and 27.5°c, and 34.3, 15.6 and 21.6°c, respectively in the summer and autumn. data collection during the cultivation period, every two-three days oxygen concentration and temperature of the nutrient solution were measured using a portable dissolved oxygen meter hi 9146 (hanna instruments, padova, italy). at the same time, ph and electric conductivity (ec) were also checked by the portable ph and conductivity meter hi 991300 (hanna instruments, padova, italy). at harvest, yield and produce quality were evaluated by collecting the following data: leaf and root fresh weight (fw, determined by weighing the whole production from each tank), leaf dry weight (dw, by ovendrying one sample of 100 g of fresh leaves per tank at 80°c until constant weight), leaf area (measured on five plants per tank using the leaf area meter li-3000 by li-cor, lincoln, ne, usa), leaf colour (estimated on 10 plants per tank using the chlorophyll meter spad-502, konica minolta, tokyo, japan) and nitrate content, which was measured spectrophotometrically on two samples per tank using the salicylic-sulphuric acid method (cataldo et al., 1975). statistics the treatments were arranged in a two-way randomized block design with three replications (one replication=one tank). data were subjected to analysis of variance (anova) and means were compared using the snk test at p=0.05 level of significance. 3. results and discussion nutrient solution aeration of the nutrient solution affected ph and oxygen concentration, while it did not show any effect on ec and temperature (fig. 1 and 2). in the aerated nutrient solution ph increased more than in the non aerated one, although without exceeding well-tolerated values for the species. aeration kept oxygen concentration at 7-8 mg l-1 while in the non aerated solution oxygen decreased gradually reaching values below 5 mg l-1 after 17 days in summer and after 21 days in autumn, fig. 1 effect of aeration (left) and volume (l per m2 of cultivated area) (right) of the nutrient solution on its o2 concentration, temperature, ph and ec during the summer cultivation cycle of spinach. error bars (±se) are shown when larger than symbols. arrows indicate the date when the consumed solution was restored with tap water. 23 that is five and nine days before harvest, respectively. therefore, when the nutrient solution was not aerated, plants were subjected to sub-optimal oxygen concentrations only for a short period of their cultural cycle. at harvest oxygen level in non aerated tanks was on average 1.92 mg l-1 in summer and 2.83 mg l-1 in autumn. no significant differences were noticed in temperature and oxygen concentration due to the volumes of the solution, while ph and ec showed variations that were greater when the volume was lower (fig. 1 and 2). those variations (a rise in ph and a decrease in ec) were due to the water added to the tanks to restore the solution consumed by the plants (about 8 l per tank in summer and 7 l in autumn), whose effect was inversely proportional to the starting volume of the solution. spinach yield and quality in summer, temperature conditions unfavourable to germination in the first week after sowing (29.5°c as mean temperature) led to a lower plant density, justifying the lower yield in the summer cycle compared to the autumn one (on average 1.6 kg m-2 and 2.7 kg m-2 of fresh leaves, respectively). on the other hand, in summer the single plants showed a larger leaf area and more developed roots (table 1 and 2). leaf and root production and leaf area did not show any significant differences due to the treatments in both the summer and the autumn cultivation cycles (table 1 and 2). fig. 2 effect of aeration (left) and volume (l per m2 of cultivated area) (right) of the nutrient solution on its o2 concentration, temperature, ph and ec during the autumn cultivation cycle of spinach. error bars (±se) are shown when larger than symbols. arrows indicate the date when the consumed solution was restored with tap water. table 1 effect of aeration and volume (l per m2 of cultivated area) of the nutrient solution on leaf production (leaf fw and leaf dw), leaf area and root production (root fw) in spinach grown in a floating system in the summer season treatments aeration no aeration volume l 252 volume l 126 volume l 60 interaction aeration x volume leaf fw (kg m-2) leaf dw (g m-2) leaf area (cm2 plant-1) root fw (g m-2) 1.6 a 1.7 a 1.5 a 1.8 a 1.6 a ns 85.9 a 91.6 a 94.5 a 91.7 a 80.0 a ns 55.3 a 53.7 a 50.5 a 59.5 a 53.6 a ns 162.7 a 138.0 a 134.0 a 169.0 a 148.0 a ns for each factor, values in each column followed by the same lower-case letter are not statistically different (snk test, p≤0.05). ns = not significant. table 2 effect of aeration and volume (l per m2 of cultivated area) of the nutrient solution on leaf production (leaf fw and leaf dw), leaf area and root production (root fw) in spinach grown in a floating system in the autumn season treatments aeration no aeration volume l 252 volume l 126 volume l 60 interaction aeration x volume leaf fw (kg m-2) leaf dw (g m-2) leaf area (cm2 plant-1) root fw (g m-2) 2.5 a 2.8 a 2.5 a 2.9 a 2.6 a ns 112.0 a 127.7 a 109.7 a 130.2 a 119.7 a ns 53.2 a 45.2 a 47.1 a 50.9 a 49.6 a ns 93.3 a 84.0 a 85.0 a 93.0 a 88.0 a ns for each factor, values in each column followed by the same lower-case letter are not statistically different (snk test, p≤0.05). ns = not significant. 24 spinach tolerance to hypoxia in a floating system was previously observed in spring (lenzi et al., 2008; baldi et al., 2009), in summer (lenzi et al., 2008) and in autumn (tesi et al., 2003 b); on the contrary, when cultivated in summer, spinach showed a reduction in yield due to oxygen depletion (tesi et al., 2003 b) but in this case the length of the cycle was 40 days and plants were exposed to hypoxia conditions for almost twice as long compared to the present work, as well as with respect to the work described in lenzi et al. (2008). no effect on crop yield of root hypoxia in a floating system was detected in rocket (ferrante et al., 2003; lenzi et al., 2008; baldi et al., 2009) and lamb lettuce (ferrante et al., 2005). in head lettuce, roots were exposed to oxygen concentration of 2.1 mg l-1 without any significant effect on plant growth (goto et al., 1996), while oxygen levels close to 0 mg l-1 during the last two weeks of cultivation caused an evident decrease in yield (tesi et al., 2003 a). as far as the quality aspect is concerned, the imposed treatments did not cause any differences in the colour of the product (spad values), while they showed some effects on nitrate accumulation in the summer cycle (table 3). in previous experiments cultivation in a floating system without a device to oxygenate the nutrient solution reduced nitrate accumulation in lamb lettuce (ferrante et al., 2005) and head lettuce (tesi et al., 2003 a) but did not show any effect in spinach and rocket (lenzi et al., 2008; baldi et al., 2009). in the experiment of ferrante et al. (2003) rocket showed a reduction in nitrate content when plants were subjected to anoxia conditions by bubbling nitrogen gas through the nutrient solution one week before harvesting. in the present experiment spinach nitrate accumulation was even higher when the nutrient solution was not aerated, with statistically significant differences compared to the aerated nutrient solution in the summer season (table 3). probably, to obtain a decreasing effect on nitrate accumulation by the onset of oxygen stress according to the mechanism suggested by gracia-novo and crowford (1973) (nitrate used as an electron acceptor alternative to free oxygen), prolonged conditions of anoxia or severe hypoxia are necessary. in experiment presented in this work, a decrease in leaf nitrate content was obtained also by reducing the volume of the nutrient solution, with statistically significant differences again in the summer cycle (table 3). such decrease, already observed both in spinach and rocket (baldi et al., 2009), was not correlated to oxygen, as demonstrated by the non-significant interaction aeration x volume and by the fact that oxygen concentration in the solution did not change due to its volume (fig. 1 and 2). instead, since the lower volume underwent a stronger diluting effect from the water added to restore plant consumption, the nitrate decrease was probably simply due to a lower availability of nitric ions for plants. a similar result was obtained in spinach and other fresh-cut vegetables grown in a floating system by using low-concentrated nutrient solutions (alberici et al., 2008; de pascale et al., 2008). in autumn, when environmental conditions are more favourable to plant nitrate accumulation (maynard et al., 1976), nitrate content was on average higher than in the summer. as in the summer, nitrate was lower with aeration and with the lower volume, although the differences with respect to non aerated conditions and higher volumes, respectively, were not statistically significant (table 3). 4. conclusions from the literature it appears that when vegetables are grown in a floating system without devices to enrich the nutrient solution with oxygen, root hypoxia may have or not a negative effect on yield depending on the species, the season and the length of the growing cycle. the season and duration of the cycle result in different hypoxia levels and more or less prolonged exposure to it. in spinach, in the case of short cycles (20-30 days), both in summer and autumn, hypoxia was not severe enough to influence yield even with low volumes of nutrient solution (up to 60 l per m2 of cultivated area). extent and duration of oxygen deficiency are probtable 3 effect of aeration and volume (l per m2 of cultivated area) of the nutrient solution on spad and leaf nitrate content in spinach grown in a floating system in the summer and autumn seasons treatments aeration no aeration volume l 252 volume l 126 volume l 60 interaction aeration x volume spad leaf nitrate (mg kg-1 fw) spad leaf nitrate (mg kg-1 fw) 24.0 a 26.2 a 24.6 a 25.1 a 25.7 a ns 3610 b 4085 a 4090 a 3901 ab 3553 b ns 23.6 a 24.9 a 23.9 a 23.7 a 25.2 a ns 4495 a 4814 a 4822 a 4780 a 4361 a ns for each factor, values in each column followed by the same lower-case letter are not statistically different (snk test, p≤0.05). ns = not significant. summer cycle autumn cycle 25 ably the most important factors influencing also nitrate accumulation. while anoxia or severe hypoxia may reduce vegetable nitrate accumulation (ferrante et al., 2003; tesi et al., 2003 a), we found that in the case of short exposure to oxygen depletion up to 2-3 mg l-1 not aerating the nutrient solution may result in an even higher nitrate content in spinach leaves. therefore, although the necessity to aerate or not the nutrient solution in a floating system depends on the aim (yield/quality) and the specific situation, we support the use of aeration. furthermore, reduction of the volume of the nutrient solution per m2 of cultivated area, which did not cause any effect on spinach yield and reduced nitrate accumulation up to 60 l, appears an interesting strategy in order to save water and fertilizers and at the same time ameliorate produce quality. acknowledgements this research was funded by the italian ministry for education, university and research, research project prin 2006 “nitrate uptake regulation in leafy vegetables grown under limited oxygen conditions”. references alberici a., quattrini e., penati m., martinetti l., gallina p.m., ferrante a., schiavi m., 2008 effect of the reduction of nutrient solution concentration on leafy vegetables quality grown in floating system. acta horticulturae, 801: 1167-1176. baldi a., lenzi a., cillerai g., tesi r., 2009 controllo del tenore in nitrato in rucola e spinacio coltivati in floating system. proceedings “xxviii convegno sia”, florence, italy, 21-23 september, pp. 421-422. boisseau y., maertens c., morard p., 1988 influence de la température sur la respiration du blé. 7th. colloquium international association optimization plant nutrition, copenhagen, denmark. campbell w.h., 1988 nitrate reductase and its role in nitrate assimilation in plants. physiol. plant., 74: 214-219. cataldo d.a., haroon m., sehrader l.e., youngs v.l., 1975 rapid colorimetric determination of nitrate in plant tissue by titration of salicylic acid. commun soil sci. plant. anal., 6: 71-80. de pascale s., maggio a., orsini f., bottino a., barbieri g., 2008 sulphur fertilisation affects yield and quality in friarielli (brassica rapa l. subsp. sylvestris l. janch. var. esculenta hort.) grown in a floating system. j. hort. sci. biotechnol., 83: 743-748. ferrante a., incrocci l., maggini r., tognoni f., 2003 preharvest and postharvest strategies for reducing nitrate content in rocket (eruca sativa). acta horticulturae, 628: 153-159. ferrante a., quattrini e., martinetti l., schiavi m., maggiore t., 2005 per la quarta gamma. colture protette, 12: 72. garcia-novo f., crawford r.m.m., 1973 soil aeration, nitrate reduction and flooding tolerance in higher plants. new phytol., 72: 1031-1039. goto e., both a.j., albright l.d., leed a.r., 1996 effect of dissolved oxygen concentration on lettuce growth in floating hydroponics. acta horticulturae, 440: 205-210. lambers h., steingröver e., smakman g., 1978 the significance of oxygen transport and metabolic adaptations in flood-tolerance in senecio species. physiol. plant., 43: 277-281. lazzarin r., enzo m., pimpini f., 2001 floating system. in: pimpini f. (eds.) principi tecnico-agronomici della fertirrigazione e del fuori suolo. veneto agricoltura, padova, italy, pp. 204. lenzi a., baldi a., tesi r., 2008 effect of hypoxia on yield and quality of leafy vegetables grown in floating system. abstracts “first symposium on horticulture in europe”, vienna, 17-20 february, pp. 212-213. maynard d.n., barker a.v., minotti p.l., peck n.h., 1976 nitrate accumulation in vegetables. adv. agron., 28: 71-118. morard p., silvestre j., 1996 plant injury to oxygen deficiency in the root environment of soilless culture: a review. plant and soil, 184: 243-254. nicola s., hoeberechts j., fontana e., 2007 ebb-andflow and floating systems to grow leafy vegetables: a review for rocket, corn salad, garden cress and purslane. acta horticulturae, 747: 585-593. tesi r., 2002 colture fuori suolo in orticoltura e floricoltura. edagricole, bologna, italy, pp. 112. tesi r., lenzi a., lombardi p., 2003 a effect of different o2 levels on spinach (spinacia oleracea l.) grown in a floating system. acta horticulturae, 614: 631-637. tesi r., lenzi a., lombardi p., 2003 b effect of salinity and oxygen level on lettuce grown in a floating system. acta horticulturae, 609: 383-387. veen b.w., 1988 influence of oxygen deficiency on growth and function of plant roots. plant and soil, 111: 259-266. impaginato 106 adv. hort. sci., 2011 25(2): 106-111 (1) corresponding author: skuhf07@yahoo.co.in received for publication 4 april 2011. accepted for publication 10 may 2011. in vitro mutagenesis and detection of variability among radiomutants of chrysanthemum using rapd a. kaul, s. kumar*(1), m. ghani department of biotechnology, university of horticulture and forestry, solan 173 230 (h.p.), india. key words: dendranthema grandiflora, in vitro mutagenesis, radiomutants, somaclones. abstract: the present study was undertaken to induce mutations in dendranthema grandiflora cv. snow ball through in vitro mutagenesis by exposing the in vitro shoots to 5, 10, 20 and 30 gy gamma radiation. rapd analysis was used to detect genetic polymorphism among the variants and the control. morphological variations were not observed with the 5 gy gamma dose during the first season. shoot regeneration, rooting and survival of shoots were affected by gamma ray doses. 10 gy of gamma irradiation was the most effective in inducing mutations in flower colour through direct in vitro mutagenesis. the shoots irradiated with 20 and 30 gy gamma radiation did not root and died. twenty rapd primers were used to amplify dna segments from the genomic dna of the control and its 10 variants, and the genetic similarity among them ranged from 0.06 to 0.79 revealing high genetic diversity. 1. introduction chrysanthemum is a major horticultural crop and it is the second largest in terms of cut flowers after rose, among the ornamental plants traded in the global flower market (kumar et al., 2006). the common garden chrysanthemum is hexaploid with 54 chromosomes (wolff, 1996). it is propagated vegetatively and has a strong self incompatibility system (richard, 1986), hence new cultivars are difficult to obtain by crossing. traditionally, new cultivars have been obtained from spontaneous mutations in vegetative reproduction, sports, being some variations more stable than others (miñano et al., 2009). in the last few years, induced mutations and somaclonal variations derived from the tissue culture process have been employed as a new source of variability (schum, 2003; datta et al., 2005; jain and spencer, 2006; zalewska et al., 2007; jain, 2010; barakat et al., 2010). although extensive work has been carried out to develop novelties in chrysanthemum through induced mutations using physical and chemical mutagens (broetjes and van harten, 1978), there is always a need to explore the possibility of new variety for floriculture trade. mutation breeding by radiation has been widely used to upgrade well-adapted plant varieties and also to develop new variations within improved agricultural characteristics. since most cultivated chrysanthemum cultivars are polyploids with high genetic heterogeneity, mutants with allied flower colour, shape, floral size and shape are often recovered. allied flower colours with chimeric tissue can be easily induced by radiation and can be isolated using in vitro tools (kumar et al., 2006). identification and characterization of cultivars is extremely important in horticultural crops in order to protect the plant breeder’s rights. traditionally, identification has been based on morphological characters; however the development of new technologies has made it possible to base this analysis on dna information. one approach is a pcr-based technique rapd (william et al., 1990) that has been widely used for plant germplasm characterization (wolff et al., 1994; huang et al., 2000; martin et al., 2002; martin and gonzalez-benito, 2005). the aim of the present study was to induce mutation in chrysanthemum cv. snow ball through in vitro mutagenesis by treating the in vitro shoots with gamma radiation and to apply rapd analysis for the detection of genetic polymorphism among the mutants and control. 2. materials and methods plant material and in vitro mutagenesis nodal segments (2-3 cm) of chrysanthemum grandiflora (tzelev) cv. snow ball, collected from one107 year-old mother plants maintained in the glasshouse of the department of biotechnology, university of horticulture and forestry solan (himachal pradesh), india, were used as explants. the explants were surface sterilized with 5% sodium hypochlorite (naocl2) for 10min, washed thoroughly three to four times with sterilized water and cultured on ms (murashige and skoog, 1962) medium supplemented with 2 mg/l benzyladenine (ba), 0.5 mg/l α-naphthalene acetic acid (naa), 30 g/l sucrose (w/v) and 8 g/l agar (w/v; sigmaaldrich, bangalore). the culture without growth regulators served as control. the ph of the medium was adjusted to 5.8 before autoclaving at 121oc at a pressure of 1.1 kg cm-2 for 15 min. the cultures were maintained under 16-hr photoperiod with a light intensity of 50-60 µmol cm-2 s-1 provided by cool, white fluorescent lamps at 24±2oc. after four weeks of culture, the shoots were subcultured on shoot multiplication medium consisting of ms salts supplemented with 0.5 mg/l ba, 0.1 mg/l indole acetic acid (iaa) and 1 mg/l gibberellic acid (ga3). shoots about 2.5-3.0 cm long wereirradiated in a gamma cell (60 cobalt source) with 5, 10, 20 and 30 gy doses. after four weeks, the shoots were multiplied and 3-4 cm long shoots were transferred to 1/2 strength ms medium supplemented with 0.3 mg/l indolebutyric acid (iba) and 0.2% activated charcoal for rooting. the shoots with well developed roots were removed from the culture vessel, washed with running tap water to remove the adhering agar and transferred to plastic pots (25 cm diameter) filled with soil:sand:fym in 1:1:1 ratio and kept in the glasshouse with 80-90% relative humidity. after acclimatization for four weeks, the plants were observed for any variations in morphological characters from the control and pcr analysis was carried out using the genomic dna. statistical analysis the data recorded for different parameters were subjected to completely randomized design (gomez and gomez, 1984). the statistical analysis based on mean value per treatment was made using analysis of variance (anova). the lsd multiple range test (p ≤ 0.05) was used to determine differences between treatments. polymerase chain reaction genomic dna was isolated from fresh, young green leaves of the control and gamma ray-irradiated plants following the method of doyle and doyle (1987) with some modifications. for amplification, reaction mixtures (21.8 µl) contained 1 µl oligonucleotide, 2.5 µl 10x taq polymerase buffer, 1.5 nm mgcl2, 2 µl each of dntps, 3 µl genomic dna and 3 µl taq polymerase (3 units/µl). random oligonucleotide primers were used for rapd amplification (bangalore genei, bangalore, india). amplification was performed in a thermocycler (mj research, usa) programmed for a first denaturation step of 3 min at 94oc followed by 36 cycles of 94oc for 30 s for denaturation, 50oc for 30 s for annealing, and 72oc for 2 min (extension). twenty operon random primers (operon technologies, inc., usa), ten of the opa series (1-10) and ten of the opb series (1-10), were employed for amplification using the cycling condition mentioned above. all samples were given 10 min at 73oc for postamplification. pcr products were separated on a 1.4% agarose gel using 1 x tae buffer and were stained with ethidium bromide. in all amplification reactions, a reaction mixture with water instead of genomic dna was used as negative control. 1 kb dna ladder (fermantas, lithuania) was used as the size marker. the amplified products were visualized using a uv transilluminator and photographed using gel documentation system (alpha imager, usa). amplified dna was scored as either presence (1) or absence (0) of band. pairwise comparison between the control and variants was performed to calculate similarity (j) between the samples (jaccard, 1908) using simqual programme of ntsys-pc (version 2). a dendrogram was produced from the resulting similarity using upgma method. 3. results effect of gamma radiation on in vitro cultures the data presented in table 1 reveal that the survival of shoots was affected by gamma ray doses. a decrease in survival was observed with an increase of irradiation dose; a significantly higher survival percentage was observed in the control shoots. the variation in survival percentage between 5 and 10 gy was statistically insignificant. a decrease was also observed on shoots forming roots. a lethal effect of higher doses (20 and 30 gy) was noticed on rooting of shoots. the shoots did not root, hence they turned yellowish brown and died. no variation was observed in shoots forming roots with 5 and 10 gy irradiation. a significant difference in percent root initiation was observed between the control and irradiated shoots. the rooted plants were acclimatized as explained above and allowed to grow in the glasshouse till flowering. table 1 effect of radiation doses on survival of in vitro shoots in d. grandiflora dose strength survival(%) number of shoots producing roots (%) 0 5 10 20 30 lsd0.05 90.47 (79.93) 66.60 (59.25) 52.38 (46.39) 42.85 (40.83) 19.05 (20.15) 25.11 (21.89) 100.00 (90.90) 82.60 (65.48) 82.35 (65.40) 0(0) 0(0) 6.25 (4.88) figures within parentheses are arc sine transformed values. 108 effect of gamma radiation on morphological characters morphological variations were not observed in the acclimatized plants exposed to 5 gy gamma radiation. all the plants flowered true to the mother floret colour/shape. therefore, selections were made among the plants exposed to 10 gy gamma radiation for agronomic and molecular characterization of mutants. at flowering stage, plants in the glasshouse were observed for any variations in morphological characters. results revealed that there were 10 variants with different morphological characters compared to the control. the data presented in table 2 reveal that morphological characters of d. grandiflorum cv. snow ball and its mutant were statistically, highly significantly affected by gamma ray doses. plant height plant height was significantly reduced in the variants compared to control (table 2). a significant variation in plant height was also observed among the variants (table 3). variant v10 gave the significantly highest value for plant height (69.00 cm), compared with the control (62.10 cm). variant v8 showed the lowest value for plant height, followed by variant v2. the plant height was significantly lower in most of the variants as compared to the control. leaf number and leaf area (cm2) the number of leaves was significantly reduced in the variants compared to control (table 2). there was a significant increase in the number of leaves in variant v4. in general, the number of leaves in the variants was lower compared to the control. variant v2 had the fewest number of leaves, followed by variants v7 and v8. statistically leaf area did not differ between the variants and control (table 2), however leaf area differed among the variants (table 3). variant v7 had significantly higher mean values of leaf area, followed by variants v10, v2 and v9 in comparison to the control. number of flower buds and flowers table 2 reports that the number of flower buds was significantly different between the variants, whereas the difference in the number of flowers was not significant. the number of flower buds and flowers varied among the variants (table 3) with variants v3 and v8 having a significantly lower number of flower buds and flowers, followed by variant v7. it was observed that in most of the variants about 82-83% of buds opened into flowers while in variant v3 only 50% of the buds opened into flowers. flower diameter and flower colour the variation in flower diameter between the control and the variants was statistically not significant (tables 2 and 3). the original flower colour of the cv. snow ball was white with incurve ray florets (fig. 1a), whereas one branch of variant v9 produced yellowcoloured flowers with incurve ray florets (fig. 1b). the results indicate that the irradiation dose of 10 gy was an effective dose in inducing mutations in flower colour, but no changes were observed in flower shape and size. table 2 analysis of variance of the morphological characters of control and variants of chrysanthemum treatment plant height (cm) number of leaves leaf area (cm2) number of buds number of flower flowers diameter (cm2) control variants lsd0.05 62.10 54.80 2.97 38.70 31.60 4.18 8.60 10.93 2.35 15.10 11.70 4.05 11.40 8.10 3.44 6.60 6.50 1.58 table 3 effect of gamma radiation (10 gy) on morphological characters in d. grandiflora treatment plant height (cm) number of leaves leaf area (cm2) number of buds number of flower diameter flowers (cm2) control v1 v2 v3 v4 v5 v6 v7 v8 v9 v10 62.10 56.00 40.00 60.00 58.00 60.00 55.00 58.00 37.00 55.00 69.00 38.70 23.00 16.00 40.00 49.00 38.00 35.00 19.00 19.00 40.00 37.00 8.61 7.39 13.00 8.48 7.87 10.85 10.48 15.16 9.32 12.56 14.15 15.10 15.00 5.00 14.00 16.00 14.00 10.00 7.00 5.00 13.00 18.00 11.40 10.00 4.00 7.00 9.00 11.00 7.00 5.00 3.00 10.00 15.00 6.60 5.50 6.00 8.00 5.50 6.60 7.30 6.00 7.50 6.80 6.60 109 characterization twenty decamer primers (table 4) were used for the amplification of genomic dna of control and mutants of dendranthema grandiflora snow ball. the number of dna fragments amplified ranged from one to five depending upon the primer and the dna sample with a mean value of 2.90 bands per primer (table 4). the amplification product ranged from 250 to 3000 bp. a total number of 58 markers were produced by the 20 primers. a total of 100% of the 58 scored bands were polymorphic in 11 genotypes (one control and its ten fig. 1 induced somatic mutations in chrysanthemum cv. snow ball after 10 gy gamma radiation treatment. (a) control, whitecoloured flowers; (b) mutated, yellow-coloured flower. table 4 nucleotide sequences and rapd amplification results of the primer used in the pcr amplification primer sequence (5’-3’) scored bands polymorphic bands polymorphism (%) opa-1 opa-2 opa-3 opa-4 opa-5 opa-6 opa-7 opa-8 opa-9 opa-10 opb-1 opb-2 opb-3 opb-4 opb-5 opb-6 opb-7 opb-8 opb-9 opb-10 total gtttcgctcc tgatccctgg catccccctg ggactggagt tgcgcccttc tgctctgccc ggtgacgcag gtccacacgg tgggggactc ctgctgggac caggcccttc tgccgagctg agtcagccac aatcgggctg aggggtcttg ggtccctgac gaaacgggtg gtgacgtagg gggtaacgcc gtgatcgcag 3 4 3 1 4 5 4 4 0 2 4 1 1 3 2 2 4 3 3 5 58 3 4 3 1 4 5 4 4 0 2 4 1 1 3 2 2 4 3 3 5 100 100 100 100 100 100 100 100 0 100 100 100 100 100 100 100 100 100 100 100 58 fig. 2 rapd profile with primer opb-4 showing polymorphism in control and its variants in chrysanthemum cv. snow ball. lanes 110: variants (v1 to v10); lane 11: control; lane 12: dna ladder (1kb). variants). figure 2 shows the amplification profiles, generated by primer opb-4 across the chrysanthemum genotypes. the rapd markers produced by the 20 primers were used to construct a similarity matrix (table 5). five clusters can be observed. the first cluster includes only table 5 jaccard’s similarity matrix in control (c) and mutated (v) plants based on rapd analysis c v1 v2 v3 v4 v5 v6 v7 v8 v9 v10 c 1.00 0.32 0.31 0.31 0.28 0.37 0.20 0.29 0.20 0.06 0.28 v1 v2 v3 v4 v5 v6 v7 v8 v9 v10 1.00 0.79 0.67 0.76 0.37 0.30 0.65 0.50 0.27 0.62 1.00 0.54 0.65 0.37 0.26 0.68 0.54 0.26 0.61 1.00 0.67 0.37 0.32 0.53 0.37 0.28 0.44 1.00 0.42 0.32 0.72 0.50 0.29 0.51 1.00 0.37 0.40 0.26 0.24 0.31 1.00 0.45 0.37 0.26 0.32 1.00 0.62 0.33 0.62 1.00 0.25 0.59 1.00 0.35 1.00 110 yellow-coloured flowers (group 5c, fan 1) (british colour council, 1938). in order to check the stability of flower colour, the mutated branch was propagated vegetatively. the plants were grown in a glasshouse, where they expressed the same colour/shape. over recent years, rapd analysis has been used in ornamental breeding to characterize genotypes and to identify genes controlling important traits (huang et al., 2000; rumińska et al., 2004; kumar et al., 2006; miñano et al., 2009). in the present study, a high level of polymorphism was observed in 10 radiomutants of chrysanthemum. wolff and van rijn (1993) also noticed a high degree of polymorphism in chrysanthemum cultivars using rapd markers. this high level of polymorphism may be due to the strict out-crossing, resulting in a higher level of heterozygosity in chrysanthemum (wolff et al., 1994). rapd markers were used to construct a similarity matrix and the results of characterization analysis revealed a high diversity between the control and its somaclones. the greatest genetic distance between control and variant 9 (v9) may be due to the fact that v9 is a somaclone where one branch was mutated and produced yellow-coloured flower heads. although the plants differed in flower colour, bands specific for flower colour could not be distinguished due to the resolution capacity of the tested primers. wolff (1996) reported that the choice of the primers may be an important factor in obtaining a rapid discrimination between samples. barakat et al. (2010) reported that mutants with different flower colour could be identified at the molecular level using rapd technique, holding promise to identify unique genes as scar markers. bhattacharya and teixeira da silva (2006) attempted to understand the molecular systematic and genetic difference between 10 original chrysanthemum cultivars and 11 mutants and reported that similarity ranged from 0.17 to 0.90 using rapid analysis. kumar et al. (2006) reported genetic distances from 0.43 to 0.96 between 13 chrysanthemum cultivars. a great genetic distance among the different cultivars showed the existence of introgressing new and novel genes from the chrysanthemum gene pool. it may be suggested, with regard to the present study, that by using rapd markers, it is possible to differentiate newly evolved chrysanthemum cultivars from their parents which can be a useful tool to supplement the distinctness, uniformity and stability analysis for plant variety protection in future. our results are in accordance with those of other workers who reported rapd as a powerful tool in the assessment of genetic variability as well as for genetic characterization, allowing differentiation of chrysanthemum mutants/cultivars (huang et al., 2000; rumińska et al., 2004; kumar et al., 2006; chatterjee et al., 2006; miñano et. al., 2009). therefore, it may be concluded that 10 gy gamma irradiation was most effective in inducing variations in flower colour and other morphological characters. morphological variations were not observed with the 5 fig. 3 dendrogram of control and mutants of chrysanthemum cv. snow ball variant 9 (v9); the second includes the control (c); the third includes variants 5 and 6 (v5 and v6); the fourth includes variants 8 and 10 (v8 and v10), and the last cluster includes variants 3, 4, 7, 2 and 1 (v3, v4, v7, v2, v1). it can be seen from figure 3 that the shortest genetic distance was found between variant 1 (v1) and variant 2 (v2), whereas the greatest distance was observed between the control and variant 9 (v9). 4. discussion and conclusions analysis of explant sensitivity is one of the basic requirements for an effective use of mutation induction in plant breeding programmes (walther and sauer, 1986). in the present study, the radio sensitivity of shoots was assessed by survival and rooting of shoots after irradiation in order to select a suitable dose of gamma irradiation. predieri (2001) also reported the necessity to identify an appropriate dose to apply in mutagenic treatments. the effect of gamma radiation on in vitro cultures in crop breeding has been studied by many workers (shen et al., 1990; charbaj and nabul, 1999; predieri and gatti, 2000; datta et al., 2005; barakat et al., 2010). reductions in survival, plant height, number of leaves and flowers were recorded after gamma irradiation. mutation in flower colour was detected as chimera in one branch of the plant, which produced 111 gy gamma dose and a lethal effect of 20 and 30 gy doses was observed on rooting and survival of in vitro shoots. one hundred percent polymorphism was observed among the radiomutants using pcr technique. the present results also indicate the applicability of gamma radiation in crop breeding, and the assessment of genetic variability and characterization of radiomutants at genomic level by rapd can be a useful tool in breeding programs aimed to improve ornamental characters of chrysanthemum cultivars. references british colour council, 1938 horticultural colour chart i and ii. the british colour council, london, pp. 1-200. barakat m.n., abdel-fattah r.s., badr m., eltorky g., 2010 “in vitro” mutagenesis and identification of new variants via rapd markers for improving chrysanthemum morifolium. african j. agric. res., 5: 748-757. bhattacharya a., teixeira da silva j.a., 2006 molecular systematics in chrysanthemum x grandiflorum (ramat) kitamura. sci. hort., 109: 379-385. broetjes c., van harten a.m., 1978 application of mutation breeding methods in the improvement of vegetatively propagated crops. an interpretative literature review. elsevier, amsterdam, the netherlands, pp. 324. charbaj i., nabul s., 1999 mutational analysis of racial differentiation in oryza sativa. curr. sci., 32: 451-453. chatterjee j., mandal a.k.a., ranade s.a., teixeira da silva j.a., datta s.k., 2006 molecular systematics in chrysanthemum x grandiflorum (ramat) kitamura. sci. hort., 119: 373-378. datta s.k., mishra p., mandal a.k.a., 2005 “in vitro” mutagenesis a quick method for establishment of solid mutant in chrysanthemum. curr. sci., 88: 155-158. doyle j.j., doyle j.l., 1987 a rapid dna isolation procedure for small amounts of fresh leaf tissue. phytochem. bull., 19: 11-15. gomez k.a., gomez a.a., 1984 statistical procedure for agricultural research. john wiley and sons, new york, usa. huang s.c., tsai c.c., sheu c.s., 2000 genetic analysis of chrysanthemum hybrids based on rapd molecular markers. bot. bull. acad. sin., 41: 257-262. jaccard p., 1908 nouvelles recherches sur la distribution florale. bull. soc. vaud. sci. natl., 44: 223-270. jain s.m., 2010 mutagenesis in crop improvement under the climate change. romanian biotechnol. lett., 15: 88-106. jain s.m., spencer m.m., 2006 biotechnology and mutagenesis in improving ornamental plants, pp. 589-600. in: teixeira da silva j.a. (ed.) floriculture, ornamental and plant biotechnology: advances and topical issues, vol. i. global science books, isleworth, uk, pp. 2481. kumar s., prasad k.v., choudhary m.l., 2006 detection of genetic variability among chrysanthemum radiomutants using rapd markers. curr. sci., 90: 1108-1113. martin c., gonzález-benito m.e., 2005 survival and genetic stability of dendranthema grandiflora tzvelev shoot apices after cryopreservation by vitrification and encapsulation-dehydration. cryobiology, 51: 281-289. martin c., uberhuaga s., perez c., 2002 application of rapid markers in the characterization of chrysanthemum varieties and assessment of somaclonal variation. euphytica, 127: 247-253. miñano h.s., gonzález-benito m.e., martin c., 2009 molecular characterization and analysis of somaclonal variation in chrysanthemum cultivars using rapd markers. sci. hort., 122: 238-243. murashige t., skoog f., 1962 revised medium for the rapid growth and bioassay with tobacco tissue culture. physiol. plant., 15: 473-479. predieri s., 2001 “in vitro” propagation and breeding of ornamental plants: advantages and disadvantages of variability. euphytica, 115: 12-14. predieri s., gatti j., 2000 pear mutagenesis: “in vitro” treatment with gamma-rays and field selection for vegetative form trials. euphytica, 114: 79-90. richard a.j., 1986 plant breeding systems. george allen and unwin, london, uk. rumińska j.l., zalewska m., sadoch z., 2004 radiomutants of chrysanthemum (dendranthema grandiflora tzvelev) of the lady group rapd analysis of the genetic diversity. plant breeding, 123: 290-293. schum a.r., 2003 mutation breeding in ornamentals: an efficient breeding method? acta horticulturae, 612: 47-60. shen t., li w., dai c., chen j.y., 1990 the induction of sports in chrysanthemum by gamma radiation. theor. appl. genet., 88: 129-136. walther f., sauer a., 1986 “in vitro” mutagenesis in roses. acta horticulturae, 189: 37-46. william g.k., kubelik a.r., livak k.l., rafalski j.a., tingey s.v., 1990 dna polymorphisms amplified by arbitrary primers are useful as genetic markers. nucleic acid res., 18: 159-164. wolff k., 1996 rapd analysis of sporting and chimerism in chrysanthemum. euphytica, 88: 159-164. wolff k., van peter r.j., hofstra h., 1994 rflp analysis in chrysanthemum l. probe and primer development. theor. appl. genet., 88: 472-478. wolff k., van rijn p.j., 1993 rapid detection of genetic variability in chrysanthemum (dendranthema grandiflora tzvelev) using random primers. heredity, 71: 336-341. zalewska m., rumińska l.j., miller n., 2007 “in vitro” propagation using adventitious bud techniques as a source of new variability in chrysanthemum. sci. hort., 113: 70-73. 275 adv. hort. sci., 2022 36(4): 275­281 doi: 10.36253/ahsc­12816 combining humic acid with npk fertilizer improved growth and yield of chili pepper in dry season b. ichwan, e. eliyanti, i. irianto, z. zulkarnain (*) department of agroecotechnology, faculty of agriculture, university of jambi, indonesia. key words: capsicum annuum, organic substance, soil improvement, sustainable agriculture. abstract: this research aimed to study the effect of humic acid and npk fertiliz­ er (15:15:15) on growth and yield of red chili, and to obtain the most suitable composition of humic acid and npk fertilizer which gave the best growth and yield. the study used a randomized block design with five replications. the treatments tested were the composition of humic acid and npk fertilizer: 100% humic acid; 75% humic acid + 25% npk; 50% humic acid + 50% npk; 25% humic acid + 75% npk; and 100% npk. data on plant growth and yield were processed by analysis of variance, and means were compared using fisher’s least significant difference test. in addition, data on plant biochemical and soil chemical parameters were determined compositely by mixing leaves taken from sample plants or soil samples into one homogenous sample. results showed that there was no significant difference in growth and yield of plants treated with 100% humic acid in comparison with those plants treated with 100% npk. however, in comparison with 100% humic acid, the application of different ratios of humic acid/npk increased plant chlorophyll contents by 65% ­ 82% and total sugar by 28% ­ 71%. the application of humic acid/npk increased soil fertility by improving soil ph as well as n, p and k. in the combi­ nation of humic acid/npk, the best growth and yield were obtained with the application of 25% humic acid + 75% npk fertilizers. therefore, for the sustain­ ability of chili cultivation, the use of humic acid needs to be accompanied with npk fertilizers at a reduced amount, along with the increase in the dose of humic acid. 1. introduction in indonesia chili (capsicum annuum l.) is one of vegetable crops with high economic value due to its large domestic and export demaind and, from time to time, price fluctuations may cause consumer unrest. the national inflation rate is significantly influenced by the increase in chili prices in certain seasons. to control the price fluctuations the indonesian government is increasing the planting area in rainy season, and control­ ling the planting area as well as production during dry season. (*) corresponding author: dr.zulkarnain@yahoo.com citation: ichwan b., eliyanti e., irianto i., zulkarnain z., 2022 ­ combining humic acid with npk fertili‐ zer improved growth and yield of chili pepper in dry season. ­ adv. hort. sci., 36(4): 275­281. copyright: © 2022 ichwan b., eliyanti e., irianto i., zulkarnain z. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 16 february 2022 accepted for publication 3 october 2022 ahs advances in horticultural science adv. hort. sci., 2022 36(4): 275­281 276 increasing production in the dry season normally is managed by application of inorganic fertilizers and use of chemicals to control pests and diseases. however, this practice has a negative impact on agri­ c u l t u r a l e c o s y s t e m a n d t h e e n v i r o n m e n t . additionally, implementing principles of sustainable agriculture such as the application of humic acid, a soil conditioner, has the potential to improve this conventional agricultural practice. humic acid has a complex molecular structure and capability to stimu­ late and activate biological and physiological process­ es in soil organisms. the study of abdellatif et al. (2017) showed that the application of 14.4 kg ha­1 humic acid to chili plants grown under heat stress could increase plant’s average height, number of flower buds, number of flowers, number of fruits per plant, as well as fruit weight. the application of 7.5­12 ml of humic acid into 10 kg of sterile soil increased the population of soil microorganisms azotobacter beijerinckii and aspergillus niger. foliar spray of 50 ml l­1 humic acid significantly increased leaf area, yield and total solu­ ble solids in grapes (popescu and popescu, 2018). onion plants sprayed with 1000 mg l­1 humic acid produced the highest growth rate and yield (al­ fraihat et al., 2018). in addition to the improvement in cultivation, chili production can be increased by growing them during the dry season. whilst a lot of land is available for cropping during the dry season the production is con­ s t r a i n e d b y t h e l i m i t e d a v a i l a b i l i t y o f w a t e r . application of humic acid as a soil conditioner could remediate water shortage in plants due to increasing leaf water content, increasing photosynthesis, antioxidant metabolism and enzyme activity; thus improving plant tolerance to stress (al­shareef et al., 2018). previous studies have demonstrated that humic acid improved plant growth and yield in a number of plant species. hermanto et al. (2013) reported that application of 20 kg ha­1 humic acid along with 100% dose of npk fertilizer resulted in maximum growth and yield as well as increased the availability and uptake of n, p, k, zn and fe in maize. furthermore, moraditochaee (2012) showed that the application of humic acid at 40 mg l­1 and 75 kg ha­1 n produced the highest harvest index in peanut. sustainable production of plants can be achieved b y r e d u c i n g t h e u s e o f i n o r g a n i c f e r ti l i z e r s . application of humic acid could help achieve this important goal. whilst humic acid has been widely applied to various plant species; the search of litera­ ture showed that no research has focused on the effect of humic acid in combination with npk fertiliz­ er on growth and yield of chili pepper. therefore, this study aimed at assessing the effect of humic acid in combination with npk fertilizer on growth and yield of red chili, and to obtain the most suitable ratio of humic acid/npk for best growth and yield in chili plants grown in dry season in indonesia. 2. materials and methods the trial was conducted at the teaching and research farm within the faculty of agriculture at the university of jambi in indonesia during the 2020 dry season. the study used commercially available humic acid (ah­90, with 90% humic acid content) and inorganic npk fertilizer (15:15:15). there were 5 combinations tested: 100% humic acid; 75% humic acid + 25% npk; 50% humic acid + 50% npk; 25% humic acid + 75% npk and 100% npk. a randomized block design with 5 replications was used in this study. humic acid was applied at the recommended dose (5 kg ha­1) and so was npk fertilizer (700 kg ha­1). the humic acid and npk were applied one week before planting in between rows. seeds were sown on a standard seedling medium, then transferred to nursery at 7 days after sowing and left in the nursery for 21 days. the medium used during the nursery growth was a mixture of soil, com­ post, and sand (2:1:1). seedlings were sprayed every 7 days with foliar fertilizer bayfolan­d at 2 ml l­1 and i n s e c ti c i d e ( a c ti v e i n g r e d i e n t a m a b e k ti n a n d mesurol) at 2 ml l­1. the one­month­old seedlings were transplanted in the field onto the growing media supplemented with humic acid and npk fertilizer at different rates as per treatments described above. a silvercoated black plastic was placed on the beds before trans­ planting the seedlings. prior to planting, a fungicide (carbofuran) was applied into planting holes at 1.5 g per plant. the planting space was 50 cm x 50 cm. a liquid npk fertilizer (15:15:15) was applied at a con­ centration of 2 g l­1 with a volume of 100 ml per plant at 6, 8 and 10 weeks after transplanting. harvesting was carried out during the first flower­ ing period (10 to 13 weeks after transplanting), with t h e c r i t e r i o n t h a t 8 0 % o f f r u i t s b e c o m i n g r e d . observation of vegetative growth was carried out during fruit enlargement period (9 weeks after trans­ ichwan et al. ‐ humic acid and npk fertilizers improved growth and yield of chili pepper 277 planting) on the following parameters: plant height, number of productive branches, leaf area, and plant total dry weight. the leaf area was measured using gravimetric method with the following formulae (sitompul and guritno, 1995): leaf area (cm2) = (x/y)·z where x = total leaf dry weight, y = sub sample leaf dry weight, and z = sub sample leaf area. observation of plant biochemical measurements were made on chlorophyll content, total sugar, and relative leaf water content (rlwc). composite leaf samples were made by physically mixing individual leaves taken from 3 sample plants of 3 replicates into one homogenous sample. compositing reduced the n u m b e r o f a n a l y s e s t o b e p e r f o r m e d a n d w a s designed to provide a representative sample of the treatment. ten youngest mature leaves on main stem were collected at 9 weeks after transplanting (wat). clean and dry leaf samples were placed in a sample bag prior to laboratory analyses. chlorophyll analysis was carried out using acetone method (arnon, 1949), and total sugar was analyzed using anthrone method (yoshida et al., 1976). relative leaf water content (rlwc) was determined according to gonzález and gonzález­vilar (2001) method. data on yields in terms of number of the fruits and fruit weight per plant were collected at harvest time. in addition, soil chemical analyses were per­ formed before transplanting and after the last har­ vest. soil analyses included ph, moisture content, organic c based on walkley­black method (walkley and black, 1934), total n using kjeldahl method (kjeldahl, 1883), available p applying bray­i method (bray and kurtz, 1945) and exchangeable k (nh4oac 1n extraction at ph 7) using method as described in (warncke and brown, 1998). the data were scrutinized employing an analysis of variance (anova) from microsoft excel application (version 16.63.1, 2022, microsoft corporation, usa). fisher’s least significant difference (flsd) at 5% level of probability was used to compare means of treat­ ments. 3. results analysis of variance showed that the application of humic acid (ha) and npk fertilizer did not signifi­ cantly affect plant height, number of branches, total leaf area, and dry weight. however, application of 25% ha in conjunction with 75% npk fertilizer result­ ed in enhanced growth compared to other treat­ ments (fig. 1). in addition, the application of 25% ha + 75% npk improved growth compared with 100% ha application whereby plant height, number of branches, leaf area and plant dry matter increased by 29.46%, 21.96%, 23.59% and 28.42% respectively. the measurement of biochemical parameters showed that the application of 100% ha resulted in the lowest chlorophyll content (2.40 mg l­1) and total sugar (0.07%). furthermore, the lowest relative leaf water content (rlwc) was found on 100% npk appli­ cation (table 1). fig. 1 ­ growth of chili on various ratios of humic acid (ha)/npk at 9 weeks after transplantation (a = plant height, b = number of branches, c = leaf area, d = total dry weight). error bars indicate the standard error (n=5). humic acid/npk chlorophyll content (mg·l­1) total sugar (%) rlwc (%) 100% ha 2.40 0.07 92.6 75% ha/25% npk 3.97 0.12 93.3 50% ha/50% npk 4.15 0.12 93.7 25% ha/75% npk 4.37 0.09 93.0 100% npk 4.30 0.11 91.3 table 1 ­ effect of different ratios of humic acid) ha / npk on chlorophyll content (*), total sugar, and relative leaf water content (rlwc) at 9 weeks after transplantation (*) chlorophyll content and total sugar were determined compo­ sitely by physically mixing individual leaves taken from each 3 sample plants of 3 replicates into one homogenous sample. adv. hort. sci., 2022 36(4): 275­281 278 analysis of variance showed that the application of different ratios of ha/npk had no significant effect o n t h e n u m b e r a n d w e i g h t o f f r u i t s p e r p l a n t . however, among the combinations tested the 25% ha + 75% npk resulted in the highest number of fruits, and the 50% ha + 50% npk produced the largest fruit weight (fig. 2). the results of soil chemical analyses (table 2) showed that the application of various ratios of ha/npk had altered soil chemical properties such as ph, organic c, total n, p, k, and soil water content. generally, the application of ha along with npk fer­ tilizer improved n, p and k content. whereas soil ph and organic carbon are highest when 100% ha is applied (table 2). 4. discussion and conclusions the application of different humic acid/npk ratios had no significant effect on growth and yield of chili as measured by plant height, total leaf area, number of branches and total dry weight, as well as number of fruits and average fruit weight. this means that humic acid can substitute npk fertilizer. however, when compared to the use of only humic acid or npk fertilizer, the application of different ratios of humic acid/npk improved plant height, total leaf area, num­ ber of branches, total dry weight, fruit numbers and fruit weight. npk is widely used to improve plant growth and yield of many horticultural crops (nafiu et al., 2011; agbede et al., 2017; achiri et al., 2018; adekiya et al., 2019; nwokwu et al., 2020). however, the use of inorganic fertilizers such as npk to help increase plant growth and yield need to be carefully consid­ ered, due to their detrimental impact on soil struc­ ture and soil biota. according to adekiya et al. (2019) npk fertilizer did not increase soil organic matter, nor reduced soil bulk density. krestini et al. (2020) claimed that npk fertilizer did not provide carbon compounds that contributed to the improvement of soil physical and biological properties. in addition, agbede et al. (2017) reported that continued applica­ tion of inorganic fertilizers can increase soil acidity and soil degradation because inorganic fertilizers release nutrients more quickly. therefore, balanced fertilization is recommended to increase the efficien­ cy of fertilizer use and help improve physical, chemi­ cal, and biological properties of soil. the results obtained here show that application of 100% humic acid provided less growth and yield in fig. 2 ­ effect of different ratios of humic acid (ha)/npk on num­ ber of fruits (a) and fruit weight (b). error bars indicate the standard error (n=5). humic acid/npk ph c­organic (%) n­total (%) p (ppm) k (cmol+/kg) water content (%) 100% ha 6.09 1.55 0.22 104.50 0.06 7.70 75% ha/25% npk 6.02 1.45 0.29 124.80 0.19 7.56 50% ha/50% npk 5.47 1.28 0.28 152.30 0.47 7.61 25% ha/75% npk 5.52 1.20 0.24 144.70 0.62 7.67 100% npk 5.32 1.22 0.23 227.30 1.07 7.50 table 2 ­ the effect of different ratios of humic acid (ha)/npk on soil chemical properties ichwan et al. ‐ humic acid and npk fertilizers improved growth and yield of chili pepper 279 c h i l i p l a n t s c o m p a r e d w i t h m i x t u r e o f h u m i c acid/npk during the one growth season of this research. however in a longitudinal experiment on peanut li et al. (2019) demonstrated that significant increase in n, p and k content of soil was found after three years continuous application of humic acid. the reason for this increase is due to humic com­ pounds high stability rate, and therefore the mineral­ ization process can occur very slowly. as a conse­ quence, humic acid cannot be used as a source of plant nutrition (canellas et al., 2015). in research reported here the chemical analyses indicated soil that was treated with 100% humic acid was lower in n, p and k content, but higher in organic carbon and soil moisture content in comparison with those treat­ ed with different humic acid/npk ratios (table 2). this was in accordance with (li et al., 2019; 2020) findings that the application of humic acid­based fer­ tilizer could increase soil nutrient contents, including the total soil nitrogen, phosphorus, and potassium. increase in soil water content was aligned with increase in relative leaf water content (rlwc) in chili plants (table 1). al­shareef et al. (2018) reported that the application of humic acid as a soil enhancer could overcome water shortage in plants by increas­ ing rlwc, thereby increasing plant tolerance to stress. further, xu et al. (2015) stated that the appli­ cation of soil enhancers such as humic acid could increase plant growth by retaining available water and nutrients, therefore reducing the impact of drought stress and nutrient loss. the application of 100% humic acid also increased soil ph from 4.43 to 6.09, resulting in better plant growth and development due to improved availabili­ ty of various nutrients in the soil at ph of 6.09. wahyudi (2007) reported that the application of humic acid might reduce the amount of exchange­ able aluminum and increase chelating aluminum in soil, thereby increasing soil ph. hasanudin (2003) suggested that the reduction in exchangeable alu­ minum was due to the formation of complex organo­ metal compound (al­chelate). increase in soil ph will increase the availability of soil nutrients needed by plants (gardner et al., 1985). in peanut, li et al. (2019) found that compared with control, humic acid significantly increased the yield and quality of peanut in continuous cropping. their results showed that application of humid acid significantly increased the soil total nitrogen, total phosphorus, total potassium, available n, available p, available k, and organic mat­ ter from second year, with maximum effect displayed in third year. chili plants treated with different humic acid/npk ratios showed better growth and yields in compari­ son to those plants treated with humic acid or npk alone. the composition of 25% humic acid + 75% npk fertilizer produced the highest growth (plant height, leaf area, number of branches and plant dry matter), as well as the highest yield (number of fruits) com­ pared to other compositions. furthermore, applica­ tion of 50% humic acid + 50% npk fertilizer resulted in the highest fruit weight. this result is in line with the increase in plant chlorophyll content, where the composition of 25% humic acid + 75% npk fertilizer caused higher plant chlorophyll content. an increase in chlorophyll content intensifies the ability of plants to carry out photosynthesis, thereby increasing plant growth (ferrara and brunetti, 2010). increase in soil k content due to humic acid / npk application caused better k uptake, increasing plant g r o w t h a n d y i e l d i n p o t a t o ( x u e t a l . , 2 0 1 5 ) . potassium is crucial in plant photosynthetic activity (marschner, 2012) and assimilate translocation effi­ ciency (sawan, 2018). this is in line with our results where application of humic acid and npk (75% ha and 25% npk or 50% ha and 50% npk) increased sugar content in plant tissue (table 1). the composition of 50% humic acid + 50% npk and 25% humic acid + 75% npk in this study provided better growth and yield of chili plants than 100% humic acid or 100% npk applications. this indicates a positive effect of the use of humic acid in partially replacing the role of npk fertilizer. the ability of humic acid to replace npk fertilizer is probably due to the ability of humic acid to modify the plant root sys­ tem architecture as well as improving the capacity of soil to bind water, thus enabling plants to acquire water and nutrients more easily (suwahyono, 2011; canellas and olivares, 2014), reducing plant stress in dry land cultivation during periods of drought (xu et al., 2015). the analysis of soil water content for different ratios of humic acid/npk showed medium treated with humic acid has a better water content than those treated with 100% npk (7.56­7.7 versus 7.5). in addition, the relative leaf water content (rlwc) at various ratios of humic acid / npk was higher than that of 100% npk application (table 1). our findings are consistent with a number of pre­ vious studies indicating that the application of humic acid needs to be accompanied by npk fertilizer to help improve plant productivity (for example in properties and growth and yield of carrot. ­ j. hortic. res., 25(1): 81­88. al­fraihat a.h., al­tabbal j.a., abu­darwish m.s., alhrout h.h., hasan h.s., 2018 ­ response of onion (allium cepa) to foliar application of humic acid under rain‐fed conditions. ­ int. j. agric. biol., 20(5): 1235­ 1241. al­shareef a.r., el­nakhlawy f.s., ismail s.m., 2018 ­ enhanced mungbean and water productivity under full irrigation and stress using humic acid in arid regions. ­ legume res., 41(3): 428­431. arnon d.i., 1949 ­ copper enzymes in isolated chloroplas‐ t s . p o l y p h e n o l o x i d a s e s i n b e t a v u l g a r i s . ­ p l a n t physiology, 24(1): 1­15. bray r.h., kurtz l.t., 1945 ­ determination of total, organic, and available forms of phosphorus in soils. ­ soil sci., 59(1): 39­46. c a n e l l a s l . p . , o l i v a r e s f . l . , 2 0 1 4 ­ p h y s i o l o g i c a l responses to humic substances as plant growth pro‐ moter. ­ chem. biol. technol. agric., 1(3): 1­11. canellas l.p., olivares f.l., aguiar n.o., jones d.l., nebbioso a., mazzei p., piccolo a., 2015 ­ humic and fulvic acids as biostimulants in horticulture. ­ sci. hortic., 196: 15­27. ferrara g., brunetti g., 2010 ­ effects of the times of application of a soil humic acid on berry quality of table grape (vitis vinifera l.) cv italia. ­ span. j. agric. res., 8(3): 817­822. gardner f.p., pearce b., mitchell r., 1985 ­ physiology of crop plants (1st edition). ­ iowa state university press, ames, ia, usa, pp. 327. gonzález l., gonzález­vilar m., 2001 ­ determination of relative water content, pp. 207­212. ­ in: roger m.j.r. (ed.) handbook of plant ecophysiology tech‐ niques. ­ kluwer academic publishers, the netherlands, pp. 468. hasanudin, 2003 ­ the increasing of nutrient availability and uptake of n and p and yield of corn through innoc‐ ulation of mycorrhiza, azotobacter, and organic matter on ultisol. ­ j. ilmu pert. indon., 5(2): 83­89. hermanto d., dharmayani n.k.t., kurnianingsih r., kamali s.r., 2013 ­ the influence of humic acid as fer‐ tilizer supplement to nutrient availability and uptake on maize plant in unirrigated land of kec. bayan‐ntb. ­ ilmu pertanian, 16(2): 28­41. kjeldahl j., 1883 ­ neue methode zur bestimmung des stickstoffs in organischen körpern. ­ zeitschrift für analytische chemie, 22(1): 366­383. krestini e.h., susilawati a., hermanto c., 2020 ­ effect of npk fertilizer and microbial consortium to growth and production of garlic (allium sativum l.), pp. 03010. ­ in: mursyidin d.h., and e. badruzsaufari (eds.) first international conference on tropical wetland biodiversity and conservation (icweb 2019). edp sciences, web of conferences, les ulis cedex, france, 1­2 november, 2019. 280 adv. hort. sci., 2022 36(4): 275­281 peanut (moraditochaee, 2012), tomato (sarno et al., 2015) and cocoa (santi, 2016)). the slow rate of humic acid mineralization needs to be followed by the application of npk fertilizers. this is aimed to increase the availability of nutrients for plants, espe­ cially in relation to the relatively short lifespan of the chili plant which is only 3 ­ 4 months. in addition, it is also necessary to consider the amount of humic acid and the time of application, so that the use of npk fertilizer as a source of plant nutrition can be further reduced. data presented here indicate that the application of different ratios of humic acid / npk could greatly increase the growth and yield of chili pepper grown in dry land cultivating, where the composition of 25% humic acid + 75% npk fertilizer resulted in improved plant growth and yield. it was found that the applica­ tion of humic acid could reduce the use of npk fertil­ izers by up to 50%. thus, for sustainable chili produc­ tion in dry land cultivation, application of humic acid in conjunction with npk fertilizer is recommended at a reduced amount. acknowledgements this research was supported by university of jambi through internal competitive research grant (contract no. 410/un.21.18/pg/spk/2020 dated 20 april 2020). the authors would like to thank the rector of university of jambi for financial support provided to conduct this research. references abdellatif i.m.y., abdel­ati y.y., abdel­mageed y.t., hassan m.a.­m.m., 2017 ­ effect of humic acid on growth and productivity of tomato plants under heat stress. ­ j. hortic. res., 25(2): 59­66. achiri t.d., michele t.z.s., konje c.n., njualem d.k., 2018 ­ effect of npk fertilizer and animal manure on some biometric parameters of irish potato (solanum tuberosum l.) in bougham, west region cameroon. ­ asian j. crop sci., 2(1): 1­10. adekiya a.o., agbede t.m., aboyeji c.m., dunsin o., ugbea j.o., 2019 ­ green manure and npk fertilizer on soil properties, growth, yield, mineral and vitamin c composition of okra (abelmoschus esculentus (l.) moench). ­ j. saudi soc. agric. sci., 18(2): 218­223. agbede t.m., olasekan a.a., eifediyi k., 2017 ­ impact of poultry manure and npk fertilizer on soil physical ichwan et al. ‐ humic acid and npk fertilizers improved growth and yield of chili pepper 281 li y., 2020 ­ research progress of humic acid fertilizer on the soil. ­ journal of physics: conference series, 1549: 022004. li y., fang f., wei j., wu x., cui r., li g., zheng f., tan d., 2019 ­ humic acid fertilizer improved soil properties and soil microbial diversity of continuous cropping peanut: a three‐year experiment. ­ sci. rep., 9: 12014. marschner p., 2012 ­ marschner’s mineral nutrition of higher plants. ­ elsevier, london, uk, pp. 651. moraditochaee m., 2012 ­ effects of humic acid foliar spraying and nitrogen fertilizer management on yield of peanut (arachis hypogaea l.) in iran. ­ arpn j. agric. biol. sci., 7(4): 289­293. nafiu a.k., togun a.o., olabiyi a.m., chude v.o., 2011 ­ effect of npk fertilizer on growth, drymatter produc‐ tion and yield of eggplant in southwestern nigeria. ­ agric. biol. j. n. am., 2: 1117­1125. nwokwu g.n., ogbonna l.c., egwu p.n., 2020 ­ effect of npk fertilizer rates on growth and yield components of eggplant (solanum melongena) cultivars. ­ niger. agric. j., 51: 530­537. popescu g.c., popescu m., 2018 ­ yield, berry quality and physiological response of grapevine to foliar humic acid application. ­ bragantia, 77(2): 273­282. santi l.p., 2016 ­ effect of humic acid on the growth of cocoa (theobroma cacao) seedlings and microbial pop‐ ulation in the humic dystrudept. ­ j. tanah dan iklim, 40(2): 87­94. sarno s., saputra a., rugayah r., pulung m.a., 2015 ­ pengaruh pemberian asam humat (berasal dari batu bara muda) melalui daun dan pupuk p terhadap per‐ tumbuhan dan produksi tanaman tomat (lycopersicum esculentum mill). ­ j. agrotek. trop., 3(2): 192­198. sawan z.m., 2018 ­ mineral fertilizers and plant growth retardants: its effects on cotton seed yield; its quality and contens. ­ cogent biol., 4: 1459010. sitompul s.m., guritno b., 1995 ­ analisis pertumbuhan tanaman. ­ gadjah mada university press, yogyakarta, pp. 412. suwahyono u., 2011 ­ prospek teknologi remediasi lahan kritis dengan asam humat (humic acid). ­ j. teknol. lingk., 12(1): 55­65. wahyudi i., 2007 ­ peran asam humat dan fulvat dari kompos dalam detoksifikasi aluminium pada tanah masam. ­ buana sains, 7(2): 123­130. walkley a., black i.a., 1934 ­ an examination of the degtjareff method for determining soil organic matter and proposed modification of the chromic acid titration method. ­ soil science, 37(1): 29­38. warncke d., brown j.r., 1998 ­ potassium and other b a s i c c a ti o n s , p p . 3 1 ­ 3 3 . ­ i n : b r o w n j . r . ( e d . ) recommended chemical soil test procedures for the n o r t h c e n t r a l r e g i o n . ­ m i s s o u r i a g r i c u l t u r a l experiment station sb 1001, university of missouri, columbia, usa. xu s., zhang l., mclaughlin n., mi j., chen q., liu j., 2015 ­ effect of synthetic and natural water‐absorbing soil amendments on photosynthesis characteristics and tuber nutritional quality of potato in a semi‐arid region. ­ j. sci. food agric., 96: 1010­1017. yoshida s., forno d.a., cock j.h., gomez k.a., 1976 ­ laboratory manual for physiological studies of rice (3rd edition). ­ the international rice research institute, los baños, laguna,the philippines, pp. 83. basi di pedologia. cos’è il suolo, come si forma, come va descritto e classificato. certini g., and f.c. ugolini. edagricole, edizioni agricole il sole 24 ore, bologna, 2010. pp. 196. isbn 978-88-506-5286-0. € 19.00. at a time when the study of crops is increasingly refers to the soil characteristics in which they grow, the text by giacomo certini and fiorenzo cesare ugolini is unanimous and enthusiastically welcomed by the scientific world, and agricultural world in general. knowing soil texture, its constitution and origin is the first step to set up and rationally manage each crop. furthermore, in the context of fruit tree species, the optimal balance between vegetation and production depends largely on the relationships between rootstock and soil and, therefore, between plant and peculiar soil characteristics which for many years will host the root system. the work of the aforementioned authors is, therefore, a long-awaited, scientific and technical reference for those who want to deepen their knowledge in the “horizon” of soil. the text is well articulated and the essential themes, accurately and deeply treated, reflect the rigorous expertise of the authors in the field. the volume includes the following eight chapters. 1. che cos’è il suolo, a cosa serve e come si forma (what is soil, what is it for how does it forms); 2. le fasi del suolo (soil phases); 3. il profilo del suolo e la sua descrizione (the soil profile and its description); 4. le forme di humus (the forms of humus); 5. i fattori della formazione del suolo (factors of soil formation); 6. i processi pedogenetici (pedogenic processes); 7. la soil taxonomy. gli orizzonti diagnostici e i regimi pedoclimatici (soil taxonomy. diagnostic horizons and soil and climate regimes); 8. i dodici ordini della soil taxonomy (the twelve orders of soil taxonomy). the volume is completed by an ample and rich list of references and a useful analytic index. furthermore, the wide range of tables, figures in black and white and color make it easy to read and support the display of topics. the text is an indispensable tool for students, to whom is addressed, but there is no doubt about the value it can bring to those working in agriculture, either as scholars or direct operators. cinzia silori oleum. manuale dell’olio da olive. ricci a. (ed.) edagricole, edizioni agricole il sole 24 ore, bologna, 2011. pp. 320. isbn 978-88-506-5276-1. € 45.00. with the release of “oleum”, this interesting and qualified volume adds to those concerning the complex and brood agro-industries topic. the text, written in the specific context of the processing of olives into oil, has makes use of the collaboration, synergistic and additive, of the most talented scholars in the field. the result obtained by the editor, antonio ricci, is that of a harmonic fusion of scientific knowledge that, right from the first chapter, gives the reader a broad and updated view of knowledge and acquisitions. each chapter, cleverly arranged in a crescendo of technical and scientific horizons, deals with the specific issues by providing, in addition to an up-to-date view of the topic, also a large repertoire of bibliographic references. the topics are organized as follows. 1. la chimica dell’olio da olive (chemistry of olive oil) (g. lercker); 2. normativa sui requisiti chimici, fisici e organolettici degli oli di oliva (regulatory requirements on chemical, physical and organoleptic characteristics of olive oil) (l. conte); 3. i contaminanti e la filiera produttiva degli oli di oliva (contaminants and the olive oil production process) (l. conte, s. moret, g. purcaro, t. populin, a. ermacora, and m. marega); 4. caratteristiche chimico-fisiche dell’oliva e dell’olio con riferimenti alle cultivar più diffuse (physical and chemical characteristics of olive and oil with reference to the most cultivated cultivars) (g. panelli); 5. l’olivo e l’olio nell’emisfero sud (olive and oil in the southern hemisphere) (g. panelli); 6. caratterizzazione molecolare delle varietà di olive e degli oli di oliva (molecular characterization of olive varieties and of olive oils (l. baldoni, r. mariotti, and n.g.m. cultrera); 7. tecnologie di estrazione, conservazione, packaging e loro riflessi sulla qualità dell’olio vergine di oliva (extraction, storage and packaging technologies and their impact on virgin olive oil quality) (m. servili, s. esposto, a. taticchi and r. sacchi); 8. utilizzazione dei reflui oleari (use of oil wastewater production) (m. servili, s. esposto, s. urbani, a. taticchi, and m. petruccioli); 9. caratteristiche meccaniche e funzionali degli impianti di estrazione dell’olio di oliva (mechanical and functional characteristics of olive oil extraction machinery and wastewater mills) (p. amirante); 10. modelli strutturali di frantoi e collocazione logistica degli impianti (structural models of mills and logistic location of facilities) (p. amirante); 11. oli aromatizzati (aromatic oils) (l. cerretani); 12. utilizzo dell’olio nella preparazione dei prodotti agroalimentari (using oil in the preparation of foodstuffs) (l. cerretani); 13. valutazione sensoriale degli oli vergini di oliva e loro classificazione (sensory evaluation of virgin olive oils and their classification) (a. giomo); 14. la valutazione sensoriale degli oli vergini di oliva (sensory evaluation of virgin olive oils) (b. alfei); 15. normativa relativa alla commercializzazione dell’olio di oliva (legislation on olive oil marketing) (r. filo della torre). the elegant typographical presentation and the impressive range of tables and images in black and white and color make the work not only attractive but also make reading it a pleasent and fuild experience. for its peculiar characteristics, the text addresses a broad spectrum of readers: from student to scientists of the subject, from the technical crusher to the manufacturer of machinery for olive oil extraction, and from traders to the legislators. enrico rinaldelli 123 book reviews 25 1. introduction cherry fruit size and quality is an important factor in production and sales of sweet cherry fruit (proebsting and mills, 1981). sweet cherry trees are typically upright, vigorous and non-precocious (lang et al., 2004) so orchard management practices focus on achieving high yields of premium quality fruit through balancing reproductive and vegetative growth. manipulation of the number of fruits (crop load) on the tree, and leaf area, can be used to encourage larger and sweeter fruit through balanced carbohydrate supply and demand (lang et al., 2004; spayd et al., 1986; whiting and lang, 2004). however, in many of these studies, yield losses due to cracking have not been presented (proebsting and mills, 1981) even when the economic losses due to cracking can be significant (hanson and proebsting, 1996). given that cracking can be induced by internal vascular flow (measham et al., 2009), it is posited that higher crop loads will reduce the incidence of cracking through increased competition between fruit for assimilate supply. it has been previously hypothesised that higher crop loads increase competition between fruit for carbohydrates and that lower crop loads result in higher assimilate supply for individual fruit (spayd et al., 1986), and that there can be a resultant increase in size (spayd et al., 1986) and concentration of sugars (proebsting and mills, 1981). it has also been noted however, that lower crop loads are associated with increased vegetative growth (kappel, 1991) and that current season’s vegetative growth is a strong sink for carbohydrates. diurnal translocation of sugars from leaves to fruit can be variable (richardson, 1998), and therefore it is difficult to assess relationships between sugars and cracking as a result of internally supplied excess water. cherry fruits are strong sinks (ayala and lang, 2008) and it has been noted that removal of spur leaves had little effect on fruit quality because alternative supplies of carbohydrates were sourced (whiting and lang, 2004). fruit and leaf ratio can be manipulated for optimum quality. two flower buds per spur has been suggested as the ideal (whiting and lang, 2004). an interaction between fruit and leaves was also implicated in the development of cracking, in a study by measham et al. (2010), which showed that leaf removal decreased the development of side cracks in cherry fruit during the few weeks prior to harvest. furthermore, diurnal water potential gradients and evaporative demands on the leaf influenced vascular flow to the fruit demonstrating a local fruit and leaf interaction. thus, given that fruit size (simon, 2006) and sugar levels (christensen, 1996) have been associated with the development of cracking, and simon (2006) cites two studies (bullock, 1952; way, 1967) that found trees with high loads that showed little cracking within variety, the potential for crop load manipulation to influence fruit cracking warrants investigation. the aim of this present study is to further examine this relationship between crop load and cracking. crop load manipulation and fruit cracking in sweet cherry (prunus avium l.) p.f. measham*, s.a. bound**, a.j. gracie*, s.j. wilson* * tasmanian institute of agriculture, university of tasmania, private bag 98, hobart, tasmania, 7001 australia. ** tasmanian institute of agriculture, 13 st johns avenue, new town, tasmania, 7008, australia. key words: fruit crop load, incidence of cracking, sweet cherry. abstract: yield loss from rain-induced fruit cracking is a perpetual risk associated with the production of sweet cherries, and is difficult to manage due to the unpredictability of fruit responses to late season rainfall. the aim of this five-year study was to investigate the relationship between fruit crop load and incidence of cracking. the results showed a negative correlation between crop load and incidence of fruit cracking, and it was found in both natural and manipulated crop load trials for all varieties studied and in all seasons assessed. the effect of crop load on final cracking levels are determined post cell division. results from this study showed that fruit width was positively correlated with cuticular cracking but, contrary to what has been purported in literature, no relationship between concentration of soluble sugars or firmness with the incidence of cracking was found. this study has confirmed that crop load should be a major consideration in orchard practices in developing strategies to manage fruit cracking. adv. hort. sci., 2012 26(1): 25-31 received for publication 23 october 2011 accepted for publication 3 february 2012 26 2. materials and methods plant material mature trees, grown on f12/1 rootstock, were used in all field trials. trials were undertaken from late october to late january during seasons 2005/06, 2006/07, 2007/08 and 2010/11 in two commercial orchards in huonville and old beach, tasmania (australia). all orchards were subjected to standard industry management practices. to investigate the effect of crop load on fruit cracking and type, five manipulated crop load trials were undertaken in years with late summer rainfall; trials 1 and 2 in seasons 2005/06 and 2006/07 respectively, trials 3, 4 and 5 in season 2010/11. a study of fruit properties from trial 5 was undertaken. in addition, a survey of natural crop load and fruit properties over three years (2005/06, 2006/07, 2007/08) was performed. the relationship between levels of cracking in situ and the cracking potential using the cracking index (christensen, 1972) was also evaluated. manipulated crop load trials to assess the impact of crop load on crack development, manipulated crop load trials were undertaken on one variety ‘simone’ at one site (huonville) in two seasons: 2005/06 (trial 1) and 2006/07 (trial 2). three further manipulated crop load trials were undertaken on different varieties and sites in one season, 2010/11; on variety ‘sweetheart’ at huonville and old beach (trial 3 and 4 respectively), and on variety ‘regina’ at huonville (trial 5). these varieties were chosen due to the variety of crack types they had previously displayed in earlier studies (measham et al., 2009) in tasmania; ‘simone’ showed a tendency for cuticular cracks, ‘regina’ for side cracks’ and ‘sweetheart’ for both. in trials 1 and 2, treatments included a low, medium and high crop load, which aimed for 2, 5 or 8 fruit per cm2 trunk cross sectional area (tcsa) respectively in a randomised complete block design with three replicates (whole tree plots). treatments were applied at pit-hardening during stage ii of fruit growth and development which occurred post bloom at 4 weeks after full bloom (4wafb). where the high crop load specified could not be reached, natural crop load was determined and used. in trials 3, 4 and 5, treatments included a low, medium and high crop load, applied at three different growth stages in a factorial design with five replicates (whole tree plots). crop load was achieved by thinning each bud to 1, 2 or 4 floral buds per spur and applied pre bloom (prb) at dormant bud stage, full bloom (fb) and post bloom (pob) at four weeks after full bloom. in addition, for trial 3, a sub sample of 30 non-cracked blemish-free fruits were randomly selected from each replicate tree for individual fruit assessments for size, total soluble solids, and firmness. mean fruit property values were used to assess relationships with crop load, and with the incidence of cracking in situ. in all trials cracking incidence was determined at harvest. fruits from each treatment were additionally assessed for cracking index (using 50 non cracked fruits per variety). in all manipulated crop load trials, the actual crop load achieved for all trial trees was recorded. natural crop load and fruit properties survey natural crop load was recorded at harvest over three seasons on three randomly selected whole trees of available varieties which included ‘kordia’, ‘lapin’, ‘regina’, ‘simone’, ‘sweetheart’, ‘sylvia’ and ‘van’. all fruits were harvested and cracking levels recorded. cracking incidence recorded at harvest was assessed in relation to natural crop load. in addition, for each season non-cracked blemish-free fruit from each variety was grouped, and a sub sample of 30 non-cracked fruits were selected for individual fruit assessments for size, weight, total soluble solids, stem length and skin thickness. mean fruit property values were used to assess the relationship with the incidence of cracking in situ for each variety. also in 2007/08, fruits from available varieties were assessed for cracking with the cracking index immersion method (christensen, 1972). the relationship between the cracking index and the level of cracking recorded in the field was investigated. given that this immersion method depends on water uptake across the skin, the impact of stem removal on uptake and the subsequent index value was also investigated using additional sub samples from variety ‘simone’ with stems either removed or left intact. measurements cracking incidence was determined as per measham et al. (2009), but with apical-end cracks and stem-end cracks combined to give a level of cuticular cracks. measham et al. (2010) concluded that these crack types were likely to be induced through the same mode of water uptake. cracking index was determined using the immersion method developed by verner and blodget (1931) as cited in and refined by christensen (1972). all fruit were harvested between 7 a.m. and 12 noon and cracking assessments, morphological measurements and laboratory-based measurements were undertaken on the same day as harvest. climate data for the months preceding and during harvest was obtained from the australian bureau of meteorology stations at huonville (situated less than 5 km from the trial site) and at old beach using a pm-k208 pm-11 phytomonitor weather station. determination of crop load prior to treatment application in manipulated crop load trials, tree girth was measured in centimetres at a point 5 cm above the graft union. tcsa was calculated for each tree for the area (a) of a circle using the formula (a = c2/4π), where c = circumference (cm) as described in measham et al. (2009). crop load was determined as total fruit number per tcsa. to determine natural crop load, all fruits from each tree were counted and crop load expressed as number of fruit per tcsa. 27 fruit property tests fruit size, weight and total soluble solid (tss) concentration (brixo) were measured as described in measham et al. (2009). fruit firmness was measured using a bioworks inc. firmtech 2 with values recorded using controlsoft software. stem length (mm) was measured using vernier callipers and skin thickness was recorded microscopically using a nikon smz800 dissecting microscope. statistical analyses to assess the relationship between crop load and cracking incidence, and crop load with mean fruit properties, data were subject to linear regression tests and anova. interactions between crop load and timing of thinning to desired load were determined prior to assessing main effects. analysis of proportion data was performed on transformed data in order to meet the assumptions of analysis. to assess the effect of natural crop load on fruit properties after accounting for variety, mean fruit property data were subject to anova using variety as a fixed factor, and then to ancova (crop load as the covariate) using proc glm (spss version 17). unless specified, all results identified as ‘significant’ are at probability level of 0.05. 3. results manipulated crop load trials all manipulated crop load trials received rainfall in the three weeks prior to harvest. in 2005/06 and 2006/07 there was a similar amount of rainfall (37 mm and 41 mm respectively). trials 3 and 4 in 2010/11 experienced 49 mm and 50 mm rainfall respectively and trial 5 received 42 mm rainfall (differing due to harvest dates). a negative linear relationship between actual crop load and total cracking incidence was recorded for variety ‘simone’ over both seasons (2005/06 and 2006/07) (fig. 1). the effect was greater in season 2005/06 than 2006/07, as indicated by the significantly greater magnitude of the slope for each crack type (slope (b) = -4.69 and -0.77 for total and side cracks and -0.69 and -0.30 for cuticular cracks). in 2010/11 a significant interaction (p = 0.045) between level and timing of crop load on total cracking in variety ‘sweetheart’ at huonville (trial 3) (fig. 2), but not at old beach (trial 4). at huonville (trial 3), within thinning times, no significant effect of crop load at the dormant (prb) or full bloom (fb) thinning times was seen, but there was a significant effect on total (p = 0.025) and side (p = 0.029) cracks (but not on cuticular cracks) when fig. 1 percentage of total cracked fruit (a), cuticular-cracked fruit (b) and side-cracked fruit (c) with actual crop load (tcsa) for variety ‘simone’. each point is for an individual tree. a significant relationship was found between actual crop load and total cracking incidence in 2005/06 (r2 = 0.903, p<0.001) and 2006/07 (r2 = 0.511, p = 0.03), cuticular cracking 2005/06 (r2 = 0.907, p<0.001) and 2006/07 (r2 = 0.540, p = 0.02) and side cracking in 2006/07 (r2 = 0.575, p = 0.02). a significant difference between seasons was found for total cracking and cuticular cracking. slope (b) of the linear regressions calculated for crop load and cracking incidence were significantly different between years. fig. 2 incidence of total cracking at huonville for variety ‘sweetheart’ under low, medium and high crop loads applied at dormant bud stage (pb), full bloom (fb) and four weeks after full bloom (pob). a significant interaction between crop load level and application timing was evident; low crop load resulted in a significantly higher level of cracking within the post bloom application time only. 28 thinning was applied post bloom (pob) (table 1). high and medium crop load levels set by post bloom thinning resulted in significantly fewer cracked fruit than low crop loads. at old beach (trial 4), there was no interaction between crop load level and timing of thinning; there was, however, a significant main effect of crop load on both total (p = 0.01) and side (p = 0.01) cracks, but not on cuticular cracks. a significant main effect of crop load on total (p < 0.001) and side (p < 0.001) cracks was also seen in variety ‘regina’ at huonville (trial 5) (fig. 3). there was an interaction of crop load level and timing of thinning on fruit size in ‘regina’ (fig. 4) where low crop loads resulted in smaller fruit when thinned pre bloom or post bloom. thinning at full bloom gave smaller fruit with medium loads. no interaction was found for fruit soluble solids or firmness; furthermore, no main effect of crop load or timing was found for fruit firmness. there was a significant main effect of crop load (p = 0.008) and timing of thinning (p = 0.013) on soluble solids with fruit from medium crop loads, and post bloom thinning displaying the highest soluble solids. no fruit properties were correlated with cracking levels except for fruit size, which was positively correlated (p = 0.01) with cuticular cracks only. cracking indices determined for all treatments in trials 3, 4 and 5 are given in table 2. there was a significant relationship (p = 0.017) between index and total cracking fig. 3 percentage of total cracked fruit (a) (r2 = 0.69) and side-cracked fruit (b) (r2 = 0.59) with actual crop load (tcsa) for variety ‘regina’ and percentage of total cracked fruit (c) (r2 = 0.33) and side-cracked fruit (d) (r2 = 0.32) with actual crop load (tcsa) for variety ‘sweetheart’. each point is for an individual tree. significant relationships were found between crop load and total cracking incidence and between crop load and side cracking. table 1 incidence of total and side cracking in ‘sweetheart’ at huonville under high, medium and low crop loads applied post bloom crack type crop load cracking incidence total high 15.22 a medium 16.62 a low 27.40 b side high 11.30 a medium 12.44 a low 22.89 b for each crack type, values followed by different letters indicate a significant difference (p<0.05). 29 recorded in situ for trail 3 only; no relationship was evident between cracking index and cracking in situ for trial 4 or 5. natural crop load and fruit property trials lower natural crop loads had higher levels of cracking incidence (fig. 5). cracking incidence remained low (less than 5%) for crop loads higher than ten fruit per cm2 tcsa in all years and for all crack types (fig. 5). when only using data points of less than 10 fruit per cm2 tcsa, relationships between cracking and crop load were found to be significant for all crack types in 2005/06 (total, r2 = 0.893, p < 0.001; cuticular, r2 = 0.853, p < 0.001; side, r2 = 0.540, p = 0.006) and for total and cuticular cracks in 2006/07 (total, r2 = 0.576, p = 0.005, cuticular, r2 = 0.463, p = 0.03). across all varieties and seasons no significant relationship was found between any of the fruit property values with total cracking incidence or individual crack type incidence. little difference can be observed between either weight or total soluble solids and changes in crop load, except perhaps a slight trend in variety ‘sylvia’ where a decrease in weight, but not in total soluble solids, occurs with a dramatic increase (three fold to 24 fruit per tcsa) table 2 cracking index (n = 50) determined for fruit from the three manipulated crop load trials (trials 3, 4 and 5) time of application crop load cracking in situ (%) cracking index trial 3 trial 4 trial 5 trial 3 trial 4 trial 5 dormant high 18 38 7 5 4 24 medium 18 32 11 10 6 16 low 14 59 28 11 15 21 full bloom high 14 31 6 8 8 3 medium 18 39 11 9 7 7 low 16 69 26 8 14 7 4wafb high 15 28 7 6 9 4 medium 17 32 11 9 4 7 low 27 50 26 18 24 12 mean incidence of total cracking is also given. a significant correlation was found between cracking index and total cracking in trial 3 only. fig. 4 fruit size (mm) determined for fruit from variety ‘regina’ under low, medium and high crop loads applied at dormant bud stage (prb), full bloom (fb) and four weeks after full bloom (pob). a significant interaction between crop load and thinning time was found. fig. 5 the percentage of total cracked fruit (a), cuticular-cracked fruit (b) and side-cracked fruit (c) with natural crop load (tcsa). each point is for an individual tree. a significant relationship was found between crop load and total cracking incidence in 2005/06 and 2006/07, and between crop load and cuticular cracking in 2005/06, and between crop load and side cracking in 2005/06 and 2006/07. 30 in crop load in the third season, 2007/08 (fig. 6). a significant variety effect was found in fruit weight (p = 0.02), total soluble solids (p = 0.001) and stem length (p < 0.001). after accounting for variety, and analysing data using crop load as a covariate, a significant effect of crop load was found for fruit weight only (p = 0.03). fig. 6 mean fruit weight and total soluble solids for seven varieties over three seasons (error bars represent sem, n = 30) with mean crop load levels (n = 3) and cracking incidence for the same varieties and seasons. for cracking incidence, expressed as percentage of total yield, columns represent total incidence, where light areas represent incidence of cuticular-cracked fruit, and dark areas represent incidence of side-cracked fruit. horizontal axis labels represent variety by letter (k kordia, l lapin, r regina, si simone, sw sweetheart, sy sylvia, v van) and season by number (1 2005/06, 2 -2006/07, 3 2007/08). a significant relationship between cracking index with total cracking incidence (p = 0.011) and incidence of sidecracked fruit (p<0.001) was found. no significant relationship was found between cracking index and incidence of cuticular-cracked fruit. no differences were found between values for stemless fruit and fruit with stems attached (both had index values of 25). 4. discussion and conclusions lower crop loads resulted in a greater incidence of fruit cracking in sweet cherry. this was seen in both manipulated crop loads and in the natural crop load survey. responses to rainfall and the difference in magnitude of cracking between 2005/06 and 2006/07 confirm a strong seasonal impact on crack development, consistent with the findings of measham et al. (2009). however, rainfall per se did not account for the differences in cracking between seasons, suggesting that other environmental parameters and fruit growth patterns are also important in the development of cracks. fruit development is important in crack susceptibility as all post bloom thinning showed increased cracking with lower crop loads. this implies that the effect of crop load on final cracking levels are determined post cell division, and cracking is therefore more likely to be attributable to cell expansion during the later stages of growth. cell expansion is a function of internal water entry, which has been linked to increased rates of side-cracked fruit (measham et al., 2010). this also supports the findings of yamaguchi et al. (2002) who linked cracking susceptibility at harvest to cell length. pre bloom thinning would therefore be the preferable option for manipulating crop load for size whilst minimising the risk of cracking. in addition, the development of the cuticle during early growth stages should be investigated with regard to cuticle integrity during the later periods of development. the number of cuticular-cracked fruits in low fruit load trees increased significantly in ‘simone’. during cell expansion, relative canopy cover on a whole tree basis in low fruit load trees, compared to high load trees, may prevent moisture loss from the fruit surface through reduced airflow around fruit bunches in a timely and effective manner, confirming the importance of leaf:fruit ratio in quality management decisions (whiting and lang, 2004). in contrast to other studies (spayd et al., 1986; whiting and lang, 2004) cracking susceptibility in this study did not seem to be related to fruit quality properties, nor did increased crop load limit fruit size or sugar accumulation, or enhance firmness (christensen, 1996) in any of the manipulated crop load trails. in the natural crop load survey, variation in fruit properties was mostly influenced by variety, with crop load only further influencing fruit weight, but not size, sugar level, stem length or skin thickness. cracking incidence was also not significantly correlated with the fruit properties recorded. this is in contrast with accepted views that both fruit size (simon, 2006) and sugar levels (christensen, 1996) are closely linked with cracking. studies suggesting vegetative growth provides a strong photoassimilate sink in apricots (costes et al., 2000) support these results whereby fruit crop load may not strongly influence source:sink relationships. results from both the manipulated and natural crop load trials do confirm studies that report no differences in sugar levels between varieties of varying cracking susceptibility (moing et al., 2004). 31 furthermore, the results of the present study can be explained by the level of crop load achieved under normal orchard practice. fruit loads were relatively low in trees from this study with the majority being lower than 15 fruits per cm2 tcsa; the highest value reached was about 27 fruits per cm2 tcsa, or the equivalent of just over 2000 fruits on a tree with a trunk circumference of 30 cm. it is possible that fruit quality (size and sugars) was not diminishing under this scenario as there were still available resources within the tree from which to draw. this finding highlights the strong potential for encouraging good fruit set, and subsequent crop load, as a practical and viable management tool in mitigating yield losses from rain-induced cherry fruit cracking, given the significant reduction in cracked fruit with increased crop loads. the incidence of cracking recorded in situ was correlated with the cracking index for varieties in the natural crop load survey but for only one of the manipulated crop load trials. the cracking index procedure may not necessarily be reliable for predicting cracking susceptibility given the differences found in cracking incidence with crop load, with seasons (measham et al., 2009), and when compared to other growing regions (christensen, 1996; greco et al., 2008). the strong correlation between cracking index and the incidence of side cracks recorded in situ supports the build up of turgor within the fruit as a likely driver of side cracking (sekse, 1995), which can be somewhat mitigated by skin and cuticular properties. this could be due to differences in shape; curvature of the skin has been related to cracking susceptibility (sawada, 1934), and could also explain why size was the only fruit parameter positively correlated with cuticular cracking. this study has confirmed that crop load management can be successfully used to mitigate cracking without compromising fruit size. the results from this study did not confirm the relationship between fruit size, or sugar, and the incidence or cracking, but highlight the importance of skin properties in crack development. references ayala m., lang g.a., 2008 c-13-photoassimilate partitioning in sweet cherry on dwarfing rootstocks during fruit development, pp. 625-632. in: eris a., and m. burak (eds.) proceedings of the v international cherry symposium. international society for horticultural science, vols. 1 and 2. christensen j.v., 1972 cracking in cherries. iii. determination of cracking susceptibility. acta agriculturae scandinavica, 22: 128-136. christensen j.v., 1996 rain-induced cracking of sweet cherries: its causes and prevention, pp. 297-330. in: webster a.d., and n.e. looney (eds.) cherries: crop physiology, production and uses. cab international, pp. 513. costes e., fournier d., salles j.c., 2000 changes in primary and secondary growth as influenced by crop load in ‘fantasme®’ apricot trees. j. of hortic. sci. & biotec., 75: 510-519. greco p., palasciano m., mariani r., pacifico a., godini a., 2008 susceptibility to cracking of thirty sweet cherry cultivars. acta horticulturae, 795: 379-382. hanson e.j., proebsting e.l., 1996 cherry nutrient requirements and water relations, pp. 243-258. in: webster a.d., and n.e. looney (eds.) cherries: crop physiology, production and uses. cab international, wallingford, uk, pp. 513. kappel f., 1991 partitioning of above-ground dry matter in ‘lambert’ sweet cherry trees with or without fruit. j. amer. soc. for hortic. sci., 116: 201-205. lang g.a., olmstead j.w., whiting m.d., 2004 sweet cherry fruit distribution and leaf populations: modeling canopy dynamics and management strategies, pp. 591-599. in: webster a.d. (ed.) key processes in the growth and cropping of deciduous fruit and nut trees. international society for horticultural science, pp. 715. measham p.f., bound s.a., gracie a.j., wilson s.j., 2009 incidence and type of cracking in sweet cherry (prunus avium l.) are affected by genotype and season. crop and pasture science, 60: 1002-1008. measham p.f., gracie a.j., wilson s.j., bound s.a., 2010 vascular flow of water induces side cracking in sweet cherry (prunus avium l.). adv. hort. sci., 24(4): 243-248. moing a., renaud c., christmann h., fouilhaux l., tauzin y., zanetto a., gaudillere m., laigret f., claverie j., 2004 is there a relation between changes in osmolarity of cherry fruit flesh or skin and fruit cracking susceptibility? j. amer. soc. for hortic. sci., 129: 635-641. proebsting e.l., mills h.h., 1981 effects of season and crop load on maturity characteristics of ‘bing’ cherry. j. amer. soc. for hortic. sci., 106: 144-146. richardson d.g., 1998 rain-cracking of ‘royal ann’ sweet cherries: fruit physiological relationships, water temperature, orchard treatments, and cracking index, pp. 677682. in: ystaas j., o. callesen (eds.) third international cherry symposium, vols. 1 and 2. sawada e., 1934 a physical consideration of the mechanism of the cracking of sweet cherries. transactions of the sapporo natural history society, xii: 365-376. sekse l., 1995 fruit cracking in sweet cherries (prunus avium l.). some physiological aspects a mini review. scientia horticulturae, 63: 135-141. simon g., 2006 review on rain induced fruit cracking of sweet cherries (prunus avium l.), its causes and the possibilities of prevention. international journal of horticultural science, 12: 27-35. spayd s.e., proebsting e.l., hayrynen l.d., 1986 influence of crop load and maturity on quality and susceptibility to bruising of ‘bing’ sweet cherries. j. amer. soc. for hortic. sci., 111: 678-682. whiting m.d., lang g.a., 2004 effects of leaf area removal on sweet cherry vegetative growth and fruit quality, pp. 467-472. in: webster a.d. (ed.) key processes in the growth and cropping of deciduous fruit and nut trees. international society for horticultural science, pp. 715. yamaguchi m., sato i., ishaguro m., 2002 influences of epidermal cell sizes and flesh firmness on cracking susceptibility in sweet cherry (prunus avium l.) cultivars and selections. j. of the japanese soci. for hortic. sci., 71: 738-746. 179 1. introduction slender spindle bush types are commonly used for compact-sized fruit trees such as apple cultivars grafted on dwarfing rootstocks. peach trees can be dwarfed and trained as slender spindle types when they are grafted on prunus tomentosa and p. japonica ( mizutani et al., 1985; yaegaki et al., 2008 ). however, these rootstocks often show graft-incompatibility for some peach scion cultivars several years after grafting (nakano and shimamura, 1983; yaegaki et al., 2008). it is difficult to maintain trees grafted on vigorous rootstocks as slender spindle types by winter pruning. their shoot growth is so great that the inside parts of the tree are shaded, resulting in poor growth and even death of shoots near the trunk. in relation to shading, neri et al. (2003) reported that shading caused leaf wilting, necrosis and abscission earlier under water-stressed conditions. it is important to maintain shoots and buds alive near the trunk to maintain the slender spindle types. otherwise, shoots extend outward resulting in the crown type of tree. however, when the tree vigor is so great, severe annual winter pruning only repeats imbalanced vegetative-oriented growth cycles each year. commercial fruit production is difficult under such conditions. many reasons have been given to support the practice of summer pruning in peach trees. it has been reported that summer pruning reduces vegetative growth, improves light penetration, enhances fruit quality, concentrates fruit maturation and increases the number of flower buds. in general, it is considered that the time of flower bud formation in deciduous fruit trees is around late july and august in the temperate zones of the northern hemisphere. thus, the time of summer pruning is very important in relation to flower bud formation, especially when heading back pruning is conducted. after summer heading back pruning, new shoots are regenerated from remaining twig parts. even thinning out pruning sometimes enhances branching and burst of buds which otherwise remain quiescent. in relation to shoot regrowth after summer pruning, neri et al. (1992) reported that it was induced only when the whole root system was well irrigated. after summer pruning the regenerated shoots are considered physiologically young compared with the spring flushes. in apple trees, the earlier the pruning time, the greater the number of flower buds (mizutani et al., 2000). apple flower buds tend to bear in the apical buds of shoots. when summer pruning is carried out earlier, new plural shoots come out and bear flower summer pruning to maintain slender spindle bush type of peach trees grafted on vigorous rootstocks f. mizutani faculty of agriculture, ehime university, matsuyama, ehime prefecture, 790-8566 japan. key words: peach, pruning, shading, shoot heading back, summer shoot thinning. abstract: the slender spindle bush type system is commonly used for compact-sized trees, especially grafted on dwarfing rootstocks. it is difficult to apply this system to trees grafted on vigorous rootstocks by winter pruning. such practices only cause the trees to repeat imbalanced vegetative-oriented growth every year. therefore, in the current work summer pruning was applied to slender spindle bush type of early maturing peach trees grafted on vigorous rootstocks. three trials were conducted: summer shoot thinning, shoot heading back trials in the field and a shoot heading back and shading trial in the pot experiment. summer shoot thinning reduced tree growth and recovered proper balance between vegetative and reproductive growth. the following season such shoot thinning enhanced bud burst and initial growth of new shoots but the final tree growth was less in the summer-pruned trees compared with winter-pruned trees. the fruit matured earlier and soluble solids content was greater and titratable acidity was lower in the summer-pruned trees. the summer shoot heading back trial revealed that it regenerates shoots, although they bear fewer flower buds compared with winter-pruned trees. such heading back is effective to keep alive shoots and buds near the trunk in slender spindle bush type systems. summer shoot heading back and shading experiments in the pot showed that shading reduced the number of regenerated shoots and flower bud formation and delayed flower blooming in the following year. thus summer shoot thinning and heading back are applicable to early maturing peach cultivars grafted on vigorous rootstocks to maintain the slender spindle bush type because thinning favors reducing tree vigor and light penetration near the trunk, and heading back keeps alive shoots and buds near the trunk which otherwise weaken or die back due to apical dominance and/or shading. adv. hort. sci., 2011 25(3): 179-186 received for publication 31 march 2011 accepted for publication 31 august 2011 180 buds in each shoot apex resulting in greater flower bud numbers (mizutani et al., 2000). however, later summer pruning diminishes such effects. it seems likely that a certain period is required for regenerated shoots to bear flower buds. erez (1982) also reported that in the meadow orchard system of peach trees, four to five months are required for sufficient shoot regeneration and flower bud formation. therefore he recommends that such systems are only applicable to early maturing cultivars, with a long enough growing season after fruit harvest. three trials (summer shoot thinning, heading back in the field, and heading back and shading in the pot) were conducted in the present work to develop new methods to maintain slender spindle bush type peach trees grafted on vigorous rootstocks. 2. maintaining tree shapes as slender spindle bush type in peach trees grafted on vigorous rootstocks by summer shoot thinning a. objectives at first we tried to maintain tree shapes as slender spindle bush type with ‘ab-1’ (‘akatsuki’x unknown peentao) peach trees grafted on vigorous rootstock (prunus persica batsch, wild form) by winter pruning. the trees grew well in the orchard (previously vineyard) in spite of the fact that chemical fertilizers were not applied. it was difficult to maintain tree shape as slender spindle type while producing quality fruit annually. to keep shoots and buds near the leader trunk, excessive severe winter pruning was practiced. such practices resulted in an improper balance between vegetative and reproductive growth. the vegetative-oriented growth is represented by vigorous shoot growth, poor fruit set, much june drop and delayed fruit maturation. when the peach trees are vigorous, severe winter pruning only repeats such a tree cycle every year. therefore, the objective of the first trial in the field was to determine whether summer shoot thinning can alter such imbalanced tree growth to the proper balance in the slender spindle type of peach trees grafted on vigorous rootstocks. b. materials and methods the orchard used in the experiment was formerly a vineyard to which chemical fertilizers had been applied according to a standard instruction in the experimental farm, faculty of agriculture, ehime university. for the purpose of dwarfing peach trees grafted on vigorous rootstocks, we planned no application of chemical fertilizers during the course of experiments. by using nine-year-old ‘ab-1’ peach trees which had so far received winter pruning, we tried summer pruning to maintain the tree as slender spindle bush type. the harvest time of ‘ab-1’ peach is mid july. summer pruning was conducted just after fruit harvest; most of it was conducted as thinning out methods not heading back. the weight of pruned shoots and leaves was measured. the following year new shoot growth, flowering, leaf mineral content, fruit growth and quality of harvested fruit were determined. c. results figure 1 shows summerand winter-pruned trees just after summer pruning on 24 july. the weight of shoots removed by summer pruning was less than that removed by winter pruning (fig. 2). however, in the case of winter-pruned trees, there were no leaves at pruning time so that the actual biomass removed from the winter-pruned trees was much fig. 1 peach trees just after summer pruning on 24 july (left: tree after summer pruning; right: tree without summer pruning) fig. 2 weight of shoots removed by summer and winter pruning from peach trees. data are presented as mean ± standard error (se). s h o o t w e ig h t (k g f .w . / tr e e ) summer pruning winter pruning 181 greater than the shoot weight pruned. three or four days earlier bud break and flowering were observed in summer-pruned trees compared with winter-pruned trees in the following year (fig. 3). the ovary size was greater in summer-pruned trees (fig. 4). the number of flowers however was less in summerpruned trees (fig. 3), while mineral and carbohydrate content in the new leaves and shoots was greater in the summer-pruned trees (figs. 5 and 6). this indicates that the shoots remaining after summer pruning received enough fig. 3 effect of summer and winter pruning on the formation of new shoots, leaves and flowers of peach trees in the following season on april 4. fig. 4 effect of summer and winter pruning on ovary size of peach flowers on 4 april. data are presented as mean ± standard error (se). o va ry le n g th w id th ( m m ) 8 6 4 2 0 length width fig. 5 effect of summer and winter pruning on mineral content of stems and leaves in peach trees on 4 april. data are presented as mean ± standard error (se). n u tr ie n t c o n te n t (% ) 5 4 3 2 1 0 summer pruning n u tr ie n t c o n te n t (% ) winter pruning 5 4 3 2 1 0 fig. 6 effect of summer and winter pruning on sugar and starch content in stems and leaves in peach trees on 4 april. data are presented as mean ± standard error (se). summer pruning c o n te n t (m g /g d w ) winter pruning 120 100 80 60 40 20 0 c o n te n t (m g /g d w ) 120 100 80 60 40 20 0 182 solar radiation to accumulate carbohydrate as reserves and mineral nutrient from the roots. on the other hand, in the winter-pruned trees, the shoots remaining near the trunk are supposed to have been shaded in the previous summer and fall. although the initial tree growth was slightly enhanced in the summer-pruned trees, the shoot growth was accelerated in the winter-pruned trees in the middle growing season and final tree size became greater in the latter group (fig. 7). fruit number and yield per tree were greater in the summer-pruned trees (tables 1 and 2). fruit weight was similar in both treatments but fruit seemed to mature earlier in summer-pruned trees. in this regard, the total soluble solids content in the juice was greater and titratable acidity was lower in summer-pruned trees. thus, summer shoot thinning seems to be applicable to vigorous peach trees grafted on vigorous rootstocks in slender spindle bush type system to recover balanced vegetative and reproductive growth. 3. shoot regeneration and flower bud formation after summer shoot heading back a. objectives because of apical dominant nature of shoots, the terminal shoot grows well, which retards the growth of lateral shoots. whereas the apical part of buds on shoots burst and extend, the lower part of buds mostly remain quiescent. in the slender spindle bush type of tree it is very important to keep alive buds or shoots near the trunk. without shoot heading back, terminal shoots extend outward, while the inside of the crown becomes shaded and shoots and buds near the trunk die back. in the second trial in the field, we examined the effects of summer shoot heading back on shoot regeneration, leaf chlorophyll content (spad value), leaf drop and flower bud formation by using peach tree grafted on vigorous rootstocks. b. materials and methods five-year-old ‘hikawahakuho’ peach trees grafted on vigorous rootstock (prunus persica batsch, wild form) which were trained as slender spindle type were used. the harvest time of this cultivar is early july. trees had been trained as slender spindle bush type by winter pruning before the experiment started. no chemical fertilizers were applied, as mentioned above, because the orchard was formerly a vineyard where the recommended amount of fertilizers had been applied according to the standard instruction. tree vigor of ‘hikawahakuho’ peach grafted on vigorous rootstocks used in this experiment was less compared with ‘ab-1’ peach described above in the previous section. summer pruning consisted of heading back of current shoots to about 10 cm and removal of vigorous shoots, which was conducted after fruit harvest on 15 july. the number of regenerated shoots, shoot growth, flower bud formation, spad values and leaf drop were detertable 1 effect of summer pruning on peach fruit yield and quality in the following season (2001) treatment fruit/tree (no.) yield (kg/tree) fruit weight (g) fruit length (mm) fruit diameter (mm) ssc (%) titratable acidity (%) summer pruning 136.0±36.7 13.0±3.1 102.2±5.3 59.4±0.7 62.2±0.7 9.27±0.26 0.27±0.01 winter pruning 96.2±5.3 8.7±0.8 102.2±4.0 58.4±0.5 60.7±0.4 8.45±0.24 0.52±0.04 data are presented as mean ± standard error (se). table 2 effect of two-successive-year summer pruning on peach fruit yield and quality in the following season (2002) treatment fruit/tree (no.) yield (kg/tree) fruit weight (g) maturity degree (z) ssc (%) titratable acidity (%) summer pruning 76.8±15.0 8.3±2.1 108.9±4.4 3.7±0.4 12.10±0.32 0.20±0.01 winter pruning 66.4±8.0 6.6±1.0 103.5±3.5 2.4±0.3 9.31±0.29 0.45±0.08 (z) for maturity degree, the score was given to green fruit=1 and ripen fruit=5. data are presented as mean ± standard error (se). fig. 7 the photos show tree sizes on 24 july in the following year. 183 mined. the following year, tree growth and fruit yield and quality were determined. c. results figures 8 and 9 show the shoot regeneration after summer pruning. there was no regrowth of shoots in winterpruned trees. spad values of leaves were greater in summer-pruned trees than winter-pruned trees (fig. 10). leaf retention was prolonged by summer pruning (fig. 11). mierowska et al., (2002) also reported that in apple spur leaf total chlorophyll content was higher in summer-pruned than non-pruned trees. these facts indicate that regenerated shoots are physiologically young compared with spring flushes. the percent of flower buds were lower in summerpruned trees than winter-pruned trees (table 3); total shoot length in the following year was less in summer-pruned trees (table 3). fig. 8 effect of summer pruning on regenerated shoot numbers of peach trees. data are presented as mean ± standard error (se). fig. 9 effect of summer pruning on the regenerated shoot length of peach trees. data are presented as mean ± standard error (se). table 3 effect of summer pruning on flower bud formation and total shoot length of peach trees in the following year treatment flower buds (%) total shoot length in the following year (m) summer pruning 17.2±4.5 21.8±4.9 winter pruning 65.7±8.7 45.0±9.0 data are presented as mean ± standard error (se). fig. 10 effect of summer pruning on spad (chlorophyll content) in the leaves of peach trees in september and november. data are presented as mean ± standard error (se). fig. 11 effect of summer pruning on leaf drop in peach trees from september to late january. data are presented as mean ± standard error (se). 184 therefore it can be said that the tree size of summerpruned trees was reduced compared with winter-pruned trees. figure 12 shows single year and two-successiveyear summer pruning on the weight of shoots pruned. two-successive-year summer pruning reduced the weight of pruned shoots. fruit quality of harvested fruit as affected by single year and two-successive year summer pruning is presented in table 4. summer pruning enhanced maturation and increased soluble solids contents and reduced titratable acidity. 4. shoot regeneration and flower bud formation after summer shoot heading back under shaded conditions a. objectives it is important to keep alive shoots and buds near the trunk to maintain peach trees as a slender spindle type. since shoots have apical dominance, terminal shoots extend outward so that inner parts of the crown become shaded without pruning. we further examined the effects of shading and summer shoot heading back on shoot regeneration and flower bud formation by using peach trees grafted on vigorous rootstocks in the pot trial. b. materials and methods potted (30 cm diameter) one-year-old ‘hikawahakuho’ peach grafted on vigorous rootstock (wild form) were used in the trial. fertilizers (n, p2o5, k2o=15%, 15%,15%) were applied at the rate of 30 g and 15 g per pot in mid march and early september, respectively. treatments consisted of shading with white and black cheesecloth, shoot heading back (to 5 cm length from shoot base) and their combinations. summer shoot heading back was conducted on 1 september. the number and length of regenerated shoots, leaf drop, spad values, flower bud formation and flowering time were determined. c. results no shoot regeneration was found without summer shoot heading back under either non-shaded or shaded conditions (fig. 13). summer shoot heading back regenerated shoots but shading reduced their number (fig. 13). heavy shading (black cheesecloth) accelerated shoot growth as compared to light shading (white cheesecloth) and non-shading (fig. 14). the number of flower buds was reduced by summer pruning and the tendency was accelerated by shading (fig. 15). shading tended to delay bloom in the following spring (fig. 16). this indicates that the inner side shoots of the tree crown delayed bud burst and initial shoot growth as described in winter-pruned ‘ab-1’ peach in the previous section (fig. 3). fig. 13 effect of late summer pruning and shading on the number of regenerated shoots of peach trees. sp = summer pruning; wp = winter pruning, wc = white cheesecloth; bc = black cheesecloth. data are presented as mean ± standard error (se). fig. 12 effect of summer and winter pruning on pruned shoot weight of peach trees. summer-pruned shoots include leaves. spⅰ, wpⅰ and spⅱ, wpⅱ indicate single-year and two-successiveyear pruning, respectively. data are presented as mean ± standard error (se). table 4 effect of summer and winter pruning on fruit yield and quality in peach trees treatment fruit no./tree fruit weight (g) yield (kg/tree) maturity degree (z) ssc (%) titratable acidity (%) summer pruning i (y) 28.1±4.1 131.0±0.5 3.7±0.7 3.5±0.4 12.8±0.2 0.27±0.03 winter pruning i(y) 41.1±5.8 128.1±0.4 5.3±1.0 2.7±0.2 11.8±0.2 0.39±0.03 summer pruning ii 22.2±3.6 136.2±6.0 3.0±0.6 3.9±0.3 13.7±0.3 0.24±0.02 winter pruning ii 30.4±4.3 128.6±0.4 3.9±0.8 2.9±0.3 12.7±0.2 0.38±0.03 (z) for maturity degree, the score was given to green fruit=1 and ripen fruit=5. (y) indicate single-year and two-successive-year pruning, respectively. data are presented as mean ± standard error (se). 185 5. discussion and conclusions slender spindle bush type training systems have been easily adopted in compact-sized trees grafted on dwarfing rootstocks (mizutani et al., 1985). however, when this system is applied to trees grafted on vigorous rootstocks, imbalanced vegetative-oriented cycle between vegetative and reproductive growth occurs. for commercial quality fruit production, a good balance between vegetative and reproductive growth is necessary. as suggested in the present work, application of summer shoot thinning can reduce vegetative growth in such vegetative-oriented trees of spender spindle type trees grafted on vigorous rootstocks. neri et al. (2010) also reported that summer pruning reduced vigor in apricot trees. to maintain the slender spindle type, it is crucial to keep alive shoots and buds near the trunk. however, shoots exhibit apical dominance by nature so that they extend outward, thus the space near the trunk becomes shaded. shading accelerates dieback of shoots and buds inside the crown. with regard to shading, neri et al. (2003) reported that shading caused leaf wilting, necrosis and abscission earlier under water-stressed conditions. thinning of shoots is effective for the penetration of solar radiation near the trunk in the crown. furthermore, heading back of shoots near the trunk induces shoot regeneration from the buds at the base, which are otherwise quiescent or died back. unless heading back is conducted, the base parts of shoots become bare without alive shoots or buds. summer shoot heading back is efficient to keep such shoots and buds near the trunk, even under shaded conditions. readers interested in further detailed information for current work are also referred to hossain et al. (2004, 2005, 2006). in conclusion, summer thinning is effective for reducing tree vigor and light penetration near the trunk, whereas summer heading back is essential for keeping alive shoots and buds near the trunk in slender spindle bush type peach trees grafted on vigorous rootstocks. references erez a., 1982 peach meadow orchard: two feasible systems. hortsci., 17: 138-142. hossain s.a.b.m., mizutani f., onguso j.m., 2004 effects of summer pruning on maintaining the shape of slender spindle bush of peach tree grafted on vigorous rootstock. j. jap. soc. agr. tech. man., 11: 55-62. hossain s.a.b.m., mizutani f., onguso j.m., elshereif a.r., 2005 effects of late summer pruning and cheesecloth shades on shoot regeneration, flower bud formation and blooming time of peach trees. bull. exp. farm fac. agr. ehime univ., 27: 11-17. hossain s.a.b.m., mizutani f., onguso j.m., el-shereif a.r., rutto k.l., 2006 effects of summer pruning on shoot growth and fruit quality in peach trees trained as slender spindle bush type. mem. fac. agr. ehime univ., 51: 9-13. fig. 14 effect of late summer pruning and shading on regenerated shoot growth of peach trees. data are presented as mean ± standard error (se). fig. 15 effect of late summer pruning and shading on flower bud formation of peach trees. sp=summer pruning; wp= winter pruning; wc=white cheesecloth; bc=black cheesecloth. data are presented as mean ± standard error (se). fig. 16 effect of late summer pruning and shading on flower blooming of peach trees in the following year. data are presented as mean ± standard error (se). 186 mierowska a., keutgen n, huysamer m., smith v., 2002 photosynthetic acclimation of apple spur leaves to summer pruning. sci. hort., 92: 9-27. mizutani f., kogami t., moon d.g., bhusal r.c., rutto k.l., akiyoshi h., 2000 effects of summer pruning on the number of apical buds near the trunk in slender-spindle-trained apple trees grafted on semi-dwarfing rootstocks. bull. exp. far. coll. agr., ehime. univ., 22: 1-10. mizutani f., yamada m., taniguchi, t., koizumi k., sugiura a., tomana t., kaddya k., 1985 dwarfing effect of p. japonica and p. tomentosa rootstocks on ‘ohkubo’ peach trees. j. jap. soc. hort. sci., 54 : 327-335. nakano m., shimamura k., 1983 growth and yield of peaches on rootstocks of prunus tomentosa and prunus persica. sci. rep. fac. agr. okayama univ., 61: 67-75. neri d., castagnoli m., poni s., corelli l., 2003 diversified response to drought of light exposed and shaded leaves of potted grapevine, peach and pear trees. j. fruit orn. plant res., 11: 5-15. neri d., morini f., massetani f., 2010 pruning: how to manage shoot growth. acta horticulturae, 862: 355-363. neri d., sansavini s., sugiyama n., 1992 summer and root pruning of split-root potted peach trees. acta horticulturae, 322: 177-189. yaegaki h., inomata y., murase s., ito a., yoshioka h., takatsuji t., suzuki k., 2008 influence of the combination of scion peach cultivars and prunus japonica thumb. and p. tomentosa thumb. as a dwarfing rootstock on growth and fruit production. bull. natl. inst. fruit tree sci., 7: 1-12. impaginato 44 adv. hort. sci., 2011 25(1): 44-50 received for publication 24 february 2011. accepted for publication 8 march 2011. shoot-tip vitrification protocol for red chicory (cichorium intybus l.) lines c. benelli*, a. previati**, a. de carlo*, m. lambardi* * istituto per la valorizzazione del legno e delle specie arboree (ivalsa), consiglio nazionale delle ricerche, via madonna del piano, 10, 50019 sesto fiorentino (fi), italy. ** centro sperimentale orticolo “po di tramontana”, veneto agricoltura, via moceniga, 7, 45010 rosolina (ro), italy. key words: cryopreservation, genetic resources, pvs2, rapd, red chicory, vitrification. abstract: shoot tips from in vitro stock plants of red chicory ‘rosso di chioggia’ line were cryopreserved by one-step vitrification. after two days of cold-hardening on hormone-free ms medium and loading for 30 min in a mixture of 2 m glycerol and 0.4 m sucrose at 25°c, shoot tips were dehydrated with pvs2 vitrification solution at 0°c for 60 min and plunged directly into liquid nitrogen. the post-thaw survival of shoot tips was achieved 79% when was cultured on recovery medium containing 0.5 mm ba. observed regrowth, after six weeks of culture in the same medium composition, was 100%. rooted cryopreserved microshoots showed good quality when transferred to the greenhouse. preliminary results proved that the genetic fidelity of the cryopreserved line was maintained. the same vitrification protocol was then applied to three other red chicory lines, ‘rosso di treviso precoce’, ‘rosso di treviso tardivo’ and ‘castelfranco’. a simple and effective protocol for the cryopreservation of red chicory shoot tips has been successfully developed as a result of this study. 1. introduction in italy, the major production area of red chicory (cichorium intybus l. var. intybus), covering about 9 thousand hectares, is located in the veneto region, one of the most economically important areas for vegetable production. an ancient typology, ‘rosso di treviso tardivo’, that was progenitor of the present varieties of red chicory, was introduced into italy in the 15th century. over time, growers selected those plants having good production, while in recent years this leafy vegetable has undergone intense selection and breeding work, and several improved typologies have been produced (such as ‘rosso di treviso precoce’, ‘rosso di verona’, ‘rosso di chioggia’, ‘variegato di castelfranco’, and others) (veneto agricoltura, 2002), which are highly appreciated for their quality and productivity. at the “po di tramontana” experimental farm in rosolina (veneto agricoltura, rovigo, italy) a specific breeding program to select high-performance lines has been continuing for many years. every year after infield evaluation, the most valuable lines are introduced and maintained in vitro by subculturing every three weeks. the stock plants obtained from these lines are transferred to the greenhouse to produce high quality seeds to be used for the production of high quality red chicory. the costs of stock culture maintenance and the risks of contamination and decay of lines can be reduced by introducing cryopreservation as a tool for a long-term preservation. cryopreservation involves the maintenance of plant propagules at ultra-low temperatures (-196°c, ln): under these conditions, biochemical and most physical processes are completely arrested and as such, plant material can be stored for unlimited periods. cryopreservation studies have been reported for belgian endive (cichorium intybus l. var. foliosum, cvs. flash, rumba and carolus) by controlled-rate freezing (demeulemeester et al., 1992; 1993) and encapsulation-dehydration techniques (vandenbussche et al., 1993). controlled-rate freezing is regarded as the traditional approach to plant cryopreservation. although controlled-rate freezing has been effective for cryopreserving differentiated tissues (reed and uchendu, 2008), reports on cryostorage using this technique are limited. one of the practical limitations of the controlled-rate freezing approach is the need for an expensive programmable freezer (engelmann, 2000). however, applying traditional (controlled-rate freezing) and new procedures (one-step freezing technique), the first examples of “cryogenic banks” are available today in several countries (reed, 2001; sakai and engelmann, 2007). at the same time, the development 45 of a vitrification system has made it possible to increase the number of plant species that can be cryopreserved (towill and bajaj, 2002; lambardi and de carlo, 2003, 2009; sakai et al., 2008). in the present study, a vitrification/one-step freezing procedure was developed using ‘rosso di chioggia’ shoot tips, with exposure of explants to the vitrification solution and rapid cooling by directly immersing in liquid nitrogen (ln). this procedure was then applied to three different red chicory typologies. in addition, validation of this method using molecular markers is presented with regard to maintenance of genetic stability. 2. materials and methods plant material microshoots of red chicory ‘rosso di chioggia (mp c7 4/212) were adventitiously induced from leaf portions through direct organogenesis (fig. 1 a). the lines were obtained by isolating each adventitious shoot and transferring them onto a semi-solid ms (murashige and skoog, 1962) medium, supplemented with 30 g l-1 sucrose, 1.0 µm 6-benzyladenine (ba) and 7 g l-1 agar (proliferation medium). the ph of the medium was adjusted to 5.8 before autoclaving for 20 min at 121°c. the shoot cultures were placed at 21±1°c under fig. 1 cryopreservation of red chicory “rosso di chioggia” line (a-f). a microshoots induced adventitiously from a leaf portion; b a shoot tip just after excision from a shoot bud; c shoot tip survival after three weeks and a well-formed shoot tip after cryopreservation with 60 min of pvs2 treatment (detail); d regrowth in plastic cylinder on ms medium with 0.5 µm ba; e cryopreserved rooted microshoot; f plantlets from cryopreservation, after potting and acclimation in the greenhouse. 46 12-hr photoperiod conditions (60 µmol m-2 s-1 photosynthetically active radiation) and subcultured every three weeks (standard culture conditions). short-day conditions avoided floral initiation. vitrification procedure before bud excision, stock plants were exposed to 4°c with an 8-hr photoperiod for a three-week coldhardening period. shoot tips (2-3 mm long), consisting of the apical meristem and 4-5 leaflets, were then excised from microshoots (fig. 1b) under a stereo microscope in aseptic conditions, and were precultured on hormone-free ms medium supplemented with 0.09, 0.3 or 0.7 m sucrose for two days at 4°c under an 8-hr photoperiod to determine the effect of sucrose pretreatment on cryopreserved explant survival. to induce dehydration, the shoot tips were loaded with a cryoprotectant (loading solution, ls; 2 m glycerol and 0.4 m sucrose) (matsumoto et al., 1994) for 30 min at 25°c in 2-ml nalgene® cryovials (10 shoot-tips per cryovial) and subsequently incubated at 0°c with the pvs2 (plant vitrification solution 2; 30% w/v glycerol, 15% w/v ethylene glycol, 15% w/v dmso in ms medium containing 0.4 m sucrose) (sakai et al., 1990). in order to choose the longest possible exposure time of shoot tips to the vitrification solution while avoiding toxic effects, shoot tips were exposed to pvs2 for 30, 60, 90 or 120 min and evaluated for shoot tip survival. after dehydration, the samples were suspended in 0.6 ml of fresh pvs2 solution and rapidly frozen to –196°c by direct immersion in ln, where they were stored for at least 2 hr. for recovery, they were quickly rewarmed by plunging the cryovials into a 40°c waterbath (warming rate: about 150°c min-1), unloaded from the pvs2 solution, washed for 20 min at 25°c in a liquid ms medium containing 1.2 m sucrose and finally plated onto the proliferation medium and maintained under standard culture conditions. cryopreserved shoot tips were assessed for survival after three weeks. in addition to proliferation medium, five different recovery media were tested to improve the regrowth of cryopreserved shoot tips: ms added with 0.5, 1.0 or 5.0 µm ba, ms with 1 µm thidiazuron (tdz) and ms with 0.5 g l-1 activated charcoal (ac). the role of ac has been discussed in many reports on plant tissue culture with different effects (thomas, 2008). survival was defined as a percentage of green shoot tips after three weeks from thawing. explants were transferred to plastic tubes (one shoot for each tube) and six weeks after ln treatment the percentage of plants demonstrating regrowth was assessed. explants treated with pvs2 but not exposed to ln were used as controls. in vitro rooting of cryopreserved shoots cryopreserved shoots were transferred to jars containing 100 ml of ms rooting medium with 20 g l-1 sucrose, 7 g l-1 agar and supplemented with 2.5 µm indole butyric acid (iba). the jars were kept in a climatic chamber, at 21°c, under a light intensity of 40 µmol m-2 s-1 and a 12-hr photoperiod. after four weeks, the rooted microplantlets were transferred to ex vitro conditions. the plantlets were transferred to the greenhouse into pots with substrate containing both sterilised peat and sand in a ratio of 2:1. after five weeks, the plants were transferred to the field and their morphological stability was evaluated. cryopreserved chicory lines the cryopreservation procedure, optimized for ‘rosso di chioggia’ shoot tips, was then tested on shoot tips from in vitro plants of three red chicory lines, ‘rosso di treviso precoce’ (tvp s5), ‘rosso di treviso tardivo’ (tvt) and ‘variegato di castelfranco’ (c90 s6’). all three lines are included in a program to safeguard and conserve red chicory in the veneto region. encapsulation-vitrification procedure ‘rosso di chioggia’ shoot tips, cold-hardened at 4°c for two days, were encapsulated into 2% na-alginate beads and treated with ls solution for 1 hr on a rotary shaker at 25°c. when this solution was removed, the beads were dehydrated by exposure to pvs2 for 2, 3 or 4 hr at 0°c, then placed in cryovials (five beads per cryovial) and plunged immediately into ln. encapsulated shoot tips were thawed in a waterbath at 40°c for 3 min and placed on ms proliferation medium. survival was assessed after three weeks. data analyses a minimum of 30 samples were used for each treatment and each experiment was repeated twice. statistical analysis of percentages was carried out by the χ2 test or non-parametric statistical test, the post hoc multiple comparison test (marascuilo and mcsweeney, 1977), both at p ≤ 0.05. genetic stability assessments of cryopreserved plantlets random amplified polymorphic dna (rapd) analysis was applied on red chicory in vitro shoots to evaluate the genetic stability of cryopreserved and non-cryopreserved shoots (untreated control) collected from the same stock cultures. genomic dna was extracted from approximately 100 mg of leaves with a ‘dneasy plant mini kit’ (quiagen). dna concentration was determined using a spectrophotometer at 260 nm, and an aliquot of dna was diluted to a working concentration of 20 ng µl-1. rapd profiles were generated using 24 arbitrary 10-mers as primers, of which 10 primers were then selected for the reproducibility, the legibility and the 47 stability of the rapd pattern: 1253, 1247, m2, m3, m10, m13, a1, a5, a9 and c7 (table 1). the amplification of dna was performed according to vettvori et al. (1996). fragments were separated on 2% agarose gels by electrophoresis and visualized by ethidium bromide staining under uv light. for each primer, amplification reactions were repeated at least twice and only those having reproducible band partners were used. minor fragments, which tend to be unstable in staining intensity, and therefore not reliable, were not considered. 3. results influence of pvs2 times and preculture treatments prolonged exposure at 0°c to the vitrification solution appeared to be harmful for explants. indeed, shoottip survival decreased after exposures of 90 min or more, while 30-min treatment was not enough to protect the explants during ultra rapid freezing (table 2). when the incubation time was limited to 60 min, more than 72% of cryopreserved (+ ln) shoot tips survived after plating onto the proliferation medium (fig. 1c). incubation for 90 and 120 min in the pvs2 solution showed even a slight decrease in percentage survival of shoot tips without freezing (ln). this result highlights the importance of incubation time with vitrification solution for shoot tip survival. to enhance the shoot tips’ osmotolerance to the vitrification solution, preculture treatments with different sucrose concentrations for two days at 4°c was applied before the ls and pvs2 treatments. however, frozen explant survival exhibited a significant decline when the shoot tips were pre-cultured on media containing high sucrose concentrations (table 3), indicating that sucrose treatments are not appropriate to improve red chicory shoot tip survival. all three red chicory lines can be successfully cryopreserved by loading shoot tips in pvs2 for 60 or 90 min, prior to freezing in ln. among the lines, maximum explant survival ranged between 65% in ‘rosso di treviso precoce’ and 76% in ‘rossso di treviso tardivo’ and ‘variegato di castelfranco’, respectively with 60 and 90 min of pvs2 treatment (table 4). recovery media the addition of ba to the recovery medium was found to be beneficial for post-thaw recovery of the shoot tips, even if with an increase of the concentration callus formation is stimulated. the highest post-thaw survival of ‘rosso di chioggia’ shoot tips was obtained with 0.5 µm ba (79%) (table 5). when 1 µm tdz was used, 33% of shoot tips survived, but after the first subculture (21 days), they stopped growing, after which no shoots developed. there was no survival of shoot tips on hormone-free medium supplemented with activated charcoal. table 1 nucleotide sequences of dna primers used for rapd analysis primer primer sequence 1253 1247 m2 m3 m10 m13 a1 a5 a9 c7 gtt tcc gcc c aag agc ccg t aca acg cct c ggg gga tga g tct ggc gca c ggt ggt caa g cag gcc ctt c agg ggt ctt g ggg taa cgc c gtc ccg acg a table 2 survival of cryopreserved shoot tips of ‘rosso di chioggia’ after exposure to pvs2 for different time periods. data were recorded three weeks after thawing (ln, liquid nitrogen) pvs2 exposure time (min) shoot tip survival (%)(z) 30 60 90 120 100 a 100 a 80 b 80 b ln + ln 33.5 b 72.5 a 40.0 b 30.0 b (z) in each column, percentages followed by different letters are significantly different at p ≤ 0.05 by the post hoc multiple comparison test. table 3 effect of sucrose concentration in preculture medium (two days at 4°c) on ‘rosso di chioggia’ shoot tips cryopreserved following 60 min pvs2 treatment sucrose concentration (m) shoot tip survival (%)(z) 0.09 0.3 0.7 68.0 a 47.5 a 17.5 b (z) percentages followed by different letters are significantly different at p ≤ 0.05 by the post hoc multiple comparison test. table 4 shoot tip survival percentage of three selected red chicory lines, following incubation and immersion in ln red chicory line loading time with pvs2 (min) (z) for each select line and each column, percentages followed by different letters are significantly different at p ≤ 0.05 by the χ2 test. shoot tip survival (%)(z) ‘rosso di treviso precoce’ ‘rosso di treviso tardivo’ ‘variegato di castelfranco’ ln + ln 60 90 60 90 60 90 80.0 a 70.0 a 80.0 a 90.0 a 90.0 a 100 a 65.0 a 55.0 a 66.7 a 76.7 a 50.0 a 76.0 b table 5 effect of recovery media on ‘rosso di chioggia’ shoot tip survival (three weeks) and regrowth (six weeks) after thawing recovery medium 0.5 µm ba 1.0 µm ba 5.0 µm ba 1.0 µm tdz 0.5 g/l ac 79.1 a 47.6 b 47.8 b 33.3 c 0 shoot tip survival (%) shoot tip regrowth (%)(z) 100 a 95.0 a 95.4 a 0 -(z) in each column, percentages followed by different letters are significantly different at p ≤ 0.05 by the post hoc multiple comparison test. 48 a further increase of regrowth was easily achieved by transferring the explants in tubes containing proliferation medium with ba. the best concentration for the development of ‘rosso di chioggia’ chicory shoots was again 0.5 µm. indeed, the plantlets obtained from this cytokinin treatment were generally taller and produced more shoot apices (fig. 1d). after in vitro rooting (fig. 1e), the microplantlets were transplanted into pots and successfully acclimated in vivo (fig. 1f). field observations of the plantlets confirmed the morphological stability of acclimatized plants with the original mother plants (fig. 2). vitrification vs encapsulation-vitrification cryopreservation by shoot-tip vitrification, using the pvs2 solution, was compared with the encapsulation-vitrification procedure in ‘rosso di chioggia’ line. the highest survival percentage of cryopreserved shoot tips was obtained by vitrification procedure (table 6). fig. 2 experimental field, the arrow indicates the plot with cryopreserved ‘rosso di chioggia’ plants. cryogenic procedure shoot tip survival (%)(z) vitrification encapsulation vitrification 76.0 a 35.2 b (z) percentages followed by different letters are significant at p ≤ 0.05 by the post hoc multiple comparison test. the encapsulation-vitrification procedure proved to be less effective for the cryopreservation of the selected red chicory line. however, only a maximum of 35% explant survival was achieved when the longest treatment of the beads with pvs2 was applied; this percentage is markedly lower than that reported in the literature using an encapsulation-dehydration procedure in chicory (vandenbussche et al., 1993). assessment of molecular stability by rapd to assess the genetic fidelity of plantlets regrown from cryopreserved shoot-tips, the rapd patterns were compared with untreated samples of the same red chicory lines. out of 24 primers screened, 10 selected primers produced clear and reproducible bands. the number of bands for each primer varied from five to twelve. each primer generated a set of amplification products of a size ranging between 350 bp and 3000 bp. preliminary results proved that the genetic fidelity of the cryopreserved lines was maintained. for all 10 primers tested, rapd fragment patterns of plantlets from cryopreserved shoot-tips did not show differences with respect to untreated shoots. figure 3 represents amplified band patterns produced by two primers (1253 and 1247). genetic fidelity was confirmed also by the other primers. 4. discussion and conclusions ‘rosso di chioggia’ shoot-tips cryopreserved using ls treatment for 30 min, pvs2 for 60 min at 0°c, and recovery on medium containing 0.5 µm ba resulted with a high survival rate (79%). this result is slightly inferior to that obtained in belgian endive, cv. flash (83%) where demeulemeester et al. (1992) used a more complex procedure, consisting of cooling the explants to -40°c at a rate of 0.5°c min-1 prior to immersion in ln. in the present study, the vitrification protocol applied to different red chicory lines showed high survival percentages, even if tolerance to the pvs2 varied in different lines. differences among lines can be considered to be genotypedependent; these results are consistent with other experiences on cryopreservation in other plant species (de boucaud et al., 2002; kim et al., 2006; benelli et al., 2009). table 6 shoot tip survival percentage of ‘rosso di chioggia’ shoottips after two different cryogenic procedures fig. 3 rapd profiles generated with primers 1253 (left) and 1247 (right) from untreated and cryopreserved plantlets of ‘rosso di chioggia’ line. in both images, lanes m correspond to the 100-bp ladder; lanes 1-9 correspond to the amplification products from untreated samples, lanes 10-17 correspond to the amplification products from cryopreserved plantlets. 49 in many cases, preculture with sucrose can be very efficient to improve cryopreservation (matsumoto et al., 1998), but such treatments applied on apical buds of several species did not induce increases in recovery after vitrification (sakai, 2000). sugar treatments can influence the membrane and protein composition (ramon et al., 2002; carpentier et al., 2005), influencing flexibility and permeability of the membrane. in red chicory shoot tips, preculture treatment on ms with 0.09 m sucrose for two days before pvs2 loading resulted in the best survival with respect to 0.3 and 0.7 m sucrose, but it did not improve the post-thaw survival percentage in the cryogenic procedure. the type and concentration of growth regulators in the recovery medium is important for survival and regrowth of cryopreserved explants (turner et al., 2001). red chicory shoot tip survival (79%) was improved when the recovery medium contained 0.5 µm ba; callus formation was induced when the ba concentration was increased, in particular with 5 µm ba. callus is not desired and represents a limitation in development of the cryopreserved explants. this phenomenon is observed in several species when a high concentration of ba was used (wang et al., 2003). demeulemeester et al. (1993) held the explants for one week on medium supplemented with plant growth regulators and then transferred them to hormone-free medium to avoid a callus phase in three varieties of chicory (flash, rumba and carolus) after cryopreservation. this protocol was effective for restricting callus formation, but led to a decrease in survival percentage. in the present study, with the ‘rosso di chioggia’ line, we obtained the highest survival rate without callus formation by applying the lowest ba concentration and the best regrowth when the single cryopreserved shoot was transferred in tube. addition of activated charcoal in post-thaw recovery medium resulted deleterious for shoot tip survival and regrowth; whereas thidiazuron gave a low survival of explants after three weeks but no subsequent growth. rooted microshoots of ‘rosso di chioggia’, obtained from cryopreserved shoot-tips, exhibited high survival and vigour in the greenhouse. application of the encapsulation-vitrification procedure showed a limited survival of cryopreserved explants and further experimentation is needed to optimize the protocol for these red chicory lines. a few genetic studies using molecular markers have been carried out on red chicory, mainly for the genetic characterization of commercial varieties (barcaccia et al., 2003), and particularly rapd markers were used to construct the genetic map of c. intybus (de simone et al., 1997) to identify and to evaluate the phylogenetic relationships among cultivars of chicory (koch and jung, 1997; van stallen et al., 2000, 2001; barcaccia et al., 2003). assessment of the genetic stability of cryopreserved shoots can be performed with different techniques (harding, 2004). in this study, rapd markers were adopted because of their simplicity, rapidity and ability to screen a randomly large part of the genome. genetic stability was reported after rapd analysis of cryopreserved plantlets and the untreated shoots. comparison of the dna patterns of control and frozen material did not reveal any variations caused by the cryoprotectant treatments or cryostorage. the results obtained are confirmed by numerous studies that demonstrated that cryopreservation did not affect the genetic stability in various species, such as potato (harding and benson, 2000), grape and kiwi (zhai et al., 2003), chrysanthemum (martin and gonzalez-benito, 2005), citrus spp. (lambardi et al, 2007), and apple (liu et al., 2008). moreover, the morphological analysis carried out in the field confirmed that there were no significant differences between plants derived from control and cryopreserved shoots. acknowledgements the authors are grateful to mr. francesco da re (veneto agricoltura) for his technical assistance in this research. references barcaccia g., pallottini l., soattin m., lazzarin r., parrini p., lucchin m., 2003 genomic dna fingerprints as a tool for identifying cultivated types of radicchio (cichorium intybus l.) from veneto, italy. plant breed., 122: 178-183. benelli c., de carlo a., giordani e., pecchioli s., bellini e., kochanova z., 2009 vitrification/one-step freezing procedure for cryopreservation of persimmon dormant buds. acta horticulturae, 833: 163-168. carpentier s., witter e., laukens k., deckers p., swennen r., panis b., 2005 preparation of protein from recalcitrant plant tissues: an evaluation of different methods for two-dimensional gel electrophoresis analysis. proteomics, 5: 2497-2507. de boucaud m.t., brison m., helliot b., herve’paulus v., 2002 cryopreservation of prunus. in: towill l.e. and y.p.s. bajaj (eds.) cryopreservation of plant germplasm ii. vol. 50. spring-verlag, berlin, heidelberg. de simone m., morgante m., lucchin m., parrin p., marocco a., 1997 a first linkage map of cichorium intybus l. using a one-way pseudo-testcross and pcr-derived markers. mol. breed., 3: 415-425. demeulemeester m.a.c., panis b.j., de proft m.p., 1992 cryopreservation of in vitro shoot tips of chicory (cichorium intybus l.). cryoletters, 13: 165-174. demeulemeester m.a.c., vandenbussche b., de proft m.p., 1993 regeneration of chicory plants from cryopreserved in vitro shoot tips. cryoletters, 14 : 57-64. engelmann f., 2000 importance of cryopreservation for the conservation of plant genetic resources, pp. 8-20. in: engelmann f., and h. takagi (eds.) cryopreservation of tropical plant germplasm. ipgri, rome, pp. 496. harding k., 2004 genetic integrity of cryopreserved plant cells: a review. cryoletters, 25: 3-22. harding k., benson e.e., 2000 analysis of nuclear and 50 chloroplast dna in plants regenerated from cryopreserved shoot-tips of potato. cryoletters, 21: 279-288. kim h.h, lee j.k., yoon j.w., ji j.j., nam s.s., hwang h.s., cho e.g., engelmann f., 2006 cryopreservation of garlic bulbil primordial by the droplet-vitrification procedure. cryoletters, 27: 143-153. koch g., jung c., 1997 phylogenetic relationships of industrial chicory varieties revealed by rapds and aflps. agronomie, 17: 323-333. lambardi m., de carlo a., 2003 application of tissue culture to the germplasm conservation of temperate broad-leaf trees, pp. 815-840. in: jain s.m., and k. ishii (eds.) micropropagation of woody trees and fruits. kluwer ac. pub., dordrecht. lambardi m., de carlo a., 2009 tecniche ed applicazione della criogenia alla conservazione ed al risanamento del germoplasma vegetale. italus hortus, 16: 79-98. lambardi m., halmagyi a., benelli c., de carlo a., vettori c., 2007 seed cryopreservation for conservation of ancient citrus germplasm. adv. hort. sci., 21: 198-202. liu y.g., liu l.x., wang l., gao a.y., 2008 determination of genetic stability in surviving apple shoots following cryopreservation by vitrification. cryoletters, 29: 7-14. marascuilo l.a., mcsweeney m., 1977 post hoc multiple comparison in sample proportions for test of homogeneity, pp. 141-147. in: marascuilo l.a., and m. mcsweeney (eds.) non parametric and distribution-free methods for the social science, brooke/cole publishing company, california, usa. martin c., gonzalez-benito m.e., 2005 survival and genetic stability of dendranthema grandiflora tzvelev shoot apices after cryopreservation by vitrification and encapsulation-dehydration. cryobiology, 51: 281-289. matsumoto t., sakai a., yamada k., 1994 cryopreservation of in vitro-grown apical meristems of wasabi (wasabia japonica) by vitrification and subsequent high plant regeneration. plant cell rep., 13: 442-446. matsumoto t.a., sakai a., nako y., 1998 a novel preculturing for enhancing the survival of in vitro-grown meristems of wasabi (wasabia japonica) cooled to –196°c by vitrification. cryoletters, 19: 27-36. murashige t., skoog f., 1962 a revised medium for rapid growth and bioassays with tobacco tissue cultures. physiol. plant., 15: 473-497. ramon m., geuns j., swennen r., panis b., 2002 polyamines and fatty acids in sucrose precultured banana meristems and correlation with survival rate after cryopreservation. cryoletters, 23: 345-352. reed b.m., 2001 implementing cryogenic storage of clonally propagated plants. cryoletters, 22: 97-104. reed b.m., uchendu e., 2008 controlled rate cooling, pp. 77-92. in: b.m. reed (ed) plant cryopreservation: a practical guide. springer verlag, berlin. sakai a., 2000 development of cryopreservation techniques, pp. 1-7. in: f. engelmann and h. takagi (eds) cryopreservation of tropical plant germplasm. ipgri, rome, pp. 496. sakai a., engelmann f., 2007 vitrification, encapsulationvitrification and droplet-vitrification: a review. cryoletters, 28: 151-172. sakai a., kobayashi s., oiyama i., 1990 cryopreservation of nucellar cells of navel orange (citrus sinensis var. brasiliensis tanaka) by vitrification. plant cell rep., 9: 3033. sakai a., hirai d., niino t., 2008 development of pvsbased vitrification and encapsulation-vitrification protocols, pp. 33-58. in: b.m. reed (ed.) plant cryopreservation: a practical guide. springer verlag, berlin. thomas t.d., 2008 the role of activated charcoal in plant tissue culture. biotech. adv., 26: 618-631. towill l.e., bajaj y.p.s., 2002 biotechnology in agriculture and forestry. cryopreservation of plant germplasm ii. springer-verlag, berlin, heidelberg. turner s.r., touchell d.h., senaratna t., bunn e., tan b., dixon k.w., 2001 effects of plant growth regulators on survival and recovery growth following cryopreservation. cryoletters, 22: 163-174. van stallen n., noten v., demeulemeester m., de proft m., 2000 identification of commercial chicory cultivars for hydroponic forcing and their phenetic relationships revealed by random amplified polymorphic dnas and amplified fragment length polymorphisms. plant breed., 119: 265270. van stallen n., noten v., neefs v., de proft m., 2001 the phylogenetic relationship between different cichorium intybus cultivars and cultivar groups, as revealed by rapds. plant breed., 120: 425-428. vandenbussche b., demeulemeester m.a.c., de proft m.p., 1993 cryopreservation of alginate-coated in vitro-grown shoo -tips of chicory (cichorium intybus l.) using rapid freezing. cryoletters, 14: 259-266. veneto agricoltura, 2002 il radicchio rosso di chioggia, di verona, di castelfranco. veneto agricoltura, vol. 3. legnaro (padova), italy. vettori c., paffetti d., pietramellara g., stotzky g., gallori e., 1996 amplification of bacterial dna bound on clay minerals by the random amplified polymorphic dna (rapd) technique. fems microbiology ecology, 20: 251-260. wang q., li p., batuman o., ron g., mawassi m., 2003 effects of benzyladenine on recovery of cryopreserved shoot tips of grapevine and citrus cultured in vitro. cryoletters, 24: 293-302. zhai z., wu y., engelmann f., chen r., zhao y., 2003 genetic stability assessments of plantlets regenerated from cryopreserved in vitro cultured grape and kiwi shoot-tips using rapd. cryoletters, 24: 315-322. 51 1. introduction asparagus (asparagus officinalis l.) is widely cultivated in italy and the total surface area devoted to this crop is as much as 6347 ha, mainly located in veneto (1610), campania (1347) and apulia (1070); notably, campania is the only italian region where the species is significantly grown in greenhouse (1052 ha) (istat, 2012). in the temperate area of the northern hemisphere, asparagus harvest begins after winter rhizome dormancy, when soil temperature is favourable to hypogeous bud resumption (mccormick and geddes, 1996; heiβner et al., 2006). therefore, the latter is earlier at lower latitudes and gradually delayed towards the north: in fact, it starts in january in california desert valleys, in february in italian southern regions and in south carolina, in may in holland, end of june in michigan and in washington districts (dufault, 1994 a). the spring harvest duration in greenhouse in southern italy is usually extended to ninety days, as it occurs in other areas with similar climatic conditions (shou et al., 2007). indeed, in such cases the favourable season length allows the plants to completely recover rhizome reservoirs, insuring crop vigour and longevity (takatori et al., 1970). however, as in italy the production period is exclusively concentrated in spring, in the other seasons of the year the product demand is satisfied by imports from warmer areas (south africa, chile, mexico, southern california). as an alternative, asparagus production can be achieved by summer forcing, through total aerial biomass cutting and subsequent irrigation a few days before the scheduled harvest. the possibility of annual double harvest has also been proposed in south carolina (dufault, 1991, 1995 and 1996) and its convenience seems to be mainly dependent on the favourable season duration. particularly, the crop forcing achieved at the end of spring or summer could reveal, respectively, too early or late and therefore damage the rhizome reservoir and consequently plant longevity. instead, a second harvest effects of crop method and harvest seasons on yield and quality of green asparagus under tunnel in southern italy g. caruso*1, g. villari**, c. borrelli*, g. russo*** * dipartimento di scienze del suolo, della pianta, dell’ambiente e delle produzioni animali, università degli studi di napoli federico ii, via università, 100, 80055 portici (na), italy. ** stazione sperimentale per l’industria delle conserve alimentari, via nazionale, 121, 84012 angri (sa), italy. *** ph.d., università degli studi di napoli federico ii, via università, 100, 80055 portici (na), italy. key words: annual double harvest, amino acids, asparagus officinalis l., fiber, organic management, sugars, vitamin c. abstract: a three-year study (2007-2009) was carried out on green asparagus under tunnel in campania (southern italy) with the purpose of verifying both the possibility to practise organic management and annual double harvest in order to extend the eco-compatible production period and to avoid expensive imports. comparisons among eight experimental treatments were made. treatments were obtained by factorial combination of two crop methods (conventional and organic) and four spring and summer harvest periods of 90-day total duration (75 days in spring plus 15 in summer; 60 days in spring plus 30 in summer; 45 days in spring plus 45 in summer; as a control, 90 days in spring), arranging a split plot design with three replicates. the conventional management led to the highest yield, as a consequence of the higher spear number per plant, while the organic management resulted in both spear calibre and mean weight increase. organic spears showed a higher level of residues and sugars but a lower content of nitrate and fibre. the treatment with 75-day harvest in spring and 15 in summer proved the best double harvest combination, leading to the highest comprehensive yield (11.5 t·ha-1), not different from the control harvested only in spring for 90 days. summer spears showed higher values of optical residue, glucose, fructose, vitamin c and some mineral nutrients; instead, spring spears attained lower nitrate and average fibre content. asparagus annual double harvest revealed economically interesting results, but the profits are strictly related to the prices of summer spears, which were evidently higher in summer than in spring in the three years of research. adv. hort. sci., 2012 26(2): 51-58 (1) corresponding author: gcaruso@unina.it received for publication 20 march 2012. accepted for publication 4 may 2012. 52 period managed in july-august could allow the plants to reconstruct the aerial biomass both after the spring production and before the winter quiescence. in comparing the traditional spring three-month yield and the comprehensive production derived from the spring and summer harvest, it should be taken into account that the double harvest period can satisfy the consumer requirement in both seasons. particularly, in summer higher economic profits could be achieved due to both a lack of asparagus spear offer at italian markets and the potential product demand by tourists. also, harvest season influences the quality of asparagus spears, which are considered a good source of essential minerals, vitamins, amino acids and dietary fibers (lopez et al., 1996). in fact, spear nutritional features are highly affected by environmental factors, especially temperature and light (makus, 1994, 1995; papadopoulou et al., 2003). in addition, bhowmik and coworkers (2001) proved that carbohydrate and organic acid contents in asparagus spears were season-dependent. also fibrousness, though being a varietal peculiarity (poll and van kuistrum, 1990; simón, 1990; billau et al., 1990; gast et al., 1991; sanchez, 1996), is influenced by crop environment and seasonal climate. in particular, it was inversely correlated with rainfall (keulder and riedel, 1990) and temperature (sosa coronel et al., 1976; keulder and riedel, 1990; poll, 1996), which regulate the spear growth rate. fibrousness is the consequence of wall lignification of the pericycle cells and vascular bundles (baxter et al., 1987). according to haard et al. (1974), spear removal causes endogenous ethylene development at an amount stimulating the peroxidase activity involved in lignin synthesis. also spear diameter influences fibrousness (clore et al., 1976; billau et al., 1990; poll, 1996) because the fibrous bundle number does not change and thus the fibre has a lower relative incidence in higher calibre spears (herner, 1973). with the purpose of studying the different aspects mentioned above, research was carried out in salerno province (southern italy) on green asparagus under tunnel with the aim of evaluating the effects of crop method and annual double harvest on spear yield and quality. 2. materials and methods research was carried out in the period 2007-2009 in fisciano (salerno province) under tunnel (40°46’ n, 14° 48’ e, 150 m a.s.l.). the asparagus crop was planted in 2004 on clay-loam soil (table 1), using cultivar desto crowns spaced 1.20 m between rows and 0.40 m between the plants along rows. temperature time course of the three research years are shown in figure 1. eight experimental treatments were compared. treatments were obtained by factorial combination of two crop methods (conventional and organic) and four spring and summer harvest periods of 90-day total duration (75 days in spring plus 15 in summer, labelled as 75+15; 60 days in spring plus 30 in summer, labelled as 60+30; 45 days in spring plus 45 in summer, labelled as 45+45; as a control, 90 days in spring). a split plot design was used, with three replicates, assigning the crop methods to plots and the harvest periods to sub-plots; the latter had a 17.3 m2 surface area. polytunnels, covered by thermal polyethylene, were made of three structural units, each of them 20 m long, 8 m wide, 2 m high at wall and 3.5 m at roof. the organic system was managed according to ec regulation 834/2007, using only substances permitted under the law. for the crop, three fertilizations per year were achieved, before and after spring harvest and after summer harvest, each with 60 kg·ha-1 of n, 40 of p 2 o 5 and 70 of k 2 o, using mineral fertilizer for the conventional method and manure for the “organic”. moreover, weed control was undertaken using linuron chemical treatment (0.75 l·ha-1 of a. p.), at spear pre-emergence, in conventional plots and by hand in organic. drip irrigation was activated at 80% soil available water consumption. harvest was carried out twice a year, in spring and in summer, except for the control harvested only in spring. spear emission in summer was urged by cutting the crop aerial biomass and, at the same time, activating irrigation. in particular, these interventions were made at the beginning of july for the 45+45 treatment, at mid-july for 60+30 and at the end of july for 75+15. table 1 soil characteristics constituents coarse sand % 24.27 fine sand % 29.87 silt % 27.27 clay % 18.97 composition organic matter (walkley and black method) % 3.47 total nitrogen (n) kjeldahl method % 0.21 available phosphate (p 2 o 5 ) olsen method ppm 12.57 available potassium (k 2 o) ammonium acetate method ppm 146.97 reaction ph 6.57 fig. 1 trend of monthly temperatures in greenhouse. 53 harvest was practised by hand when the spears emerged 150 mm out of soil and it began on 28 february, 3 march and 2 march, respectively in 2007, 2008 and 2009. at each picking, the plot product was separated in marketable and waste fractions. the latter included the folded, damaged or less than 10 mm thick spears. after harvesting, marketable spears were cut at 30 mm from the base and their calibre was measured on 30-unit random samples. moreover, with the aim of estimating product prices during spring harvests, data published by infomercati (www.infomercati.com) were used. informercati is a consortium of the main italian fruit and vegetable wholesale markets, which records and processes the daily prices of each product. in july and august the prices were detected by local sellers to whom the research spears were given, as in summer no asparagus transactions were recorded. in order to evaluate spear qualitative characteristics, random 30-unit samples were collected in each plot (for three replicates) both in conventional and organic treatments. in the control, this procedure was achieved 45 days after beginning of harvest; in the 75+15, 60+30 and 45+45 treatments sample collection was made 37, 30 or 22 days, respectively after spring harvest and 7, 15 or 22 days after summer harvest. spears were immediately transferred to the experimental station for the food preserving industry, angri (salerno) branch, where the following determinations were made: dry residue: in an oven at 70°c under vacuum until steady weight; optical residue (expressed in °brix): on spear sap after squeezing, at a temperature of 20°c, by means of a digital refractometer, model r.f.m81, from bs (bellingham+stanley); reducing sugars (glucose and fructose) and sucrose: by hplc, using the 600e waters chromatographic system and a column sugar-pak waters at 85°c, edta-ca in water solution as eluent (50 mg·l-1); titratable acidity: expressed as grams of monohydrate citric acid per 100 g of product in agreement with the official analysis methods for vegetable preserves of the italian ministry of agriculture and forestry (mipaf, 1973); proteins: with the kjeldahl method, utilising a foss tecator digestor with a kjeltec 2300 distiller; lipids: measured in accordance with the official analysis methods for vegetable preserves of the italian ministry of agriculture and forestry (mipaf, 1973); vitamin c: by hplc using the model 600e waters chromatographic system, equipped with a 486 waters uv detector set to 410 nm λ and a column biorad mod. hpx87h at 35°c; fibre: the sigma chemical company enzymatic kit was used. the samples were weighed, dried (105°c), gelatinized in the presence of heat-resistant α–amylase and digested enzymatically by proteases and amydoglucosydase, to remove proteins and starch, whereas soluble fibre was precipitated by ethanol. the residue, filtered, washed with ethanol and acetone, dried and weighed, was split into two fractions used to determine, respectively, proteins and ash. fibre content was obtained by the difference between the weights of the residue and the proteins and ash; free amino acids: by means of hplc with a waters 600e chromatographic system, connected to a personal computer using millenium32 software, version 3·05·01, and equipped with a waters 717 autosampler and a fluorescence detector set at a λ of 205 nm-395 nm. the measurements were carried out utilising: a waters accqtag column (spherical c-18, 4 µm 150 x 3.9 mm, at a temperature of 35°c; in condition of gradient with eluent a, or a 140 mm sodium acetate trihydrate buffer, tea, edta-na2, sodium azide, eluent b, consisting of acetonitrile, and eluent c, which consists of water and with an injection volume equal to 5 μl; mineral anions (chlorides, nitrates, phosphates): by hplc with 600e waters system, a mod. 717 autosampler and a dionex column (mod. as11, 4 x 250 mm); mineral cations: (calcium, magnesium, potassium, sodium, iron, copper, zinc): by atomic adsorption spectrophotometry, after sulpho-nitric mineralization, with a model 1100 perkin-elmer spectrophotometer. data were processed by analysis of variance and duncan multiple range test was used for mean separation at 0.05 and 0.01 probability levels (n = 3). 3. results and discussion from the data reported in table 2 it can be stated that there are no significant differences among the research table 2 asparagus yield results as influenced by crop method and harvest seasons treatment marketable spears yield t·ha-1 no. per plant mean weight g calibre mm year 2006 10.3 14.3 35.8 15.6 2007 10.1 14.6 34.4 15.5 2008 10.2 15.1 33.9 15.3 ns ns ns ns crop method conventional 10.7 15.6 34.0 15.1 organic 9.7 13.7 35.3 15.8 * * * * harvest seasons spring 90 days 11.7 a 16.4 a 35.4 a 15.8 a spring 75 days + summer 15 days 11.5 a 16.5 a 35.1 a 15.6 a spring 60 days + summer 30 days 9.5 b 13.7 b 34.7 a 15.5 a spring 45 days + summer 45 days 8.1 c 12.1 c 33.4 b 14.9 b ns= not significant; * significant at p≤ 0.05. means followed by different letters are significantly different according to the duncan test at p≤0.05 (n=3). 54 years in terms of yield. this means that spring harvest did not condition the subsequent summer plant productive behaviour and summer harvest did not condition the crop performances in following year’s spring. moreover, no plant density reductions were recorded. conventional asparagus management caused the highest yield, as a consequence of the higher spear number per plant (table 2). this is consistent with poll and van kruistum (1987) referring to a correlation between spear number and yield. organic management, instead, resulted in spear calibre and mean weight increase. these differences between the two crop methods are the consequence of the yield differences recorded both in spring and summer (table 3), though the conventional method resulted in higher production than the organic one by 14.2% in spring but only by 6% in summer. contrary to our results, in previous research (warman, 1991) no significant difference was detected between the two crop methods. as for the comparison among the annual double production periods (table 2), the 75+15 treatment (75-day spring harvest plus 15 days in summer) caused the highest comprehensive yield (11.5 t·ha-1), owing to the highest spear number, calibre and mean weight. however, it was not different from the control harvested only in spring for 90 days. the lowest production was attained by the combination of 45-day spring harvest plus 45 days in summer (8.1 t·ha-1). notably, the comprehensive yield obtained by the double harvest crops was influenced to a greater extent by spring results than by summer ones, as in the first season more remarkable differences among the treatments were recorded (table 3). in fact, in spring the production increases caused by the 75+15 treatment were, respectively, by 54% and 170% in comparison with 60+30 and 45+45, though correspondingly the harvest period was 25% and 67% longer. in summer, however, an opposite yield trend was shown by the annual double harvest treatments, but unable to balance the spring results. in fact, in summer the 45+45 treatment had a longer harvest period than 60+30 and 75+15, respectively, by 100% and 200%, to which the same percentage yield increases corresponded. the productive results were, therefore, affected by the lower temperatures recorded in march which, in agreement with previous reports (heiβner et al., 2006), conditioned the shooting rate. the latter was, instead, favoured by the gradual temperature increase, which allowed in the 75+15 treatment to get more than 60% of the whole spring production in the last 30 days of harvest. in fact, quick spear emission is promoted by an air temperature of about 24°c (bouwcamp and mccully, 1975) or soil temperature of about 20°c and it is correlated with the total yield (poll and van kruistrum, 1987; keulder and riedel, 1996). therefore, summer temperature was always very favourable to spear immediate and continuous emission, in agreement with the reports of dufault (1994 b), thus differences among the harvest treatments were not so remarkable as those recorded in spring. but in summer, yield results were conditioned by spear calibre, with values showing an inverse relation with temperature, consistent with previous findings (wagenvoort et al., 1980; pill et al., 1993). notably, both in spring and summer spear calibre and mean weight decreased from the shortest to the longest harvest period, confirming the effects of harvest pressure on these variables (mcgrady and tilt, 1990). table 3 asparagus spring and summer yield results as influenced by crop method and harvest seasons marketable spears yield (t ha-1) no. per plant mean weight (g) calibre (mm) spring summer spring summer spring summer spring summer year 2006 8.0 d 3.0 10.6 d 4.9 38.2 31.0 16.5 14.1 2007 7.9 d 3.0 11.0 d 4.9 36.4 30.6 16.3 13.9 2008 8.0 d 2.9 11.5 d 4.8 35.7 30.4 16.2 13.8 ns ns ns ns ns ns ns ns crop method conventional 8.4 d 3.1 11.8 d 5.1 36.0 30.1 15.9 13.7 organic 7.6 d 2.9 10.2 d 4.6 37.6 31.2 16.8 14.1 * * * * * * * * harvest seasons spring 90 days 11.7 a 16.4 a 35.4 c 15.8 c spring 75 days + summer 15 days 10.0 b 1.5 c 14.1 b 2.4 c 35.7 c 31.4 a 15.7 c 14.6 a spring 60 days + summer 30 days 6.5 c 3.0 b 8.8 c 4.9 b 37.1 b 30.6 b 16.5 b 13.8 b spring 45 days + summer 45 days 3.7 d 4.4 a 4.8 d 7.4 a 38.9 a 30.0 c 17.2 a 13.4 c ns = not significant; * significant at p≤ 0.05. means followed by different letters are significantly different according to the duncan test at p≤0.05 (n=3). 55 our research showed that the goal of annual double harvest is pursuable in terms of yield in irrigation regime, if the traditional spring harvest is reduced from 90 to 75 days and integrated with 15 days of summer harvest. alternative double harvest combinations, represented by 60-day spring harvest plus 30-day summer harvest or by 45-day harvest both in spring and summer, caused significantly lower yields than the control. nevertheless, their economic profits are strictly related to the prices of summer spears. in this regard, the mean prices of green asparagus spears recorded in the three years of research were evidently higher in summer (5.5 €·kg-1 in july and 6.0 in august) than in spring (3.45 €·kg-1 in march, 2.68 in april and 2.36 in may). from the statistical processing of spear quality data no significant differences were recorded both between the examined years (2008 and 2009) and among the different harvest durations within the same season. therefore, only the results concerning the comparison between conventional and organic methods and between spring and summer harvest are reported (tables 4, 5, 6 and 7). with regard to quality indicators (table 4), organic management resulted in higher values of dry and optical residues, glucose, fructose and sucrose than conventional cultivation, whereas acidity, proteins and lipids were not affected by crop method. also the harvest season had significant influence on quality, as summer spears showed higher values of optical residue, glucose, fructose and vitamin c than the spring ones; instead, no significant change was detected for dry residue, sucrose, titratable acidity, lipids and proteins. the latter result was similar to that of shou et al. (2007) but, differently from our findings, these authors reported a decreasing trend for dry residue and reducing sugars and an increasing trend for acidity. among the sugars, fructose attained higher incidence than glucose and sucrose, as also it resulted in our research. with respect to ascorbic acid, both esteve et al. (1995) and shou et al. (2007) found a content decrease in summer spears compared with spring ones. fibre content was affected by crop method (table 5), as organic spears attained lower values than conventional ones. also significant differences were recorded between spring and summer spears, as the latter showed higher average fibrousness, consistent with previous reports (shou et al., 2007). presumably, in summer the spear fibre increase caused by calibre reduction prevailed the opposite effect of temperature; in fact, the latter is inversely correlated with fibre content during spear growth, as reported by sanchez (1996). with respect to the fibre content of each 5 cm fraction obtained by spear split, the organic method led to higher values than the conventional in all the comparisons (table 5). regarding harvest season, it was found that summer spears showed higher fibre content than the spring ones only from the basal to the middle fraction. as for fibre content along the spear, the highest value was detected in the white basal fraction and it was as much as 2.6 times higher than the one recorded in the green tip (respectively, 28.1 and 11.0%). haard et al. (1974) stated that fibre reduction towards the spear tip is the consequence of isoperoxidase changes, which start from the cut surface and gradually extend to the tip. table 4 asparagus spear quality as influenced by crop method and harvest seasons treatment dry residue (g·100 g-1 fw) optical residue (°brix) glucose (g·100 g-1 dw) fructose (g·100 g-1 dw) sucrose (g·100 g-1 dw) acidity (g·100 g-1 dw) protein (g·100 g-1 dw) lipids (g·100 g-1 dw) vitamin c (mg·100 g-1 dw) crop method conventional 8.38 6.72 4.54 7.54 1.55 4.93 31.4 6.20 659.9 organic 9.11 7.26 5.40 8.41 1.77 5.02 32.4 6.26 673.0 * * * * * ns ns ns ns harvest seasons spring 8.64 6.81 4.83 7.78 1.63 4.87 31.7 6.13 627.0 summer 8.85 7.18 5.11 8.17 1.69 5.08 32.1 6.33 705.9   ns * * * ns ns ns ns * ns= not significant; * significant at p≤ 0.05 (n=3). table 5 asparagus spear fibrousness as influenced by crop method and harvest seasons treatment spear fibre content fibre content per spear section from tip to base (cm) 0-5 g·100 g-1 dw 5-10 g·100 g-1 dw 10-15 g·100 g-1 dw 15-20 g·100 g-1 dw 20-25 g·100 g-1 dw crop method conventional 18.8 11.7 12.8 14.1 24.8 30.7 organic 15.9 10.3 11.1 11.7 20.8 25.5 * * * * * * harvest seasons spring 16.6 11.0 11.6 12.1 21.6 26.5 summer 18.1 10.9 12.3 13.7 24.0 29.7 * ns ns * * * ns= not significant; * significant at p≤ 0.05 (n=3). ( )( )( )( )( ) 56 concerning the amino acids, we have reported only the mean composition and the statistical differences among the individual compounds (table 6), as both crop method and harvest season did not cause any significant effect on this variable. with regard to composition, glutamine and asparagine showed the highest content in the asparagus spears, whereas cystine, methionine, tyrosine and histidine displayed the lowest. these results are consistent with those reported by other authors (shou et al., 2007; slupski et al., 2010). as for spear chemical composition (table 7), sodium and copper showed significant higher contents in organic crops, whereas the conventional method led to higher nitrate accumulation; the other ions examined were not affected by crop management. in previous research (warman, 1991) no significant differences were recorded between conventional and organic methods with regard to spear mineral nutrients. with respect to harvest season, summer spears showed higher sodium, iron and zinc than the spring ones. in the latter season, contrary to the findings of other authors (shou et al., 2007), a significantly higher nitrate content was detected, which was however lower than the one recorded in other research (shalaby et al., 2004). 4. conclusions a three-year investigation (2007-2009) was carried out on green asparagus under tunnel in salerno province (campania, italy) in order to investigate the effects of crop method and annual double harvest on spear yield and quality. organic crops did not give as high yields as the conventional, but produced better spear quality in terms of higher level of residues and sugars and lower content of nitrate and fibre. asparagus summer forcing, achieved through the plant aerial biomass cut and irrigation, did not limit the crop productivity but revealed interesting economically. compared with the traditional 90-day harvest period, the annual double production practised for 75 day in spring and 15 in summer did not significantly differ in terms of yield. however, production decreased with the other spring-summer combinations, made up of 60-day harvest in spring plus 30 in summer or of 45-day harvest in both seasons. nevertheless, the annual double harvest effectiveness also depends on asparagus market receptivity, which in summer is potentially interesting owing to tourist demand. this perspective is supported by the investigation conducted during our research: higher spear prices in summer than in spring (on average, respectively 5.75 €·kg-1 vs 2.83) due to the lack of italian product offer. from a qualitative point of view, summer spears showed higher table 7 asparagus spear chemical composition as influenced by crop method and harvest seasons treatment calcium mg·100 g-1 dw magnesium mg·100 g-1 dw potassium mg·100 g-1 dw sodium mg·100 g-1 dw iron mg·100 g-1 dw copper mg·100 g-1 dw zinc mg·100 g-1 dw chlorides mg·100 g-1 dw phosphates mg·100 g-1 dw nitrates mg·100 g-1 dw crop method conventional 25.0 22.2 249.6 3.5 1.19 0.12 0.95 57.1 62.4 45.9 organic 23.5 21.7 263.9 4.2 1.16 0.15 0.93 57.7 66.7 14.2 ns ns ns * ns * ns ns ns * harvest seasons spring 24.0 21.7 254.0 3.7 1.11 0.13 0.89 55.2 63.9 43.2 summer 24.5 22.2 259.5 4.0 1.24 0.14 0.99 59.6 65.3 16.9   ns ns ns * * ns * ns ns * ns= not significant; * significant at p≤ 0.05 (n=3). ( ( ( ( ( ( ( ( ( () ) ) ) ) ) ) ) ) ) table 6 asparagus spear amino acid composition (average of crop methods and harvest seasons) amino acids content (g·100 g-1 dw) alanine 1.18 bd arginine 1.24 bc asparagine 3.60 ac cystine 0.16 hc glutamine 3.97 ac glycine 0.76 cf histidine 0.40 fh isoleucine 1.03 ce leucine 1.11 bd lysine 1.23 bd methionine 0.29 gh phenylalanine 0.62 eg proline 1.56 bc serine 1.05 cd threonine 0.74 df tyrosine 0.37 fh valine 0.97 ce means followed by different letters are significantly different according to the duncan test at p≤0.05 (n=3). 57 values of optical residue, glucose, fructose, vitamin c and some mineral nutrients, compared with the spring product. nevertheless, the latter attained lower fibre content, though only from the middle to the basal fraction. acknowledgements the authors thank mr. roberto maiello for his assistance with laboratory analyses. references baxter l., waters l. jr., breenew w., 1987 a quick method for determining fibrousness of vegetable tissue. hortscience, 22(2): 315. bhowmik p.k., matsui t., kawada k., suzuki h., 2001 seasonal changes of asparagus spears in relation to enzyme activities and carbohydrate content. sci. hortic., 88: 1-9. billau w., buchloh g., hartmann h.d., 1990 the influence of temperature, cultivar, soil-type and stalk diameter on the lignification of white asparagus. acta horticulturae, 271: 173-184. bouwkamp j.c., mccully j.e., 1975 effects of simulated non-selective mechanical harvesting on spear emergence of asparagus officinalis l. sci. hortic., 3(2): 157-162. clore w.j., carter g.h., drake s.r., 1976 preand postharvest factors affecting textural quality of fresh asparagus. j. amer. soc. hort. sci., 101: 576-578. dufault r.j., 1991 response of spring and summer-harvested asparagus to harvest pressures. hortscience, 26(7): 845-847. dufault r.j., 1994 a impact of forcing summer asparagus in coastal south carolina on yield, quality and recovery from harvest pressure. j. amer. soc. hort. 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j.t.k., van kruistrum g., 1987 some aspects of asparagus forcing. asparagus research newsletter, 5(2): 27-34. poll j.t.k., van kuistrum g., 1990 the effect of temperature on yield and quality of forced asparagus. acta horticulturae, 267:151-152. sanchez m.t., 1996 influence of variety and preharvest factors on the texture of white and green asparagus. alimentaria, 34(276): 29-34. shalaby t., sator j., haneklaus s., schnug e., 2004 influence of variety and cultivation on mineral elements and protein content of asparagus (asparagus officinalis l.). acta horticultuare, 629: 313-320. shou s., lu g., huang x., 2007 seasonal variations in nutritional components of green asparagus using the mother fern cultivation. sci. hortic., 112: 251-257. simón a., 1990 fibrousness of white asparagus varieties. investigación agraria, producción y protection vegetales, 5(2): 239-246. 58 slupski j., korus a., lisiewska z., kmiecik w., 2010 content of amino acids and the quality of protein in aseaten green asparagus (asparagus officinalis l.) products. intern. j. food sci. & techn., 45(4): 733-739. sosa coronel j., vest g., herner r.c., 1976 distribution of fiber content in asparagus cultivars. hortscience, 11: 149-150. takatori f.h., stillmann j.i., souther f.d., 1970 asparagus yields and plant vigor as influenced by time and duration of cuttings. calif. agric., 24(4): 8-10. wagenvoort w.a., van os e., simonse l., 1980 forcing asparagus in water in forcing trays. groenten en fruit, 36(23): 64-67. warman p.r., 1991 effects of manures and fertilizers on asparagus yield, fern mineral content and soil fertility. sci. hortic., 47(3-4): 231-237. 253 1. introduction root-restricted cultivation is an effective technique for saving resources, to control root environment, to anticipate yield and to regulate the quality of vegetables. for all these reasons, its use has significantly increased during the last decades in vegetable nurseries (shi et al., 2008). root restriction (rr) may occur when container size and/ or rooting volume is physically constrained (tschaplinski and blake, 1985; ismail and noor 1996; saito et al., 2008; mugnai et al., 2009), especially in greenhouse-grown horticultural crops (thomas, 1993). a reduced container volume stimulates the formation of a denser root mass, with decreased root growth (ismail and noor, 1996). together with a strong limitation of the soil available to the root system for water and nutrients uptake, rr also reduces canopy growth (ismail and noor, 1996; shi et al., 2008) by affecting many plant physiological and biochemical processes. the mechanism behind the reduced shoot growth is not yet fully understood. several hypotheses have been proposed: water and nutrient stresses (hameed et al., 1987), decrease in root respiration (shi et al., 2007), reduction in photosynthesis (shi et al., 2008), and synthesis and translocation of plant hormones (liu and latimer, 1995). however, contradictory results are reported as to which of these factors plays a significant role in the response of aerial plant parts to restricted root growth, with strong differences between species. leaf photosynthesis strongly depends on environmental conditions such as radiation, co 2 concentration and temperature. in addition to these environmental conditions, photosynthesis is subjected to internal regulation associated with sink demand for assimilates (marcelis, 1991). in the presence of a physical restriction to root growth, a major metabolic sink for photosynthetically fixed carbon at seedling stage (thomas and strain, 1991) may result in feedback inhibition mechanisms (shi et al., 2008). this investigation was therefore planned to determine the morphological and physiological changes induced in response to rr conditions and to determine the time frame within which these changes occurred. in particular, this research aims to study the link between leaf gas exchange parameters and carbohydrate production in regulating growth in pepper (capsicum annuum l.) plants. 2. materials and methods plant material experiments were carried out at the department of plant biology, university of pisa (italy). seeds of pepper (capsicum annuum l.) cv. sienor were sown in seedling flats filled with vermiculite and placed in a germinating room at constant temperature (25°c) and light intensity (300 mol m-2 s-1 ppfd). after germination, seedlings with the first true leaves were selected for uniformity and single plants were transplanted into 7 ml (root restricted, rr) starch accumulation in the leaves of root-restricted pepper affects plant growth by a feedback-inhibition of the photosynthesis h. al-debei*, s. mugnai** * department of horticulture and crop science, university of jordan, amman 11942, jordan. ** dipartimento di scienze delle produzioni vegetali, del suolo e dell’ambiente agroforestale, università degli studi di firenze, viale delle idee, 30, 50019 sesto fiorentino (fi), italy. key words: capsicum annuum l., carbohydrates, leaf gas exchange, root restriction. abstract: the mechanism behind the reduced growth occurring in plants subjected to root restriction is still not fully understood. therefore, this investigation was planned to determine the morphological and physiological changes induced in response to root volume reduction and to determine the time frame within which these changes occurred. in particular, this research focused on the effect of root restriction on growth, leaf gas exchange parameters, carbohydrate production and water relations in pepper (capsicum annuum l.). our results show that the reduced growth is mainly linked to a feedback inhibition of the photosynthesis, caused by a concurrent limited stomatal conductance (probably driven by both stomatal factors and hormonal substances) together with a strong accumulation of starch in the leaves. adv. hort. sci., 2011 25(4): 253-259 received for publication 30 september 2011 accepted for publication 26 october 2011 254 and 230 ml (control) speeding flats filled with vermiculite. flats were placed in a greenhouse and suspended 15 cm above the benches to facilitate air pruning of roots and to induce rr treatment throughout the experiment period. in each flat 24 seedlings were planted regardless of the original number of cells per flat to minimize the effect of mutual shading, to avoid light competition between plants and to allow for uniform plant density. in order to avoid any water or nutrient stress, a closed fertirrigation system controlled by a timer was established to supply water and nutrients at frequent and regular intervals. the nutrient solution was composed as follows: 10 mm no 3 -, 1 mm h 2 po 4 -, 8 mm k+, 4 mm ca2+, 1.5 mm mg2+, 1 mm so 4 2-, 0.04 mm fe2+ and microelements (ph 6.0, ec=1.2 ms/ cm). the nutrient solution was renewed every week. growth measurements five plants per treatment were sampled at weekly intervals. roots were carefully washed, and then plants were separated into leaves, stems and roots. leaf area was measured with an area meter (delta t-devices ltd., cambridge, uk), plant height was estimated using a ruler, and dry weight for each organ was obtained after oven drying (48 h at 70°c). leaf gas exchange measurements net co 2 assimilation (a), stomatal conductance (g) and transpiration (e) measurements were performed weekly (n=5) on the central sector of the youngest fully expanded leaf by using an open system (cms 400, heinz walz, effeltrich, germany) connected to an assimilation chamber and equipped with a high sensitivity irga (binos, leybold haeraeus, germany) under temperature (24°c) and growing light (400 μmol/m*s par) conditions provided by a mercury vapour lamp (osram hqi-ts 250 w/ndl). calculation of all the parameters was performed following von caemmerer and farquhar (1981) using a specific software (diagas 2.02, walz, effettrich, germany). water use efficiency (wue) was calculated as the ratio between a max and e max . for each crop species, e, a and g were also measured under different light intensities (0, 20, 50, 100, 200, 400, 600, 800 and 1000 μmol/m*s) as described above. a piece of black cloth was used to provide complete darkness, whereas different layers of wire mesh with very small holes were used to provide the required light intensity. chlorophyll content five leaf disks (10 mm diameter) were randomly taken from the uppermost fully expanded leaves at weekly intervals, and extracted in 2 ml of n,n-dimethylformamide for 24 h in the dark. absorbance was then determined for each sample using a spectrophotometer at 647 and 663 nm. chlorophyll a and b contents, and a/b ratio were calculated according to moran (1982). determination of total, osmotic and turgor potentials leaf water potential measurements were taken on the same leaf immediately after measuring gas exchange (n=5). total water potential (ψw) was determined using a pressure chamber (pardossi et al., 1991). osmotic potential (ψs) of the leaf xylem sap was determined using an osmometer (precision system, usa) by determining the freezing point depression of the sample. leaf turgor potential (ψp) was calculated using the following equation (eq. 1): ψp = ψw ψs (eq. 1) measurement of sugar content leaf, stem, and root samples (approx. 50 mg each) were taken at weekly intervals (n=5) and directly freeze-dried in liquid nitrogen. samples were homogenized and extracted with 1 ml hot 80% ethanol, boiled for 5 min, centrifuged at 12000 rpm for 15 min and then the supernatant was collected. the pellet was extracted again as described above, and the supernatant was collected again. at the end, the pellet was evaporated to remove any excess of ethanol. particulates including starch were suspended in 1 ml of koh 20 mm, boiled and centrifuged at 8000 rpm for 15 min and the supernatant was collected. the extract from ethanol was used for sucrose, glucose and fructose determinations, and the extract from koh was used for starch determination. for sugar determination, two 200 μl aliquots from the ethanol extract were taken, one incubated for 30 min at 37°c with 100 μl solution containing invertase (1 mg invertase/ml na-acetate 50 mm at ph 4.6), and the other one with 100 μl solution containing na-acetate 50 mm at ph 4.6, they were then both brought to the final volume (1 ml) with a solution containing 100 mm tris-hcl, ph 7.6, 3 mm mgcl 2 , 2 mm atp, 0.6 mm nadp, 1 unit hexokinase and 1 unit glucose-6-p-dehydrogenase (incubated at 37°c for 30 min). absorbance at 340 nm was then measured by a spectrophotometer. the concentration of glucose in each solution was determined from glucose standard curves according to guglielminetti et al. (1995). the solution without invertase was used to calculate the amount of free glucose in the sample and the difference between the two gave the amount of sucrose (as glucose equivalent). for each of them, 10 μl of solution containing 15 μl of phosphoglucoisomerase in 150 μl of tris-hcl 300 mm at ph 7.6 were incubated at 37°c for 15 min, then absorbance at 340 nm was determined. the difference between the one without invertase and treated with phosphoglucoisomerase and the other without invertase at the first determination gave the amount of free fructose (as glucose equivalent). for starch determination, 100 μl of extract was incubated at 37°c for 1 h with 100 μl solution of na-acetate 100 mm ph 5.2/10 u α-amylase. this solution was then incubated with 100 μl of na-acetate 100 mm ph 4.6/10 u amyloglucosidase at 55°c for 1 h. finally, the solution was boiled and centrifuged to eliminate denaturated protein from α-amylase and amyloglucosidase. 100 μl of this solution was taken and brought to 300 μl with distilled water; starch analysis (as glucose equivalent) was then carried out as mentioned above for glucose. 255 statistical analysis data were analyzed by one-way anova, and means (n=5) were separated using duncan’s multiple range test (p≤0.05). statistical analysis was performed using graphpad prism 4.0 (graphpad software). 3. results and discussion root volume reduction greatly affected growth parameters, confirming several previous results concerning growth depression induced by rr in many horticultural crops (see for example kharkina et al., 1999; saito et al., 2008; shi et al., 2008), but scarcely in pepper (ismail and davies, 1998). total dry weight significantly decreased starting from day 30 after emergence with reducing container size (fig. 1a). in detail, rr pepper plants showed a 3.85-fold lower total dry weight compared to control at the end of the experiment. leaf area was also greatly affected by volume reduction: rr plants showed a 4.15-fold reduction (fig. 1b) at the end of the experiment. rr plants appeared to be smaller (reduced height values) (fig. 1c), denoting a slackened development compared to control plants, with a preferred allocation of dry matter in the root system than in the aerial system, as demonstrated by a slight increase in the root:shoot ratio (fig. 1d). rr generally caused an increase in root:shoot ratio (mugnai et al., 2000), with roots growing in smaller volume forming a highly branched mat. the increased root:shoot ratio reported by some researchers for many crop species subjected to rr might be attributed to an increased substrate temperature in smaller containers in conjunction with a possible temperature dependence of root elongation, as suggested by hurley et al. (1998). during the first month, no significant differences were noticed in leaf gas exchange parameters. stomatal conductance (g) significantly decreased in rr plants (fig. 2a) table 1 chlorophyll content (a, b and a/b ratio) measured at weekly intervals from day 22 to the end of the experiment in leaves collected from control and root-restricted (rr) plants day control plants root-restricted plants (rr) chl a (mg cm-2) chl b (mg cm-2) a/b chl a (mg cm-2) chl b (mg cm-2) a/b 23 12.469 4.485 2.780 11.453 4.170 2.746 30 13.387 4.756 2.814 13.107 4.642 2.823 37 14.671 5.639 2.601 13.845 5.259 2.632 44 16.274* 6.347* 2.564 14.058* 5.589* 2.515 51 17.784* 7.450* 2.387 11.493* 4.768* 2.410 * indicates significantly different values between the two treatments for the same parameter and date for p≤0.05 (n=5), when means were separated by duncan’s test. fig. 1 growth parameters measured at weekly intervals from day 23 to the end of the experiment in both control and root-restricted (rr) plants:total dry weight (a), leaf area (b), plant height (c) and root:shoot ratio (d). * indicates significantly different values for p≤0.05 (n=5), when means were separated by duncan’s test. fig. 2 leaf gas exchange parameters measured at weekly intervals from day 23 to the end of the experiment in both control and root-restricted (rr) plants: stomatal conductance (a), net co 2 assimilation (b) and transpiration (c). * indicates significantly different values for p≤0.05 (n=5), when means were separated by duncan’s test. 256 from day 36, leading to a significant reduction in both net co 2 assimilation (a, fig. 2b) and transpiration (e, fig. 2c) until the end of the experiment. the reduction in a was not related to a decrease in the chlorophyll content of rr plants (table 1), as significant differences between the two treatments were noticed for chlorophyll a, b, and a/b ratio only after 43 days from the beginning of the experiment. also, rr plants showed increased instantaneous water-use efficiency values (wue) (fig. 2d), as reported for several species under stress (blum, 2009). rr treatment also affected leaf gas exchange parameters’ response to light (fig. 3). all the parameters (g, a and e) strongly decreased their values, leading to less pronounced response curves. in details, photosynthetic parameters, such as dark respiration, light compensation point and maximum co 2 assimilation, started to significantly decrease after 37 days (table 2). leaf water status did not seem to be the cause of the stomatal closure in rr plants, as total water potential (fig. 4a) and turgor potential (fig. 4c) did not show any significant difference in either of the treatments, even if slight reductions in total water potential and osmotic potential were measured at the end of the experiment in rr plants. table 2 dark respiration (dr, µmol co 2 m-2 s-1), light compensation point (lcp, µmol m-2 s-1 par), maximum co 2 assimilation (amax, µmol co 2 m-2 s-1), water-use efficiency (wue, µmol co 2 mmol h 2 o-1) measured every two weeks from day 22 to the end of the experiment in leaves collected from control and root-restricted (rr) plants day control plants root-restricted plants (rr) dr lcp amax wue dr cp amax wue 23 -1.07 14.06 17.31 5.47 -0.9 12.83 15.11 5.51 37 -3.31* 41.56* 24.87* 5.59* -2.75* 71.50* 6.67* 6.34* 51 -2.93 42.17* 15.71* 5.55* -2.87 85.56* 6.46* 6.65* * indicates significantly different values between the two treatments for the same parameter and date for p≤0.05 (n=5), when means were separated by duncan’s test. fig. 3 light saturation curves for the three leaf gas exchange parameters (g, a and e) measured every two weeks from day 23 to the end of the experiment in both control and root-restricted (rr) plants. 257 this result also confirmed that no symptom of water stress ever occurred during the experimental period, leading to a positive feedback about our experimental system. our results reveal that rr significantly reduces g, as previously noticed by other authors on different species (ismail and noor, 1996; kharkina et al., 1999), and that g is the primary cause of the reduction in a in rr plants, suggesting a stomatal factor limiting the photosynthetic rate under rr conditions (shi et al., 2008). however, the decline in g was not correlated to a concurrent decline in total water potential, as leaf tissues were able to maintain a high level of turgor during the whole experiment. therefore, other factors should be involved in the stomatal closure. it has been suggested that rr induces a reduction in g through a decrease in the supply of growth substances from roots to shoots and/or an imbalance in root and shoot hormones. for example, shi et al. (2008) reported that shoot growth suppression might be caused by the influence of aba originating from the restricted roots. ismail and davies (1998) found that the slight increase in xylem sap [aba] measured in pepper plants could not account for the reduction in leaf growth and g. they suggested that insufficient aba synthesis occurred to trigger the processes that cause reductions in leaf growth and g. sugar content determination led to interesting results. sucrose content significantly increased in rr plants starting from day 37 (fig. 5a). also, rr treatment led to a clear increase in glucose content (fig. 5b) and a concurrent decrease in fructose content (fig. 5c) together with a great accumulation of starch (fig. 5d). in particular, starch accumulation in the tissues began early in the developmental process (day 29). starch was mainly compartmentalised in the leaves (fig. 6a) of rr plants, whereas no significant differences were noticed both in stems (fig. 6b) and in roots (fig. 6c) between control and rr plants, except for day 33. the decline in a observed in rr conditions was often interpreted as a feedback inhibition by carbohydrate accumulation (pezeshki and santos, 1998). plant fig. 4 leaf water status determined at weekly intervals from day 23 to the end of the experiment in both control and root-restricted (rr) plants:total water potential (a), osmotic potential (b) and turgor (c). * indicates significantly different values for p≤0.05 (n=5), when means were separated by duncan’s test. fig. 5 sugar content measured at weekly intervals from day 23 to the end of the experiment in both control and root-restricted (rr) plants:total sucrose (a), total glucose (b), total fructose (c) and total starch (d). * indicates significantly different values for p≤0.05 (n=5), when means were separated by duncan’s test. 258 growth is strongly affected by leaf photosynthetic activity, since photosynthates are essential either as the source of carbon used for the build-up of organic compounds or as the source of energy needed for biochemical reactions involved in growth and maintenance processes. growth rate may regulate photosynthesis either through effects on the supply of growth substances translocated into leaves or through effect on the translocation rate of photosynthates from leaves to the growing organs (carmi et al., 1983). the accumulation of photosynthates is influenced by the rate of their translocation to the sink organs (sonnewald and willmitzer, 1992), and sink demand for photosynthates has a marked influence on source leaf photosynthesis, which is greatly dependent on sink strength, considered as a product of sink size and sink activity (sonnewald and willmitzer, 1992). however, sink size is determined by different parameters. roots are recognized as a metabolic sink that influences the partitioning of photosynthetically fixed carbon (gifford and evans, 1981; robbins and pharr, 1988). sink limitation caused by rr can greatly reduce leaf photosynthetic rate in many crop species (hameed et al., 1987; ismail and noor, 1996; whiley et al., 1999; shi et al., 2008), and reduced translocation of assimilates from leaves (robbins and pharr, 1988; kharkina et al., 1999). rr often promotes an accumulation of non-structural carbohydrates in the stem and leaves in response to the lack of the active sinks (nishizawa and saito, 1998), meaning that the difference in the growth rate between rr and control treatments was not due to a decrease in assimilates’ supply to the organs whose growth was restricted (mandre et al., 1995). our results suggest that the role of the leaves as sink organs may increase when root growth is extremely limited by volume restriction and a relatively larger amount of carbohydrate may accumulate in the canopy. a new shoot to root equilibrium may be established for an increased function of leaves and stem, together with a concurrent diminished function of the roots. therefore, it can be concluded that as a result of reduced vegetative growth an excess of assimilates was produced which could not be used for growth, and thus accumulated in the form of starch, as also indicated by shi et al. (2008). accumulation of nonstructural carbohydrates in the leaves in response to rr could provide a feedback mechanism that reduces carbon metabolism (thomas and strain, 1991). starch accumulation may reduce net photosynthetic rate by avoiding intracellular co 2 transport (shi et al., 2008). however, contradictory results were obtained by rieger and marra (1994), suggesting that reduced co 2 assimilation cannot always be explained by a feedback inhibition of carbohydrates. the relatively low maximum assimilation (a max ) rates for container-grown plants compared to field-grown plants may be attributed to containers restricting the root sink, thus causing the photo assimilate supply to exceed the capacity of demand (i.e. end-product inhibition of photosynthesis) as indicated by whiley et al. (1999). in conclusion, our results show that growth reduction by rr is mainly linked to a photosynthetic limitation, caused by a reduced stomatal conductance (probably driven by both stomatal factors and hormonal substances) and a strong accumulation of starch in the leaf tissues, which led to a feedback inhibition of the photosynthetic process. acknowledgements the authors are particularly indebted to prof. franco tognoni for scientific support. references blum a., 2009 effective use of water (euw) and not water-use efficency (wue) is the target of crop yield improvement under drought stress. field crops research, 112(2-3): 119-123. fig. 6 starch content in the different plant organs measured at weekly intervals from day 23 to the end of the experiment in both control and root-restricted (rr) plants:leaf (a), stem (b) and roots (c). * indicates significantly different values for p≤0.05 (n=5), when means were separated by duncan’s test. 259 carmi a., hesketh j.d., enos w.t., peters d.b., 1983 interrelationships between shoot growth and photosynthesis, as affected by root growth restriction. photosynthetica, 17: 240-245. gifford r.m., evans l.t., 1981 photosynthetic carbon partitioning and growth. annual review plant physiology, 32: 485-509. guglielminetti l., perata p., alpi a., 1995 effect of anoxia on carbohydrate metabolism in rice seedlings. plant physiology, 108: 735-741. hameed m.a., reid j.b., rowe r.n., 1987 root confinement and its effects on the water relations, growth and assimilate partitioning of tomato. annals of botany, 59: 685692. hurley m.b., rowarth j.s., trought m.c.t., barnes m.f., rowe r.n., 1998 variations in water availability and temperature in the root environment during root volume restriction studies. new zealand journal of crop and horticultural science, 26: 127-134. ismail m.r., davies w.j., 1998 root restriction affects leaf growth and stomatal response: the role of xylem sap aba. scientia horticulturae, 74: 257-268. ismail m.r., 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carbon dioxide. plant physiology, 96: 627-634. thomas t.h., 1993 effects of root restriction and growth regulator treatments on the growth of carrot (daucus carota l.) seedlings. plant growth regulation, 13: 95-101. tschaplinski t.j., blake t.j., 1985 effects of root restriction on growth correlations, water relations and senescence of alder seedlings. physiologia plantarum, 64: 167-176. von cammerer s., farquhar g.d., 1981 some relationships between the biochemistry of photosynthesis and the gas exchange of leaves. planta, 153: 376-387. whiley a.w., searle c., schaffer b., wolstenholme b.n., 1999 cool orchard temperatures or growing trees in containers can inhibit leaf gas exchange of avocado and mango. j. of amer. soc. for hortic. sci., 124: 46-51. impaginato 75 adv. hort. sci., 2023 37(1): 75­82 doi: 10.36253/ahsc­13943 non­destructive determination of ripening in melon fruit using time­resolved spectroscopy m. vanoli 1 (*), g. cortellino 1, v. picchi 1, m. buccheri 1, m. grassi 1, f. lovati 1, l. marinoni 2, p. levoni 2, a. torricelli 2, 3, l. spinelli 3 1 consiglio per la ricerca in agricoltura e l’analisi dell’economia agraria, centro di ricerca ingegneria e trasformazioni agroalimentari (crea‐ it), via g. venezian, 26, 20123 milano, italy. 2 politecnico di milano, dipartimento di fisica, piazza leonardo da vinci, 32, 20133 milano, italy. 3 istituto di fotonica e nanotecnologie, consiglio nazionale delle ricerche (ifn‐cnr), piazza leonardo da vinci, 32, 20133 milano, italy. key words: carotenoids, cucumis melo l., ethylene, juiciness, quality, trs. abstract: the aim of this work was to explore the feasibility of time­resolved reflectance spectroscopy (trs) in determining the ripening degree and the quality of orange­fleshed melons. sixty ‘honey moon’ melons were measured by trs in the 540­1064 nm range and classified as less (lem), medium (mem), and more (mom) mature according to increasing values of μa540. mom fruit showed yellower peel color, slightly more orange pulp, higher juiciness and higher carotenoid contents than lem ones. mom fruit also showed higher internal ethylene concentration and lower firmness than lem ones, even if the differences were not significant. the μa540 was positively related to internal ethylene, carotenoid accumulation, and juiciness, indicating that μa540 was linked to different ripening processes in melons. however, the relationship between μa540 and total carotenoid content was not as high as expected due to the low variability of pulp color and of carotenoid content. changes in flesh color toward a more orange shade were accompanied by increased juiciness and ethylene production and by carotenoid accumulation, while changes in peel color were associated with changes in flesh firmness and juiciness. in con­ clusion, the absorption coefficient measured at 540 nm (µa540) by trs could be used to sort melons in different ripening degrees; however, its applicability will need to be evaluated on a larger number of fruits and on other varieties. 1. introduction melon (cucumis melo l.) fruit are particularly appreciated by con­ sumers for their sweetness, flavour, texture, and attractive flesh color, as well as for their nutritional and phytochemical properties. melons are a good source of carotenoids, ascorbic acid, and phenolic compounds (gómez­garcía et al., 2020; manchali et al., 2021; singh et al., 2022). melon quality depends on the balance between sugars, organic acids, volatiles, and texture characteristics (kyriacou et al., 2018). these traits are strongly affected by genotype, agro­climate conditions, harvest matu­ (*) corresponding author: maristella.vanoli@crea.gov.it citation: vanoli m., cortellino g., picchi v., buccheri m., grassi m., lovati f., marinoni l., levoni p., torricelli a., spinelli l., 2023 ­ non‐ destructive determination of ripening in melon fruit using time‐resolved spectroscopy. ­ adv. hort. sci., 37(1): 75­82. copyright: © 2023 vanoli m., cortellino g., picchi v., buccheri m., grassi m., lovati f., marinoni l., levoni p., torricelli a., spinelli l. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 10 november 2022 accepted for publication 20 january 2023 ahs advances in horticultural science https://doi.org/10.36253/ahsc-13943 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2023 37(1): 75­82 76 rity, post­harvest handling, and storage conditions (kyriacou et al., 2018). melons picked at the optimal harvest maturity have premium quality as maturity at harvest has a large impact on sugars, volatiles, and texture. during ripening, rind color changes from green to yellow, orange, or creamy yellow, depend­ ing on melon genotype; mesocarp color turns from pale green to orange in orange­fleshed melons, along with carotenoid accumulation; firmness and acidity decrease, while sucrose as well as soluble solids, vita­ min c and ph markedly increase (beaulieu and lea, 2007; tristan et al., 2022). melons harvested mature develop the strongest flavour, whereas fruit harvest­ ed early develop a less aromatic flavour: esters are predominant in ripe melons, aldehydes are present at higher concentration in immature fruit while a sharp increase of alcohols is typical of overripe mel­ ons (senesi et al., 2005; beaulieu and lea, 2007; vallone et al., 2013; lignou et al., 2014). during ripening, the sensory scores for color intensity, fruity aroma, juiciness, and sweetness increased while the sensory scores for firmness and sourness generally decreased (vallone et al., 2013). in climacteric mel­ ons, ethylene was produced at higher levels in fully ripe fruits and was negatively related to firmness and positively related to sensory juiciness and fruity flavour and aroma (senesi et al., 2005; vallone et al., 2013). for more than two decades, several efforts have been made to measure quality characteristics of mel­ ons in a nondestructive way (zeb et al., 2021). in industrial fruit sorting, accuracy, cost, and detection speed are important factors. thus, spectroscopic techniques being fast, simple, and cost­effective, have been widely studied. in melons, vis­nir spec­ troscopy (sanchez et al., 2014; lu et al., 2015; khurnpoon and sirisomboon, 2018; zeb et al., 2021), computer vision (calixto et al., 2022) and hyperspec­ tral imaging (sun et al., 2017; cho et al., 2022) were studied with less or more successful results for pre­ dicting soluble solids, moisture, pulp color, and firm­ ness and for classifying fruit according to different sweetness degree or as suitable or not for harvesting. among spectroscopic techniques, time­resolved reflectance spectroscopy (trs) is gaining increasing interest in assessing fruit quality. due to its accuracy in measuring optical properties in deep tissues it allows the evaluation of maturity and internal defects of horticultural products with a relatively thick sur­ face layer (lu et al., 2020). trs allows the complete o p ti c a l c h a r a c t e r i z a ti o n o f a d i ff u s i v e m e d i u m through the measurements of the absorption (μa) and of the scattering (μs) coefficients by probing flesh at a depth of 1­2 cm with no or limited influence from the skin (cubeddu et al., 2001; rizzolo and vanoli, 2016). while scattering is related to the struc­ tural properties of fruits and vegetables, absorption depends on the chemical composition of the tissue, mainly on the presence of pigments such as chloro­ phylls, anthocyanins, and carotenoids (lu et al., 2020). trs has been mainly applied in post­harvest studies on fruit and vegetables for estimating fruit ripening, for the detection of internal defects, and for discriminating fruit with different texture and senso­ ry characteristics (rizzolo and vanoli, 2016). the absorption coefficient measured at 670 nm (μa670) at harvest was shown to be a maturity index for various fruit, such as peaches, nectarines, plums, pears, and apples, as it declines with fruit ripening (vanoli and buccheri, 2012). the μa540 (carotenoid tail) has been used as a non­destructive maturity index for man­ goes, as it was able to classify intact mangoes of dif­ ferent cultivars according to pulp color and to the contents of total and individual carotenoids (rizzolo and vanoli, 2016; vanoli et al., 2018). trs has not yet been studied on melons, so the aim of this work was to explore the feasibility of trs in determining the ripening degree and the quality of orange­fleshed melons. 2. materials and methods fruit the experiment was carried out on ‘honey moon’ (cucumis melo l. cantalupensis) melons. ‘honey moon’ fruits have round shape, medium size, a smooth grey­green skin that turns yellow when fully ripe. fruits has firm, deep orange flesh and show high sugar content and a pleasant and almost exotic aroma. in order to have high variability in fruit matu­ rity, 60 melons were picked and selected in a pack­ inghouse in sermide (mantova­italy) on a peel color basis (15 fruit for four color stages: blue, gray­green, yellow­green and yellow). then fruit were immedi­ ately transported to the crea­it lab in milan, mea­ sured by trs in the 540­1064 nm range and classified as less (lem), medium (mem) and more (mom) mature according to increasing μa540 values (low μa540=less mature fruit; high μa540=more mature fruit). all the 60 melons were also individually evalu­ ated for color (peel and flesh) and for standard matu­ vanoli et al. ‐ ripening determination of melons by trs 77 rity indices (flesh firmness, soluble solids content, acidity). among the 60 melons, 20 fruits covering the whole range of μa540 (i.e., the highest, the lowest and 18 intermediate values of μa540), were chosen for internal ethylene concentration, total carotenoid content and juiciness analyses. time‐resolved reflectance spectroscopy (trs) whole melons were measured by trs on two opposite sides in the fruit equatorial region. a portable compact setup working at discrete wave­ lengths developed at politecnico di milano (zhao et al., 2022) was employed. the light source is a super­ continuum fiber laser (sc450­6w, fianium, uk) pro­ viding white­light pulses, with duration of a few picoseconds. two custom­made filter wheels loaded w i t h o v e r a l l 1 4 b a n d ­ p a s s i n t e r f e r e n c e fi l t e r s (maxline series, semrock, ny, usa, and techspec series, edmund optics, new jersey, usa) are used for spectral selection in the range 540­1064 nm. light is delivered to the sample by a 200 μm core step­index fiber and collected by a 1 mm core graded­index fiber; interfiber distance was set to 1.5 cm. a pair of filter wheels identical to the previous one is used for cutting off the fluorescence signal originated from the sample when it is illuminated in the visible spec­ tral region. the light then is detected by a cus­ tomized silicon photomultiplier module (martinenghi et al., 2016) and the photon time­of­flight distribu­ tion is measured by a time­correlated single­photon counting board (spc­130, becker&hickl, germany). the instrumental response function has a full width at half maximum of about 100 ps and the typical acquisition time is 1 s per wavelength. a semi­infinite model for photon diffusion in a turbid medium was used to analyze trs data to assess the bulk optical properties of the samples (martelli et al., 2009) to obtain the estimates of µa and µs at each wavelength. peel and flesh color color was measured on the equatorial region on two opposite side of whole fruit and, after cutting longitudinally the melons, on two opposite portions of the flesh, 1.5 cm from the fruit edge. a cm2600d spectrophotometer (konica minolta sensing, inc., osaka, japan) with the primary illuminant d65 and 2° observer, was used to perform the color measure­ ments in the l* (lightness) a* (green­red), b* (yellow­ blue) color space. from a* and b* values, hue (h°) and chroma (c*) were computed according to h° = arctangent (b*/a*) × 360/(2×3.14) and c*= (a*2 + b*2)­2. in the flesh also the color spectra (350­740 nm) were considered. color readings were averaged for each fruit. standard maturity indices and juiciness flesh firmness after having cut the melons into two parts along the longitudinal axis, flesh firmness was measured on two opposite sides in the equatorial part of the fruit (around 1,5 cm from the fruit edge) using an 8 mm diameter plunger mounted on a ta­xt plus texture analyzer (stable micro systems, godalming, uk) at a crosshead speed of 200 mm min­1. the two measure­ ments were averaged for each fruit. soluble solids content (ssc) and titratable acidity two longitudinal slices around 4 cm thick, taken from two opposite sides of each fruit (in the same places of the firmness measurements) were frozen for the evaluation of soluble solids content (ssc) and of titratable acidity. after thawing, ssc was deter­ mined on few juice drops from each slice by using an automatic refractometer (rfm81, bellingham­stanley ltd., england). titratable acidity (ta) was measured by titrating 10 g of juice plus 50 ml of distilled water with 0.1 n naoh to ph 8. juiciness juiciness was measured on pulp cylinders (diame­ ter = 15 mm; height = 10 mm) taken from two oppo­ site sides of the equatorial part of the fruit. each cylinder was compressed between two plates with a ta­xt plus texture analyzer (stable micro systems, godalming, uk) at a deformation rate of 100 mm min­1 by a compression of 50% of the original height of the cylinder (eccher zerbini et al., 1999). this method correlated with sensory analysis, and only the juice that could be easily and quickly released by the pulp cylinder was measured. juiciness was calcu­ lated as the ratio between the juice weight spilled out after compression and the cylinder’s original weight. internal ethylene concentration 1 ml sample of internal gas was taken from the seed cavity of each melon using a syringe equipped with a 15 cm long, 15­gauge needle. the sample was injected in a dani gs 86.10 gas chromatograph and analyzed for the ethylene content following the con­ ditions reported by rizzolo et al. (2005), using a deac­ tivated aluminum oxide f1 (80­100 mesh) column (1/8 in × 200 cm alltech italia, sedriano, italy), col­ umn temperature 100°c, injection temperature 100°c, and a flame ionization detector temperature adv. hort. sci., 2023 37(1): 75­82 78 of 225°c. ethylene was identified and quantified by relating the peak area of the sample to that of a 10 μl l­1 external standard and was expressed as ppm. total carotenoid content t w o s l i c e s p e r f r u i t w e r e f r o z e n f o r t o t a l carotenoid content analysis. 1 g of frozen sample was r a p i d l y h o m o g e n i z e d ( u l t r a ­ t u r r a x , i k a ­ w e r k , germany) in 5 ml of an ice­cold solution of hexane: acetone:ethylacetate (2:1:1) containing 1% of 1% butylated hydroxitoluene (bht) in methanol. the homogenate was sonicated for 10 min, centrifuged at 10000 g for 15 min at 4°c and the organic phase was collected. the absorbance of the organic phase, after proper dilution, was measured spectrophotometri­ cally at 450 nm. total carotenoid content was esti­ mated using the molar extinction coefficient for b­ carotene in hexane (139200 l mol­1 cm­1), as reported in craft and soares (1992). statistical analysis data were submitted to analysis of variance (statgraphics ver. 7, manugistic inc., rockville, md, usa) considering trs maturity class as a source of variation, and means were compared by tukey’s test at p≤0.05%. the relationships between μa540 and quality characteristics and the relationships among quality parameters were studied using regression analysis. for each parameter, the model with the higher performance was considered. only the models with correlation coefficient r>0.50 were considered. 3. results trs absorption spectra trs absorption spectra of ‘honey moon’ melons (fig. 1a) showed a maximum at 980 nm (correspond­ ing to water), high absorption values at 540 nm (cor­ responding to the tail of carotenoids) and very high variability in the chlorophyll absorption range (610­ 690 nm). the variability in the chlorophyll absorption was not linked to the presence of chlorophyll in the pulp but depended on the presence of a green layer (about 3.5­7 mm) between the rind and the pulp, which interfere with trs measurements that pass through the pulp, up to 2 cm. the color spectra of the pulp confirmed the trs data, showing a very low absorption in the chlorophyll range and a very high absorption in the carotenoid range (400­500 nm) (fig. 1b). then, the absorption coefficient measured at 540 nm (µa540) was chosen as a possible ripening i n d e x f o r m e l o n s d u e t o i t s c o r r e l a ti o n t o t h e carotenoid content in the pulp, as previously found by rizzolo and vanoli (2016) and vanoli et al. (2018) in mangoes. the µa540 ranged from 0.194 to 0.663 cm­1. melons were ranked according to increasing µa540 (increasing maturity) and sorted into three maturity classes: low (µa540 = 0.383±0.008 cm ­1), m e d i u m ( µ a5 4 0 = 0 . 4 5 1 ± 0 . 0 0 4 c m ­ 1) a n d m o r e mature (µa540 = 0.,537±0.014 cm ­1). peel and flesh color peel and pulp color significantly changed with trs maturity classes (table 1). peel color turned from green to yellow­green with advancing trs maturity class, as b* and c* values increased and h° values decreased from lem to mom melons. pulp color became slightly more orange, as b* and h° were lower in mom fruit than in lem ones. there was no fig. 1 ­ trs absorption spectra (a) and pulp color spectra (b) of ‘honey moon’ melons. vanoli et al. ‐ ripening determination of melons by trs 79 main change in the pulp color of melons, as b* ranged from 29.3 to 38.1 and h° from 64.5 to 70.2. standard maturity indices, juiciness, internal ethylene and total carotenoids content flesh firmness ranged from 7.09 to 27.55 n, ssc from 7.4 to 12.2% and acidity from 1.70 to 5.57 g l­1 citric acid. flesh firmness, ssc and acidity were not significantly affected by trs maturity classes (fig. 2 a, b, c). however, firmness and acidity were lower and ssc was higher in mom fruit than in lem ones. juiciness ranged from 6.86 to 22.27% and was signifi­ cantly higher in mom fruit than in the lem ones (fig. 2 d). internal ethylene concentration ranged from 51.9 to 153.3 ppm. it did not significantly change with trs maturity class, even if the lem fruit showed the lowest concentration and mom ones the highest (fig. 2e). total carotenoids content ranged from 16.95 to 28.25 mg kg­1 fw; it was significantly higher in mom fruit than in lem ones (fig. 2f). regression analysis the results of regression analysis between µa540 and quality parameters are summarized in table 2. the µ a540 was significantly related to juiciness (r=0.53), internal ethylene concentration (r=­0.86) and total carotenoid content (r=0.66). the µa540 was also positively related to skin color (except for a*) table 1 ­ peel and pulp color parameters of 'honey moon' melons in relation to trs maturity classes means in the same column followed by different letters are statistically different at p≤0.05 (tukey's test). fig. 2 ­ flesh firmness (a), soluble solids content (b), acidity (c), juiciness (d), internal ethylene concentration (e) and total carotenoid content (f) in ‘honey moon’ melons in relation to trs maturity classes. bars refer to se. maturity stage l* a* b* c* h° peel less mature 72.4 a ­6.1 a 23.3 b 24.2 b 106.5 a medium mature 73.4 a ­5.4 a 26.0 ab 26.7 ab 103.4 ab more mature 76.0 a ­5.4 a 28.8 a 29.4 a 101.2 b pulp less mature 62.1 a 14.0 a 33.9 ab 36.7 a 67.6 a medium mature 63.2 a 14.4 a 34.7 a 37.6 a 67.5 a more mature 60.0 a 14.4 a 33.3 b 36.3 a 66.6 b 80 adv. hort. sci., 2023 37(1): 75­82 with r<0.5. no significant correlation was found between µa540 and pulp color, flesh firmness, ssc and acidity. significant relationships were also found among peel color, firmness and juiciness (table 3). firmness was related to b* peel (r=0.62), c* peel (r=0.60) and to h° peel (r=0.58); juiciness was related to b* peel (r=0.57), c* peel (r= 0.55) and to h° peel (r= 0.54). as for pulp color, total carotenoid content was positive­ ly related to a* pulp (r=0.68) and negatively to h° pulp (r= −0.88); internal ethylene was related to a* pulp (r= −0.61) and juiciness to h° pulp (r= 0.58). significant relationships were also found between juiciness and total carotenoids (r= 0.64) and between juiciness and firmness (r= 0.73). 4. discussion and conclusions ‘honey moon’ melons used in this experiment showed pulp color similar to that of fruit of the same cultivar picked at commercial maturity, even if firm­ ness, acidity and ssc had lower values (cavicchi and pasotti, 2004). total carotenoid content showed val­ ues typical of orange­fleshed fruit (saladie et al., 2015; singh et al., 2022). internal ethylene concen­ tration confirms that ‘honey moon’ is a climacteric genotype, as there was an increase in ethylene pro­ duction with advancing fruit maturity (senesi et al., 2005; vallone et al., 2013; saladie et al., 2015). trs measurements of melons showed some prob­ lems. in fact, the absorption in the chlorophyll range cannot be used as a maturity index as already done for other fruits, such as apples, pears, peaches and nectarines (rizzolo and vanoli, 2016), since chloro­ phyll was almost absent in the melon pulp while it was present in a layer just under the peel. on the other hand, µa540 was able to distinguish mom mel­ ons from lem ones, as mom fruit showed yellower peel color, slightly more orange pulp, higher juiciness and higher carotenoid contents, in agreement with kyriacou et al. (2018), senesi et al. (2005) beaulieu and lea (2007), vallone et al. (2013), saladie et al. (2015) and tristan et al. (2022). mom fruit also showed higher internal ethylene concentration and lower firmness than lem ones, even if the values showed high variability and the differences were not significant. the µa540 was also positively related to internal ethylene, carotenoid accumulation and juici­ ness, indicating that µa540 is linked to different ripen­ ing processes in melons. however, the relationship between µa540 and total carotenoid content was not as high as expected (r=0.66) and as previously found in mangoes when r ranged from 0.78 to 0.91 depend­ ing on the cultivar (vanoli et al., 2018). it seems that in ‘honey moon’ melons, the range of µa540 (0.194­ 0.663 cm­1) is quite similar to that of mangoes (0.117­ 0.835 cm­1), while the variability in total carotenoids table 2 ­ regression models between µa540, juiciness, internal ethylene concentration and total carotenoid contents for each significant regression, the following data are given: r= correlation coefficient; p=significance of the model (***, p<0.001; **, p<0.01; *, p<0.05), and model type. dr= double reciprocal; rx=reciprocal x. table 3 ­ regression models between firmness, juiciness, inter­ nal ethylene concentration, total carotenoid content and pulp color for each significant regression, the following data are given: r= correlation coefficient; p=significance of the model (***= p<0.001; **= p<0.01; *= p<0.05) and mt= model type. ry=reciprocal y; lin=linear; rx=reciprocal­x; dr=double recipro­ cal. r p model type juiciness 0.531 * dr internal ethylene ­0.864 *** rx total carotenoids 0.664 ** dr r p mt firmness b* peel 0.616 *** ry c* peel 0.604 *** ry h° peel 0.576 *** lin juiciness firmness 0.733 *** rx b* peel 0.565 * dr c* peel 0.548 * dr h° peel 0.538 * rx h° pulp 0.577 * rx ethylene a* pulp ­0.617 * ry carotenoids juiciness 0.640 * dr a* pulp 0.679 ** dr h° pulp ­0.881 *** lin vanoli et al. ‐ ripening determination of melons by trs 81 (16.95­28.25 mg kg­1 fw) and in pulp color (h° pulp= 64.5­70.2) was narrower in melons than in mangoes (total carotenoids= 5­56 mg kg­1 fw; h° pulp=71­104). m o r e o v e r n o c o r r e l a ti o n w a s f o u n d i n m e l o n s between µa540 and pulp color while in mangoes µa540 was negatively related to h° pulp with r= 0.83­ 0.98 (vanoli et al., 2018). in ‘honey moon’ fruit, with advancing trs ripening degree, the changes in flesh color toward a more orange shade were accompa­ nied by increased juiciness values and ethylene pro­ duction and by carotenoid accumulation, while changes in peel color toward a yellow shade were associated with fruit softening. in conclusion, the absorption coefficient mea­ sured at 540 nm (µa540) by the trs technique could be used to sort melons in different ripening degrees; however, its applicability will need to be evaluated on a larger number of fruits and on other varieties. acknowledgements this study was funded from the italian ministry of agriculture, agridigit project, subproject agrofiliere (d.m. 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university of yasouj, yasuj, iran. 2 horticulture crops research department, natural resources research and education center of hormozgan, areeo, bandar abbas, iran. key words: abiotic stress, membrane integrity, tropical fruit. abstract: guava fruit is a tropical fruit thus sensitive to the chilling injury. in this study the effects of melatonin (known to protect membrane integrity and to help to face abiotic and biotic stress) is evaluated for reduction of chilling injury during postharvest. guava fruits were dipped into 10, 100 and 1000 μmol l­1 melatonin solutions, then kept at cold storage (10±1°c and 90% relative humidity) for 21 days. several parameters including chilling injury, malondi­ aldehyde content, electrolyte leakage and increased total phenolic compounds and antioxidant activity, phospholipase d and lipoxygenase activity were mea­ sured after treatment. measurements were made every 7 days during the stor­ age. results showed that melatonin decreased chilling injury, malondialdehyde content, electrolyte leakage and increased total phenolic compounds and antioxidant activity compared to the control. also, results indicated that chill­ ing injury of guava fruit by using melatonin decreased through increasing integrity of membrane and reducing phospholipase d and lipoxygenase activity. thus, melatonin can be a useful treatment for decreasing postharvest chilling disorder of guava fruit. 1. introduction guava (psidium guajava l.) is one of the most important fruits of tropical and sub­tropical regions in the world. the fruits are delicious, rich in vitamin c and minerals (deepthi et al., 2016). there is a great demand of guava fruits in both domestic and international markets for fresh and processing purposes. cold storage is one of postharvest technologies for maintaining quality of horticultural crops until human consumption. however, guava is sensitive to chilling disorder of cold storage (temperature of below 12°c). signs of chilling injury include irregular ripening and surface pitting on the fruit which decreases quality of fruit (etemadipoor et al., 2020). resistance to the chilling temperature related to several factors. one of t h e m o s t i m p o r t a n t f a c t o r s i s m a i n t a i n i n g m e m b r a n e i n t e g r i t y (*) corresponding author: a_mirshekari@yu.ac.ir citation: mirshekari a., madani b., 2022 ­ decreasing postharvest chilling injury of guava fruit by using melatonin treatment. ­ adv. hort. sci., 36(1): 37­ 42. copyright: © 2022 mirshekari a., madani b. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 27 october 2021 accepted for publication 13 february 2022 ahs advances in horticultural science https://doi.org/10.36253/ahsc-12222 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2022 36(1): 37­42 38 (wongsheree et al., 2009). membrane integrity can be measured using leakage, malondialdehyde con­ tent, lipoxygenase and phospholipase d (aghdam et al., 2014). several methods used to decrease chilling injury symptoms of fruit rely on the use of hot water and uv­c (pongprasert et al., 2011). melatonin plays in fruit ripening and senescence and membrane integrity and protection against abiotic and biotic stresses (rastegar et al., 2020). melatonin treatment maintained quality of in pear (liu et al., 2019), peach (gao et al., 2016), and grape (xu et al., 2018) and tomato (aghdam et al., 2019) fruits during cold storage. however, melatonin effects on reducing chilling injury of guava fruit have not been evaluated during cold storage. therefore, the purpose of this study was to investigate melatonin effects on chilling injury reduction. 2. materials and methods the guava fruits (green stage maturity) were bought from a commercial orchard in hormozgan province, iran and the uniform sized fruits were transferred to the laboratory. twelve fruits per four replications were dipped into 10 (t2), 100 (t3) and 1000 (t4) μmol l−1 melatonin solutions for 10 min. distilled water was used as the control (t1). fruit were kept at cold storage (10±1°c and 90% relative humidity) for 21 days. several parameters were mea­ sured soon after treatment, and then the measure­ ments were made every 7 days during the storage. finally, the parameters were checked again one day after exposing to the ambient temperature (25±1°c). the measurements included chilling injury index assessment, percentage of ion leakage, malondialde­ hyde content, weight loss, titratable acidity, soluble solids concentration (ssc), ascorbic acid, total pheno­ lic content (tpc), antioxidant activity, phospholipase d and lipoxygenase activity. chilling injury (ci) index assessment, percentage of ion leakage (el) and malondialdehyde (mda) content ci index was assessed subjectively with a scale from 1 to 5, where 5= >50% surface pitting area, 4= 31­50% surface pitting area, 3= 16­30% surface pit­ ting area, 2= 1­15% surface pitting area, 1= 0% no chilling symptoms. el was measured by using method described by madani et al. (2016) and results expressed as per­ centage. the mda content was determined based on the method described by wang et al. (2015). the content of mda was expressed as nmol g­1 fw. weight loss, soluble solids concentration (ssc), titratable acidity (ta), ascorbic acid, total phenolic content (tpc) and total antioxidant activity weight loss was measured based on initial and final experiment at 7­day intervals during storage using a digital balance, and results were expressed as percentage. soluble solids concentration (ssc) and ta of pulp tissues were measured by using the method of ali et al. (2011) and the results were expressed as %ssc and %ta, respectively. ascorbic acid was measured using dye method described by ranggana (1986) and results were expressed as mg 100 g­1 fresh weight (fw). tpc were assayed using folin­ciocalteu procedure (singleton and rossi, 1965). results were expressed as mg of gallic acid equivalents (gae) per gram of fresh weight (mg gae g­1 fw). the dpph assay was measured according to the method described by mirshekari et al. (2019). results were expressed as percentage. phospholipase d and lipoxygenase activity phospholipase d and lipoxygenase assay was determined based on the method described by aghdam and mohammadkhani (2014). one unit of phospholipase d was defined as the amount of enzyme that catalyzed the formation of 1 nmol d­ nitrophenol h­1. one unit of lipoxygenase was defined as the amount of enzyme which causes an increase in absorption of 0.01 min­1 at 234 nm and 25°c when linoleic acid was used as the substrate. protein con­ tent was estimated according to bradford (1976). enzymes activities were expressed as units per mil­ ligram of protein. statistical analysis experiments were carried out using completely randomized design. four replications per treatment used for this study. data were analyzed using (sas) version 8.2 (sas institute inc., cary, nc, usa). variation sources were storage days and treatments and means were compared with duncan’s multiple range test (dmrt) at significance level of 0.05. mirshekari and madani ‐ decreasing chilling injury of guava by melatonin 39 3. results and discussion chilling injury (ci), electrolyte leakage (el), and malondialdehyde (mda) i n t h e p r e s e n t s t u d y , m e l a t o n i n t r e a t m e n t reduced chilling injury index of guava fruit after 7 days of chilling storage when compared with the con­ trol (table1). moreover, ci increased with storage time (table 1). at the end of storage day control (t1) had the severe chilling injury index (4.8) with highest pitting signs, while t4 had the lowest chilling injury index of 3.2. usually, ci happens at the cell mem­ brane, and maintaining its integrity reduces ci (mirdehghan et al., 2007; mirshekari et al., 2020). accordingly, electrolyte leakage has been used as an indicator of membrane damage. in this study, el increased during storage for control and treated melatonin fruits (table 1). however, at the end of storage day el was lower in t3 and t4 compared to the t2 and t1 (table 1). these results showed the role of melatonin in maintenance of membrane integrity. comparable results have been stated for sapota fruit by mirshekari et al. (2020). researches have shown that melatonin treatments affect elec­ tron flow acceleration in mitochondria to maintain membrane integrity (tan et al., 2013). a s s h o w n i n t a b l e 1 , m d a o f c o n t r o l f r u i t increased during storage. also, lower mda content were observed in all melatonin treated fruits com­ pared to the (t1) after 21 days of chilling storage (table 1). one of the first events in the ci is mem­ brane lipid peroxidation. mda is the final product of lipid peroxidation (imahori et al., 2008). lower tem­ peratures are the main inducers of oxidative damage which produce higher ros and change the ratio of unsaturated fatty acids to saturated forms (antunes and sfakiotakis, 2008). melatonin treatments low­ ered mda accumulation of sapota fruit (mirshekari et al., 2020). weight loss, ssc, ta, ascorbic acid, tpc and total antioxidant activity weight loss was at the highest rate (14.3 %) in the control fruits (t1) after 21 days. treated fruit (t4) showed lower weight loss (6.7%) compared to the t1 and t2 at the end of storage day (table 2). weight loss is an index for assessing quality of fruits during storage (yaman and bayonidirli, 2002). skin strength properties of fruit by using melatonin treatment might lower weight loss. our results are comparable with rastegar et al. (2020) who indicated that the weight loss was decreased by using melatonin treatment in mango. ssc and ta concentration are main factors for fruit quality judgment. the initial ssc value of this study was 5.2% in control fruits (t1) and increased table 1 ­ melatonin treatments (0, 10, 100 and 1000 μmol l−1) effects on chilling injury index (ci), electrolyte leakage (el) and malondi­ aldehyde (mda) content in guava fruit stored at 10°c for up to 21 days (z) small and capital letters show significant differences by dmrt at p= 0.05 between treatments in columns, and storage time for each parameter, respectively. treatment (μmol l­1) storage (day) 0 7 14 21 ci 0 (t1) 0 a d z 2.3 a c 3.5 a b 4.8 a a 10 (t2) 0 a d 2.1 a c 3.2 ab b 4.1 b a 100 (t3) 0 a d 1.1 b c 2.5 bc b 3.8 b a 1000 (t4) 0 a d 1.3 b c 2.1 c b 3.2 c a el (%) 0 (t1) 6.5 a d 18.0 a c 32.3 a b 49 a a 10 (t2) 6.5 a d 16.5 a c 30.2 a b 47 a a 100 (t3) 6.5 a d 12.5 b c 22.2 b b 31.7 b a 1000 (t4) 6.4 a d 10.5 b c 20.5 b b 32.3 b a mda (nmol g‐1 fw) 0 (t1) 5.5 a d 8.3 a c 12.3 a b 15.6 a a 10 (t2) 5.3 a d 7.2 b c 11.6 a b 13.1 b a 100 (t3) 5.0 a b 5.6 c b 7.6 b a 8.5 c a 1000 (t4) 5.6 a b 5.9 c b 8.2 b a 9.1 c a adv. hort. sci., 2022 36(1): 37­42 40 ta content compared to the t1 and t2 (table 2). the higher amounts of ta in melatonin treated fruit can be related to the reduction of respiration rate during storage (han et al., 2004). ascorbic acid decreased during storage. after 21 days of storage, ascorbic acid was higher in t3 and t4 compared to the t1 (table 2). one of the most signifi­ cant signs of the nutrient value of fruits is ascorbic acid. the ascorbic acid reduction during storage can be related ascorbic acid oxidase (choudhary et al., 2016). melatonin treatment increases oxidative stress resistance by increasing ascorbic acid (gao et al., 2016). our results are comparable with gao et al. (2016) who stated that melatonin treatment main­ (z) small and capital letters show significant differences by dmrt at p= 0.05 between treatments in columns, and storage time for each parameter, respectively. table 2 ­ melatonin treatments (0, 10, 100 and 1000 μmol l­1) effects on weight loss, soluble solids content (ssc), titratable acidity (ta), ascorbic acid, total phenolic content (tpc) and total antioxidant activity (taa) in guava fruit stored at 10°c for up to 2 treatment (μmol l−1) storage (day) 0 7 14 21 weight loss (%) 0 (t1) 0 a d z 4.5 a c 9.5 a b 14.3 a a 10 (t2) 0 a d 3.2 b c 8.3 b b 13.4 a a 100 (t3) 0 a d 2.6 c c 5.2 c b 8.6 b a 1000 (t4) 0 a d 2.1 c c 4.2 d b 6.7 c a ssc (%) 0 (t1) 5.2 a d 8 a c 12.1 a b 13.4 a a 10 (t2) 5..1 a c 7.3 ab b 11.7 a a 12.6 a a 100 (t3) 5.2 a d 6.6 bc c 8.2 b b 9.2 b a 1000 (t4) 5.2 a c 5.8 c bc 6.9 c ab 7.5 c a ta (%) 0 (t1) 0.9 a a 0.6 a b 0.4 b c 0.3 bc c 10 (t2) 0.9 a a 0.7 a b 0.4 b c 0.2 c c 100 (t3) 0.9 a a 0.8 a a 0.7 a a 0.5 ab b 1000 (t4) 0.9 a a 0.7 a ab 0.6 a bc 0.5 a c ascorbic acid (mg 100 g‐1 fw) 0 (t1) 135.5 a b 145.6 b a 121.5 b c 118.3 c c 10 (t2) 136.4 a b 149.2 ab a 126.5 b c 122.4 bc c 100 (t3) 136.2 a b 151.2 a a 132.7 a c 127.2 ab d 1000 (t4) 134.5 a b 150.3 ab a 133.8 a b 129.1 a c tpc (mg gae g−1 fw) 0 (t1) 175 a d 210.6 c a 189.7 b b 181.5 d c 10 (t2) 172 a c 201.2 d a 191 b b 188.5 c b 100 (t3) 173.2 a d 233 a a 213.2 a b 200.7 b c 100 (t4) 174 a d 224.2 b a 218.7 a b 214.2 a c taa (%) 0 (t1) 52.3 a c 62..1 b b 70 c a 65.2 b ab 10 (t2) 52 a c 64.2 b b 72.7 c a 67 b b 100 (t3) 53.2 a c 72.4 a b 78.5 a a 74.2 a b 1000 (t4) 52.7 a b 75 a a 77.2 ab a 75.5 a a during storage (table 2). however, at the end of stor­ age, t3 and t4 melatonin treatments decreased ssc content compared to untreated (t1) and t2 treat­ ments. lower ssc content in this research is in agree­ ment with results of liu et al. (2018) who showed that ssc decreased with melatonin treatments during storage. lower amounts of ssc might be due to the slower respiration rate and a weaker metabolic activ­ ity due to the reduced rate of carbohydrate hydroly­ sis. table 2 shows ta content of treated and control fruits. ta content was the highest at harvest day (0.9%). however, ta content decreased during stor­ age. at the end of storage t3 and t4 showed higher mirshekari and madani ‐ decreasing chilling injury of guava by melatonin 41 tained ascorbic acid content of peach. tpc and antioxidant activity decreased during chilling storage. however, melatonin treated fruit had higher tpc and antioxidant capacity compared with control (table 2). it has been shown that mela­ tonin treatment increased tpc by regulating gene expression in phenyl propanoid pathway (zhang et al., 2016). moreover, liu et al. (2018) stated that melatonin treatment increased tpc and dpph scav­ enging capacity of strawberry. this indicated that melatonin showed positive effect on antioxidant activity of guava fruit. phospholipase d and lipoxygenase activity p h o s p h o l i p a s e d a n d l i p o x y g e n a s e a c ti v i t y increased during chilling storage. however, mela­ tonin treatment decreased their activities during storage (table 3). similarly, melatonin treatment decreased ci signs and inhibited phospholipase d and lipoxygenase activity of sapota fruit (mirshekari et al., 2020). studies indicated that ci was achieved by the activities of membranous lipolytic enzymes like phospholipase d and lipoxygenase which catalyse peroxidation of polyunsaturated fatty acids and are believed to be major contributors to ci in plant tissue (aghdam and mohammadkhani, 2014). 4. conclusions results of this study showed that melatonin treat­ ments during cold storage of guava fruit decreased chilling injury, soluble solids concentration, malondi­ aldehyde content, electrolyte leakage, phospholipase d and lipoxygenase activity and increased titratable acidity, ascorbic acid, total phenolic compounds and a n ti o x i d a n t a c ti v i t y c o m p a r e d t o t h e c o n t r o l . accordingly, we found that melatonin application reduced ci of guava fruit with enhancing membrane integrity and decreasing phospholipase d and lipoxy­ genase activity. concerning the different treatments, we found out that while 10 μmol l­1 produced results which were not significantly different from control treat­ ment, 100 μmol l­1 and 1000 μmol l­1 were signifi­ cantly different for ci, weight loss, soluble solids con­ centration, total phenolic compounds and phospholi­ pase d activity at the end of storage. in conclusion, melatonin application at 1000 μmol l­1 can be recom­ mended to be used to decrease ci in guava fruit under cold storage. references a g h d a m m . s . , m o h a m m a d k h a n i n . , 2 0 1 4 ­ 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(ed.). cab international, wallingford, oxfordshire, uk, 2012, pp. 280. isbn 978-1-84593-767-6. € 110.00. the purpose of the book (which contains 19 chapters) is to provide a general description of the world of peppers. even though difficulties exist with pepper taxonomy, the book presents a good introduction where all disputes are clearly elucidated and discussed. botany and cultivation techniques are widely described and throughout the book several unanswered questions relating to the lack of information about peppers emerge, keeping up the reader’s interest. some information can result a bit redundant in the chapters, which are written by different authors, but this offers the reader the opportunity to easily jump from one part to another. moreover, all the chapters are clearly written and present a thorough bibliography, making the book suitable for both scientists and common devotees of peppers. the book can be used as a manual for cultivation, starting from a single plant to a massive production of peppers, with both biotic and abiotic stresses properly described. particular attention is given to cultivation problems such as pests and weed control and for descriptions and reports, causes and effects of various types of diseases are also listed. other common problems relating to peppers and analyzed in the book include irrigation, fertilization and soil composition, and economic and logistic problems regarding field and greenhouse production of plants. from the molecular side, the book is a good anthology of the majority of molecules involved in peppers and underlying their functions and physiology. while a large part of the work is fully dedicated to field production, a brief chapter is dedicated to tissue culture of peppers despite their recalcitrant nature. finally, germination and transplant techniques are illustrated, making this book complete and useful for a wide audience. diego comparini stolo. bibliographische findmitell zur gartenkultur. band i. italien. strumento bibliografico sulla cultura dei giardini. volume 1. italia. (bibliographic tools for the culture of gardens. volume 1. italy) schneider u., and g. gröning. wernersche verlgasgesellschaft mbh, worms, germany, 2009. pp. 576. isbn 978-3-88462-248-3 this volume, dedicated to italy, is the first of nine in the series stolo on the history and cultural theory of gardens and related topics in the center-west of europe. each volume treats a single country and can be consulted individually, as the material presented pertains to the subject country. at the same time, the ample literature reported opens up international connections, presenting to the reader the geographic impact and extension of the treated topic. the work required to compile the bibliographic material was carried out between 2004 and 2007, and required numerous trips to public and private libraries. in its complex, this work by stolo is a notable tool to access the history of gardens in various european countries, and it is presented in a way that is easy-to-use and in-depth. this particular work on italy is divided into eight chapters: sull’importanza sistematica delle conclusioni letterarie nella cultura dei giardini. (the systematic importance of literary conclusions in terms of garden culture) 2. strumenti bibliografici generali e documentazione letteraria. (general bibliographic tools and literary documentation) 3. bibliografie specifiche e strumenti bibliografici sulla cultura dei giardini e materie affini. (specific bibliographies and bibliographic tools regarding the culture of gardens and related topics) 4. manuali e opere generali sulla cultura dei giardini come contributi e temi speciali. (manuals and general works on the culture of gardens as contributions and special themes) 5. materiale primario. (primary material) 6. periodici e collane (periodicals and series) 7. ricerca in internet. (internet research) 8. organizzazioni, federazioni, associazioni, biblioteche nella cultura dei giardini. (organizations, federations, associations and libraries for the culture of gardens). enrico rinaldelli impaginato 67 adv. hort. sci., 2023 37(1): 67­73 doi: 10.36253/ahsc­13838 co2 modified atmosphere packaging: stress condition or treatment to pre­ serve fruit and vegetable quality? m. cefola 1, i. capotorto 1, v. lippolis 2, s. cervellieri 2, a. damascelli 2, r. cozzolino 3, b. de giulio 3, b. pace 1 (*) 1 institute of sciences of food production, national research council (cnr) c/o cs‐dat, via m. protano, 71121 foggia, italy. 2 institute of sciences of food production, national research council (cnr), via g. amendola, 122/o, 70126 bari, italy. 3 institute of food science, national research council (cnr), via roma, 64, 83100 avellino, italy. key words: carbon dioxide, fermentative metabolites, modified atmosphere packaging, respiration rate, short­term treatment. abstract: in addition to the adoption of proper temperature and relative humidity, the selection of an atmosphere surrounding packaged fresh produce with reduced o2 and/or increased co2 is one of the most widely used and use­ ful tools to prolong the shelf­life of horticultural crops. however, as o2 and/or co2 values that might cause injury are strictly related to the commodity, they should be optimized for each product. here three study cases are reported about the application of modified atmospheres (ma), with different co2 con­ centrations (0­40 kpa), to table grapes (cv. italia) and sweet cherries (cv. ferrovia) and, as a short­term treatment (48 h at 0°c), to fresh­cut artichokes (cv. violet de provence). in each trial, the effect of high co2 treatment on quali­ ty parameters was observed during cold storage. concerning table grape ‘italia’, our results show that low co2 (up to 10 kpa) ma preserved the quality and sensory parameters of the fruit, whereas high co2 (> 20 kpa) caused a fer­ mentative metabolism. as for sweet cherries ‘ferrovia’, 20 kpa co2 ma helped to maintain the quality traits during storage. on the other hand, this fruit proved to be sensitive to co2 accumulation (over 20 kpa) in hypoxic conditions, since it caused an increase in respiration rate and the biosynthesis of volatile fermentative metabolites. finally, for fresh­cut artichokes, a short­term co2 treatment, up to 10kpa, reduced respiration rate and browning index, preserv­ ing the volatile profile, while high co2 (40 kpa) may have caused fermentative metabolism. in conclusion, the application of a ma enriched in co2 has been shown to have different effects on the quality parameters of the three prod­ ucts, in agreement with the fact that co2 sensibility depends on each specific fruit or vegetable under study. 1. introduction fruits and vegetables are perishable products, and extending the keeping quality during their postharvest life represents one of the main (*) corresponding author: bernardo.pace@ispa.cnr.it citation: cefola m., capotorto i., lippolis v., cervellieri s., damascelli a., cozzolino r., de giulio b., pace b., 2023 ­ co2 modified atmo‐ sphere packaging: stress condition or treatment to preserve fruit and vegetable quality? ­ adv. hort. sci., 37(1): 67­73. copyright: © 2023 cefola m., capotorto i., lippolis v., cervellieri s., damascelli a., cozzolino r., de giulio b., pace b. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 4 october 2022 accepted for publication 26 january 2023 ahs advances in horticultural science https://doi.org/10.36253/ahsc-13838 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2023 37(1): 67­73 68 goals of researchers in this field. it is widely known that, together with the proper temperature and rela­ tive humidity management, the gas composition sur­ rounding the product during storage, is one of the main factors that affect the quality of horticultural crops (kader, 2003). in general, the decrease in oxy­ gen, the increase in co2, or the association of both conditions are useful to preserve the physiological state of fruits and vegetables, reducing the rate of respiration, oxidative processes, and decay, thus pro­ longing their shelf­life. in contrast, inappropriate gas concentrations outside safe limits can cause stress conditions that lead to physiological disorders, devel­ o p m e n t o f o ff ­ o d o u r s d u e t o f e r m e n t a ti v e metabolism, or increases in susceptibility to decay (mangaraj and goswami, 2009). although low o2 and high co2 have similar effects, under modified atmo­ sphere packaging (map) conditions, elevated co2 is a major factor influencing the quality of fruits and veg­ etables (watkins, 2000). in addition, the sensitivity to elevated high co2 and/or low o2 levels depends on the commodity (toivonen and deell, 2002). it is influ­ enced by pre and postharvest factors, such as culti­ vars or stage of maturity, and by processing, since the oxygen consumption and the consequent co2 accumulation in fresh­cut produce is faster than in corresponding intact produce (francis et al., 2012). to obtain the beneficial effect of map, gas conditions should be optimized for each product. starting from these considerations, the aim of the present work was to compare the effect of different co2 concen­ trations in map on the quality of table grapes, sweet cherries and fresh­cut artichokes as case studies. 2. materials and methods table grapes (vitis vinifera l., cv. italia), sweet cherries (prunus avium l., cv. ferrovia), and artichokes (cynara cardunculus (l.) subsp. scolymus hayek, cv. violet de provence) were provided by local farms located in noicattaro and foggia (italy) and processed at the postharvest laboratory of cnr­ispa the day of harvest. selected bunches of table grapes (about 1 kg each) were placed in polyethylene terephthalate (pet) trays (model cl1/135 carton pack, rutigliano, italy). they were sealed with a vacuum sealer (model boxer 50 lavezzini vacuum packaging system, fiorenzuola d’arda, italy) in 30 x 40 cm polyamide/polyethylene (pa/pe) bags (orved s.p.a., musile di piave, italy) applying two modified atmosphere (ma) mixtures with different initial co2 concentrations plus 1 kpa of o2: 1.0/0.03 o2/co2 kpa (1 kpa­o2), and 1.0/20.0 o2/co2 kpa (1 kpa­o2 + 20 kpa­co2). unsealed bags were used as control (air). all samples (4 replicates per treatment) were analyzed at harvest and after 20 days of storage at 5°c for respiration rate (rr), rachis browning (rb), ethanol, and acetaldehyde contents. for sweet cherries, about 200 g of fruits, without defects or diseases, were placed in pet trays and sealed in 30 x 40 cm pa/pe bags with three ma mix­ tures: 1.0/0.03 o2/co2 kpa (1 kpa­o2), 16.0/20.0 o2/co2 kpa (16 kpa­o2 + 20 kpa­co2), and 1.0/20.0 o2/co2 kpa (1 kpa­o2 + 20 kpa­co2). samples stored in unsealed bags (air) were used as controls. all sam­ ples (3 replicates per treatment) were analyzed at harvest and after 21 days of storage at 5°c for rr, relative water content of peduncles (rwc), and volatile organic compounds (vocs). as for artichokes, the heads were trimmed, elimi­ nating the external bracts and cutting the stem. the obtained artichoke hearts were then cut into quar­ ters and dipped for 5 min in a solution of 1% ascorbic acid + 0.2% citric acid (w:v), drained, and randomly selected for different treatments. in particular, three replicates of 16 artichoke quarters were kept for the initial determinations, while the remaining quarters were closed in 50 x 50 polypropylene (pp) bags (carton pack® rutigliano, italy), about 600 g per bag, applying 4 ma mixtures with different initial co2 con­ centrations plus 10 kpa of o2: 10.0/10.0 o2/co2 kpa (co2­10kpa), 10.0/20.0 o2/co2 kpa (co2­20kpa), 10.0/30.0 o2/co2 kpa (co2­30 kpa), 10.0/40.0 o2/co2 kpa (co2­40 kpa). unsealed bags were used as con­ trol (air). after 48 h of storage at 0°c, all bags were opened, artichoke quarters were placed in open poly­ ethylene bags and analyzed after 48 h at 0°c plus 7 days of storage at 5°c for rr, browning index (bi) and vocs profile. the headspace gas composition (o2 and co2) within each ma package was monitored daily using a gas analyzer (checkpoint, pbi dansensor, ringsted, denmark). rr was measured initially (fresh) and at the end of the storage for each prod­ uct using a closed system, as reported by kader (2002 a). samples were put into 6 l sealed plastic jars, allowing the accumulation of co2 up to 0.1 kpa. for co2 analysis, 1 ml of gas sample was collected from the headspace of each jar and injected into a gas chromatograph (p200 micro gc, agilent, santa clara, ca, usa) equipped with dual columns and a thermal conductivity detector. carbon dioxide was analysed cefola et al. ‐ response of fruit and vegetable to different co2 levels in map 69 with a retention time of 16 s and a total run time of 120 s on a 10 m porous polymer (ppu) column (agilent, santa clara, ca, usa) at a constant tempera­ ture of 70°c. rr was expressed as ml co2 kg ­1 h­1. in table grapes, rb was scored on a rating scale from 1 to 5 (1= absence, 2= light; 3= moderate; 4= severe; 5= extreme) as reported by lichter et al. (2011), whereas for acetaldehyde and ethanol analysis, the procedure reported by cefola et al. (2018) was used. in sweet cherries, the rwc of peduncles was cal­ culated in percentage, as reported by cefola et al. (2018), while the vocs analysis was carried out, as reported by cozzolino et al. (2019). in fresh­cut artichokes, bi and vocs were evalua­ ted, as reported by capotorto et al. (2020). 3. results and discussion starting from the gas composition inside ma pack­ ages described above for each product, during stor­ age the concentrations of o2 and co2 changed due to the respiration of the products and gas permeation through packaging material, and the final gases com­ position were reported in table 1. as for table grapes, o2 concentrations decreased from 1 kpa to about 0.2 kpa or 0.3 kpa, in 1k pa­o2 and in 1 kpa­o2 + 20 kpa­co2 respectively, while co2 concentrations increased from 0.03 kpa to roughly 10 kpa in 1 kpa­o2 packages, and from 20 kpa to about 30 kpa in 1k pa­o2 + 20 kpa­co2 ma. for sweet cherries, in 16 kpa­o2 + 20 kpa­co2 bags, the o2 concentration gradually decreased, reaching the mean value of about 1 kpa after 21 days of storage. in 1 kpa­o2 and 1 kpa­o2 + 20 kpa­co2 samples, the initial o 2 concentration remained unchanged during the storage. on the other hand, the amount of co2 increased during conservation, reaching the final mean values of 25.7 kpa, 45.3 kpa and 42.4 kpa in 1 kpa­o2, 16 kpa­o2 + 20 kpa­co2 and 1 kpa­o2 + 20 kpa­co2 packages, respectively. finally, as for fresh­cut artichokes, no significant changes in gas composition inside bags were observed. results on table grapes are reported in table 2. in the fresh samples the rr measured was equal to 4.2 (± 0.4) ml co2 kg ­1 h­1; after 20 days of the storage, a reduction in rr was measured in air samples (3.0 ± 0.2 ml co2 kg ­1 h­1), while it remained almost con­ stant in table grapes samples treated with 1 kpa o2 (4.7 ± 0.6 ml co2 kg ­1 h­1). on the contrary the use of high co2 concentrations (>20 kpa) in the ma mixture (1 kpa­o2 + 20 kpa­co2) increased the value of rr resulting more than a 2­fold higher than fresh sam­ ple. significant differences were observed, for sample stored in air and 1 kpa­o2 + 20 kpa­co2 (table 2). as shown in table 1, browning of the table grapes rachis was found in all treatments after 20 days of storage. however, higher browning was observed in air and in 1 kpa­o2 + 20 kpa­co2 samples, whereas the use of 1 kpa­o2 was able to keep a light to moderate brown­ ing of the rachis (mean value of 2.5). after storage ethanol and acetaldehyde concentrations did not change from their initial values (4.2 and 0.6 mg l­1, respectively) in the air samples, whereas they signifi­ cantly increased in table grapes exposed to low (<10 kpa) or high co2 (>20 kpa) concentrations (table 2). moreover, samples exposed to 1 kpa­o2 + 20 kpa­co2 s h o w e d h i g h e r a c c u m u l a ti o n s o f e t h a n o l a n d acetaldehyde than table grapes packed in 1 kpa­o2. treatment initial kpa final kpa o2 co2 o2 co2 1 kpa­o2 1.0 0.04 0.2 10.0 1 kpa­o2 + 20 kpa­co2 1.0 20.0 0.3 30.0 air 20.0 0.03 20.0 0.03 table 1 ­ initial and final concentration of o2 and co2 for each treatment on table grape treatment respiration rate (ml co2 kg ­1 h­1) rachis browning (1­5) ethanol (mg l­1) acetaldhyde fresh 4.2 b 1.0 c 4.2 c 0.6 c 1 kpa­o2 4.7 b 2.5 b 2142 b 8.9 b 1 kpa­o2 + 20kpa­co2 9.0 a 3.6 a 3606 a 17.6 a air 3.0 c 4.4 a 5.8 c 0.6 c table 2 ­ effect of co2 treatments on respiration rate, rachis browning, ethanol and acetaldehyde contents of table grapes (vitis vinifera cv. italia) after 20 days of storage at 5°c mean values followed by different uppercase and lowercase letters indicate significant differences between fresh and treated sample, and within treatments at day 20, respectively, according to lsd test (p≤0.05). adv. hort. sci., 2023 37(1): 67­73 70 sweet cherries (table 3) showed an initial respira­ tion rate of 8.2 (± 0.3) ml co2 kg ­1 h­1 which increased 1.5 fold in air and more than 5 times in the other ma treatments. the highest rr was observed in 1 kpa­o2 + 20 kpa­co2 (48.9 ± 0.7 ml co2 kg ­1 h­1) followed by 16 kpa­o2 + 20 kpa­co2 (44.4 ± 0.6 ml co2 kg ­1 h­1) and 1 kpa­o2 (43.2 ± 0.1 ml co2 kg ­1 h­1). the rwc % of peduncle increased in all ma treat­ ments, maybe due to the high relative humidity inside the packages. the highest rwc % values were observed in low o2 treatments, probably thanks to the lower respiration rate of these samples. among vocs analysed, 1­pentanol, marker of sen­ sory alteration, was closely associated with negative aroma intensity which resulted directly described as pungent, and fermented flavour. whereas the reduc­ tion of hexenal and 2­hexenal were indicators of lost in freshness (cozzolino et al., 2019). as reported in table 2, 1­pentanol was not detect­ ed in fresh and air samples, while a significant increase of this alcohol was observed in the other ma treatments. samples treated with 1 kpa­o2 + 20 kpa­co2 had the highest value of 1­pentanol, while 1 kpa­o2 and 16 kpa­o2 + 20kpa­co2 ma treatments had similar values (table 2). as for hexanal, (table 3) statistical analysis did not show significant changes after 21 days at 5°c excepted for the treatment, 1 kpa­o2 + 20 kpa­co2 which showed a reduction with respect to the fresh sample. in contrast, 2­hexenal decreased significantly during storage, but no differ­ ences in its concentration were observed when com­ paring ma treatments (table 3). results of fresh­cut artichokes are reported in table 4. rr of the fresh sample was 120.8 (± 0.2) ml co2 kg ­1 h­1. after the short­term co2 treatments (48 h, 0°c) and 7 days of storage at 5°c, rr decreased significantly in all samples, except in artichokes treat­ ed with 40 kpa of co2. the lowest rr was detected in fresh­cut artichokes treated with co2­10 kpa (44.5 ± 4.3 ml co2 kg ­1 h­1), followed by co2­20 kpa and co2­ 30 kpa, which reported similar values (68.1 ± 1.1 and 63.9 ± 5.2 ml co2 kg ­1 h­1, respectively), and air (93.6 table 3 ­ effect of co2 treatments on respiration rate, relative water content (rwc) of peduncles, 1­pentanol, hexanal and 2­hexenal relative peak area (rpa) contents of sweet cherries (prunus avium cv. ferrovia) after 21 days of storage at 5°c mean values followed by different uppercase and lowercase letters indicate significant differences between fresh and treated sample, and within treatments at day 21, respectively, according to lsd test (p≤0.05). table 4 ­ effect of short­term co2 treatments on respiration rate, browning index, ethanol and hexanal relative peak area (rpa) con­ tents of fresh­cut artichokes (cynara cardunculus cv. violet de provence) after 7 days of storage at 5°c mean values followed by different uppercase and lowercase letters indicate significant differences between fresh and treated sample, and within treatments at day 7, respectively, according to lsd test (p≤0.05). treatment respiration rate (ml co2 kg ­1 h­1) relative water content of peduncles (%) 1­pentanol relative peak area (%) hexanal 2­hexenal fresh 8.2 b 53.9 b 0.0 b 112.3 ns 366.6 a 1kpa­o2 43.2 ac 65.4 aa 6.0 ab 47.3 ns a 118.0 b ns 16kpa­o2 + 20kpa­co2 44.4 ab 58 ab 7.6 ab 26.4 ns ab 99.9 b ns 1kpa­o2 + 20kpa­co2 48.9 aa 60.5 ab 18.6 aa 14.3 bb 55.7 b ns air 12.1 ad 53.6 ns c 0.0 ns c 40.5 ns ab 125.2 b ns treatment respiration rate (ml co2 kg ­1 h­1) browning index ethanol relative peak area (%) hexanal fresh 120.8 a 0.0 b 34.5 ns 41.7 ns co2­10 kpa 44.5 bd 131 ad 114.0 ns c 5.4 ns a co2­20 kpa 68.1 bc 137 abc 379.1 ab 5.0 ns a co2­30 kpa 63.9 bc 135 acd 482.5 ab 4.3 ns a co2­40 kpa 121.5 aa 140 ab 363.5 ab 2.1 bb air 93.6 bb 152 aa 886.3 aa 3.8 ns a cefola et al. ‐ response of fruit and vegetable to different co2 levels in map 71 ± 1.7 ml co2 kg ­1 h­1), while the highest rr was observed in co2­40 kpa (121.5 ± 0.2 ml co2 kg ­1 h­1). as expected, all samples developed browning after 7 days of storage, regardless of treatment. however, d i ff e r e n c e s i n t h e s e v e r i t y o f b r o w n i n g w e r e observed when comparing treatments at the end of the storage: the application of short­term co2 treat­ ments (from 10 kpa to 40 kpa) significantly reduced the incidence of browning compared to air samples and, among the co2 treatments, co2­10 kpa had the lowest browning index (table 4). considering all the vocs identified by hs ­spme/gc­ms analysis, ethanol and hexenal were, respectively, the most representative compounds of negative and positive aspects of fresh­cut artichokes (capotorto et al., 2020). a s s h o w n i n t a b l e 4 , e t h a n o l s i g n i fi c a n t l y increased during storage, except for the co2­10kpa treatment, where its concentration was similar to that of fresh samples. the highest ethanol concentra­ tion was found in air samples, followed by treat­ ments added with co2 at 20, 30, and 40 kpa that showed similar values. as for hexanal, it was signifi­ cantly lower only in fresh­cut artichokes treated with co2­40 kpa (table 4). for table grapes, data related to ethanol and acetaldehyde, together with the rr results, indicate that high co2 concentrations (>20 kpa) on this com­ modity may cause physiological injury and the induc­ tion of anaerobic metabolism. the present results are supported by similar find­ ings on the effect of high co2 concentrations (>20 kpa) on table grapes by cefola and pace (2016). high co2 concentrations also negatively influence the acceptability of table grapes by consumers: rachis browning, is, in fact, the main issue that limits the acceptability of table grapes by consumers (cefola et al., 2018). a similar effect of high co2 concentrations (>20 kpa) on the acceleration of rachis browning was previously observed on table grapes (crisosto et al., 2002; deng et al., 2006) and is a consequence of the stress induced by exposure to high co2 concentra­ tions (crisosto et al., 2002; liguori et al., 2015). for sweet cherries, considering that the highest rr was observed when ma with 20kpa co2 was applied, these results indicate that this co2 concen­ tration, especially when associated with low oxygen, can cause stress, as confirmed by voc analysis. similar behaviour in the production of c5 volatiles, s u c h a s 1 ­ p e n t a n o l , w a s p r e v i o u s l y o b s e r v e d (contreras et al., 2017; mastrandrea et al., 2017), and it seems to be favoured under low o2 and high co2 atmospheres. the present results on the cv. ferrovia are in contrast with previous results on other sweet cherries cultivars (kader et al., 1989; esturk et al., 2012), but those cultivars have better tolerance to high co2. it has been stated that the physiological suscepti­ bility of commodities to high co2 can be cultivar­ dependent, and is generally seen with vegetables and other fruit (watkins, 2000). results on fresh­cut artichokes indicate that the application of high co2 concentrations (around 40 kpa) has a negative effect on the shelf­life. similar results on the detrimental effect of high co2 were previously observed on fresh­cut arti­ chokes during storage (la zazzera et al., 2012, 2015). as observed for table grapes, sweet cherries, and fresh­cut artichokes, the exposure to elevated co2 atmospheres can stimulate respiration and ethylene production rates, indicating a stress response (kader, 2002 a). these increases in respiration might be related to the inhibition by high co 2 of several enzymes of the krebs cycle, including succinate dehydrogenase, which triggers anaerobic respiration or causes the accumulation of succinic acid, which is potentially toxic to cells (kays, 1991; varoquaux, 1991; kader, 2002 b). furthermore, for fresh­cut arti­ chokes, the intolerance to high co2 concentration and mechanical wounding enhances a different array of enzymatic pathways, many of which are associat­ ed with volatile accumulation, which lead to devel­ opment of off­flavors (salunkhe et al., 1976; la zazzera et al., 2015). 4. conclusions for table grapes, the storage in high co2 (>20 kpa) caused a severe increase in respiration rate, ethanol and acetaldehyde accumulation, and a decline in sen­ sory quality due to the rachis browning, all probably consequences of the induction of the anaerobic metabolism. the application of co2 up to 10kpa was able, instead, to keep the good quality table grapes during storage. sweet cherry (cv. ferrovia) is very sensitive to high co2 when it is applied together with low oxygen in ma, as indicated by responses in respiration rate, rel­ ative water content of the peduncles, and voc emis­ sions, with some of these responses being considered positive and some negative in relation to quality. 72 adv. hort. sci., 2023 37(1): 67­73 short­term treatment with high co2 (around 40 kpa) caused an increase in respiration rate and the induction of fermentative metabolism in fresh­cut artichoke. the application of co2 concentrations up to 10 kpa reduced respiration rate and tissue brown­ ing during storage in air at 5°c and preserved the fresh voc profile. application of short­term co2 might be a promising postharvest treatment to pre­ serve the quality and the volatile profile of fresh­cut artichokes during storage. acknowledgements the research leading to these results has received funding from puglia regional call “aiuti a sostegno dei cluster tecnologici regionali per l’innovazione”. continnova project: container innovativo isotermico intermodale equipaggiato con atmosfera controllata per il trasporto di prodotti ortofrutticoli freschi” (cod. vfqa3d0). references capotorto i., innamorato v., cefola m., cervellieri s., lippolis v., longobardi f., logrieco a.f., pace b., 2020 ­ high co2 short‐term treatment to preserve quality and volatiles profile of fresh‐cut artichokes dur‐ ing cold storage. ­ postharvest biol. technol., 160: 111056. cefola m., damascelli a., lippolis v., cervellieri s., l i n s a l a t a v . , l o g r i e c o a . f . , p a c e b . , 2 0 1 8 ­ relationships among volatile metabolites, quality and sensory parameters of ‘italia’ table grapes assessed during cold storage in low or high co2 modified atmo‐ spheres. ­ postharvest biol. technol., 142: 124­134. cefola m., pace b., 2016 ­ high co2‐modified atmosphere to preserve sensory and nutritional quality of organic table grape cv. ‘italia’ during storage and shelf‐life. ­ eur. j. hortic. sci., 81: 197­203. c o n t r e r a s c . , s c h w a b w . , m a y e r s h o f e r m . , gonzález­agüero m., defilippi b.g., 2017 ­ volatile compound and gene expression analyses reveal tempo‐ ral and spatial production of lox‐derived volatiles in pepino (solanum muricatum aiton) fruit and lox speci‐ ficity. ­ j. agric. food chem., 65: 6049­6057. cozzolino r., martignetti a., cefola m., pace b., capotorto i., de giulio b., montemurro n., pelli­ cano m.p., 2019 ­ volatile metabolites, quality and sensory parameters of “ferrovia” sweet cherry cold stored in air or packed in high co2 modified atmo‐ spheres. ­ food chem., 286: 659­668. crisosto c.h., garner d., crisosto g., 2002 ­ carbon dioxide‐enriched atmospheres during cold storage limit losses from botrytis but accelerate rachis browning of ‘red globe’ table grapes. ­ postharvest biol. technol., 26: 181­189. deng y., wu y., li y., 2006 ­ physiological responses and quality attributes of ‘kyoho’ grapes to controlled atmo‐ sphere storage. ­ lwt ­ food sci. technol., 39: 584­590. esturk o., ayhan z., ustunel m.a., 2012 ­ modified atmosphere packaging of “napoleon” cherry: effect of packaging material and storage time on physical, chemical, and sensory quality. ­ food bioprocess technol., 5(4): 1295­1304. francis g.a., gallone a., nychas g.j., sofos j.n., colelli g., amodio m.l., spano g., 2012 ­ factors affecting quality and safety of fresh‐cut produce. ­ critical rev. food sci. nutrition, 52(7): 595­610. kader a.a., 2002 a ­ methods of gas mixing, sampling and analysis, pp. 145­148. in: kader a.a. 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( e d . ) , postharvest technology of horticultural crops. univ. calif. danr, publication, 3311, oakland, ca, usa. kader a.a., 2003 ­ a perspective on postharvest horticul‐ ture (1978‐2003). ­ hortscience, 38(5): 1004­1008. kader a.a., zagory d., kerbel e.l., 1989 ­ modified atmosphere packaging of fruits and vegetables. ­ crit. rev. food sci. nutr., 28(1): 1­30. kays s.j., 1991 ­ postharvest physiology and handling of perishable plant products. ­ van nostrand reinhold inc., new york, ny, usa, pp. 526. l a z a z z e r a m . , a m o d i o m . l . , c o l e l l i g . , 2 0 1 5 ­ designing a modified atmosphere packaging (map) for fresh‐cut artichokes. ­ adv. hort. sci., 29(1): 24­29. la zazzera m., rinaldi r., amodio m.l., colelli g., 2012 ­ influence of high co2 atmosphere composition o n f r e s h ‐ c u t a r ti c h o k e q u a l i t y a tt r i b u t e s . ­ a c t a horticulturae, 934: 633­640. lichter a., kaplunov t., zutahy y., daus a., alcha­ natis v., ostrovsky v., lurie s. 2011 ­ physical and visual properties of grape rachis as affected by water vapor pressure deficit. ­ postharvest biol. technol., 59(1): 25­33. liguori g., sortino g., de pasquale c., inglese p., 2015 ­ effects of modified atmosphere packaging on quality parameters of minimally processed table grapes during cold storage. ­ adv. hortic. sci., 29(2­3): 152­ 154. mangaraj s., goswami t.k., 2009 ­ modified atmo‐ sphere packaging of fruits and vegetables for extending shelf‐life‐a review. ­ fresh produce, 3(1): 1­31. mastrandrea l., amodio m.l., pati s., colelli g., 2017 ­ effect of modified atmosphere packaging and temper‐ ature abuse on flavor related volatile compounds of rocket leaves (diplotaxis tenuifolia l.). ­ j. food sci. cefola et al. ‐ response of fruit and vegetable to different co2 levels in map 73 technol., 54(8): 2433­2442. salunkhe d.k., do j.y., maga j.a. 1976 ­ biogenesis of aroma constituents of fruits and vegetables. ­ crit. rev. food sci. nutr., 8: 161­190. toivonen p.m.a., deell j.r., 2002 ­ physiology of fresh‐ cut fruits and vegetables, pp. 99­131. ­ in: lamikanra o. (ed.) fresh‐cut fruits and vegetables. crc press, boca raton, ca, usa, pp. 480. varoquaux p., 1991 ­ ready‐to‐use fresh fruits and veg‐ etables. ­ revue générale froid, 81: 33­43. watkins c.b., 2000 ­ responses of horticultural commodi‐ ties to high carbon dioxide as related to modified atmosphere packaging. ­ horttechnology, 10(3): 501­ 506. 65 adv. hort. sci., 2011 25(1): 65-68 received for publication 20 october 2010. accepted for publication 18 february 2011. short note performance of tomato under greenhouse and open field conditions in the trans-himalayan region of india m.s. kanwar sher-e-kashmir university of agricultural sciences and technology of kashmir, regional agricultural research station, leh 194 101, ladakh, jammu and kashmir, india. key words: greenhouse, ladakh, open field, solanum lycopersicon, tomato. abstract: production of tomato is limited by harsh climate and a short growing season in the trans-himalayan ladakh region of india. the performance of five tomato genotypes was compared under polyhouse and open field conditions. the study revealed that the performance of all tested tomato genotypes is far superior in the polyhouse, as compared to open field conditions, for all the considered characters. ‘shivalik’ performed best with respect to yield characters followed by ‘pusa rohini’ under polyhouse conditions. however, in the open field, ‘pusa rohini’ showed the highest values, followed by ‘shivalik’. cultivation of tomato under the polyhouse produced 136.12% more yield per ha and 188.93% more fruits per plant compared to open field cultivation. therefore, tomato cultivation under protected conditions is advised for ladakh growing conditions, employing specific polyhouse-responsive varieties. 1. introduction tomato (solanum lycopersicon l.) is available throughout the year in india. however, in the state of jammu and kashmir, with the exception of the jammu region, it is mostly confined to the summer season. in the trans-himalayan ladakh region, production of tomato is limited by climate and a short growing season. ladakh has a harsh climate and extreme temperature fluctuations ranging from -37°c to +38°c. in ladakh, tomato can be grown in open conditions but yield remains poor with low quality and it remains weather-dependent. therefore, protected cultivation is a feasible answer for successful cultivation of tomato in this region. singh and asrey (2005) also recommended that cultivation of tomato in a greenhouse would help obtain high productivity and better return. therefore, it is useful to study tomato production potential in the ladakh region with respect to yield and horticultural traits under protected conditions (preferably in a zero-energy polyhouse) in comparison to the open field. 2. materials and methods the experiment was conducted under naturally ventilated polyhouse and open field conditions at the experimental farm, stakna (leh) of the regional agricultural research station (skuast-k) located at 3319 m amsl with latitude 33°58.551’ ns and longitude 77°41.995’ew. the climate of the area is typically dry temperate. five genotypes including four hybrids (ph5, shivalik, jaya and naveen 2000+) and one op variety (pusa rohini) were transplanted in a naturally ventilated polyhouse and the open field. planting distance was 60 x 30 cm. the design of the experiment was factorial rbd and material was replicated thrice. individual data of each location were also subjected to statistical analysis in rbd to have more authentic information with regard to tomato genotypes. data recorded on 13 characters were subjected to statistical analysis as per snedecor and cochran (1967). 3. results and discussion there were significant differences among tomatoes grown under polyhouse and open field condition for all the characters, except for locules per fruit, confirming thereby the certain role of polyhouse in the cultivation of tomato in the trans-himalayan region. similar 66 pu sa ro hi ni ph -5 sh iv ali k ja ya na ve en 20 00 + m ea n cd 0.0 5 (l ) cd 0.0 5 (g ) cd 0.0 5 (l xg ) pl an th eig ht (c m ) va rie tie s 91 .12 98 .12 12 5.7 0 12 2.9 0 16 9.2 0 12 1.4 0 10 .43 29 .95 ns 77 .50 56 .25 96 .25 95 .50 11 5.0 0 88 .10 12 .63 84 .31 77 .19 11 1.0 10 9.2 14 2.1 ns 10 .25 9.5 0 10 .00 10 .00 10 .00 9.9 5 1.1 4 ns ns 6.2 5 5.5 0 6.0 0 5.7 5 6.5 0 6.0 0 ns 8.2 5 7.5 0 8.0 0 7.8 8 8.2 5 ns 91 .5 89 .5 89 .5 91 .00 88 .50 95 .65 3.3 0 ns ns 95 .25 96 .25 93 .75 99 .00 94 .00 90 .00 ns 93 .37 92 .87 91 .62 95 .00 91 .25 ns 10 .25 9.5 0 10 .00 10 .00 10 .00 9.9 5 0.5 1 ns ns 6.2 5 5.5 0 6.0 0 5.7 5 6.5 0 6.0 0 0.6 7 8.2 5 7.5 0 8.0 0 7.8 8 8.2 5 ns 68 .50 67 .25 68 .75 71 .50 74 .50 70 .05 4.0 7 ns ns 39 .00 35 .50 38 .25 36 .75 39 .50 37 .80 ns 53 .75 51 .37 53 .50 54 .12 56 .88 ns l1 l2 m ea n l1 l2 m ea n l1 l2 m ea n l1 l2 m ea n l1 l2 m ea n st em gi rth (m m ) da ys to fir st ha rv es t no .o fh ar ve sti ng ha rv es td ur ati on (d ay s) ch ar ac ter s lo ca tio n ta bl e1 pe rfo rm an ce of to m ato ge no ty pe su nd er po ly ho us ea nd op en co nd iti on sf or pl an tc ha ra cte rs l1 = po ly ho us e. l2 = op en fie ld . results were obtained by singh and asrey (2005) as they found excellent tomato crops in polyhouses compared to the outside environment. significant differences were observed among tomato genotypes pooled over locations for number of fruits per plant, yield per plant, yield per ha, fruit length, tss and locules per fruit. however, no polyhouse x genotype interaction was noted. comparative performance of tomato genotypes is presented here. plant characters in polyhouse and open conditions, ‘naveen 2000+’ had significantly higher plant length. results are in conformity with those of singh and singh (2000) under open field conditions. ‘naveen 2000+’ also had a significantly greater number of harvests, followed by ‘pusa rohini’ in open field conditions. under the polyhouse, the number of harvests ranged from 9.50 to 10.25. cargnelutti et al. (2004) obtained 11-14 harvests under plastic greenhouse. the polyhouse had a significant effect on all the plant characters under study (table 1). ganesan (2002) also reported better plant height in polyhouses as compared to open field conditions. yield characters yield per plant, yield per ha and number of fruits per plant were highest in ‘shivalik’ followed by ‘pusa rohini’ under greenhouse conditions (table 2). gualberto et al. (2007) also recorded significant differences for yield per ha. however, in open field conditions, ‘pusa rohini’ gave the highest value for all three characters, followed by ‘shivalik’. however, the differences were non-significant for yield per ha. the performance of ‘pusa rohini’ was the best, followed by ‘shivalik’, for all these characters when data was pooled from the different locations. the performance of tomato was statistically superior in polyhouse cultivation compared to open conditions. ganesan (2002) observed similar trends for yields per plant in polyhouses respect to open field conditions. fruit characters the highest recorded value for fruit length under polyhouse and open conditions, as well as for pooled data, came from ‘naveen 2000+’ however this genotype was at par with ‘jaya’ and ‘shivalik’ in the polyhouse (table 3). eklund et al. (2005) recorded fruit weight of 147.35 g, fruit length of 57.67 mm, fruit diameter of 69.75 mm and 5.25 locules per fruit for an elite hybrid in a controlled protected structure. all these values were higher than those found in the present study with the probable reason being fluctuating environment at fruit development as the present experiment was conducted under a naturally ventilated polyhouse. in open field and pooled data, ‘naveen 2000+’ had statistically superior fruit length. tss was statistically the highest in ‘pusa rohini’ under polyhouse conditions and in pooled data, while in open conditions ‘shivalik’ exhibited the highest tss, which was at par 67 pu sa ro hi ni ph -5 sh iv ali k ja ya na ve en 20 00 + m ea n cd 0.0 5 (l ) cd 0.0 5 (g ) cd 0.0 5 (l xg ) no .o ff ru its pe rp lan t yi eld pe rp lan t( kg ) yi eld pe rh a( q) l1 l2 m ea n l1 l2 m ea n l1 l2 m ea n 25 .03 22 .64 26 .81 20 .72 22 .34 25 .31 1.6 1 3.9 5 13 .00 4.4 9 9.9 6 7.3 6 8.9 7 8.7 6 2.7 6 ns 19 .02 13 .56 18 .39 14 .04 15 .65 2.5 3 1.2 43 1.0 66 1.2 82 0.9 94 0.9 83 1.1 13 0.2 37 ns 0.6 57 0.2 13 0.5 37 0.3 54 0.4 62 0.4 44 0.0 87 0.1 48 0.9 5 0.6 4 0.9 1 0.6 7 0.7 2 0.1 37 69 0.4 2 59 2.3 6 71 2.2 2 55 1.2 5 54 5.8 3 61 8.4 0 13 1.4 3 ns 36 5.1 4 11 8.3 4 29 8.2 0 19 6.3 9 25 6.3 9 26 1.9 0 57 .34 ns ns 52 7.8 39 2.8 50 5.2 37 3.8 40 1.1 90 .65 ta bl e2 pe rfo rm an ce of to m ato ge no ty pe su nd er po ly ho us ea nd op en co nd iti on sf or yi eld ch ar ac ter s l1 = po ly ho us e. l2 = op en fie ld . pu sa ro hi ni ph -5 sh iv ali k ja ya na ve en 20 00 + m ea n cd 0.0 5 (l ) cd 0.0 5 (g ) cd 0.0 5 (l xg ) fr ui tw eig ht (g ) va rie tie s 78 .00 78 .10 67 .40 71 .05 74 .70 73 .85 11 .49 ns ns 60 .06 54 .09 59 .23 57 .86 57 .22 57 .69 ns 69 .03 66 .09 63 .32 64 .45 65 .96 ns 48 .97 44 .75 49 .61 49 .59 53 .74 49 .33 2.3 5 4.5 2 ns 44 .51 40 .47 46 .49 47 .36 52 .82 46 .33 4.7 0 46 .74 42 .61 48 .05 48 .47 53 .28 3.7 2 52 .23 57 .18 48 .97 51 .89 49 .27 51 .91 3.4 0 ns ns 47 .63 48 .10 48 .43 48 .08 45 .45 47 .56 ns 49 .99 52 .64 48 .70 49 .98 47 .36 ns 6.7 5 5.1 0 5.4 0 5.9 5 5.0 5 5.6 5 0.1 2 0.2 6 ns 5.3 5 5.1 0 5.5 5 5.1 0 5.4 0 5.3 0 0.3 0 6.0 5 5.1 0 5.4 8 5.5 3 5.2 3 0.1 8 3.0 3.5 3.0 3.0 2.2 5 2.9 5 ns 0.5 6 ns 3.0 3.0 2.5 3.0 2.5 2.8 0 ns 3.0 0 3.2 5 2.7 5 3.0 0 2.3 8 0.3 7 l1 l2 m ea n l1 l2 m ea n l1 l2 m ea n l1 l2 m ea n l1 l2 m ea n fr ui tl en gt h (m m ) fr ui td iam ete r( m m ) ts s (°b ) lo cu les /fr ui t ch ar ac ter s lo ca tio n ta bl e3 pe rfo rm an ce of to m ato ge no ty pe su nd er po ly ho us ea nd op en co nd iti on sf or va rio us fru it ch ar ac ter s l1 = po ly ho us e. l2 = op en fie ld . ch ar ac ter s va rie tie slo ca tio n with ‘naveen 2000+’ and ‘pusa rohini’. the statistically lowest number of locules per fruit in polyhouse conditions was recorded for ‘naveen 2000+’, while in pooled data ‘naveen 2000+’ and ‘shivalik’ were at par. performance improvement perusal of data in table 4 reveals that mean yield per ha, number of fruits per plant, fruit weight, plant height, harvest duration and number of harvests were 136.12, 188.93, 16.16, 37.80, 85.32 and 65.83% more, respectively, under polyhouse conditions compared to the open field. these findings demonstrate the suitability, as well as economic feasibility, of polyhouses in the pusa rohini ph-5 shivalik jaya naveen 2000+ mean percent increase in yield per ha number of fruits per plant 89.08 400.56 138.73 180.79 112.89 136.12 fruit weight plant height harvest duration no. of harvest 52.54 404.23 169.18 181.52 149.05 188.93 29.87 44.39 13.79 22.80 30.55 16.16 17.57 74.44 30.60 28.69 47.13 37.80 75.64 89.44 79.74 94.56 88.61 85.32 64.00 72.73 66.67 73.91 53.85 65.83 genotype table 4 percent improvement in tomato performance under polyhouse versus open conditions for economic characters trans-himalyan ladakh region for tomato cultivation. gualberto et al. (1998) also reported 40-45 % higher marketable yield in greenhouses than with open field conditions. growth and yield attributes were also recorded as poor in the open field condition. therefore, it may be concluded that naturally ventilated polyhouses are a good and less expensive option for tomato cultivation in the trans-himalayan region to obtain higher yield, number of fruits per plant and longer harvest duration. varieties like ‘shivalik’ and ‘pusa rohini’ are responsive to protected cultivation in this region and may be used for cultivation after further testing to increase the return per unit area. references cargnelutti filho a., radin b., matzenauer r., storck l., 2004 number of harvest and comparison of tomato genotypes cultivated under plastic greenhouse. pesquisa agropecuaria brasileira, 39(10): 953-959. eklund c.r.b., caetano l.c.s., shimoya a., ferreira j.m., gomes j.m.r., 2005 performance of tomato genotypes under protected cultivation. horticultura brasileira, 23(4): 1015-1017. ganesan m., 2002 effect of poly-greenhouse models on plant growth and yield of tomato (lycopersicon esculentum) indian journal of agricultural science, 72(10): 586-588. gualberto r., de oliveira p.s.r., de guimaraes a.m., 1998performance of fresh market tomato cultivars under protected cultivation. horticultura brasileira, 25(2): 244-246. gualberto r., resende f.v., de guimaraes a.m., ambrosio c.p., 2007 performance of long-life salad tomato cultivars grown in a protected environment and under field conditions. unimar ciencias, 7(2): 133-138. singh a.k., singh a., 2000 performance of tomato hybrids under sub-montane and low hills subtropical condition of h.p. crop. res., 20(3): 539-540. singh r., asrey r., 2005 performance of tomato and sweet pepper under unheated greenhouse. haryana j. hort. sci., 34(1-2): 174-175. snedecor g.w., cochran w.g., 1967 statistical methods. oxford and ibh publishing co. pvt. ltd., new delhi, india, pp. 593. 68 59 1. introduction pear psylla is one of the most important pests affecting production of pears of pyrus communis parentage. a sucking insect that causes severe wilting and defoliation, which reduces yields and weakens the trees. at least seven species of pear (pyrus)-feeding psyllids in the genus cacopsylla (formerly psylla) are recognized, but there are three major species which occur primarily west of china: c. pyricola foerster is the only species found in north america; c. pyri l. and c. pyrisuga foerster are also endemic to europe. c. pyricola probably originated in western eurasia in contact with wild pyrus (bell et al., 1996). all of the major cultivars of the european pear are susceptible to this homopteran insect, varying only slightly in the degree of infestation and tolerance to feeding. major cultivars of p. pyrifolia parentage are slightly less susceptible, while those of p. x p. bretschneideri or p. ussuriensis origin (e.g., ya li and tzu li) appear to be moderately resistant (beutel, 1985). 2. morfology and life of psylla c. pyri is characterized by a seasonal dimorphism, which is strong enough that the two morphotypes were at one time considered to be distinct species (slingerland, 1892). the winter form is a large dark red overwintering adult with wide blackish longitudinal and traverses scratches, that is quite larger (2.6-2.9 mm) than the smaller and light-colored summerform adult (2.1-2.7 mm). pear psylla spends much of the winter in reproductive diapause, characterized by immature ovaries and a lack of mating. dispersal of winterforms from the orchard in autumn begins in early september (civolani and pasqualini, 2003), and peaks during late-october and early-november, coinciding with leaf fall in pear. this winterform adults overwinter both alone or in small groups in bark crevices, branch intersections and at the base of shoots on the pear host plant (priore, 1991) and away from the host plant, in the earth, or under rocks and clods which are exposed to sun irradiance (nguyen, 1962). so, some individuals spend the entire winter on pears, others recolonize pear early in spring before bud break, long before any sign of green foliage, since they would feed on a plant other than pear but not complete develop (fye, 1983). cool, wet autumns result in a reduction in dispersal out of the orchard. as soon as the weather conditions become favourable, winterforms reach the apical twigs and pierce the plant by inflicting their stylets at the bud base. in sicily overwintering nymphs from which adults flutter in february have been noticed as well (nucifora, 1969; tremblay, 1995). the egg maturation is very slow in overwintering females and seems to be accelerated when psylla adults ceases to disperse among orchards (rieux et al., 1992; lyoussoufi et al., 1994). generally diapause terminate in mid december, but in the more precocious females this event may occur starting from the end of november (rieux et al., 1990). at the end of january all females are mature pear resistance to psilla (cacopsylla pyri l.). a review s. nin*, a. ferri*, p. sacchetti**, e. giordani* * dipartimento di scienze delle produzioni vegetali, del suolo e dell’ambiente agroforestale, università degli studi di firenze, viale delle idee, 30, 50019 sesto fiorentino (fi), italy. ** dipartimento di biotecnologie agrarie, università degli studi di firenze, via maragliano, 75-77, 50144 firenze, italy. key words: breeding, germplasm, pest control, p. communis abstract: pear psylla, cacopsylla pyri l., is one of the most important insect pests in european pear production areas. control measures are directed specifically at controlling pear psylla and require accurate and timely information about insect densities in the orchard. thus, there is a widespread interest in the search for suitable biological control agents and in breeding for resistance to pear psylla. modes of host plant resistance to pear psylla damage have been studied extensively by several authors and the susceptibility of many european pear genotypes have been investigated in order to detect cultivars resistant or highly tolerant to this pest useful in breeding programs. this review presents an update of published results and knowledge on psylla life, host finding for feeding and oviposition, type of damages, monitoring and control strategies with renewed and improved efficacies, resistance characterization and breeding, with particular regard to the identified sources of resistance and the screening methods. adv. hort. sci., 2012 26(2): 59-74 received for publication 18 may 2012. accepted for publication 26 june 2012. 60 and inseminated, but for the beginning of egg-laying by overwintered winterforms a temperature over 10°c for 2 consecutive days (thermic quiescence) is required (nguyen, 1975). in italy egg-laying begins in late february in campania (priore, 1991), early march in emilia-romagna (giunchi, 1959) or early april in veneto (terza and pavan, 1988). because of the absence of foliage at this time, the first eggs are deposited directly on wood, generally at the base of unopened buds (spurs), in a number of 300-400 per female. as foliage becomes available in midto latemarch, oviposition shifts to occur primarily on expanding leaves and flowers: eggs are deposited along mid-veins and petioles of developing leaves and on stems and sepals of blossoms. first nymphal instars escape from wintereggs in concurrence with bud opening and leaves sprouting and infest the new vegetation. the first generation of summerform adults appears in april and fecundity of females appears to be quite higher compared to winterforms due to their major longevity (on average about 600 eggs per female) (stratopoulou and kapatos, 1995), although high temperatures can cause a substantial reduction in fecundity. springand summer-deposited eggs require ca. 6-10 days to hatch, depending upon temperature (mcmullen and jong, 1977). there are 5 nymphal instars. nymphs require 3-4 weeks to complete development at moderate (21-27°c) temperatures (georgala, 1956; mcmullen and jong, 1977). male and female progeny are produced in equivalent numbers (burts and fischer, 1967). afterwards (may) feeding nymphs of second generation develop on growing shoots with an aggregate distribution on leaves and internodes (deronzier and atger, 1980; pasqualini et al., 1997) immersed in pools of honeydew, which they produce in extremely large amounts. further generations overlap with all the ontogenetic stages and phases till autumn. in the warmer periods aestivation phenomenons may occur (stratopoulou and kapatos, 1995). c. pyri develops 5-7 generation per year. 3. host finding, probing and feeding behaviour host finding for feeding and oviposition contemplates a sequence of phases of hierarchic nature. the first, out of the three principal phases, consist of a host selection that the insect makes from distance using visual and olfactive impulses. the second phase occurs when the insect takes contact with the plant surface getting information about its physical structure. these impulses may strongly influence female egg-laying. the third phase regards the discrimination between host and non host plant through gustative impulses perceived during the survey of the internal tissues, the so called ‘probing behaviour’. although most of the homopteran insects have been reported to make little use of volatile compounds for the long and mid distance host finding and acceptance, the chemical characteristics are thought to be much more plant specific than the quality of the visual spectrum (prokopy and owens, 1983; dethier, 1982). the role of olfactive and gustative sensilli existing in the antennas and tarsi in not yet known, whereas that of gustative sensilli in the mouth apparatus is evident. psylla rarely initiates oviposition activities immediately upon leaf contact. rather, oviposition activities tend to be preceded by settling-probing activity, as evidence that plant cues received on initial contact are insufficient to release oviposition activity but that plant cues received during settling-probing activity release oviposition activities. thus plant cues received during oviposition activities ultimately affect whether the egg is depositated (horton and krysan, 1991). deprivation or habituation may result in higher number of eggs oviposited on less suitable genotypes than in free-choice tests. indeed, c. pyricola adult has been found to be more selective in oviposition activities than in its settling-probing activities, i.e. probing is not likely to be an indicator of a variety’s acceptability, since c. pyricola is able to colonize and feed nonhosts like pyrus calleriana and malus spp., but without laying eggs (horton and krysan, 1990). as for plant cues that mediate host acceptance it has been reported that pear psylla readily settled on nonhosts to an extent that initially unacceptable species eventually receive eggs, thus suggesting either that plant cues that release settling attivity differ from those that release abdomen bend activity and oviposition, or that thersholds for these activities differ. yet, there is evidence that settling is partially mediated by leaf surface characteristics for winterform psylla. first, settling-probing activity differs between upper and lower leaf surfaces, suggesting that cues received at the leaf surface affect activity. second, at leaf contact, pear psylla scrape the leaf surface with their tarsi (ullman and mclean, 1988; horton and krysan, 1990). finally, despite the tendency to settle readily on apple, the amount of time between initial leaf contact and onset of settling-probing was smaller for psylla encountering bartlett pear than for those encountering apple, suggesting that leaf surface cues affected behaviour. psylla adults ingest more frequently xylematic tissue, while nymphs prefer phloematic tissue or at least that of vascular fasces. a good knowledge on the different c. pyri feeding phases by means of epg may allow to discriminate between susceptible and resistant selections and to locate the mechanisms of resistance within plant tissues (civolani et al., 2010). up to know only little differences have been found in the feeding behavior of psylla adults on the susceptible william and the resistant ny10353 pears. however, lasting of the first and second non-probing is longer on william compared to ny10353, and c. pyri needs less time to reach the phloematic fasces in the susceptible plant, in accordance wih the assumption of horton and krysan (1990, 1991). 4. types of damage pear psylla causes three primary types of damage: fruit russet, psylla shock, and pear decline (burts, 1970; westi61 gard et al., 1979; beers et al., 1993). fruit russet is caused by the feeding activities of nymphs and is of most concern to growers, and control programs are generally directed at preventing this injury, since it can be caused by relatively low population densities (burts, 1988). as in other homoptera, pear psylla ingests excessive quantities of plant juices and other plant products that must be eliminated (as honeydew) during the digestive process. in fact, lymph in the phloematic tissue is rich of carbohydrates and poor of nitrogenous substances, due to this deficiency the insect has to absorb a great quantity of lymph that it afterwards excretes through his digestive apparatus producing honeydew. adult psylla excrete these waste products as small, waxy pellets that cause no harm to the plant or fruit. conversely, the immature form of the insect secretes copious quantities of honeydew, a sugary, sticky substance. if nymphal-produced honeydew is in contact with fruit for a significant period of time it causes dark blotches or streaks on the surface of the fruit (russetting), which in turn results in downgrading of the fruit at harvest (burts, 1970). honeydew allows a black, sooty mold fungus (antennaria, aureobasidium, capnodium, ceratocarpia, cladosporum, torula, ulocladium) to grow on both fruit and leaves, not only reducing the quality of the fruit, but also blocking sunlight from the leaves and decreasing photosynthesis. a second type of injury, also caused by the sucking nymphs at high densities, is of a more indirect nature than that previously mentioned. infected leaves turn brown and often fall and the fruits drop prematurely or are small and of poor quality, thus suppressing root growth and reducing tree vigor and yield. (westigard and zwick, 1972). these symptoms have collectively been termed psylla shock, and are caused by a toxin in the saliva of feeding nymphs (beers et al., 1993). symptoms of the injury can be similar in appearance to those associated with pear decline disease. psylla shock can be particularly damaging because the effects are not always restricted to the year of infestation, but symptoms may carry-over into a second year even if densities are not high the second year (beers et al., 1993). cultivars that are less preferred by psylla, such as some red pears or pears of asian origin, tend less likely to experience this type of damage. finally, adult pear psylla vector the mycoplasma-like organism (hibino and schneider, 1970) that is the causal agent of pear decline disease especially during vegetative growth (carraro et al., 1998; davies et al., 1998; guerrini et al., 2000). the feed and phytoplasma are assumed together from a diseased plant and transmitted to a healthy plant during the salivation of phloematic feedings (carraro et al., 1998). the way of transmission is persistentdispersive, as pear decline phytoplasma propagate in the insect body. disease acquisition and inoculation require at least 1-2 hours of phloematic feeding; thereafter the vector undergoes to a period of latency (about 1-2 weeks) during which the phytoplasma circulates and propagate within its body till he reaches the slave glands. both winterform and summerform pear psylla can be important in the transmission of pear decline (blomquist and kirkpatrick, 2002). this pathogen causes sieve-tube necrosis at or below the graft union (batjer and schneider, 1960; westigard et al., 1979), preventing tree-synthesized nutrients from reaching the roots and resulting in starvation of the roots (wilde and mcintosh, 1964). symptoms of the disease include a slow to abrupt decline or collapse in growth and vigor, causing a reduction in yield and (often) death of the tree. certain affected pear trees may recover if psylla densities are kept low or during winter quiescence thanks to the degeneration of epigeous phloematic tubes (giunchedi and refatti, 1997; davies et al., 1998). severity of the disease depends upon psylla density and type of rootstock (beers et al., 1993). cultivars that have been grafted onto p. communis rootstock are less susceptible than those grafted onto p. pyrifolia or p. ussuriensis rootstock. quince (cydonia oblongata) rootstocks posses a limited aptitude to allow phytoplasma survival between one vegetative cycle and another. resistant rootstock has largely remedied this problem in various pear growing regions. 5. monitoring and control tactics the psyllid c. pyri, along with its natural enemies, needs to be carefully monitored for correct integrate pest management and biological pest control decision making. moreover, timing of spray application against c. pyri is crucial because recommended insecticides are only efficient at certain stages. monitoring should provide starting from spring density of eggs, nymphs, adults and presence of the principal antagonist. a simple method for estimating densities of pear psylla is desirable. monitoring pear psylla is made difficult by the uneven distribution of insects (eggs and nymphs) on the trees, a distribution that may in fact change seasonally. densities of psylla may also vary with height in the tree canopy (and sex) both for c. pyricola (brunner, 1984; horton, 1994) and c. pyri (stratopoulou and kapatos, 1995). currently, sampling of the adult population is necessary to determine the onset of reentry in late winter or the popolation density, and sampling of fruit spurs for eggs is often the easiest way to determine the beginning of egglaying. counts have been obtained in usa using frappage, sticky traps, beat trays and open-ended organdy bags, the last one providing direct estimates of psylla numbers per leaf but being extremely time consuming (horton, 1994; horton and lewis, 1997). effectiveness of yellow stickyboard traps have been examined by several authors and seasonality of the catch and flight activity of pear psylla (c. pyricola) according to weather conditions have been reported (krysan and horton, 1991; horton, 1994; civolani and pasqualini, 2003; erler, 2004), as well as diurnal difference (horton, 1993) and intraorchard changes in distribution associated with leaf fall (horton et al., 1993). laboratory study have shown that males of both the sum62 merform and winterform morphotypes in c. pyricola are attracted to volatiles given off by females, whereas in the field male has shown a clear preference for sticky traps that have been baited with live females compared with traps baited with live males or left unbaited (brown et al., 2009). limb beating or limb jarring to collect arthropod specimens from trees has been known for a long time and in several variations: frappage or beating tray (two-dimensional) and beating umbrella (three-dimensional). the first of this procedure has been applied by several authors and has been reported by jenser et al. (2010) to depend considerably on weather condition, while the second one to be much less temperature and wind-sensitive, due to its vertical extension, and much more suitable for collecting fast moving or flying beneficial organisms than the twodimensional method. in contrast with the hypothesis that any data collected for the adults using a beating umbrella would be influenced more by weather conditions than those gathered using funnels, sanchez and ortìn-angulo (2011) have found a higher efficacy of the net in relation to the funnel. the same authors also have stated a low efficacy of the beating techniques for sampling nymphs that may be due to the fact that they hold tight to the substrate and are not easily removed by the act of beating. both the application of the sticky board traps and beating tray provide accurate information about the changes of pear psylla population density (jenser et al., 2010). in particular the capture of adults using either the funnel or the net may be used to estimate the absolute number of c. pyri nymphs on trees, thanks to the high correlation found by sanchez and ortìn-angulo (2011) between nymphs counted on shoots and the capture of adults using either of the beating techniques. several authors have reported the same relationship for other psyllid species (horton, 1994; jenser et al., 2010). the beating techniques also have the advantage of being less time-consuming than the sampling of leaves and, for beating over a net or tray, samples may be processed directly in the field, although the amount of collected insects using beating umbrella some times makes necessary the laboratory process. in europe the dinamic of c. pyri populations have been studied using frappage by various authors (deronzier, 1984; rieux at al., 1992). according to civolani and pasqualini (2005) frappage is the sampling method which best represents the dinamics of populations of psylla and its predators (antocoridis, coccinellids, lacewings). predators overwintering in bark cervices may be estimated by using corrugated cardboard traps (bogya et al., 1999; horton et al., 2002; civolani and pasqualini, 2003; jenser et al., 2010). alternatively the psylla eggs have been counted on the shoots and leaves using a binocular dissecting microscope by several authors (jenser et al., 2010) and a few of the authors investigated and counted both the eggs and larvae. this method provides real data, but it’s time consuming; the sample must be taken into the laboratory and the counting completed within a short time. berlese funnel is a widespread technique for extracting arthropods mainly from soil and litter samples (stäubli et al., 1992). moreover the mite brushing machine or leaf brushing machine developed by henderson and mcburney (1943) is a technology that can reduce the time required to obtain either absolute counts or estimates of arthropods on leaves from samples. recently developed, the wash-down method described by jenser et al. (2010) offers the advantages of the independence of the weather conditions (temperature, wind, rain) and the daily rhythm of the examined psylla stages. since practically every larvae developing on the flowers and shoots are extracted, it provides suitable data about the pear psylla population density and its changes, as well as about the effectiveness of the insecticides. this method has been suggested to provide also significant data to judge the susceptibility or tolerance of the pear cultivars to pear psylla species. since observing the population development of pear psylla is time-consuming and prone to error, phenological models could assist growers in the timing of monitoring and control measures, as they simulate and predict, by means of driving variables (usually temperature), the timing of natural events. there have been modest attempts to develop degree-day models that predict onset of egglaying and appearance of first generation nymphs (westigard and zwick, 1972; brunner, 1984; beránková and kocourek, 1994) and timing of reentry (horton et al., 1992), with aims toward improving timing of the dormant spray. morgan and solomon (1993) have provided a phenological model for c. pyricola which have been integrated into a multipest forecasting system. further on, a phenological model for c. pyri based on biological mechanisms, in particular the emergence of juvenile instars of the second generation, has been developed by schaub et al. (2005). in italy the defence against c. pyri is mainly based on integrated pest management (ipm), supported by natural control aimed to equilibrate the complex biological relationships of the field community (civolani, 2012). among the basic strategies there are the ‘good agricultural practice’ (gap) techniques that reduce tree suitability for growth and reproduction of pear psylla by avoiding overuse of fertilizers, incorrect or over pruning, and reducing excessive plant vigor (beers et al., 1993; civolani, 2012). suckers or water sprouts should be removed from scaffold limbs (beers et al. 1993), because these are a source of rapidly growing and highly nutritious foliage. also the strategies to control other pest species, such as the technique of mating disruption and the use of granulosis virus (cpgv) employed to control codling moth (cydia pomonella), may influence psylla and assist in chemical control. however, in the last decade commercial pear growers have relied primarily on the use of synthetic products to control pear psylla, and the advantages and disadvantages of the main strategies performed in the last 20 years in integrated and conventional farms have been described by civolani (2012). unfortunately, these methods are not always entirely effective, as their efficiency depends both on the active in63 gredients employed and the weather conditions at the time of treatment and moreover pear psylla has developed resistance to several classes of commonly used insecticides (riedl et al., 1981; follett et al., 1985; burts et al., 1989; croft et al., 1989). current control recommendations emphasize destruction of the overwintered generation, or offspring of the overwintered generation with insecticides. a typical control program for overwintered adults is performed at leaf fall, commonly in france on c. pyri and in north america on c. pyricola, with the application of pesticides belonging to the pyrethroid family (with or without mineral oil added), repeated as necessary in late winter (at bud swelling stage or bud break) to break down the population of females emerging from winter shelters and about to lay eggs. these pyrethtoids are completely non-selective but broad spectrum and therefore dangerous for the beneficial insects. for this reason the treatment must be performed only at complete leaf fall (late november or early december), when a. nemoralis populations have already found shelter in bark crevices while c. pyri adult winter forms are still active on plants. efficiency of treatments may vary considerable upon seasonal conditions. for example most psylla adults take shelter early and survive to the late autumn treatments when an early frost occurs at the beginning of autumn. similarly the efficiency of chemicals is reduced after frost waves at the end of winter which interrupt and delay the emergence of adults, while activity of pesticides is best after a mild winter when almost all adults leave their shelters at the time of treatment (civolani, 2000; civolani and pasqualini, 2003). in italy the dormant sprays are discouraged since the pest population, after an initial sharp decline, soon recovers in spring because the natural control by its predator a. nemoralis is limited, then increases again in may, reaching the economic threshold for spring-summer treatments (civolani, 2012). the main side effect of the use of pyrethroids in late winter is that they sharply reduces the psylla first generation and therefore could starve the anthocorids, interfering with their settlement during early plant growth in spring. various alternative biorational solutions to synthetic pesticides have been tested against the overwintering generations, and among them kaolin and some oily compounds. kaolin, a white, non-abrasive, fine-grained allumosilicate mineral that is purified and sized so that it can be easily dispersed in water, creates a mineral barrier on plants that prevents oviposition and insect feeding (puterka et al., 2000). treatment with kaolin has been reported to hinder egg anchorage on the leaf surface and inhibiting host-plant acceptance. moreover, some insects have been found to be less mobile and unable to reach the laying site (host location) on plants, as their body and wings have became soiled (pasqualini et al., 2003; daniel and wyss, 2006). further on, puterka et al. (2005) investigated the effects of particle film type (hydrophobic versus hydrophilic) and formulation determining that there are a number of biological effects particle films have on pear psylla beyond the deterrence of adult settling and oviposition. alternatively, mineral oils and oily compounds could also be used to interfere with egg deposition by psylla adults. a good reduction of the number of eggs laid has been obtained in northern italy with pure mineral oil alone (‘dormant oil’) (pasqualini et al., 2003) and in turkey with fish-liver oil and summer oil (erler, 2004). some growth regulators have proved to possess a good activity against eggs and nymphs of first and second generation by interfering on the cuticle transport and deposition during larvae development (erler and cetin, 2005). at the beginning of the second generation growers can assess the risk to their orchard and still target specific stages. therefore, treatments target mostly eggs and/or young larvae of the second generation. the treatments against summer generations can be performed towards eggs or nymphs. chitin inhibitors, usually employed against c. pomonella, have shown a secondary effect on second generation eggs, usually laid in the first decade of may, especially when they are applied on newly laid eggs (white eggs) or on eggs laid in a short time after the treatment. however, control strategies against juvenile stages are of most relevance, and were performed in the past with generic organophosphorates, whereas are based in present times on specific synthetic active ingredients, often acaricides. among these, abamectin (produced by the soil bacterium streptomyces avermitilis) is the basic chemical employed today against young nymphs of second generation (usually in may) and included in the italian disciplinary of integrated management. the best results are obtained when yellow eggs are mostly present and when the hatching peak has not yet achieved (pasqualini and civolani, 2006). abamectin is allowed only once in a year or twice in case of young orchards; since it’s not systemic, the addition of mineral oil may improve its penetration within 24 hour time. a new broad spectrum acaricides, namely spirodiclofen (baj2740, trade name: envidor®), belonging to the new chemical class of tetronic acid derivatives, has been discovered by bayer cropscience during the 1990s and is commercially available since 2007. spirodiclofen has a new original mode of action (interference with lipid biosynthesis) and shows no cross-resistance to any resistant mite or whitefly field population, representing an invaluable new tool to manage insecticide resistance in rotation with abamectin. it’s efficiency is best on yellow eggs some days before the hatching of first instar nymphs and is improved by addition of mineral oil, although often lower than that of abamectin (pasqualini and civolani, 2007; boselli and cristiani, 2008; marčić et al., 2009). besides summer mineral oils, whose main action is that of dissolving honeydew, sodium dioctyl sulphosuccinate or other vegetal free fatty acids may be used for washing the trees (briolini et al., 1989). recently some other novel compounds have been used, similar to liquid glue and capable of controlling almost all juvenile instars of c. pyri. these products are synthetic sugar esters (sucrose oc64 tanoate) and represent a relatively new class of insecticidal compounds that are produced by the reaction of sugars with fatty acids, valuable in crop integrated pest management programs (puterka et al., 2003). it’s important to keep in mind that summer psylla infestation depend on the antagonists development in spring, first of all the most important one, a. nemoralis, which has to be protected. for first instar nymphs control threshold is given by the ratio between number of infested shoots and number of shoots with the antagonist antocoride, which have to be ≥ 5 (marani and reggidori, 2007). also the relevant effect of weather conditions on pest populations should not be underestimated. in fact, the development of psylla is strongly reduced by high summer temperatures that cause the death of eggs and the slowdown of juvenile growth. on the other hand, cold and rainy periods during blossoming and petal fall encourage nymph spreading on plants, often clustering in flower calyxes, sometimes causing russet blotches or young fruit drop (civolani, 2012). climate conditions, such as wind, have been demonstrated to have an impact on the clustering of psyllids, wheras spatial factors, such as distance from a mixed hedge have been found to be related to beneficial arthropod community (debras et al., 2008). localized resistance cases to organophosphorates insecticides, pyrethroids and carbamates pesticide families and chitin inhibitors family have been developed and have been largely documented, especially for c. pyricola in north america since 1960 (harries and burts, 1965). resistance rates among the active ingredients has been reported to be very variable in laboratory tests and probably there are different mechanisms involved in the resistance to different pesticide families, as reported by civolani (2012). in italy, cases of loss of efficiency of abamectin have been noticed in some orchards in emilia-romagna region, indicating that there is a high risk of selection for resistance to abamectin, especially if the number of treatments per year is high. up to now, the tests data indicate that no apparent resistance to abamectin has been developed in c. pyri populations of that region, but may rather be related to incorrect pest defence management (civolani et al., 2007). control strategies should be based on a limited use of pesticides, possibly selective ones, in order to foster the development of a. nemoralis populations, which become a relevant factor to control the pest, preying on both eggs and nymphs of psylla. in emilia-romagna a. nemoralis generally shows three generations and may feed also on other insects, for example aphids and the pear sawfly hoplocampa brevis. laboratory tests have shown an average predation of about 300 psylla nymphs during the entire life of an adult, which lasts about 60 days (civolani, 2012). one problem is that the populations of this anthocorid grow rapidly in spring only if there is psylla of first generation in the orchard for feeding, therefore in may-june some amounts of the pest have to be tolerated. a further weakening of the wild a. nemoralis populations may be caused by the large amount of active ingredients used against other pests, having significant toxic effects on a. nemoralis. among these, thiacloprid, the most frequently pesticide used against the codling moth, c. pomonella, as well as the neonicotinoids, not employed in italy and europe as specific psyllicides, but against aphids and the pear sawfly h. brevis. the artificial introduction of the antagonist antocoride at the end of march beginning of april is a very useful mean for controlling eggs and young nymphs of the first generation. the flow is made with about thousand individuals per hectar fractioned in 3 times at weekly intervals. resettlement is much more feasible much wider the cultivated area is (minimum 1 hectar). some authors however retain that 500 individuals would be sufficient for each introduction (beninato and morella, 2000). good natural equilibrium have been obtained in veneto with the introduction of 500-600 psylla adults per ha in a sole time in may (mori and sancassani, 1984). in france the introduction of a. nemoralis has been performed by the distribution of pelargonium stems containing 2.940 eggs of the psylla antagonist (fauvel et al., 1994; rieux et al., 1994). some authors indicated that the efficacy of this predator is not strongly mediated by plant quality, at least at tree scale, thus, for systems where pest population growth is strongly tied to plant vigor or quality, the reduction of fertilizers to the minimum level required for proper fruit set is likely to improve the success of pest biocontrol (daugherty et al., 2007). 6. new pesticides and strategies for integrated pest management in the last years new pesticides have been developed with generally low toxicity towards beneficial insects. aksebio2 is a mixture of various aromatic plant essential oils, edible plant extracts and a bacterium tr 2000 which decreases oviposition and immature stages of the pest (erler et al., 2007). spirotetramat (movento®) is a new, fully systemic and ambimobile active ingredient particularly effective against a broad range of sucking pests, similar to the tetronic acid derivate spirodiclofen. its singularity depends upon its unique translocation property, which allows the protection of new shoots or leaves appearing after foliar application, in fact after foliar uptake the insecticidal activity is translocated within the entire vascular system (nauen et al., 2008). due to the lack of any cross-resistance to existing chemical classes of insecticides, spirotetramat is a very interesting alternative to be used in rotation schedules. natural plant compounds, fungal pathogens and different orchard ground cover all seem promising controls. among nontoxic plant compounds, sugar-ester extracted from wild tobacco has proved to be most successful in psylla control (usa), killing most nymphs within 2 hours. even nymphs that hatched 3 to 5 days after spraying die as soon as they walk on leaves (stanley, 1993). rapeseed 65 oil and petroleum oil as well have showed a total efficacy against eggs laid by winterforms females of c. pyri (marčić et al., 2008, 2009). several naturally occurring fungal pathogens (spores of beauveria, verticillium, and paecilomyces mixed with either oil or water) have given 100-percent control as well within 5 days. the advantage is that fungi can last indefinitely compared to the sugarester that may persist on the plant for about a week. they are host-specific, completing their life cycle on infected insects on the plant, and therefore nontoxic to humans, animals and beneficial insects. after killing their host, the fungi release hundreds of spores, each capable of infecting another pear psylla. since pear psylla also have several predators (table 1), planting ground covers with perennial crops between tree rows to attract them could provide a measure of control (stanley, 1993). new strategies for integrated pest management of psylla may be offered in the future by the optimization of the recently identified sex attractant pheromone, the 13-methylheptacosane, for c. pyricola winterforms males (guédot et al., 2009). 7. resistance to pear psylla all of the main cultivars of the european pear grown commercially (abate fétel, william, conference, doyenne de comice, kaiser, etc.) (bellini and nin, 2002) are susceptible to this arthropod pest and biological controls are becoming of limited effectiveness since resistance to insecticides has developed rapidly. host plant resistance would therefore be a valuable control strategy. resistance to the pear psylla has been demonstrated in the east asian pear species, p. betulifolia bunge, p. calleryana decne., p. fauriei schneid., p. ussuriensis maxim., and p. x bretschneideri redh. (westigard et al., 1970; quamme, 1984; moore and ballington, 1991). hybrids of p. ussuriensis x p. communis have been found to be resistant to c. pyricola (harris, 1973; harris and lamb, 1973; quamme, 1984) as well as to c. pyri (robert et al., 2004). different interspecific hybrids between p. communis and p. longipes or p. pyrifolia have shown high levels of resistance to c. pyri, too (robert et al., 2004). resistance has been reported also for a few genotypes of p. nivalis jacq. and sorbopyrus (westigard et al., 1970; bell, 1992). small fruit size of the pure species and gritty or coarse texture of both the pure species and interspecific hybrids may limit the utility of some of this germplasm for rapid transfer of resistance into cultivars with p. communis type fruit. within p. communis, moderate resistance has been demonstrated in the old italian cultivar spina carpi (quarta and puggioni, 1985), and in eleven ‘primitive’ cultivars from yugoslavia and hungary (bell and stuart, 1990; bell, 1992). all of these genotypes have relatively poor fruit quality but are important sources of resistance within the primary gene pool available for improvement of p. communis cultivars. in many countries ex situ pear collections have been established in some important pear growing areas with a great diversity of national, local and foreign cultivars, mainly for evaluation of resistance to major disease and insects, to be used as potential parents in breeding (quarta and puggioni, 1985; braniste et al., 1994; braniste and militaru, 2008; benedek et al., 2010). more than 200 pear cultivars of tuscan, national and international origin, intable 1 natural enemies associated with pear psylla in europe natural enemy taxonomic group species predators arachnida, araneae unidentified spiders dermaptera forficula auricularia linnaeus heteroptera, anthocoridae anthocoris nemoralis (fabricius) orius spp. heteroptera, nabidae nabis spp. heteroptera, miridae several species heteroptera, lygaeidae several species neuroptera, chrysopidae chrysoperla carnea (stephens) chrysopa formosa brauer chrysopa septempunctata wesmael anisochrysa prasina (burmeister) coleoptera, coccinellidae several species belonging to different genus diptera, syrphidae episyrphus balteatus (de geer) epistrophe spp. parasitoids hymenoptera, encyrtidae trechnites psyllae (ruschka) prionomitus mitratus (dalman) hymenoptera, pteromalidae syrphophagus mamitus (walker) entomopathogenic fungi entomophthora sphaerosperma from: armand et al., 1991; tremblay, 1995; civolani and pasqualini, 2003; erler, 2004. 66 cluding also 25 afghan accessions, are presently being evaluated in ex situ and in situ collections for psylla resistance at the department of plant, soil and environmental science of the florence university (dipsa-unifi) within the ager project ‘innovapero: management and crop innovations for high-quality pear production’. moreover, evaluation of insect preference in tunnel is being in progress on 26 local cultivars showing good pomological traits. taking earlier and present results into account almost 60 european pear cultivars being resistant or highly tolerant to pear psylla infestation and damage can actually be listed (table 2). some of these ancient or local cultivars may be exploited both in organic farming or in breeding, but further investigations are needed to estimate their yield capacity and fruit quality (benedek et al., 2010; szabó et al., 2010). moreover, some varieties considered resistant in field have shown to be susceptible, if isolated and articially infested by adults (westigard et al., 1970; harris, 1975). methods of evaluating host resistance are sufficiently developed and rapid nymphal feeding bioassays have been developed to screen pear germplasm for antibiosis-based resistance by harris (1973, 1975) and butt et al. (1989) for the evaluation of pear germplasm introduced in north america from eastern europe, and then modified by different authors (table 3). the results of tests can vary, depending on the type of assay and host phenological stage, which affects ovipositional preference (bell and puterka, 2003). genetic psylla resistance do not follow a general rule and is supposed to be often polygenically inherited (harris and lamb, 1973). lespinasse et al. (2008) found that psylla resistance was not well transmitted from the p. ussuriensis x p. communis hybrid ny10355 to its progenies, assuming that genetic resistance in ny10355 may result table 2 pear cultivars showing some degree of resistance to psylla as reported by different authors cultivar tolerant resistant moderately resistant low susceptible country reference 20th century x serbia stamenkovic et al., 1993 bartjarka x usa bell, 1992 bókoló körte x hungary benedek et al., 2010; szabó et al., 2010 bötermö kálmán x hungary benedek et al., 2010; szabó et al., 2010 bulgaresti x romania braniste et al., 1994 cantalupesti x romania braniste et al., 1994 cantari x romania sestras et al., 2009 cj16-9-13 x romania straulea et al., 1992 craiesc x romania braniste et al., 1994 cure x romania straulea et al., 1992 cure-6 x hungary benedek et al., 2010 d’aout lamer x france robert and raimbault, 2005 daoyenné de poitiers x france robert and raimbault, 2005 erabasma x usa bell and stuart, 1990 ewerd x romania braniste et al., 1994 füge alakú x hungary benedek et al., 2010 general osmanwill x romania braniste et al., 1994 haydeea x romania sestras et al., 2009 honeysweet x usa quamme, 1984 imperiale x romania braniste et al., 1994 imperiale x romania sestras et al., 2009 jerisbasma x usa bell, 2003 kajzerka x usa bell, 1992 karamanka x usa bell, 2003 karamanka x serbia stamenkovic et al., 1993 karamanlika x usa bell and stuart, 1990 katman x usa bell and stuart, 1990 katman x france robert and raimbault, 2005 kései kálmán x hungary benedek et al., 2010 kieffer seedling x romania braniste et al., 1994 67 either from the combination of several small-effect resistance genes, according to pasqualini et al. (2006), or from a combination of dominance or epistatic effects or from both. a genetic mapping approach should help researchers to understand the genetic mechanism of psylla resistance. the molecular interaction between pear tree and the piercing/sucking psylla has been investigated through the construction and characterization of cdna subtracted libraries. genes expressed upon insect infestation were identified in a susceptible and a resistant pear genotype. the two expression profiles were found to be different: in the resistant plant more genes involved in the response to biotic and abiotic stress were activated than in the susceptible one. the further characterization of the identified genes could lead to the development of molecular markers associated with tolerance/resistance to psylla (salvianti et al., 2006). the quantitative resistance to pear psylla has been analyzed recently in a progeny of the european pear angelys crossed with the resistant genotype ny10355, and by screening parents/seedlings with microsatellite markers a qtl (quantitative trait loci) that explained 15% of the phenotypic variability has been determined and mapped on the linkage group 17 (bouvier et al., 2011). cultivar tolerant resistant moderately resistant low susceptible country reference kieffer x hungary benedek et al., 2010 kieffer éd x hungary benedek et al., 2010 krupen burnusus x usa bell and stuart, 1990 krupen burnusus x usa puterka, 1997 lorencz kovacs x romania sestras et al., 2009 lorenz x romania braniste et al., 1994 lucele x usa bell, 1992 magness x romania braniste et al., 1994 magness x serbia stamenkovic et al., 1993 mednik x usa bell and stuart, 1990 mednik x usa puterka, 1997 monglow x italy quarta and puggioni, 1985 nagyasszony körte x hungary benedek et al., 2010 nyári kálmán x hungary benedek et al., 2010 obican vodenac x usa bell and stuart, 1990 obican vodenac x usa puterka, 1997 pinguoli x serbia stamenkovic et al., 1993 rocha portugheza x romania braniste et al., 1994 rozs nyári körte x hungary benedek et al., 2010 severinka x romania sestras et al., 2009 sierra x usa quamme, 1984 sirrine x usa quamme, 1984 sirrine x italy quarta and puggioni, 1985 smokvarka x usa bell and stuart, 1990 spadona x romania braniste et al., 1994 spina carpi x italy quarta and puggioni, 1985 spina carpi x france robert and raimbault, 2005 steiner x hungary benedek et al., 2010 téli kálmán x hungary benedek et al., 2010 tomnatice x romania braniste et al., 1994 topka x usa bell and stuart, 1990 triomphe de joidogne x romania braniste et al., 1994 triomphe de joidogne x romania sestras et al., 2009 vidovaca x serbia stamenkovic et al., 1993 viki körte x hungary benedek et al., 2010; szabó et al., 2010 william precoce morettini x romania braniste et al., 1994 zelinka x usa bell and stuart, 1990 zelinka x usa puterka, 1997 68 8. resistance characterization resistance is characterized by both ovipositional non-preference (antixenosis = settling and oviposition) and feeding inhibition, delayed development and increased nymphal mortality (antibiosis) (bell and stuart, 1990). while antixenosis influences the size of the initial nymphal population, antibiosis probably exerts the greatest effect on population levels over a season. so, feeding rejection is a major component of resistance and leads directly to a precocious nymphal mortality; the mechanism for feeding acceptance or rejection is probably internal to the leaf as reported by butt et al. (1988). table 3 pear resistance to psylla: assay methods adopted in controlled conditions reference number of insects used for artificial infestation site of infestation replications per cultivar/selection observations (hours or days after infestation) harris, 1975 100 adults plant 1 4 days: removal of adults, growth and development of the resultant progeny butt et al., 1988 1 nymph lower midrib of 10 fully expanded detached-leaves 3-10 2 h, 4 h, 6 h, 24 h: position of nymph and presence of honeydew butt et al., 1988 1 nymph lower midrib of 10 fully expanded leaves of potted trees 2 2 h, 4 h, 6 h, 24 h: position of nymph and presence of honeydew butt et al., 1988 10 first instars lower midrib of the 2 youngest fully expanded leaves of potted trees 2 24 h: position of nymph and presence of honeydew butt et al., 1989 25 first-instar nymphs 2 youngest and fully expanded leaves of a shoot 4 each day: feeding determined by excretion of honeydew puterka et al., 1993 2-6 females excised twig collected at different stages of bud development 8 24 h after infestation at stages of dormant bud, green tip, fully expanded leaf: adults per twig 48 h: adults per twig 72 h: eggs per twig berrada et al., 1995 15 pairs of sexually mature adults 10-16 leaves (≈300 cm2) 4 24 h: removal of adults and egg count each day: survival of eggs and larvae until they developed into adults baldassari et al., 1996 6-10 third-fourth instar nymphs 2 younger and more expanded leaves of a shoot 2-3 5 days: vitality of nymphs, amounts of produced honeydew, possible development of sooty moulds 15 days: number of deaths baldassari et al., 1996 10 first instar nymphs 2 younger and more expanded leaves of a shoot 3 every day: number and age of dead nymphs, days needed for possible development of adults puterka et al., 1997 5 nymphs 4 fully expanded terminal leaves 5 4 days: nymphal survival and development alternating 3rd and 4th day up to day 29: nymphal survival and development robert et. al., 1999 1 female plant 1 2-7 days: female removal after 50 eggs on average per plant had been laid each week: larval mortality and count of different instars bell, 2003 10 second or third instar nymphs underside of the top 2 youngest fully expanded leaves of 5 48 h: number of surviving and actively feeding nymphs robert and raimbault, 2005 4 females and 1-2 males in two times at 8 day-interval plant 7-8 15 days: number of eggs on the 8 upper leaves of shoots 36, 63 and 98 days: number of nymphs 134 days: shoot and leaf state pasqualini et al., 2006 300-400 males and females plant 5-16 10 -25-50 days: number of adults per plant 10-25 days: number of eggs per plant 25-50 days: number of nymphs producing honeydew per plant pasqualini et al., 2006 1 female upper surface of a leaf in a clip-cage 3-11 48-72 h: number of laid eggs per female bouvier et al., 2011 8 insects plant 7 presence of honeydew on the first, second and last third of the plant. the quantity of larvae present on the whole plant 69 volatile substances emitted by the leaves of different varieties are not substantially dissimilar and therefore do not probably play a basilar role in the affinity an repulsion of psylla adults (miller et al., 1989), but bioassays on this topic are still lacking. resistance of genotypes is not directly proportional to leaf cuticle thickness, the resistant genotype ny10355 for instance has a lower content of cutin compared to the susceptible william variety (gérard et al., 1993). pubescence is not a major factor in feeding deterrence according to bell and stuart (1990). however, antixenosis is influenced by both the physiological status (bigre and lefeuvre, 1982) and bud phenological phase (stuart et al., 1989; puterka et al., 1993) of pear tree. thus differences in leaf morphology may influence psylla oviposition, bearing in mind that the insect prefers to lay the eggs on prominent structures such as leaf vein or crevices at the base of fruiting spurs. resistant genotypes express antibiosis with the production of a limited amount of honeydew and a strong nymphal mortality (butt et al., 1988, 1989). however, the quantity of produced honeydew has not been denotive of the infection intensity on selections obtained by induced mutagenesis (baldassari et al., 1996). ingestion of substances belonging to the group of polyphenols (for example tannins) has been suggested to be the cause of this mortality (bell, 1984). challice and williams (1968) underlined the presence of the group of active components flavone glycosides in the asiatic pyrus ussuriensis, which is lacking in pyrus communis. braniste et al. (1994) evidenced a lower total isoperoxidase activity in resistant pear cultivar compared to susceptible ones. also sugar content in leaves differed between resistant and susceptible genotypes, an increase of sugar content due to a reduced level of starch synthesis and also its rapid degradation was noticed in susceptible cultivars. fiori and lamb (1982) found the presence of secretory cells to be much more extensive in the phloem of leaf midveins of pear genotypes with resistance against p. pyricola and suggested that average percentage of the phloem area occupied by secretory cells in may-june may provide a valid method for determining the resistance of pear trees to p. pyricola. antibiosis towards preimmaginal stages is accompanied by a reduction of feeding frequency, which may be linked to the presence of nutritional inhibitors (butt et al., 1989) or to an insufficient plant alimental appetizer (chang and philogène, 1975). later on, the ex novo induction of a phenolic compound (3-o-trans-p-cumaroyltormentic acid) has been demonstrated after 12 hours from the phytophaga attack with a pick after 30 days from infection (scutareanu et al., 1999). this induction has been recently shown to be local (conference) or systemic (william and ny10355), but there are no evidence on whether this induction can modify c. pyri behavior or not (scutareanu et al., 1999). a different effect has been attributed to other volatile substances still originating during the wounding process of the mouth apparatus. some of these essences released from infected pear leaves, i.e. the monoterpene (e,e)-α-farnesene and the phenolic compound methyl salicylate, are primarily responsible of the attraction of the main psylla predators, namely anthocoris nemoralis and anthocoris nemorum (scutareanu et al., 1997, 1999, 2001). the capacity of some plant species to emit mixture of volatile compounds, dominated by terpenes, to attract carnivorous arthropods that prey on or parasitise herbivorous insects or mites, has been well documented as plant defence strategy (degenhardt et al., 2003). finally, antixenosis and antibiosis are often associated in resistant genotypes, but are supposed to be indipendent from a genetic point of view, since only one of this two mechanisms of resistance exist in some genotypes (puterka et al., 1993). 9. breeding fortunately, pear species vary considerably in their resistance to pear psylla and breeding for resistance is possible. for breeding, the use of the larger fruited species (p. ussuriensis and p. x bretschneideri) should prove to be more efficient for combining resistance with europeantype fruit quality. in italy, the experimental institute for fruit crops, rome, forlì section (isf-fo) has been studying genetic improvement of pear for about 35 years, looking with particular regard for fire blight and pear psylla resistant cultivars. the breeding activity for the transfer of pear psylla resistance lists 22 crossing combinations, about 8,200 seedlings and 13 advanced selections, 3 of which are rather tolerant to pear psylla (baldassarri et al., 1996). praiseworthy is the selection isf.68-14-44-11, which is rather tolerant to pear psylla, although the fruit does not have sufficient eating quality (rivalta and dradi, 1998). transfer of resistance traits have been reported more recently in crosses with different ny selections (isf 94-1/174-267, isf 94-4/103-267, isf 94-5/-51-268) and selections of p. pyrifolia (isf 98-5-70-150, isf 90-12/110-149) (pasqualini et al., 2006). the department for tree crops, bologna university (dca-ubo), has been implementing a programme of both intervarietal and interspecific cross breeding, which began about 30 years ago, in order to develop diversified pears for quality and ripening calendar, without disregarding the evaluation of fire blight and psylla resistance (sansavini and rosati, 1986; sansavini, 1999). ny10353 and ny10355 have been used as male parents, while max red bartlett and doyenne de comice as female parents, and eight seedlings have been selected and are under evaluation. among these, dca 92052105-119 (ny10353 x doyenne de comice) has shown a great degree of psylla resistance in controlled growth chamber and is actually under evaluation in open field (musacchi et al., 2005; pasqualini et al., 2006). moreover, a number of 90 aflp primer combination has allowed to indentify, through a bulk segregant analysis, a first step of molecular markers linked to psylla (sansavini, pers. com.). 70 in france, a close collaboration between inra and the national institute of horticulture (inh), angers, has been recently started for the definition of precocious tests for evaluation of pear psylla resistance as well as potential parents to be used in the breeding project. now, some 10,000 resistant hybrids and 60 selections are under study using artificial inoculation tests (le lezéc, 1991; le lezéc, pers. com.). of great importance is also the breeding programme which is undergoing at the fruit research stations of pitesti-maracineni, cluj-napoca and voinesti, romania, whose goals have been focused since many years on resistance improvement to fire blight, pear psylla and scab by means of intra and interspecific hybridization followed by backcross. the initial sources concerning psylla resistance were represented by biotypes derived from p. serotina and subsequent f 1 and f 2 interspecific selections. some foreign and native p. communis cultivars were used as parents (napoca, butirra precoce morettini, butirra hardy, butirra six, doyenné d’hiver, etc.) and the psylla resistant or tolerant cultivars haydeea, euras, getica and ina estival have been promoted and named (andreis, pers. com.; braniste, pers. com.; sestras et al., 2009). in north america the breeding programmes initiated in the 1920’s and 1930’s developed in the 1960’s into two impressive programmes for disease and insect resistance at harrow in canada and at kearneysville (usda) in the united states, based on hybridisation with cultivars and selections from p. ussuriensis and p. pyrifolia (characterised by a higher resistance, probably of monogenic type), with fruit characteristics of p. communis being recovered by backcrossing to selected p. communis cultivars (bellini and nin, 1997). resistance to pear psylla represent an additional breeding objective of the pear programme at harrow (brunner, 1997; hunter, 1994; hunter, pers. com.), while has been added as specific primary objective of the united states department of agriculture (usda) breeding programme. selection methods have been developed from detailed studies of the modes of resistance (a 24-hour nimphal feeding bioassay plus choice and non choice oviposition assays for further resistance characterization), almost 4,400 seedlings have been evaluated and rapd markers associated with resistance to nymphal feeding antixenosis are in progress. among the most recent cultivars coming from united states are elliot, gourmet, potomac and blacke’s pride (bell and van der zwet, 1992; bell et al., 1996). often, parallel studies are carried on in order to support and speed up the attainment of the pursued goals. generally, the future direction of such programs will include a biotechnology component, with the objective of identifying and transferring genes for resistance to fire blight and pear psylla (bellini and nin, 1997; bellini et al., 2000). acknowledgements supported by progetto ager, grant n° 2010-2107. references armand e., lyoussoufi a., rieux r., 1991 évolution du complexe parasitaire des psylles du poirier psylla pyri et psylla pyrisuga [homoptera: psyllidae] en vergers dans le sud-est de la france au cours de la période hivernale, printanière et estivale. entomophaga, 36(2): 287-294. baldassarri n., baronio p., rocchetta g., salvaterra g., 1996 indagine sullo sviluppo di cacopsylla pyri (l.) 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composition, tuber. abstract: plectranthus edulis (vatke) agnew is a tuber crop originated and culti­ vated in ethiopia for food and medicinal uses. it has higher quality nutrient composition than other tuber crops. the cultivation of p. edulis is under threat because of introduction of exotic species, land use change, habitat destruction, population pressure, selection of only few local varieties by farmers and clima­ te change. although p. edulis is a high potential food security crop, there is no any improved variety as there has been very little research on the crop. this lack of research attention including absence of improved varieties and lack of conservation strategy may lead to significant genetic erosion of this crop. previous studies of p. edulis include documentation of indigenous knowledge, analysis of nutritional composition and the use of its starch in drug formulation, traditional cultivation practices, micropropagation, in vitro regeneration, and morphological and molecular genetic diversity. however, more extensive research in all aspects is required for its improvement, use and conservation. therefore, it is imperative to review the research results of this crop for the research community. this article reviewed research findings of this crop and presents it in a comprehensive way so that the readers will get ample informa­ tion and can refer original research findings for the details. 1. introduction plectranthus edulis (vatke) agnew (synonym. coleus edulis) is an ancient indigenous tuber crop that occurs both as cultivated and wild spe­ cies in ethiopia. although it is known by different local names depending on the place where it is cultivated, oromo dinich is the most popular local name. the cultivation of p. edulis is restricted to ethiopia. it is mainly cul­ tivated in the south and south western parts of the country at altitudes ranging between 1880 and 2200 m a.s.l. as food, and sometimes medicine (demissie, 1988; taye et al., 2007; megersa, 2010). p. edulis is a dicotyle­ donous plant that grows up to a height of 1.5 m (taye, 2008). p. edulis is a high energy food crop and the tubers contain substantial amounts of micro­ and macro­nutrients. when compared to irish potato (*) corresponding author: tileye.feyissa@aau.edu.et citation: feyissa t., 2021 ­ prospects for improvement of plectranthus edulis (vatke) agnew: a high potential food security crop. ­ adv. hort. sci., 35(3): 319­327. copyright: © 2021 feyissa t. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. author contributions: all authors contributed equally to the work. competing interests: the authors declare no competing interests. received for publication 17 january 2021 accepted for publication 16 july 2021 ahs advances in horticultural science review paper https://doi.org/10.36253/ahsc-10346 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2021 35(3): 319­327 320 (solanum tuberosum), p. edulis has higher food energy and contains twice the amount of fat and cal­ cium (ehnri, 1997). it contains a similar amount of protein to that of irish potato and almost twice that of sweet potato (ipomoea batatas) after cooking (ehnri, 1997). the cultivation of p. edulis is under threat because of a decline in diversified local food production systems as a result of introduction of exotic species, land use change, habitat destruction, population pressure, selection of only a few local varieties by far­ mers, farming system intensification and climate change (smolders, 2006; fao, 2010). the decline in cultivation of the crop may result in erosion of the genetic base preventing the use of distinctive useful traits that are used for crop adaptation and improve­ ment (padulosi et al., 2002; ibc, 2005). there is enormous untapped potential in ethiopia to exploit the rich and diverse plant genetic resour­ ces of underutilized root and tuber crops including p. edulis. in spite of this potential, very little research has been done to improve the productivity of p. edu‐ lis (ibc, 2007). currently, however, p. edulis is reali­ zed as a high potential food security crop that is attracting research attention. the poor storability of tubers is found to be the major production constraint of p. edulis. it is propa­ gated by vegetative methods so that it is susceptible to diseases that are transmitted from one generation to the next through infected planting material (taye, 2008). this problem can be solved by integration of new technologies including biotechnological approa­ ches. documentation of information on indigenous knowledge, uses, and distribution of this crop is essential for its conservation, further utilization, and improvement (mathenge, 1995). it is also important to initiate research on seed production of p. edulis. so far, there is no any improved variety of p. edulis. the objective of this article is to review some aspects of this crop, with particular regard to biotechnologi­ cal approaches and present it in a comprehensive way so that the readers will get summarized informa­ tion to design research or policy on conservation and improvement of the crop. 2. production practices farmers select seed tubers from the previous har­ vest or they buy from the market. most farmers plow the land more than three times before planting and mix farmyard manure with the soil (mekbib and weibull, 2012). farmers do not use chemical fertili­ zers and other commercial inputs for production of p. edulis. the seed tubers are planted from march to april as there is brief rain during this time. farmers commonly slice a single tuber into pieces and plant the pieces because they claim planting tuber pieces increases tuber yield (taye et al., 2007; mekbib and weibull, 2012). research findings also showed that planting tuber pieces results in higher yields than whole tubers (taye, 2008). in southern ethiopia, almost 50% of the farmers plant the tubers with their sprouts whereas the others plant after removing the sprouts (taye, 2008). most p. edulis growing farmers remove the shoot apex with one or two pairs of leaves from the main s t e m a n d b r a n c h e s t o i n c r e a s e s t e m n u m b e r . research results show that this practice significantly increases the number of stems and enhances faster stolen formation. most farmers commonly build up the soil around the stem to enhance plant growth and increase tuber yield. the absence of positive cor­ relation between frequency of removing the shoot apex and amount of the soil built up around the stem was reported, although both activities serve the same purpose (taye, 2008). weeding is carried out at least three times at dif­ ferent growing stages of p. edulis. following harvest of p. edulis, farmers plant other crops, and the yields of crops that follow p. edulis in the rotation are high. the reason for increase in yield could be due to the slowly released nutrients into the soil and improved soil structures as the result of the organic manure applied in previous cropping seasons (mekbib and weibull, 2012). depending on the type of variety, p. edulis is harvested from six to eight months after planting. although the cultivation is mainly perfor­ med by male farmers, female farmers also play important roles in selecting tubers for different end­ use criteria related to the household food require­ ments (mekbib and weibull, 2012). farmers used to maintain several local varieties of p. edulis for different purposes. however, the num­ ber of local varieties preserved by farmers has been declining. so far, the total number of local varieties all over the country is not known. however, there are at least six local varieties of p. edulis that grow in spe­ cific areas as study results on three kebeles (the smallest administrative units in ethiopia), namely delebo­atewaro, delebo­wogene and kokete­mare­ chare of sodo zuria district, south ethiopia have feyissa ‐ plectranthus edulis for food security 321 shown (mekbib and weibull, 2012). growth habits, taste, tuber skin color, days to maturity, drought tolerance, storage period and marketability are the main characteristics of the plant used by farmers to identify local varieties (mekbib and weibull, 2012). distribution and seed exchange of local varieties by farmers play key roles in conservation and utilization of local varieties (de boef, 2008). younger farmers mostly purchase the planting material from markets whereas older farmers (older than fifty years) use their own saved seed tubers from the previous har­ vest. older farmers have much richer indigenous knowledge than younger farmers about the cultural value of the crop, indicating a limitation in the docu­ mentation of knowledge on the management. these knowledge gaps may lead to cultivation of only few local varieties (negri, 2003; mekbib and weibull, 2012). this calls for more research on documentation of local knowledge and transferring this knowledge to the young farmers. one of the major challenges of p. edulis produc­ tion is seed tuber storage. the tuber is stored in the field where it is produced until used for the required purpose. this storage method results in significant tuber yield loss due to excessive heat especially during dry seasons. the land where the tubers are stored remains idle until all the tubers are removed. therefore, as farmers do not want their land to be idle, most of them often obtain seed tubers from other sources during planting season (mekbib and weibull, 2012). farmers cover p. edulis fields with mulching materials to protect the tubers from direct sunlight until the next planting season. after the pre­ paration of the land for planting, tubers are transfer­ red to the pits for a day to facilitate the germination and performance of seedlings. most seed tubers are obtained from farmers who have enough land to pro­ duce p. edulis as they produce more than a hou­ sehold requirement and save the surplus for sale (negri, 2003). although different cultural practices have been designed to overcome the production con­ straints of p. edulis, very little research has been done to tackle the problems. another important con­ straint is shortage of land for planting (mekbib and weibull, 2012). this can be solved by amending land usage policy. some efforts have been made to evaluate perfor­ mance of p. edulis with regard to yield and yield rela­ ted characters and reports show different perfor­ mance of the crop at different environments (taye et al., 2013). tuber fresh weights of 4556­4933 g m­2, or 45­49 mg ha­1 were obtained at experimental sites, which is higher than yields produced by farmers (740­1480 g m­2). this higher experimental yields compared to farmers’ yields is because farmers har­ vest the tubers early late emergence, slow develop­ ment of the canopy, full coverage of the ground in a short period of time and decline in soil cover during senescence caused poor radiation interception. radiation interception of a crop can be enhanced by increasing ground cover by the canopy. this could be a c h i e v e d b y p l a n ti n g l a r g e s e e d t u b e r p i e c e s (wiersema and cabello, 1986; lommen and struik, 1994), by planting more tuber pieces per area (spitters, 1990) or by using traditional practices that enhance the canopy development (taye et al., 2012). 3. nutritional content p . e d u l i s c o n t a i n s s u b s t a n ti a l a m o u n t s o f nutrients. moisture content of the tuber dry matter ranges from 14.1% to 17.5%, whereas the protein content ranges from 0.70% to 1.76% (hellemans et al., 2017). these values are higher than cassava tuber, which contains 0.3% protein and 0.1% to 0.3% starch (alvani, 2011; waterschoot et al., 2015). another study reported 11.2% moisture, 0.14% ash, 0.21% lipid, 0.43% protein, and 99.22% starch con­ tent on a dry weight basis (assefa, 2015) (table 1). table 1 ­ proximate composition of plectranthus edulis tubers on dry weight basis in g per 100 g (z) ethiopian health and nutrition research institute sample type moisture content (%) crude protein total ash crude fiber crude fat carboydrate energy (calories) references raw 81.9 1.5 1.1 0.70 0.20 15.3 69 ehnri (z), 1997 boiled 73.8 1.0 1.3 1.00 0.20 23.7 101 ehnri, 1997 starch 11.2 ­ 0.14 ­ 0.21 99.2 ­ assefa, 2015 starch 15.5 1.09 3.0 0.24 ­ ­ alvani et al., 2011 adv. hort. sci., 2021 35(3): 319­327 322 samples collected from different regions of ethiopia showed variation in fiber content based on their geographic location. starch extracted from t u b e r s a m p l e s c o l l e c t e d f r o m w o l a i t a z o n e (chenqoua, inuka and lofua areas) showed signifi­ cantly lower fiber content than tuber samples collec­ ted from arjo white, chencha and jarmet. moreover, the samples collected from arjo white, chencha and jarmet contain higher fiber content than cassava or potato. whether this variation is due to the environ­ ment or genetic variation needs further investigation. p. edulis contains amylase ranging from 14.2% to 23.9% (waterschoot et al., 2015). tuber samples collected from abay chomen district of oromia region, ethiopia, showed variation in mineral content may be due to genotype or envi­ ronmental variation. p. edulis is found to be rich in potassium followed by magnesium, calcium, zinc, manganese, and copper. mineral content of p. edulis is presented in table 2. the trace metals (chromium and nickel) and the heavy metals (lead and cadmium) were not detected, indicating p. edulis is safe with regard to the toxic heavy metal health concerns (lema, 2016). the phosphorus content of most p. edulis samples is twice as much as in irish potato. high phosphorus content is linked to resistant starch, paste viscosity and gel strength (lu et al., 2012). higher phosphorus content is responsible for the gra­ nular structure, and especially for the crystalline region to be less rigid and therefore enabling absorp­ tion of more water (lin et al., 2013). high phospho­ rus content was also found to impart high viscosity to the starch and increase gel strength. this indicates p. edulis starch can be used in food processing that requires high gel strength and can provide resistant starch in functional food preparation (hellemans et al., 2017). however, further research should be con­ ducted to confirm this finding. p. edulis is found to be richer in most of the mineral elements than irish potato. starch physicochemical properties scanning electron microscopy (sem) of the starch granules showed elliptical and some oval shapes. the starch showed a normal monomodal granule size distribution. x­ray diffraction pattern of the starch showed typical b­type with a distinctive peak (assefa, 2015). on the contrary, other authors reported distinct bimodal distribution pattern of starch granu­ lar characteristics, which indicates the presence of small (b­type) and large (a­type) granules (hellemans et al., 2017). the shape of b­type granules is spheri­ cal whereas that of a­type is elliptical when visuali­ zed by the cryo­sem. for starch extracted from p. edulis tuber, such bimodal distribution is not com­ mon. starch extracted from irish potato and cassava shows normal distribution pattern (waterschoot et al., 2015). therefore, p. edulis can be used in food industry and as an alternative for potato starch, which is less suitable to be used in pasteurized foods. typical water absorption (wa) pattern, swelling power and relative humidity of p. edulis is reported to be higher than irish potato starch but its solubility at all studied temperatures is lower than irish potato (assefa, 2015). the physicochemical analysis of the starch showed 1.08 g/g wa of p. edulis (hellemans et al., 2017). this means that the starch granules absorb on average of 1.08 times their own weight after it is completely hydrated. only a small difference was observed in wa among p. edulis starch samples of dif­ ferent accessions. the high water binding ability of the starch could reduce the stickiness of dough, increase moistness, improve handling and soften the texture of baked products, which makes p. edulis starches more suitable for use in baked goods (taggart, 2004). differential scanning calorimeter (dsc) thermo­ grams of p. edulis starch showed higher onset tempe­ rature (to) of 69.2°c, peak temperature (tp) of 74.3°c and ending temperature (te) of 83.3°c than the starch of irish potato (assefa, 2015). similarly, the pasting properties showed p. edulis starch has table 2 ­ mineral content of plectranthus edulis in mg per 100 g w ppm/dm­ parts per million/dry matter. sample k ca fe mg zn p references raw ­ 29.0 9.30 ­ ­ 90.0 ehnri, 1997 boiled ­ 19.0 1.10 ­ ­ 62.0 ehnri, 1997 starch w 513 397 20.8 175 4.5 1803 alvani et al., 2011 starch 3.54 0.62 ­ 0.49 0.24 ­ lu et al., 2012 feyissa ‐ plectranthus edulis for food security 323 (bryan, 1983). these problems might be solved by using the benefits of modern plant biotechnology such as plant tissue culture that can supply a large number of disease­free planting materials to growers who continually acquire it. micropropagation and in vitro shoot regeneration increased use of rapid multiplication techniques enables production of large amounts of pathogen­ free planting material. most rapid multiplication techniques in root and tuber crops involve the use of aerial portions of the plant so that contact with soil and tuber is broken and most non­systemic patho­ gens can be eliminated (bryan, 1983). meristem cul­ ture is the most popular tissue culture technique for production of disease free planting materials. it can be used alone or in combination with thermotherapy to eliminate a number of viruses and bacteria (kartha and gamborg, 1975). meristem culture technique eli­ minates systemic pathogens found in the mother stock plant. the first micropropagation protocol of p. edulis was developed from meristem culture (tsegaw and feyissa, 2014) followed by in vitro shoot regenera­ tion from leaf derived callus (aschale and feyissa, 2019). shoots were initiated from meristem explants using murashige and skoog (ms) (murashige and skoog, 1962) medium. the ms medium was supple­ mented with 1.0 mg/l gibberellin (ga3) and 0.1 mg/l a­naphthalene acetic acid (naa) in combination with different concentrations of benzylaminopurine (bap) ( t s e g a w a n d f e y i s s a , 2 0 1 4 ) . a c c o r d i n g t o t h e authors, ms medium containing the aforementioned hormones was proved to be the best in terms of shoot initiation from meristem for mother plants col­ lected from two locations, holeta and wolaita. the shoots were transferred to multiplication medium and the highest mean shoot number per explant (7.2) was obtained on medium containing 1.0 mg/l kinetin in combination with 0.1 mg/l naa for shoots derived from mother plants collected from holeta while, 6.2 shoots per explant was obtained on medium containing 3.0 mg/l kinetin in combination with 0.05 mg/l naa for shoots derived from mother plants collected from wolaita. the effect of solid and liquid media on in vitro axillary shoot proliferation was investigated by culturing shoots on ms medium containing different concentrations of bap in combi­ nation with thidiazuron (tdz) (yimam, 2013). the highest mean shoot number per explant, 5.85 and 6.07, were obtained on solid and liquid media, h i g h e r p a s ti n g t e m p e r a t u r e t h a n i r i s h p o t a t o (hellemans et al., 2017). a rapid increase in viscosity occurred at tp range of 70.7 to 74.0°c compared to irish potato starch (52.2°c). the disintegrating abilities of starch of p. edulis and irish potato were compared in paracetamol tablet formulations prepared by wet granulation method (assefa, 2015). the granules were prepared with the starches at different disintegrant concentra­ tions and characterized for particle size distribution and flow properties. the prepared tablets showed excellent flow property as manifested by the weight uniformity. after studying the crushing strength, fria­ bility, disintegration time, and dissolution rate of the tablets using standard methods, the results showed that p. edulis starch favorably competed with potato starch as a disintegrant in the paracetamol tablet for­ mulations. in other recent study, carboxymethylated p. edulis starch was evaluated as a suspending agent in metro­ nidazole benzoate suspensions by comparing with sodium carboxymethyl cellulose (nacmc) (brhane, 2020). the author reported viscosities of the formu­ lations prepared with carboxymethylated p. edulis starch were significantly lower than that of nacmc. the flowability of the suspensions was in the order of carboxymethylated p. edulis starch (cmps) greater than nacmc. at 1% concentration, carboxymethyla­ ted p. edulis starch resulted in significantly higher sedimentation volume than nacmc. potassium dihy­ drogen phosphate (kh2po4), which is employed as a flocculating agent, significantly increased the sedi­ mentation volume of the suspensions prepared with carboxymethylated p. edulis starch and nacmc. the redispersibilities of cmps were found to be better than nacmc. the author pointed out that all suspen­ sions resulted in a release of greater than 85% of drug within 1 h. all suspension formulations were found to be stable (brhane, 2020). the author con­ cluded carboxymethylated p. edulis starch can be used as alternative suspending agent. 4. prospects of biotechnology for improvement of p. edulis in addition to the shortage of seed tubers and the poor storability of the tubers, systemic diseases, viru­ ses, viroids and mycoplasma as well as several patho­ genic bacteria are the most devastating root and tuber crops including p. edulis, in terms of yield loss 324 adv. hort. sci., 2021 35(3): 319­327 liquid medium containing 0.1 mg/l iaa. the highest m e a n r o o t n u m b e r p e r s h o o t o f 1 0 . 1 5 o n m s medium supplemented with 2.0 mg/l iba was also reported (aschale and feyissa, 2019). all in vitro regenerated plants of holeta origin and 96% of wolaita origin survived after one month of acclimatization in a greenhouse. similar study repor­ ted 100% and 82.85% survival of plantlets derived from solid and liquid media, respectively, after accli­ matization in greenhouse (yimam, 2013). all in vitro rooted and 78 % ex vitro rooted microshoots survi­ ved in the greenhouse (aschale and feyissa, 2019). the development of these protocols for micropropa­ gation and in vitro regeneration of shoots from callus is the prerequisite for further advanced biotechno­ logy research such as genetic transformation and genome editing of this crop. 5. genetic diversity of p. edulis for improvement of any crop, the existence of adequate genetic diversity is imperative. this could be achieved if and only if there is appropriate conser­ vation strategy. selection and cultivation of only few local varieties of p. edulis and its current replacement by other tuber crops has been causing serious gene­ tic erosion of the local gene pool of this crop. genetic diversity analyses using morphological traits there are several studies on morphological gene­ tic diversity of p. edulis. genetic diversity of 36 acces­ sions of p. edulis using 16 morphological traits was assessed and highly significant variation among the accessions for all the analyzed traits except length of t u b e r w a s r e p o r t e d ( g a r e d e w e t a l . , 2 0 1 3 ) . i n another study, 20 p. edulis accessions that were assessed using 29 morphological traits showed 75.7% variance among the accessions (mekbib, 2007). this study showed that there are accessions collected from different regions of the country but showed genetic similarity, and some of those collected from the same geographic area showed different genetic background. this indicates geographic diversity does not necessarily represent genetic diversity. similarly, genetic diversity analysis of 20 accessions using 13 traits revealed significant variation of all traits except flower length and leaf width (soresa, 2017). recent extensive genetic diversity study of 174 accessions at three environments based on 12 qualitative and 16 quantitative traits also showed a highly significant respectively, containing 1.5 mg/l bap in combination with 0.5 mg/l tdz. developing in vitro regeneration protocol through callus phase or somatic embryogenesis is a prerequi­ site to genetically improve a crop through biotechno­ logical approaches including genetic engineering, genome editing and creating somaclonal variants. recently, in vitro regeneration protocol of p. edulis have been developed by using leaf explants (aschale and feyissa, 2019). the highest percentage of callus induction (100%) was obtained on ms medium sup­ plemented with 1.5 mg/l naa in combination with 1.0 mg/l bap and 2.0 mg/l naa in combination with 0.5 mg/l bap. the highest shoot regeneration per­ centage (46.6%) from callus was obtained on ms medium containing 0.1 mg/l bap in combination with 0.5 mg/l tdz. the highest mean shoot number per callus (1.66) was obtained on medium containing 1.5 mg/l bap in combination with 1.0 mg/l tdz. the highest mean shoot length (0.63 cm) was obtained on medium supplemented with 0.5 mg/l bap in com­ bination with 0.1 mg/l tdz. the highest mean shoot number per explant (58.68) was obtained on medium supplemented with 0.5 mg/l bap in combination with 0.4 mg/l ga3. rooting and acclimatization shoots cultured on half salt strength ms medium r o o t e d b e tt e r t h a n t h o s e c u l t u r e d o n f u l l s a l t strength (tsegaw and feyissa, 2014). shoots derived from explants collected from holeta resulted in the highest percentage of rooting (100%) and 3.12 mean root number per shoot whereas shoots whose explants were collected from wolaita resulted in 7 6 . 7 % r o o ti n g a n d 2 . 2 m e a n r o o t n u m b e r p e r explant. microshoots were also directly planted in a greenhouse for rooting and acclimatization by bypas­ sing the in vitro rooting stage and the overall best result in rooting was obtained in ex vitro rooting con­ dition. it was promising in cost reduction as it bypas­ ses the in vitro rooting stage. reducing the cost of in vitro production is a key for increasing the applica­ tion of the method. rooting of microshoots in vitro is expensive and can even double the price of the plan­ tlets (zimmerman, 1988; de klerk, 2002). in another study, shoots were cultured on half strength ms medium containing different concentrations of indole acetic acid (iaa) and indole butryic acid (iba) for roo­ ting (yimam, 2013). the highest mean root number per shoot (10.55) was obtained on solid medium and 13.11 mean root number per shoot was obtained in feyissa ‐ plectranthus edulis for food security 325 variation among the populations and non­significant environment­population interaction for most of the quantitative traits (gadissa et al., 2020). the first six principal axes of principal components analysis accounted for 77% of the total variation. the popula­ tions were grouped into four clusters but there was no strong groupings based on geographical locations from where the genotypes were collected. this indi­ cates the historical or contemporary gene flow, parti­ cularly tuber exchange among the different geo­ graphical regions. although there are reports of rela­ tively high genetic diversity, still there is a need for more collections and evaluation over multiple loca­ tions and seasons for well­refined genetic diversity estimation. however, as the analyzed traits are affec­ ted by the environment, the morphological genetic diversity results should be complemented by molecu­ lar genetic diversity studies. genetic diversity analyses at molecular level assessment of molecular genetic diversity of p. edulis was conducted by using inter simple sequence repeat (issr) markers for the first time in 2015 (shiferaw, 2015). the shannon information index ranged from 0.25 to 0.39 with overall index of 0.48. nei’s gene diversity ranged from 0.18 to 0.30 with overall mean diversity of 0.33, and 74.23% within population variation. analysis of molecular variance (amova) showed within population diversity range of 41.30% to 65.22%. another study using the same marker exhibited overall percent polymorphism of 95%, 0.62 shannon information index and 0.40 nei’s gene diversity (gebrehiwet et al., 2019). more exten­ sive study of genetic diversity assessment was con­ ducted by using expressed sequence tag simple sequence repeats (est­ssrs) derived from p. barba‐ tus (gadissa et al., 2018). the author identified 128 alleles in 12 populations that consisted of 287 indivi­ dual plants. the results showed gene diversity index range of 0.31 to 0.39 with overall mean of 0.35. a m o n g t h e 1 2 p o p u l a ti o n s , w e n b e r a , a w i a n d wolaita populations showed the highest genetic diversity, and hence these populations can be consi­ dered for in situ conservation and identification of genotypes with traits of interest that can be used in breeding programs. amova showed low population differentiation with only 3% of the total variation accounting for variation among populations. struc­ ture and cluster analyses did not group the popula­ tions into distinct clusters, which may be attributed to historical and contemporary gene flow and also the reproductive biology of the crop. 6. conclusions p. edulis is a high potential food security crop con­ taining substantial amounts of nutrients. however, there is no any improved variety of the crop, and cur­ rently its cultivation is declining. shortage of tuber seed is the major problem of this crop. recently, the farmers are turning to produce other cash crops that generate more income. with advances in plant research, it is important to integrate both conventio­ nal and modern technologies to improve this crop. unless urgent attention is given to such neglected and underutilized crops, these crops are extremely subjected to genetic erosion as the farmers shy away from producing them. using biotechnological resear­ ch tools such as plant tissue culture, genetic enginee­ ring, genome editing and plant molecular breeding might be a future hope for improvement of this crop, although the international funding opportunities for such orphan crops are highly limited. in vitro propa­ gation and in vitro regeneration protocols from diffe­ rent explants have already been developed as reviewed in this article and these protocols can be used for improvement of p. edulis. in addition, infor­ mation from the genetic diversity results reported so far could be used as a baseline for more research in the area and improve this crop using genomic tools including marker assisted breeding. acknowledgements the author acknowledges addis ababa university for providing the necessary resources during writing of this review. the author is also grateful to graduate students who have been working with him and other researchers who generated interesting information on plectranthus edulis that served as resources to write this review. references alvani k., qi x., tester r.f., snape c.e., 2011 ­ physico‐ chemical properties of potato starches. ­ food chem., 125(3): 958­965. aschale n., feyissa t., 2019 ­ in vitro regeneration of plectranthus edulis (vatke) from leaf derived callus. ­ adv. hort. sci., 2021 35(3): 319­327 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nauni lies at latitude and longitude of 300 52’n and 77° 11’ and chambaghat, 30° 55’ n and 77° 06’. all the parents except two gynoecious lines were of monoecious type. crosses among eight parents were attempted in a half-diallel fashion. the material comprising eight parents, 28 f 1 s and one check (pusa sanyog) was sown in randomized block design with three replications. spacing was 1.25x1.00 m. data were recorded on randomly selected plants for yield and horticultural characters at both the locations. griffing’s (1956) method ii model i was used to derive general and specific combining ability estimates. the analysis of variance for combining ability was based on following mathematical model: p ijk = m + g ii + g jj + s ij + b k + e ijk selecting parents for developing superior hybrids in cucumber (cucumis sativus l.) b.s. dogra* , m.s.kanwar** * regional horticultural and forestry research station, dr y s parmar university of horticulture and forestry, bhota-176041, hamirpur (hp), india. ** high mountain arid agriculture research institute (skuast-k), leh, 194101 jammu and kashmir, india. key words: cucumber, cucumis sativus, gca, hybrids, sca. abstract: estimates of general combining ability of parents and specific combining ability of the crosses help to select desidered parents for hybridization and development of superior hybrids. crosses among eight parents were attempted in a half-diallel fashion. the material comprising eight parents, 28 f1s and one check (pusa sanyog) was sown at two locations in randomized block design with three replications. the highest estimates of general combining ability (gca) were exhibited by g2 and gyn1 for most of the characters at both the locations. in general, there was close agreement between gca effects and per se performance, but in some cases it did not hold good, which may be due to a higher degree of gene action involved. the superior cross combinations which recorded high specific combining ability (sca) estimates and per se performance for yield and number of fruits were k-90 x g2 and k-90 x gyn1 and hence may be exploited for the development of f1 hybrid (s) after testing their performance at multi-locations for two to three years. adv. hort. sci., 2011 25(4): 239-244 received for publication 1 june 2011 accepted for publication 21 october 2011 240 where, p ijk = phenotypes of the hybrids between ith and jth parents in kth plots m = population mean g ii = gca effects of ith parent g jj = gca effects of jth parent s ij = sca of the crosses between ith and jth parents b k = block effects e ijk = environmental effect associated with ijkth observation 3. results and discussion analysis of variance (table 1) for combining ability revealed that the importance of gca (σ2g) was more than sca (σ2s), indicating the preponderance of additive gene action for days to first female flower appearance (dfffa) at location 1 and days to marketable maturity (dmm) at both locations. however, in all the other traits, the sca component was higher in magnitude than gca’s, indicating the preponderance of non-additive gene effects. however, mean sum of squares for gca and sca were highly significant for all the characters except tss, suggesting the importance of both additive and non-additive genetic variance in agreement with the findings of om et al. (1978). similar trends at both the locations proved that the conclusions on gene actions are authentic. the parents g 2 , gyn 1 and poinsette had negative estimates for dfffa and node at which first female flower appears (nfff) at both the locations (table 2) showing earliness in fruit bearing and were good general combiners for these characters. among f 1 ’s, the sca effects were significantly negative in 12 and 15 crosses, respectively, for these two traits at l1 (table 3) whereas significantly negative in 15 crosses for each of these two traits at l2 (table 4). the crosses lc-11 x gyn 1 (poor x high) and ec 173934 x lc-40 (poor x poor), respectively, had the highest sca effect at l1 and the crosses lc-11 x lc-40 (poor x poor) and ec 173934 x lc-40 (poor x poor), respectively, had the highest sca effects at l2 for these traits. the parents g 2 and gyn 1 (l1) and g 2 , gyn 1 and poinsette (l2) with significantly high gca estimates (with negative value) were good general combiners for dmm. crosses lc-11 x gyn 1, ec 173934 x lc-40, k-90 x g 2 and k-90 x ec 173934 had high sca estimates at both the locations for dmm. el-shawaf and baker (1978), om et al. (1978), and wang and wang (1980) also reported greater additive genetic variance for dmm. the parents g 2 and gyn 1 may be used in the hybridisation programme for developing early hybrids adapted to a wide range of climate. lc-11 x gyn 1 and ec 173934 x lc-40 may be exploited as early hybrids after further multi-locational testing. these crosses may also be exploited to produce transgressive segregants in advanced generations. with regard to fruit length, the parents gyn 1, lc-11 and k-90 were good general combiners as is evident from their high gca estimates at both locations. fourteen crosses exhibited significant sca effects. the sca effects were high in crosses poinsette x lc-40 and g 2 x poinstte involving poor x poor general combiners. k-90, k-75 and ec 173934 had the highest gca with respect to fruit width and hence were good general combiners. the sca effect was maximum in g 2 x gyn 1 involving poor x poor general combining parental lines (at l1) and in g 2 x k-75 involving poor x high general combining parental lines (at l2). in india, slicing cucumbers are preferred, therefore lengthy fruits are desirable. kupper and staub (1988) and hormuzdi and more (1989) reported contrasting results for fruit length and width due to different experimental material and environment. table 1 analysis of variance for combining ability for different characters in f 1 cucumber source of variation df character days to first female flower appearance node of first female flower days to marketable maturity fruit length fruit width tss flesh to seed cavity ratio fruit weight no. of fruits per plant yield per plant internodal length location 1 nauni gca 7 678.818 * 27.997 * 705.436 * 6.425 * 1.087 * 0.005 0.001 * 3787.657 * 9.898 * 0.735 * 9.512 * sca 28 42.264 * 3.049 * 45.029 * 3.237 * 0.243 * 0.021 0.0015 * 693.149 * 1.159 * 0.193 * 2.183 * error 70 0.557 0.228 0.562 0.004 0.002 0.0013 0.00004 62.357 0.112 0.0013 0.272 σ2g 67.826 2.777 70.487 0.642 0.108 0.0004 0.0001 372.53 0.979 0.073 0.924 σ2s 41.707 2.821 44.467 3.0233 0.240 0.020 0.002 630.79 1.047 0.191 1.911 σ2g/ σ2s 1.626 0.984 1.585 0.199 0.451 0.021 0.068 0.591 0.934 0.383 0.483 location 2 chambaghat gca 7 390.457 * 35.726 * 577.811 * 7.820 * 0.993 * 0.012 * 0.0016 * 3515.486 * 14.247 * 0.786 * 7.800 * sca 28 67.477 * 4.551 * 37.300 * 3.895 * 0.268 * 0.028 * 0.0009 * 612.551 * 1.582 * 0.181 * 1.510 * error 70 0.431 0.205 0.442 0.089 0.023 0.006 0.000035 49.232 0.148 0.0096 0.358 σ2g 39.003 3.552 57.737 0.773 0.097 0.0006 0.000159 346.630 1.409 0.078 0.744 σ2s 67.046 4.346 36.859 3.806 0.245 0.022 0.00088 563.320 1.434 0.171 1.153 σ2g/ σ2s 0.582 0.817 1.566 0.203 0.395 0.029 0.081 0.615 0.983 0.452 0.646 * significant at 5% level of significance. 241 the best general combiners for tss at both locations in order of merit were ec 173934 and lc-40. among 28 specific combinations, 16 (at l1) and 14 (at l2) crosses exhibited positive sca effects being maximum in k-90 x poinsette and poinsette x k-75 at l1 and lc-40 x gyn 1 , k-90 x poinsette and k-75 x lc-40 at l2. for flesh to seed cavity ratio (fsr), the best general combiners were poinsette, ec 173934 and gyn 1 , irrespective of locations. cross combination k-90 x k-75 at l1 and poinsette x ec 173934 at l2 had maximum sca among seven significant and positive specific combinations. in contradiction to the present results, importance of additive gene action for fsr has been reported (dogra, 1995). the parents lc-11, k-90 and k-75 depicted high per se performance with respect to fruit weight at both locations as is evident from their high gca effect (table 2). these parents had maximum concentration of favourable genes for increasing fruit weight. eleven (at l1) and 12 (at l2) specific cross combinations had significantly positive sca effects (tables 3 and 4), being maximum in k-90 x lc-11 (high x high) and k-90 x ec 173934 (high x poor). non-additive gene action for fruit weight was also obtained by ghaderi and lower (1979) in consonance with the present findings. however, gyn 1 and g 2 were identified as good general combiners for number of fruits per plant. the top specific combinations in order of merit were k-90 x g 2 , k-90 x gyn 1 and k-75 x gyn 1 involving medium high, medium x high and poor x high general combiners, respectively. the situation holds good for both the locations with respect to number of fruits. importance of non additive gene action for number of fruits per plant was also reported (om et al., 1978; ghaderi and lower, 1979; dogra, 1995). however, the present results with regard to fruit weight and number of fruits are in disagreement with el hafeez et al. (1997). this may be due to differences in the parental material used for making diallel crosses. for yield per plant, k-90 was the best general combiner in addition to gyn 1 and g 2 irrespective of location (table 2). the sca effects (tables 3 and 4) were high for k-90 x g 2 (high x high), k-90 x gyn 1 (high x high) and lc-11 x gyn 1 (poor x high). the present results on yield per plant were similar to earlier findings of om et al. (1978), ghaderi and lower (1979), wang and wang (1980) and doligibh and sidorova (1983) but in contradiction to the work of gu et al. (2004). parents such as g 2 , gyn 1 and lc-40 had negative gca effects and were considered good general combiners for internodal length. nine (at l1) and 10 (at l2) specific combinations had significant negative values with the maximum in k-90 x poinsette and poinsette x ec 173934, poor x poor general combiners at each location. as is evident from the data in tables 2, 3 and 4, environmental effect was observed as non-significant on genotable 2 estimates of general combining ability of parents for different characters in cucumber source of variation character days to first female flower appearance node of first female flower days to market-able maturity fruit length fruit width tss flesh to seed cavity ratio fruit weight no. of fruits per plant yield per plant internodal length location 1 k-90 0.000 0.367* -0.550* 0.361* 0.364* -0.016* -0.0002 20.083* 0.017 0.276* 0.021 g2 -12.133* -2.567* -12.217* -1.404* -0.041* 0.004 0.004* -25.250* 1.317* 0.302* -1.856* poinsette -2.433* -0.767* -2.0183* -0.105 -0.531* -0.031 0.014* -4.917* -0.217* -0.055* 1.048* ec173934 8.167* 1.633* 8.517* -0.390* 0.191* 0.037* 0.011* 7.417* -0.617* -0.346* 0.144 k-75 0.733* 0.733* 1.017* -0.050* 0.320* -0.004 0.017* 10.083* -0.017 0.024* 1.084* lc-11 6.600* 0.633* 6.583* 0.388* 0.136* -0.022 0.007* 32.750* -0.783* -0.089* 0.604* lc-40 9.800* 2.067* 9.950* -0.225* -0.008* 0.029* 0.005* -8.417* -1.283* -0.379* -0.593* gyn1 -10.733* -2.100* -11.117* 1.425* -0.433* 0.002 0.008* -16.917* 1.583* 0.268* -0.453 se (gi) 0.221 0.141 0.222 0.019 0.013 0.011 0.0019 2.336 0.099 0.011 0.154 cd0.05 (gi) 0.441 0.281 0.443 0.037 0.026 0.021 0.0038 4.658 0.197 0.022 0.307 location 2 k-90 0.075 0.258* -0.267* 0.208* 0.269* -0.021 -0.013* 20.492* 0.508* 0.301* 0.116 g2 -10.092* -2.908* -11.600* -1.355* 0.016 -0.015 -0.016* -23.341* 1.842* 0.285* -1.828* poinsette -1.158* -0.375* -1.133* -0.285* -0.574* -0.008 0.018* -7.141* -0.325* -0.053* 0.693* ec173934 7.642* 1.192* 7.867* -0.592* 0.196* 0.065* 0.014* -5.342* -0.858* -0.384* 0.489* k-75 2.908* 0.792* 1.100* -0.025 0.309* -0.013 -0.010* 8.825* -0.258* 0.058* 0.869* lc-11 2.875* 1.325* 6.300* 0.495* 0.083* -0.028* -0.0001 30.825* -1.092* -0.125* 0.513 lc-40 5.675* 2.358* 7.900* -0.148* 0.083* 0.045* -0.0007 -5.342* -1.358* -0.363* -0.364* gyn1 -7.925* -2.642* -10.167* 1.702* -0.381* -0.026* 0.009* -18.375* 1.542* 0.279* -0.488* se (gi) 0.194 0.134 0.197 0.088 0.044 0.023 0.0018 2.076 0.114 0.029 0.177 cd0.05 (gi) 0.387 0.267 0.393 0.175 0.088 0.046 0.0036 4.139 0.227 0.058 0.353 * significant at 5% level of significance. 242 t ab le 3 e st im at es o f sp ec if ic c om bi ni ng a bi li ty o f f 1 fo r di ff er en t ch ar ac te rs i n cu cu m be r at n au ni ( l 1) c ro ss es c ha ra ct er s d ay s to fi rs t fe m al e flo w er ap pe ar an ce n od e of fi rs t fe m al e flo w er d ay s to m ar ke ta bl e m at ur ity fr ui t l en gt h fr ui t w id th t ss fl es h to s ee d ca vi ty ra tio fr ui t w ei gh t n o. o f f ru its pe r p la nt y ie ld p er pl an t in te rno da l le ng th k -9 0x g 2 -7 .4 22 * -1 .0 59 * -7 .2 52 * -2 .4 81 * -0 .4 11 * -0 .0 82 * 0. 12 2* -1 3. 25 9* 2. 68 5* 1. 02 3* -0 .6 27 * k -9 0x p oi ns et te -4 .7 89 * -0 .5 26 * -4 .6 18 * -0 .4 40 * 0. 08 6* 0. 27 5* -0 .0 46 * -3 8. 59 3* 0. 84 8* -0 .3 24 * -2 .5 30 * k -9 0x e c 17 39 34 -7 .0 56 * -1 .2 59 * -7 .6 12 * -0 .4 45 * -0 .5 73 * -0 .0 73 * -0 .0 40 * 40 .5 74 * -0 .7 52 * -0 .0 59 * -1 .8 94 * k -9 0x k -7 5 10 .3 78 * 1. 97 4* 9. 48 4* 1. 38 2* 0. 45 1* 0. 01 1 0. 01 2* -3 5. 25 9* -0 .3 52 * -0 .5 43 * 0. 46 6 k -9 0x l c -1 1 9. 17 8* -0 .2 59 9. 94 8* -0 .4 06 * 0. 51 6* -0 .0 61 * -0 .0 31 * 55 .4 07 * -0 .2 52 -0 .3 09 * 2. 24 6* k -9 0x l c -4 0 -5 .3 56 * -1 .6 93 * -5 .0 85 * 1. 32 4* -0 .7 94 * 0. 00 6 0. 00 6* -1 .7 59 -0 .0 85 -0 .3 03 * 2. 04 3 k -9 0x g yn 1 -4 .4 89 * -0 .5 26 * -5 .3 52 * -1 .3 43 * -0 .1 82 * -0 .0 51 * -0 .0 13 * -2 3. 25 9* 2. 38 2* 0. 50 9* -0 .0 30 g 2 xp oi ns et te 2. 01 1* 1. 07 4* 3. 71 5* 2. 33 4* -0 .3 93 * 0. 00 2* -0 .0 45 * 0. 07 4 -0 .1 19 -0 .3 16 * 0. 68 0* g 2 x e c 17 39 34 3. 41 1* 0. 00 7 3. 34 8* 1. 81 9* 0. 48 2* -0 .0 32 -0 .0 29 -4 .0 93 -1 .0 52 * -0 .3 98 * 0. 88 3* g 2x k -7 5 0. 17 8 -1 .0 93 * 0. 51 5 2. 11 3* 0. 30 6* 0. 11 2* -0 .0 09 * 21 .0 74 * 1. 34 8* 0. 49 4* 1. 14 3* g 2 x l c -1 1 -3 .3 56 * 0. 67 4* -4 .0 52 * -1 .1 42 * -0 .3 67 * 0. 04 3 -0 .0 14 * 24 .0 74 * 0. 11 5 0. 26 8* -0 .0 44 g 2 x l c -4 0 9. 44 4* 0. 57 4* 9. 24 8* 0. 63 8* -0 .2 40 * 0. 16 0* 0. 00 3 -2 6. 42 6* -1 .3 85 * -0 .6 59 * 0. 45 3 g 2 x g yn 1 -0 .3 56 * 0. 74 1* 0. 98 2* 1. 42 1* 0. 75 6* 0. 09 3* -0 .0 23 * -1 4. 59 3* 0. 08 2 -0 .1 40 * 1. 01 3* po in se tte x e c 17 39 34 1. 04 4* -1 .1 26 * 1. 31 5* 2. 07 0* 0. 19 2* 0. 08 5* 0. 08 0* 5. 57 4 0. 48 2* 0. 06 5* -2 .3 87 * po in se tte x k -7 5 -0 .5 22 * 0. 10 7 -1 .1 85 * 2. 00 2* 0. 34 3* 0. 22 3* -0 .0 39 * 16 .4 07 * -0 .1 18 0. 20 1* 2. 00 6* po in se tte x l c -1 1 -5 .0 56 * -1 .1 26 * -5 .4 18 * -0 .3 25 * -0 .5 27 * -0 .0 23 * -0 .0 35 * 10 .4 07 * 0. 64 8* 0. 47 8* 1. 41 9* po in se tte x l c -4 0 -4 .9 22 * -1 .2 26 * -5 .4 52 * 2. 62 2* 0. 65 4* -0 .2 56 * 0. 03 2* -2 8. 42 6* 0. 48 1* 0. 08 8* -1 .5 17 * po in se tte x g yn 1 14 .9 44 * 0. 94 1* 15 .6 15 * -1 .9 95 * 0. 12 6* 0. 12 0* 0. 03 2* 18 .4 07 * -0 .3 85 * 0. 26 0* 1. 10 9* e c 17 39 34 x k -7 5 -1 .4 56 * 1. 04 1* -2 .8 85 * 0. 03 3 -0 .2 76 * -0 .2 05 * 0. 00 06 -7 .7 59 * -0 .7 18 * -0 .3 14 * 1. 00 9* e c 17 39 34 x l c -1 1 -0 .3 22 * -2 .5 26 * -0 .7 85 * 1. 16 1 0. 13 9* -0 .0 74 * 0. 04 4* 9. 57 4* 0. 04 8 0. 18 9* 0. 48 9* e c 17 39 34 x l c -4 0 -9 .1 89 * -3 .2 93 * -8 .8 18 * 1. 17 4* -0 .5 25 * -0 .1 07 * -0 .0 02 -5 .9 26 1. 54 8* 0. 44 6* 0. 35 3 e c 17 39 34 x g yn 1 9. 67 8* 3. 54 1* 8. 91 5* -0 .0 92 * -0 .0 59 * 0. 03 6* -0 .0 18 * -1 4. 09 3* -1 .9 85 * -0 .6 02 * 0. 17 9 k -7 5x l c -1 1 3. 77 8* 0. 37 4 3. 71 5* 1. 23 8* 0. 43 6* 0. 02 0 -0 .0 14 -3 6. 25 9* 0. 44 8* -0 .2 58 * -2 .0 84 * k -7 5x l c -4 0 4. 24 4* -2 .0 59 * 4. 68 2* 0. 90 1* -0 .6 77 * 0. 13 4* -0 .0 13 * -0 .0 93 -0 .0 52 -0 .0 28 * -1 .9 54 * k -7 5x g yn 1 -6 .5 56 * -0 .8 93 * -6 .5 85 * -1 .1 16 -0 .3 79 * -0 .2 13 * -0 .0 20 * 13 .4 07 * 1. 41 5* 0. 53 8* 0. 00 6 l c -1 1x l c -4 0 4. 04 4* 4. 04 1* 4. 11 5* 0. 49 6* -0 .5 93 * -0 .0 05 -0 .0 03 22 .2 41 * -0 .2 85 0. 04 9* 1. 25 9* l c -1 1x g yn 1 -9 .4 22 * -1 .4 59 * -9 .8 18 * -0 .4 54 * -0 .1 58 * 0. 12 8* -0 .0 30 * 25 .7 41 * 0. 18 1 0. 51 8* -0 .6 14 * l c -4 0x g yn 1 3. 37 8* -0 .2 26 2. 48 2* -1 .7 07 * 0. 17 3* 0. 21 1 -0 .0 19 * -1 6. 42 6* -1 .3 18 * -0 .3 52 * -2 .0 84 * se (i j) ± 0. 67 6 0. 43 3 0. 68 0 0. 05 8 0. 04 4 0. 03 2 0. 00 57 7. 16 0 0. 30 3 0. 03 3 0. 47 2 c d 0. 05 1. 99 4 0. 88 3 1. 35 6 0. 11 6 0. 08 8 0. 06 4 0. 01 1 14 .2 80 06 04 0. 06 6 0. 94 1 *s ig ni fi ca nt a t 5% l ev el o f si gn if ic an ce . 243 t ab le 4 e st im at es o f sp ec if ic c om bi ni ng a bi li ty o f f 1 fo r di ff er en t ch ar ac te rs i n cu cu m be r at c ha m ba gh at ( l 2) c ro ss es c ha ra ct er s d ay s to fi rs t fe m al e flo w er ap pe ar an ce n od e of fi rs t fe m al e flo w er d ay s to m ar ke ta bl e m at ur ity fr ui t l en gt h fr ui t w id th t ss fl es h to s ee d ca vi ty ra tio fr ui t w ei gh t n o. o f f ru its pe r p la nt y ie ld p er p la nt in te rno da l le ng th k -9 0x g 2 -7 .8 26 * -0 .9 15 * -7 .2 07 * -2 .2 55 * -0 .7 45 * -0 .0 95 * 0. 00 9* -8 .6 41 * 3. 69 6* 1. 05 8* -1 .0 97 * k -9 0x p oi ns et te -3 .7 59 * 0. 88 5* -3 .6 74 * -0 .0 25 0. 01 2 0. 26 5* -0 .3 90 * -2 8. 17 4* -0 .1 37 -0 .3 61 * -0 .9 17 * k -9 0x e c 17 39 34 -6 .8 93 * -2 .0 15 * -6 .6 74 * -0 .1 52 * -0 .6 25 * -0 .1 42 * -0 .0 28 * 43 .3 59 * -1 .2 70 * -0 .1 40 * -1 .5 80 * k -9 0x k -7 5 9. 17 4* 1. 71 9* 11 .4 26 * 1. 08 1 0. 29 5* 0. 07 0 0. 04 2* -2 7. 47 4* -0 .5 37 * -0 .5 65 * 1. 14 0* k -9 0x l c -1 1 11 .2 07 * -0 .1 48 7. 89 3* -0 .1 72 0. 08 8 -0 .0 49 -0 .0 14 * 33 .8 59 * -0 .3 70 * -0 .2 52 * 2. 26 3* k -9 0x l c -4 0 -1 .5 93 * -1 .5 15 * -3 .3 74 * 1. 70 5* -0 .4 12 * -0 .0 22 -0 .0 04 -1 .6 41 -0 .4 37 * -0 .2 88 * -0 .1 93 k -9 0x g yn 1 -7 .6 59 * -1 .1 82 * -5 .9 74 * -1 .5 78 * -0 .0 82 * -0 .0 50 -0 .0 07 * -1 9. 67 4* 1. 66 3* 0. 43 1* -0 .7 37 * g 2 xp oi ns et te 3. 74 1* 1. 38 5* 4. 65 9* 2. 83 8* -0 .5 02 * -0 .0 09 -0 .0 36 * -1 7. 67 4* -0 .4 70 * -0 .2 55 * 0. 02 7 g 2 x e c 17 39 34 0. 60 7* 0. 81 9* 2. 32 6* 2. 84 5* 0. 42 8* 0. 01 8 -0 .0 22 * -1 4. 47 4* -1 .9 37 * -0 .4 34 * 1. 36 3* g 2x k -7 5 -5 .6 59 * -1 .4 48 * -2 .9 07 * 2. 27 8* 0. 78 2* 0. 19 6* -0 .0 01 23 .0 26 * 0. 79 6* 0. 33 4* -0 .5 83 * g 2 x l c -1 1 -1 .2 93 * 0. 35 2 -3 .7 74 * -1 .2 75 * -0 .3 92 * 0. 04 5 -0 .0 08 * 24 .3 59 * -0 .7 04 * 0. 18 4* -0 .3 27 g 2 x l c -4 0 18 .5 74 * 1. 31 9* 7. 29 3* 0. 70 2* 0. 00 8 -0 .0 95 * 0. 02 3* -1 2. 80 7* -1 .4 37 * -0 .5 15 * 0. 71 7* g 2 x g yn 1 -0 .8 26 * 0. 65 2* 2. 02 6* 1. 01 8* 0. 73 8* 0. 14 3* -0 .0 14 * -7 .5 07 * -0 .6 70 * -0 .0 87 0. 80 7* po in se tte x e c 17 39 34 2. 67 4* -1 .0 48 * 2. 85 9* 0. 90 8* 0. 31 8* 0. 14 5* 0. 07 7* 14 .3 26 * 0. 89 6* 0. 14 7* -1 .9 57 * po in se tte x k -7 5 -3 .5 93 * -0 .3 15 -1 .3 74 * -1 .7 92 * 0. 07 2 0. 19 0* -0 .0 42 * 30 .1 59 * 0. 29 6 0. 25 5* 1. 33 0* po in se tte x l c -1 1 -2 .8 93 * 0. 48 5* -6 .5 74 * -0 .4 78 * -0 .3 02 * 0. 00 5 -0 .0 12 * 11 .4 93 * 0. 79 6* 0. 49 2* 1. 58 7* po in se tte x l c -4 0 -0 .6 93 * -1 .5 48 * -2 .8 41 * 2. 66 5* 1. 06 5* -0 .1 70 * 0. 02 9* -2 9. 00 7* 0. 72 9* 0. 06 6 -1 .9 03 * po in se tte x g yn 1 11 .5 74 * 1. 11 8* 13 .8 93 * -2 .2 52 * -0 .3 72 * 0. 06 9 0. 01 5* 9. 95 9* -0 .1 70 0. 34 4* 0. 75 3* e c 17 39 34 x k -7 5 -1 .7 26 * 3. 45 2* -0 .0 41 -0 .0 85 -0 .1 65 * -0 .2 50 * 0. 00 2 -3 .3 07 -0 .1 70 -0 .2 47 * -0 .2 00 e c 17 39 34 x l c -1 1 4. 30 7* -2 .0 82 0. 75 9* 1. 09 5* 0. 19 5* -0 .0 69 0. 04 2* 19 .6 93 * 0. 33 0* 0. 23 6* -0 .3 43 e c 17 39 34 x l c -4 0 -6 .1 59 * -4 .1 15 * -8 .5 07 * 1. 10 5* -0 .2 05 * -0 .1 75 * -0 .0 04 -1 9. 14 1* 1. 26 3* 0. 34 0* 1. 60 0* e c 17 39 34 x g yn 1 2. 77 4* 0. 21 9 4. 89 3* -0 .4 12 * -0 .1 09 * -0 .0 70 * -0 .0 14 * -1 8. 84 1* -1 .9 70 * -0 .6 18 * 1. 42 3* k -7 5x l c -1 1 5. 70 7* -1 .0 15 * 4. 19 3* 1. 06 2* 0. 58 2* -0 .1 57 * -0 .0 14 * -4 1. 14 1* 0. 39 6* -0 .3 09 * -0 .8 57 * k -7 5x l c -4 0 4. 90 7* -3 .7 15 * 4. 25 9* 0. 93 8* -0 .8 85 * 0. 20 3* -0 .0 17 * -6 .0 41 -0 .0 04 -0 .1 31 * -1 .6 13 * k -7 5x g yn 1 8. 50 7* -1 .0 48 * -5 .6 74 * -1 .5 78 * -0 .2 55 * -0 .2 42 * -0 .0 20 * 10 .3 26 * 1. 42 9* 0. 30 7* 0. 07 7 l c -1 1x l c -4 0 -2 2. 72 6* 3. 08 5* 6. 39 3* 0. 61 8* -0 .8 25 * -0 .0 15 -0 .0 14 * 23 .0 26 * 0. 16 3 0. 02 5* 0. 07 7 l c -1 1x g yn 1 -5 .7 93 * -2 .2 48 * -7 .5 41 * -0 .8 65 * 0. 23 8* 0. 12 3* -0 .0 30 * 28 .3 26 * -0 .4 04 * 0. 57 4* -1 .4 67 * l c -4 0x g yn 1 2. 07 4* 5. 05 2* 1. 85 9* -2 .3 22 * 0. 10 5 0. 31 7* -0 .0 23 * -1 2. 17 4* -1 .1 37 * -0 .3 02 * -1 .3 22 * se (i j) ± 0. 59 5 0. 41 1 0. 60 3 0. 27 1 0. 13 4 0. 07 0 0. 00 54 6. 36 2 0. 34 9 0. 08 9 0. 54 2 c d 0. 05 1. 18 6 0. 81 9 1. 20 2 0. 54 0 0. 26 7 01 39 0. 01 1 12 .6 85 0. 69 6 0. 17 7 1. 08 1 *s ig ni fi ca nt a t 5% l ev el o f si gn if ic an ce . 244 types and hybrid combinations for most of the characters. the results are similar at both locations with developed hybrid combinations and hence hybrids k-90 x g 2 and k-90 x gyn 1 can be exploited in similar types of climates. k-90, g 2 and gyn 1 may be used in hybridisation for developing high yielding hybrids with higher number of fruits per vine, long fruits and high tss on the basis of results from location 1, whereas g 2 and gyn 1 are promising for developing high yielding hybrids with higher number of fruits per vine and short inter-nodal length on the basis of results from location 2. it can be concluded that g 2 and gyn 1 may be used in hybridisation for developing high yielding hybrids with more fruits per vine and wider adaptability. the crosses k-90 x g 2 and k-90 x gyn 1 can be released as hybrids after further testing. references dogra b.s., 1995 heterosis and combining ability studies in cucumber (cucumis sativus l.) m.sc. thesis, dr y s parmar university of horticulture and forestry, solan, india, pp. 591. dogra b.s., kanwar m.s., 2011 exploiting heterosis for yield and horticultural traits in cucumber (cucumis sativus l.). indian j. plant genet. resour., 24(3): 332-339. doligibh s.t., sidorova a.m., 1983 combining ability of induced mutants and partially dioecious forms of cucumber. genetika, 19(8): 1292-1300. el hafeez a.a., el sayed s.f., gharib a.a., 1997 genetic analysis of cucumber yield and its components by diallel crossing. egyptian j. hort. sci., 24(2): 141-159. el-shawaf i.i.s., baker l.r., 1978 inheritance of yield in parthenocarpic hybrid pickling cucumber (abstract). hort. science, 13(3): 355. ghaderi l.r., lower r.l., 1979 heterosis and inbreeding depression for yield in populations derived from six crosses of cucumber. j. amer. soc. hort. sci., 104(4): 564-567. griffing j.b., 1956 concept of general and specific combining ability in relation to diallel crossing system. aust. j. biol. sci., 9: 463-494. gu x.f., zhang s.p., xu c.q., 2004 analysis of combining ability of early yield and total yield character of cucumber cultivated in open field in spring. china vegetables, 6: 1315. hormuzdi s.g., more t.a., 1989 studies on combining ability in cucumber (cucumis sativus l.). indian j.genet., 49(2): 161-165. kupper r.s., staub j.e., 1988 combining ability between lines of cucumis sativus l. and cucumis sativus var. hardwickii (r.) alef. euphytica, 38: 197-210. om y.h., choi k.s., lee c.h., choi c.i., 1978 diallel analysis of several characters in cucumber (cucumis sativus l.). korean j. breeding, 10(1): 44-50. wang y.j., wang y.s., 1980 preliminary analysis of combining ability in autumn cucumber. scientia agricultura sinica, 3: 52-57. 265 adv. hort. sci., 2022 36(4): 265­273 doi: 10.36253/ahsc­13352 arbuscular mycorrhizal fungi potentiate the root system and the quality of goldenberry fruits j.l.t. chiomento 1 (*), d. filippi 1, g.m. krasnievicz 2, j.e.c. de paula 2, m. fornari 3, t.s. trentin 2 ¹ postgraduate program in agronomy, university of passo fundo, passo fundo, rs, brazil. ² undergraduate program in agronomy, university of passo fundo, passo fundo, rs, brazil. 3 postgraduate program in agricultural engineering, federal university of santa maria, santa maria, rs, brazil. key words: berry flavor, mycorrhization, physalis peruviana l., root morphology. abstract: the lack of information on the horticultural performance of golden­ berry (physalis peruviana l.) is one of the factors that limits the expansion of the crop. still, aiming to establish a sustainable management for this culture, inoculation with arbuscular mycorrhizal fungi (amf) can be adopted. therefore, the objective of the research was to investigate whether goldenberry plants in the absence and presence of inoculation with amf differ in terms of horticul­ tural performance. the four treatments studied were the absence (control) and the presence of three inoculants based on amf (mycorrhizal community, glomus intraradices and rhizophagus clarus), arranged in a randomized block design, with five replications. goldenberry plants produced in substrate enriched with amf had a more voluminous root system and a greater amount of fine roots. additionally, the fruits were sweeter and more flavorful when produced by plants inoculated with the mycorrhizal community and with r. clarus. it is concluded that mycorrhization has no effect on fruit production. however, goldenberry plants submitted to mycorrhizal biotechnology enhance the chemical quality of fruits and present a more profuse root system. g. intraradices is most effective in colonizing the roots of the plant host. 1. introduction goldenberry (physalis peruviana l., solanaceae) is a horticultural crop native to the andean highlands that has attracted worldwide attention due to its bioactive compounds such as carotenoids, physalins and polyphenols (ramadan, 2011). in addition to promoting the health of con­ sumers, these biomolecules present in fruit extracts have antifungal action against phytopathogens, such as botrytis cinerea pers., and there­ fore can be widely used in agriculture as a bioinput (filippi et al., 2020). still, goldenberry stands out for its potential for intensive cultivation (*) corresponding author: jose­trevizan@hotmail.com citation: chiomento j.l.t., filippi d., krasnievicz g.m., de paula j.e.c., fornari m., trentin t.s., 2022 ­ arbuscular mycorrhizal fungi potentiate the root system and the quality of goldenberry fruits. ­ adv. hort. sci., 36(4): 265­273. copyright: © 2022 chiomento j.l.t., filippi d., krasnievicz g.m., de paula j.e.c., fornari m., trentin t.s. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/ index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 6 july 2022 accepted for publication 6 october 2022 ahs advances in horticultural science adv. hort. sci., 2022 36(4): 265­273 266 (etzbach et al., 2018). a single plant can produce 300 fruits and the productivity of this horticultural crop can reach from 20 to 33 tons per hectare (yildiz et al., 2015). despite the traditional establishment of crops in the open field (muniz et al., 2014), the goldenberry cultivation in greenhouse is increasing (aguilar­ carpio et al., 2018). this is because greenhouse culti­ vation can help to avoid inconveniences such as pests, diseases, rain, strong winds, hail and frost (costa et al., 2016). similar to the traditional cultivation of other solanaceae, such as tomato and pepper, in order to obtain an optimal productive yield of goldenberry, producers need to use a large amount of chemical inputs, which can contaminate the agroecosystem of cultivation (chiomento et al., 2020 a). there is no doubt, therefore, that the establishment of agroeco­ logical agriculture is an important tool for sustainable food production, with environmental and socioeco­ nomic benefits (llano et al., 2018). thus, an alterna­ tive to minimize the inconveniences in the cultivation of goldenberry and start the establishment of sus­ tainable management in this horticultural culture corresponds to the use of inoculants based on arbus­ cular mycorrhizal fungi (amf). as there is a limitation regarding the availability of commercial amf­based inoculants available in brazil (trentin et al., 2022), this reduces the use of mycorrhizal biotechnology in the production of goldenberry due to the lack of knowledge of this bioinput by the producers. the lim­ itation regarding the availability of commercial inocu­ lants is mainly due to the high cost linked to the pro­ duction technology of this bioinput. amf (phylum glomeromycota), a ubiquitous group of soil microorganisms, establish symbiotic associations with more than 70% of vascular plants (brundrett and tedersoo, 2018). the literature reporting the association between mycorrhiza and goldenberry is scarce. for example, under saline con­ ditions, amf increased fruit growth rate (miranda et al., 2011) and improved berry unsaturated fatty acid concentration in response to heavy metal stress (hristozkova et al., 2017). under water stress, arbus­ cular mycorrhiza increased root dry matter accumu­ lation and improved attributes related to plant gas exchange (reyes et al., 2019). in non­stressful envi­ ronments, it was found that goldenberry plants sub­ jected to mycorrhizal biotechnology produced less acidic and tastier fruits (chiomento et al., 2020 a). this scarcity of information demands more research to fill the existing gaps regarding the morpho­horti­ cultural performance of goldenberry and their inter­ active effects with mycorrhizas. in brazil there is only one commercial amf inocu­ lant available to farmers. the commercial scale pro­ duction of this bioinput has high costs linked to the inoculum production technology, such as the estab­ lishment of cultures of amf species and transport, handling and development of the carrier substrate (schlemper and stürmer, 2014). to avoid some of these costs, on‐farm production of inoculants is used, with indigenous or exotic amf isolates, in which the technology can be easily transferred to farmers (douds junior et al., 2012). the process of obtaining the on‐farm inoculant can be started using amf infective propagules, such as spores, hyphae and parts of colonized roots (douds junior et al., 2010). therefore, based on the hypothesis that mycor­ rhizal biotechnology enhances plant host growth and improves fruit chemical quality, here we investigate whether the horticultural performance of goldenber­ ry is influenced by the use of amf­based on‐farm inoculants. 2. materials and methods plant material the research was carried out in passo fundo (28° 15’ 46” s, 52° 24’ 24” w), rio grande do sul (rs), brazil, in greenhouses, from august (winter) 2018 to july (winter) 2019 . a commercial tray with goldenberry fruits at mat­ uration stage 5 was purchased (icontec, 1998). in august 2018, seeds from three randomly chosen fruits were selected, transferred to paper towels and kept at room temperature until dry. subsequently, these seeds were germinated in plastic gerbox boxes containing blotting paper and 0.1 molar (m) potassi­ um nitrate (kno3) solution. the boxes were stored in a b i o c h e m i c a l o x y g e n d e m a n d ( b o d ) o v e n , a t 25°c±1°c, until the plants were obtained for the pro­ duction of seedlings, which constituted the plant material for the research. the steps for obtaining the plants are shown in figure 1. experimental design the four treatments studied were the absence (control) and the presence of three inoculants based o n a m f [ m y c o r r h i z a l c o m m u n i t y , g l o m u s i n t r a r a d i c e s n . c . s c h e n c k & g . s . m r . a n d rhizophagus clarus (t.h. nicolson & n.c. schenck) c. trevizan chiomento et al. ‐ mycorrhizal biotechnology in the goldenberry cultivation 267 walker & a. schüßler], arranged in a randomized block design with five replications. each plot consist­ ed of three goldenberry plants. the amf community used came from the crop­ trap of agricultural soil collected at a reference site for strawberry cultivation in the municipality of são josé do hortêncio (29° 29’ 33” s, 51° 12’ 24” w), rio grande do sul state, brazil (chiomento et al., 2019 a), composed of ten fungal species according to the c l a s s i fi c a ti o n o f g l o m e r o m y c o t a p r o p o s e d b y redecker et al. (2013): acaulospora foveata trappe & janos, claroideoglomus aff. luteum, claroideoglomus claroideum (n.c. schenck & g.s. sm.) c. walker & a. schüßler, claroideoglomus etunicatum (w.n. becker & gerd.) c. walker & a. schüßler, funneliformis aff. geosporum, funneliformis aff. mosseae, funneli‐ formis mosseae (t.h. nicolson & gerd.) c. walker & a. schüßler, glomus aff. versiforme, glomus sp. (cae­ saris like) and glomus sp2. the isolate g. intraradices came from the commercial product myke® pro and t h e i s o l a t e r . c l a r u s w a s o b t a i n e d f r o m t h e international collection of glomeromycota culture (cicg). cultivation procedures we applied the treatments (amf) in two stages: 1) in the acclimatization of the seedlings; 2) in trans­ planting to the place of cultivation. thus, of the total amount of mycorrhizal inoculant used (10 g), we applied 5 g in the acclimatization of the seedlings and 5 g at the time of transplanting. in september (spring) of 2018, thirty days after the germination of goldenberry seeds, the plants obtained (fig. 1d) were acclimatized in 72­cell poly­ styrene trays, filled with the sterilized horta 2® sub­ strate (120°c for 20 minutes) and with treatments related to mycorrhization (1/2 of the total amount), with the purpose of seedling production. horta 2® is composed of pine bark, vermiculite, acidity correc­ tors and fertilizers (nitrogen, phosphorus and potas­ sium) in amounts not supplied by the manufacturer. a 500 g sample of the substrate was analyzed to obtain its physical (brazil, 2007) and chemical (mapa, 2014) attributes (table 1). the trays were kept on metal benches, 1.2 m from the ground surface, in a greenhouse (90 m2), installed in the northeast­southeast direction, with a semicir­ cular roof. the galvanized steel structure is covered with a low­density polyethylene film with anti­ultravi­ olet additive (150 micron thickness) and the sides are covered with an anti­aphid screen. the irrigation used during acclimatization was with sprinklers (1.8 l.min­1 per unit), in the mechanized system. the irri­ gation regime consisted of activating the sprinklers seven times a day, with total wetness of 14 minutes. the water depth supplied to the seedlings was 7.8 mm.day­1. in december (summer) 2018, after three months of acclimatization, the seedlings were transplanted fig. 1 ­ obtaining goldenberry plants. (a) separation of seeds from fruits. (b) selection of seeds. (c) germination in ger­ box. (d) plants produced. substrate physical properties d (kg m­3) tp (m3.m­3) ae (m3.m­3) raw (m3.m­3) bw (m3.m­3) rw (m3.m­3) horta 2 ® 241 0.837 0.303 0.149 0.020 0.365 chemical properties n % (m.m­1) p2o5 (m.m ­1) k2o % (m.m ­1) oc % (m.m­1) ph ec % (ms.cm­1) cec (mmolc.kg ­1) 0.36 0.39 0.00 12.60 6.1 0.45 278.60 table 1 ­ physical and chemical properties of the horta 2® substrate (z) d= density; tp= total porosity; ae= aeration space; raw= easily available water; bw= buffer water; rw= remaining water. (w) n= nitrogen; p2o5 = phosphorus pentoxide; k2o= potassium oxide; oc= organic carbon; ph= hydrogen potential; ec= electric conducti­ vity; cec= cation exchange capacity. adv. hort. sci., 2022 36(4): 265­273 268 into pots (3.6 l), filled with sterilized horta 2® (120°c for 20 minutes) and complemented with the other part of the treatments related to mycorrhization (1/2 of the total amount). the pots were kept in beds cov­ ered with mulching, in a greenhouse (430 m2), with a semicircular roof, installed in the northeast­south­ east direction. the galvanized steel structure was covered with a low­density polyethylene film (150 microns thick) and with an anti­ultraviolet additive. localized irrigation was carried out using drip rods (2.4 l.h­1 per unit), in the mechanized system. the irrigation regime consisted of activating the dripping rods six times a day, with total wetting for six min­ utes. the nutrient solutions supplied to the plants, fortnightly, were made according to furlani and fernandes júnior (2004), but with a 50% reduction in phosphorus supply. through a mini meteorological station, we verified that the average general temper­ ature recorded during the experiment was 25.66°c. the plants were conducted with three stems and were tutored with the aid of wires. no biocides were used during the crop cycle. the evaluations started after the fruiting of the plants, in february (summer) of 2019. we evaluated the root system morphology and the productive yield (number and weight) and quality of fruits. root system morphology at the end of the experiment, in july 2019, the plants roots were washed in water to eliminate sub­ strate fragments. the roots were digitized by a scan­ ner and the images obtained were analyzed by the winrhizo® software. the attributes evaluated were total length (tl, cm), surface area (sa, cm2) and vol­ ume (v, cm3). the roots were grouped by the soft­ ware into different diameter classes in relation to their total length (böhm, 1979): very thin (vt, ø<0.5 mm), thin (th, ø from 0.5 to 2 mm) and thick (tk, ø>2 mm). to verify the infective capacity of amf, root por­ tions of mycorrhizal plants were prepared according to phillips and hayman (1970) and their percentage of mycorrhizal colonization (mc) was determined according to trouvelot et al. (1986), by the equation: mc(%) = (total number of fragments with mycorrhizal roots) x 100 (total number of fragments) (1) fruit production from fruiting, in february 2019, the total number of fruits (tnf, number per plant) and the total pro­ duction of berries (tp, grams per plant) were evaluat­ ed. in addition, the average fresh fruit mass (affm, grams) was evaluated. the fruits were harvested when they were in the stages of maturation between 4 and 6 (icontec, 1998). the fruits were weighed on an electronic digital scale. chemical fruit quality the analysis of fruit quality was performed at the end of the experiment, in july 2019. the chemical characteristics of the fruits were evaluated regarding the content of total soluble solids (tss, %) and total titratable acidity (tta, % of citric acid), from 20 fruits of each treatment for each repetition. the tss con­ tent was determined in an analog refractometer, and the tta was performed according to the norms of the adolfo lutz institute (zenebon et al., 2008). to evaluate the flavor of the fruits, the tss/tta ratio was determined. data analysis the data obtained were submitted to analysis of variance (anova) and the averages of the treatments were compared by the tukey test, at 5% error proba­ bility, with the aid of the costat® program (cohort software, 2003). 3. results root system morphology we verified a significant effect of mycorrhizal inoculants only for the attributes mc, v and th. g. intraradices had a greater ability to infect plant roots than the mycorrhizal community and r. clarus (fig. 2a). the fungal structures identified inside the roots of goldenberry plants were hyphae, vesicles and arbuscules. in addition, plants inoculated with r. clarus had 41% and 42% greater root volume than non­mycorrhizal plants and those inoculated with the amf community, respectively (fig. 2b). also, plants inoculated with the mycorrhizal community had a greater amount of fine roots (+47%) compared to the control (fig. 2c). fruit production we observed a positive effect of treatments only for the affm attribute. non­mycorrhizal plants pro­ duced fruits with higher average fresh mass (+29%) compared to plants inoculated with the mycorrhizal trevizan chiomento et al. ‐ mycorrhizal biotechnology in the goldenberry cultivation 269 community, but did not differ from plants mycor­ rhizal with g. intraradices and r. clarus (fig. 3). chemical fruit quality m y c o r r h i z a l i n o c u l a n t s i n fl u e n c e d t s s a n d tss/tta attributes (fig. 4). sweeter (fig. 4a) and tastier (fig. 4b) fruits were produced by plants inocu­ lated with the mycorrhizal community and with the isolate r. clarus. 4. discussion and conclusions here, we show that goldenberry plants in the absence and presence of amf inoculation differed in horticultural performance. in the first productive cycle of the plants, we did not observe the effect of mycorrhization on the production of berries. we believe that in goldenberry the fungal species used require a period of more than one year to benefit the fruit yield, as occurs in strawberry (fragaria x ananassa duch.) cultivation (robinson­boyer et al., 2016). however, goldenberry plants inoculated with a m f s h o w e d a m o r e p r o f u s e r o o t s y s t e m . fig. 2 ­ root system morphology of goldenberry plants in the p r e s e n c e a n d a b s e n c e o f a m f i n o c u l a t i o n . ( a ) mycorrhizal colonization (%). (b) root volume (cm³). (c) amount of fine roots (cm). data presented as mean ± standard deviation. means followed by the same letter in the column did not differ significantly by the tukey test (p≤0.05). fig. 3 ­ average fresh fruit mass (grams) of goldenberry plants in the presence and absence of amf inoculation. data pre­ sented as mean ± standard deviation. means followed by the same letter in the column did not differ significantly by the tukey test (p≤0.05). fig. 4 ­ chemical quality of goldenberry fruits in the presence and absence of amf inoculation. (a) total soluble solids (%). (b) fruit flavor. data presented as mean ± standard deviation. means followed by the same letter in the column did not differ significantly by the tukey test (p≤0.05). (abbott and robson, 1981), which limits the plant’s response to mycorrhization (lambais and cardoso, 1990). although mycorrhizal colonization is impor­ tant, the percentage of root infectivity is not always c o r r e l a t e d w i t h t h e e ffi c i e n c y o f s y m b i o s i s (konvalinková and jansa, 2016). this lack of relationship between infectivity and amf efficiency in improving crop growth may be related to the time required for the establishment of root colonization (abbott and robson, 1981). under long­term, arbuscular mycorrhiza promotes more benefits to the plant host (ortas, 2012), mainly by increasing the acquisition of water and minerals to the plant. the major function of amf is suggested to be nutrient acquisition (zhang et al., 2019) and thus, under high nutrient conditions, amf can shift from a net benefit to a cost for the host (johnson et al., 2015). in our study, the nutrients supplied during cul­ tivation were not limited to goldenberry; therefore, the inoculation effect may not have been potentiated in terms of fruit yield. however, we proved the benefit of mycorrhiza­ tion on the chemical quality of the fruits through the increase in the sugar content (fig. 4a) and the better berry flavor (fig. 4b), as already reported for zucchini (cucurbita pepo l.) (rouphael et al., 2015), strawber­ ry (costa et al., 2020) and tomato (solanum lycoper‐ sicum l.) (sellitto et al., 2019). plants grown with amf showed a more developed root system (fig. 2), which allows extrarradicial hyphae to extend beyond the rhizosphere, making water acquisition more effi­ cient (xu et al., 2017). due to greater water availabili­ ty, these plants have greater stomatal opening, which increases the rate of transpiration and, thus, there is a greater supply of carbon dioxide for photo­ synthesis (vicente­sánchez et al., 2014). as a result, there is a greater production of sugars, which are the primary source of photosynthesis, and this explains the increase in the fruit sugar content and, conse­ quently, the best flavor of the berries (fig. 4). the lack of effect of g. intraradices on berries quality can be attributed to the low effectiveness of this fungal species on goldenberry. various factors modulate the arbuscular mycorrhiza effect on the performance of their associated plants and this includes the traits of the host and the fungi them­ selves (chiomento et al., 2019 a). cultivated plants vary in their responsiveness to amf due to their mor­ phology (chiomento et al., 2019 b) and amf differ in the benefits provided to the plant (werner and kiers, 2015). 270 adv. hort. sci., 2022 36(4): 265­273 furthermore, the use of the mycorrhizal community and r. clarus allowed to harvest fruits with better chemical quality. this indicates that the fungal species present in these two inoculants have an affin­ ity for this horticultural crop. the use of amf com­ patible with the host commonly provides more satis­ f a c t o r y r e s u l t s ( c h i o m e n t o e t a l . , 2 0 2 2 ) . g . intraradices was more effective in colonizing the roots of the plant host. however, the mycorrhizal community tested in this study stood out in relation to the fungal isolates for generally improving the hor­ ticultural performance of goldenberry. we verified that the root system of the mycor­ rhizal plants was more profuse, with greater volume (fig. 2b) and with a greater amount of fine roots (fig. 2c). this benefit to the roots has already been reported by reyes et al. (2019), who demonstrated that mycorrhization in goldenberry under water stress increased the accumulation of root dry matter. due to the plasticity of the roots, their characteristics can be modulated by several factors, including amf (hodge et al., 2009). during the establishment of the association between host and fungus, many molecu­ lar signals are initiated, including an amf (lipo­chito­ oligosaccharides) diffusible factor called “myc fac­ tor”, which stimulates the formation of finer roots (oláh et al., 2005), altering the morphology of the root system of plants. in addition, root modifications under mycorrhization may be related to the alloca­ tion of sugars to roots (wu et al., 2011) and hormon­ al regulation (zou et al., 2017), independent of sym­ biotic signaling (gutjahr, 2014). the more fine roots there are in mycorrhizal plants, the better their acquisition of water and nutrients (chiomento et al., 2021), as these roots are the ones that most acquire and use the available resources in the plant growth medium (costa et al., 2019). differently from what was expected, the produc­ tive performance of goldenberry was higher when the plants were not mycorrhized (fig. 3). this sug­ gests that amf initially demand carbon from the host for their maintenance and only later repay this bene­ fit to the plant symbiont. these results, however, contradict the literature. for example, miranda et al. (2011) reported benefits of amf in the production of goldenberry, under limiting conditions to plants, which were subjected to abiotic stresses, which did not happen in our study. there is not always a high relationship between fungal infectivity and efficiency in promoting crop growth due to the time required to establish bidirectional flow between symbionts trevizan chiomento et al. ‐ mycorrhizal biotechnology in the goldenberry cultivation 271 the two genera that made up the monospecific inoculants, glomus and rhizophagus, have already been reported in studies of amf diversity in golden­ berry (ramírez­gómez et al., 2019) and have also been used in applicability studies (rhizophagus) in t h i s h o r ti c u l t u r a l c r o p ( m i r a n d a e t a l . , 2 0 1 1 ) . however, in our study, the mycorrhizal community, representing a multispecific inoculant, stood out in relation to the fungal isolates for generally improving the horticultural performance of goldenberry, mainly by increasing the amount of fine roots produced by the plants (fig. 2c) and for benefiting fruit quality (fig. 4). inoculation with amf populations, such as the fungal community used in this study, generally provides more satisfactory results due to greater compatibilities at the fungus­host interface and by increasing mutualistic effects with two or more sym­ bionts instead of just one (chiomento et al., 2019 b). this mycorrhizal community was obtained through the trap culture technique, that is, produced on‐ farm. a mycorrhizal inoculant rich in propagules and produced on‐farm may be a suitable solution for large­scale inoculation of crops (agroecosystems), seedlings (nurseries) and in potting media for veg­ etable growers (soilless cultivation) (douds junior et al., 2006). the use of non­sterilized growth medium (soil and/or substrate) as a component of the on­ farm inoculant represents a source of propagules for other amf present in this growth medium, which results in an inoculant with greater taxonomic diver­ sity (schlemper and stürmer, 2014). this is strongly desired due to the functional diversity exhibited by mycorrhizal species for the promotion of plant growth, for example (chiomento et al., 2020 b). furthermore, a diversified inoculant potentiates a combination between fungal isolates and an eventual host (douds junior et al., 2006). therefore, the results of our research confirmed the potential of applying mycorrhizal biotechnology to goldenberry, as the work demonstrated that amf can be a valuable tool for the cultivation of this veg­ etable. the complex roles of amf in agroecosystems are just beginning to be understood (chiomento et al., 2022). in this way, a greater understanding of the application and benefits of amf can enable their use in the sustainable production of vegetables (trentin et al., 2022). we conclude that in the first production cycle, there is no effect of mycorrhization on the total num­ ber of fruits and total production of berries. we believe that for the goldenberry cultivation the fun­ gal species used require a period of more than one year to benefit the fruit yield. on the other hand, goldenberry plants submitted to mycorrhizal biotech­ nology have a more profuse root system and produce fruits with better chemical quality. g. intraradices is most effective in colonizing the plant host roots. however, the mycorrhizal community stands out in relation to the fungal isolates for generally improving the horticultural performance of goldenberry. thus, the application of this biotechnological tool in the goldenberry culture can be an alternative to spread and promote its sustainable cultivation. acknowledgements amf used in this work are regulated by sistema nacional de gestão do patrimônio genético e do conhecimento tradicional associado (sisgen) of the ministry of the environment, brazil, according to the registration number a198f50. references abbott l.k., robson a.d., 1981 ­ infectivity and effective‐ 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of environmental factors on communities of arbuscular mycorrhizal fungi associated with chenopodium ambrosioides revealed by miseq sequencing investigation. ­ sci. rep., 7(45134): 1­11. yildiz g., i̇zli n., ünal h., uylaşer v., 2015 ­ physical and chemical characteristics of goldenberry fruit (physalis peruviana l.). ­ j. food sci. technol., 52(4): 2320­2327. zenebon o., pascuet n.s., tiglea p., 2008 ­ métodos físi‐ co‐químicos para análise de alimentos. ­ instituto adolfo lutz, são paulo, pp. 1020. zhang s., lehmann a., zheng w., you z., rillig m.c., 2019 ­ arbuscular mycorrhizal fungi increase grain yields a meta‐analysis. ­ new phytol., 222:543­555. zou y.n., wang p., liu c.y., ni q.d., zhang d.j., wu q.s., 2017 ­ mycorrhizal trifoliate orange has greater root adaptation of morphology and phytohormones in response to drought stress. ­ sci. rep., 7: 41134. 17 1. introduction although in italy lawns are traditionally established with cool season grasses, the use of warm season species has been increasing over the last two decades (croce et al., 2004; volterrani et al., 2010). warm season grasses show several advantages over cool season species. they tolerate high temperatures and drought, show lower water consumption, tolerate higher salinity concentrations in the soil and in irrigation water, establish rapidly and present excellent recovery properties due to the abundant production of stolons and rhizomes (biran et al., 1981; carrow and duncan, 1988; beard, 1989; dudeck and peacock, 1993; beard and sifers, 1997; volterrani et al., 1997; croce et al., 2006; harivandi and marcum, 2008). bermudagrasses (cynodon spp.) are the warm season turfgrass species most widely used in the transition and warm regions of the world due to their aggressive growth habit, tolerance to a wide range of mowing heights, marked resistance to many abiotic stresses, and the high quality of the cultivars recently developed by breeding programs (wu et al., 2009). a technique recently developed for turf establishment of warm season species is based on the transplant of single plug plantlets pre-rooted in the nursery (volterrani et al., 2008). nursery activity consists of the pot cultivation of donor plants from which stolons are collected, divided into one-node sprigs about 2 cm long, then cultivated in alveolate trays until the formation of mature plantlets. the crucial point of this practice is stolon fragmentation. this operation is very time consuming and consequently labour-costly. in fact, stolons must be cut several times to obtain sprigs of the right length due to long internodes. the effectiveness of trinexapac-ethyl (te) in controlling bermudagrass growth is reported by many authors (johnson, 1994, 1997; fagerness and yelverton, 1999; lowe and whitwell, 1999; fagerness and yelverton, 2000; fagerness et al., 2002; bunnel et al., 2005; mccullough et al., 2005; baldwin et al., 2006; mccullough et al., 2007; baldi et al., 2010). in these reports, te is applied to bermudagrass turfs in order to facilitate their management (mainly by reducing mowing requirements), ameliorate their quality or enhance stress tolerance (e.g. salinity tolerance or cold tolerance). the rates of te normally adopted in bermudagrass turf management are around 0.11 kg a.i. ha-1, which is the recommended rate for bermudagrass maintained at golf fairway height (cooper, 2003). plant height, texture, density, clipping weight and root mass are the growth parameters related to plant response to te to be considered. on the contrary, since turf establishment by means of pre-rooted plantlets propagated by stolon in the nursery has been recently developed, the effects of plant growth regulators (pgrs) on grass plants grown to produce propagating material have not been investigated. furthermore, parameters such as stolon length, internode length and stolon number, important for nursery activity, have been seldom considered. traditionally, pgrs are classified into two main groups, type i and type ii, according to their biological mode of effects of trinexapac-ethyl on stolon development in potted patriot bermudagrass a. lenzi, a. baldi, m. nannicini, a. pardini, r. tesi dipartimento di scienze delle produzioni vegetali, del suolo e dell’ambiente agroforestale, università degli studi di firenze, piazzale delle cascine, 18, 50144 firenze, italy. key words: internode length, nursery production, plant growth regulators, sprigs, warm season turfgrasses. abstract: a recent technique developed for establishment of warm season turfgrasses is based on the transplant of single plug plantlets pre-rooted in the nursery. plantlets are obtained from one-node sprigs about 2 cm long derived from stolon fragmentation. usually, stolons must be cut several times to obtain sprigs of the right length because of overly long internodes. in the present study, potted plants of patriot bermudagrass grown in the nursery were treated with trinexapac-ethyl (te) at the rates 0.1, 0.2, 0.4 and 0.8 kg a.i. ha-1. te application was aimed at obtaining internode shortening in order to facilitate the stolon division practice. in fact, te-treated plants showed a decrease in the average length of internodes with respect to control at any applied rate. nevertheless, the lowest rate applied (0.1 kg a.i. ha-1) did not assure a prolonged effect while the highest rate (0.8 kg a.i. ha-1) caused a decrease in the yield of sprigs. therefore, our results suggest that te may be advantageously used and at rates of 0.2-0.4 kg a.i. ha-1 to control stolon development of patriot bermudagrass for nursery purposes. adv. hort. sci., 2012 26(1): 17-20 received for publication 20 september 2011 accepted for publication 24 january 2012 18 action. type i pgrs inhibit cell division and differentiation in the plant meristems and are excellent seedhead inhibitors; type ii compounds suppress growth by inhibiting the biosynthesis of gibberellic acid, which is needed for cell elongation (watschke et al., 1992). in turfgrass, three of the most commonly used pgrs [flurprimidol (fl), trinexapac-ethyl (te) and paclobutrazol (pbz)] are type ii chemicals (lowe and whitwell, 1999). turf plants treated with type ii pgrs usually show an altered morphology of leaves and a more compact growth habit due to a reduced internode length (rossi, 1993). therefore, for their mode of action, type ii pgrs may presumably be useful to reduce stolon length by controlling internode elongation. in this paper, the effects of te on potted plants of bermudagrass grown in the nursery are described. the aim of the te treatment was to obtain a stolon shortening to facilitate the stolon division practice and, hopefully, without reducing the overall production in sprigs, that result from the number of stolons per plant and the number of nodes per stolon. 2. materials and methods plant material, growing conditions and experimental treatments the experiment was carried out between april and june 2010 at the pacini horticultural nursery located in rigoli (pisa), central italy (45°45’ n, 10°26’ e, 6 m a.s.l.). on 7 april 2010, plantlets derived from one-node sprigs of bermudagrass [cynodon dactylon (l.) pers. x c. transvaalensis burtt-davy] cv. patriot were transplanted in 20 cm-diameter, 3.80 l volume polyethylene pots (three plantlets per pot) filled with peat (baltikum-s132; 92% organic matter, ph 5.5-6.5, electrical conductivity 0.6-1.0 ds m-1) fertilized with 2 g l-1 of osmocote-pro (scotts) 1810-11 + 2 mgo. on 12 may 2010, after stolons had started to develop, plants were trimmed down to the pot rim height and five treatment rates [0 (= control), 0.1, 0.2, 0.4 and 0.8 kg a.i. ha-1] of te [(4-cyclopropyl-α-hydroxy-methylene)-3,5dioxocyclohexanecarboxylic acid methyl ester] were applied by foliar spraying (1250 l ha-1) of the commercial product primo maxx (syngenta). during the experiment, plants were irrigated as needed by an overhead sprinkler system. during the period, average maximum and minimum temperatures were 35°c and 20°c, respectively, while relative humidity (r.h.) reached 55% and 70% as minimum and maximum values, respectively. a randomized block design was adopted with five replicates per treatment (1 replicate = 1 pot). data collection after treatment, stolon length and the number of nodes per stolon were recorded weekly for seven weeks on three stolons per pot, and average internode length was calculated. on 30 june 2010, seven weeks after treatment, fresh weight and dry weight of aerial biomass (after oven-drying at 80°c for 48 hr) and the number of stolons of each pot were determined. in addition, the total number of sprigs per pot was calculated as the product of the number of stolons per pot and the final number of nodes per stolon. statistics data were subjected to one-way analysis of variance (anova) and means were compared using the lsd test at p≤0.05 level of significance. 3. results te induced a statistically significant shortening of stolons compared to the control one and two weeks after application at the rates 0.2, 0.4 and 0.8 kg a.i. ha-1 (fig. 1). three weeks after treatment, the effect of te treatments on stolon length began to wane (fig. 1). during the entire monitoring period (seven weeks), no statistically significant differences were observed between te-treated plants and control plants in the number of nodes per stolon (fig. 2). fig. 1 stolon length in patriot bermudagrass treated with different rates of te (kg a.i. ha-1). within the same week, different letters show significantly different values for p≤0.05 (lsd test). vertical bars show se of the means (n=5); ns= not significant. fig. 2 number of nodes per stolon in patriot bermudagrass treated with different rates of te (kg a.i. ha-1). within the same week, different letters show significantly different values for p≤0.05 (lsd test). vertical bars show se of the means (n=5); ns= not significant. 19 te-treated plants showed a decrease in the average length of internodes (fig. 3). in control plants internode length varied between 2.7 and 3.0 cm; in te-treated plants stolons showed an average internode length varying from 1.8 (first week) to 2.5 cm (seventh week) at the rate 0.1 kg a.i. ha-1, from 1.4 (first week) to 2.1 cm (seventh week) at the rate 0.2 kg a.i. ha-1, from 1.2 (first week) to 1.8 cm (seventh week) at 0.4 kg a.i. ha-1, and from 1.2 (third week) to 1.5 cm (seventh week) at 0.8 kg a.i. ha-1 (fig. 3). the differences between control and te-plants were statistically significant at all the applied rates one week after treatment and afterwards at the rates 0.2, 0.4 and 0.8 kg a.i. ha-1. at the end of the monitoring period, no statistically significant differences were observed between te-treated plants and control plants in fresh matter production, while dry matter decreased significantly compared to control at 0.2 and 0.8 kg a.i. ha-1 but, surprisingly, not at 0.4 kg a.i. ha-1 (table 1). table 1 aerial biomass production in patriot bermudagrass treated with different doses of te (kg a.i. ha-1) seven weeks after treatment treatment te (kg a.i. ha-1) fresh matter (g per pot) dry matter (g per pot) number of stolons per pot number of sprigs per pot 0 128.5±5.1 ab (z) 38.3± 4.6 a 110±7.4 a 915±83.3 a 0.1 136.7±5.8 a 37.9±1.3 a 104±6.4 ab 912±138.3 a 0.2 113.2±5.2 b 31.8±1.5 b 86±2.6 b 673±48.0 ab 0.4 121.0±4.7 b 34.5±2.6 ab 89±6.3 b 786±80.6 ab 0.8 115.0±6.6 b 31.3±2.0 b 62±5.6 c 597±72.9 b (z) values in the same column followed by different letters are significantly different for p≤0.05 (lsd test). values are means (± se) of five replicates. te treatment had a detrimental effect on stolon number per pot starting from the rate 0.2 kg a.i. ha-1 upwards, but the average number of nodes derived from one pot decreased significantly only at the highest rate applied (0.8 kg a.i. ha-1) (table 1). 4. discussion and conclusions the effect of te on the number of bermudagrass stolons was previously investigated by fagerness et al. (2002). these authors found that te (0.11 kg a.i. ha-1) did not affect the number of stolons in tifway bermudagrass when plants were grown in a 22/17°c day/night temperature regime, while at 36/31°c te-treated plants showed about twice as many stolons as control plants four weeks after te application. in our experiment, temperature ranged between 20 to 35°c and 0.1 kg te ha-1 did not cause any effect on stolon number in patriot bermudagrass seven weeks after its application. on the contrary, higher rates of te (0.2, 0.4 and 0.8 kg a.i. ha-1) reduced significantly the number of stolons compared to control. an indirect evaluation of the effect of te on stolon lengthening in turfgrass species may come from percentage lateral regrowth, a parameter used to estimate the horizontal growth or recovery of turfs as described by bunnell et al. (2005). these authors applied te at 0.039 kg a.i. ha-1 to tifeagle bermudagrass every three weeks and measured percentage lateral regrowth weekly for eight weeks. averaged weekly data of te-treated plants did not show any differences compared to untreated plants. totten et al. (2006) reported that te did not affect the percentage lateral regrowth in tifway bermudagrass when used at the rate 0.052 kg a.i. ha-1, while at 0.104 kg a.i. ha-1 percentage lateral regrowth was significantly reduced compared to control two weeks after te application. in our experiment, a te rate of at least 0.2 kg a.i. ha-1 was needed for a significant decrease of stolon length in patriot bermudagrass (fig. 1). that effect lasted for two weeks after te treatment. internode length after one, two or three te applications at 0.153 kg a.i. ha-1 to a mature tifway bermudagrass turf was measured by richardson (2002). the measurements were taken ten weeks after the last te application and no effect of the pgr was detected. in our experiment internode length was significantly shortened in te-treated plants at any applied te rate one week after one application; from two to seven weeks after treatment stolon showed average internode length with statistically significant differences between control and te-treated plants at the rate 0.2 kg a.i. ha-1 and higher. in conclusion, our results suggest that te may be advantageously used to control stolon development of patriot bermudagrass for nursery purposes (shortening of the internodes in plants grown in pot to facilitate stolon division practice). in order to obtain prolonged effect, higher rates than those normally adopted in bermudagrass turf management seemed to be required (at least 0.2 kg a.i. ha-1). the highest rate applied (0.8 kg a.i. ha-1) was not advisable as it caused also almost a halving in the number of stolons per pot and a statistically significant decrease in the yield of sprigs. under the conditions in which we operated, the best result was achieved at the rates 0.2 and 0.4 kg a.i. ha-1. with such rates, although the number of stolons per pot was significantly reduced compared to control, the yield in nodes remained unvaried. fig. 3 average internode length in patriot bermudagrass treated with different rates of te (kg a.i. ha-1). within the same week, different letters show significantly different values for p≤0.05 (lsd test). vertical bars show se of the means (n=5); ns= not significant. 20 acknowledgements this research was funded by the ministry of agricultural, food and forestry policies of italy. project: “sistemi avanzati per la produzione vivaistica di tappeti erbosi di specie macroterme ad uso multifunzionale a basso consumo idrico ed energetico”. the authors wish to thank the pacini horticultural nursery for the use of the greenhouses and other facilities. references baldi a., lenzi a., nannicini m., pardini a., tesi r., 2010 plant growth regulator treatments for the management of patriot bermudagrass. proceedings of the second conference of the european turfgrass society. angers, france, april 11-14, pp. 32-34. baldwin c.m., liu h., mccarthy l.b., bauerle w.l., toler j.e., 2006 effect of trinexapac-ethyl on the salinity tolerance of two ultradwarf bermudagrass cultivars. hortscience, 41(3): 808-814. beard j.b., 1989 turfgrass water stress: drought resistance components, physiological mechanisms, and species-genotype diversity. proceedings of the sixth international turfgrass research conference, tokio, japan, pp. 23-28. beard j.b., sifers s.i., 1997 genetic diversity in dehydration avoidance and drought resistance within the cynodon and zoysia species. int. turfgrass soc. res. j., 8: 603-610. biran i., bravdo b., bushkin-harav i., rawitz e., 1981 water consumption and growth rate of 11 turfgrasses as affected by mowing height. agron. j., 79: 85-90. bunnell b.t., mccarty l.b., bridget jr. w.c., 2005 ‘tifeagle’ bermudagrass response to growth factors and mowing height when grown at various hours of sunlight. crop sci., 45: 575-581. carrow r.n., duncan r.r., 1988 salt-affected turfgrass sites. assessment and management. john wiley & sons, hoboken, new jersey, usa, pp. 185. cooper r.b., 2003 summary of 2003 cutless 50wp turfgrass growth regulator research on 419 bermudagrass fairways. sepro corp. www.sepro.com/documents/cutlesscooper.pdf. croce p., de luca a., falcinelli m., 2006 tappeti erbosi. edagricole, bologna, pp. 340. croce p., de luca a., mocioni m., volterrani m., beard j.b., 2004 adaptability of warmseason turfgrass species and cultivars in a mediterranean climate. acta horticulturae, 661: 365-368. dudeck a.e., peacock c.h., 1993 salinity effects on growth and nutrient uptake of selected warm-season turf. int. turfgrass soc. res. j., 7: 680-686. fagerness m.j., yelverton f.h., 1999 effects of trinexapac-ethyl on late season development and cold hardiness of ‘tifway’ bermudagrass. proc. northeast. weed sci. soc., 53: 63. fagerness m.j., yelverton f.h., 2000 tissue production and quality of ‘tifway’ bermudagrass as affected by seasonal application patterns of trinexapac-ethyl. crop sci., 40: 493-497. fagerness m.j., yelverton f.h., livingston d.p. iii, rufty jr. t.w., 2002 temperature and trinexapac-ethyl effects on bermudagrass growth, dormancy, and freezing tolerance. crop sci., 42: 853-858. harivandi m.a., marcum k.b., 2008 a review of salt tolerance among sports field turfgrasses. acta horticulturae, 783: 159-162. johnson b.j., 1994 influence of plant growth regulators and mowing on two bermudagrasses. agron. j., 86: 805-810. johnson b.j., 1997 growth of ‘tifway’ bermudagrass following application of nitrogen and iron with trinexapac-ethyl. hortscience, 32(2): 241-242. lowe d.b., whitwell t., 1999 plant growth regulators alter the growth of ‘tifway’ bermudagrass (cynodon transvaalensis x c. dactylon) and selected turfgrass weeds. weed technol., 13(1): 132-138. mccullough p.e., liu h., mccarty l.b., toler j.e., 2007 trinexapac-ethyl application regimens influence growth, quality, and performance of bermudagrass and creeping bentgrass putting greens. crop sci., 47: 21382144. mccullough p.e., mccarty l.b., liu h., whitwell t., 2005 response of ‘tifeagle’ bermudagrass (cynodon dactylon x cynodon 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turfgrass society. angers, france, april 11-14, pp. 223-225. watschke t.l., prinster m.g., brenninger j.m., 1992 plant growth regulators and turfgrass management, pp. 557-588. in: waddington d.v., r.n. carrow, and r.c. shearman (eds.) turfgrass. american society of agronomy, agronomy monograph, n. 32. madison, wi, usa, pp. 805. wu y., martin d.l., anderson j.a., bell g.e., anderson m.p., walker n.r., moss j.q., 2009 recent progress in turf bermudagrass breeding research at oklahoma state university. usga turfgrass and environmental research online, 8(16): 1-11. 164 1. introduction persimmon trees, having no dwarf rootstocks of commercial value, tend to grow high. lowering tree height by heavy dormant pruning has been a routine practice for efficient management of many persimmon orchards. in densely planted orchards, heavy pruning is an inevitable practice to restrict tree size. this practice, in turn, causes vigorous shoot growth resulting in an excessive crowding of the canopy. poor fruit set and excessive supply of nitrogen also stimulate the occurrence of vigorous shoots. tree crowding not only hinders orchard operations, but deteriorates fruit quality as the tree interior becomes heavily shaded. shoots of vigorous and succulent growth make the tree susceptible to anthracnose (colletorichum gloeosporioides) when humidity within the canopy is high. summer pruning is one of many options to alleviate the problems of crowding, ensuring adequate light penetration into the canopy and controlling excessive shoot growth. however, removal of shoots during growing season involves the loss of functional leaf surface, which may lead to reduced tree development and fruit growth. loss of leaf area may also reduce reserve accumulation for early growth the next season. the effect of summer pruning on physiological process and tree growth seems well documented in other deciduous fruit crops, but limited studies have been conducted in persimmon. in this paper we evaluate the effects of summer pruning on tree growth, fruit quality, and nutrient composition of perennial organs in persimmon. 2. tree response to summer pruning vegetative growth since removal of active leaf area reduces the production of dry matter during the growing season, summer pruning suppresses vegetative growth in other fruit crops, the effect of which is closely related to pruning severity, timing and cultivars (taylor and ferree, 1984; rom and ferree, 1985; marini and barden, 1987; mediene et al., 2002; zamani et al., 2006). in young ‘fuyu’ and ‘nishimurawase’ persimmons, thinning 26% of total shoot length in late july reduced dry weight of dormant one-year-old twigs, but it did not significantly affect total dry weights of aerial wood and the root (choi et al., 2003 a). the result might be related in part to a high photosynthetic activity of remaining leaves until later in the season as shown in leaf chlorophyll concentrations (fig. 1). although not significant, negative relationships were observed between the increment of trunk cross-sectional area and pruning severity in the same experiment (choi et al., 2003 a) and summer heading-back (song et al., 2001). summer pruning effect on tree growth and fruit production of persimmon s.t. choi*, d.s. park*, k.p. hong*, s.m. kang** * sweet persimmon research institute, gyeongnam agricultural research and extension services, 262-1 woodong, jinyoung, gimhae 621-802, republic of korea. ** department of horticulture, institute of agriculture and life science, gyeongsang national university, 900 gazwa-dong, jinju 660-701, republic of korea. key words: pinching, secondary shoot, topping, tree vigor, water sprout. abstract: this paper reviews the effect of summer pruning in persimmon (diospyros kaki thunb.) with regard to its positive and negative aspects on tree growth and fruit production. in order for this practice to be of a significant value, a clear understanding is needed to appreciate the fact that so many variables are involved and much remains to be substantiated. major reasons for summer pruning of persimmon are to improve fruit quality by enhancing light penetration into the tree canopy and to restrict vegetative shoot growth. summer pruning generally suppresses tree growth even though it elevates leaf activity. positive effects of summer pruning on skin color, soluble solids, and appearance of fruits are observed mainly in those orchards where the trees are heavily dormant-pruned to lower tree height and to secure space in high density plantings. secondary shoot pruning and topping could also improve fruit quality, increasing flower bud formation of remaining shoots. summer-pruning effects are highly dependent on its severity and timing to affect tree growth, shoot regrowth, reserve accumulation, and fruit quality. adv. hort. sci., 2011 25(3): 164-169 received for publication 11 april 2011 accepted for publication 13 may 2011 165 leaf activity summer pruning is known to increase photosynthesis, dark respiration, and transpiration of shoot leaves in other fruit crops (taylor and ferree, 1981; marini and barden, 1982; myers and ferree, 1983). as shoot thinning severity in late july increased in ‘nishimurawase’ persimmon, specific leaf weight tended to become higher (table 1). chlorophyll content per unit area increased by 1.9-fold and 2.3-fold in ‘fuyu’ and about 2-fold in ‘nishimurawase’ trees that had been thinned 17% and 26% of their shoots, respectively (fig. 1). results indicated that photosynthesis can hardly compensate for the leaf area that has been removed, and there would be a sizable reduction in the production of photosynthates depending on pruning time regrowth it is possible that shoot regrowth following summer pruning can have serious negative effects on the supply of photosynthates to fruit and shoot and resistance to diseases and pests (forshey et al., 1992; choi et al., 2003 b). in addition, excessive regrowth may reduce flower bud formation by gibberellins produced during shoot regrowth (forshey et al., 1992). the extent of regrowth following summer pruning was influenced by the time and severity (miller, 1982; ferree et al., 1984). when ‘fuyu’ persimmon was summer-pruned on 20 june or 4 july, the earlier pruning produced more secondary growth more than the later one (fujimura, 1932). summer pruning after late july, when buds enter physiological rest, did not result in a great problem of regrowth in persimmon trees, but heavy summer pruning may stimulate some buds to break into growth (table 1). heading-back cuts in vigorous shoots would easily induce regrowth more than thinning cuts. late july through early august is the appropriate time for thinning out vigorous shoots to avoid regrowth in south korea. reserve accumulation early loss of foliage from pruning may result in reduced carbohydrate levels of the tree, and that could adversely affect cold hardiness (marini and barden, 1987). when the current shoots were thinned up to 26% of their total length in late july in young ‘fuyu’ and ‘nishimurawase’ persimmon, the differences in non-structural carbohydrates and inorganic nutrients in fourto five-year-old wood of aboveground parts and the large root, measured on 2 april the following year, were not consistent with the pruning severity the previous summer (choi et al., 2003 a). this result might be due to the increased activity of the leaves as presented in chlorophyll concentration in figure 1. however, thinning 50% of total shoots decreased carbohydrate concentration in one-year-old shoots of field-grown ‘nishimurawase’ (table 2) but not in those of ‘fuyu’ (choi et al., 2003 b). in the same experiment, the severe summer pruning also reduced flower buds in both cultivars the following year, since low carbohydrates in the shoots (choi et al., 2011) and excessive regrowth (forshey et al., 1992) were negatively related to flower bud formation. 3. fruit quality affected by summer pruning summer pruning has been recommended to improve table 1 effect of summer pruning severity on 29 july on light penetration and tree growth of ‘nishimurawase’ persimmon (choi et al., 2003 b) shoot-thinned (%) light penetration (%) tcsa increment (%) regrowth (cm/tree) slw (mg·cm-2) leaf spad reading 0 11.3 b 6.8 a 0 a 10.14 b 51.6 a 25 24.7 a 7.8 a 63 a 10.92 ab 52.8 a 33 27.9 a 7.4 a 163 a 10.93 ab 51.8 a 50 30.9 a 6.1 a 215 a 11.18 a 54.8 a after summer pruning of seven-year-old trees, the leaf-fruit ratio was changed from 20 of 0% thinning to 10 of 50% thinning in an orchard planted at 3.5 x 2 m. tcsa= trunk cross-sectional area. slw= specific leaf weight. mean values in each column with the same letter are not significantly different by duncan’s multiple range test at p≤ 0.05. fig. 1 effect of different severities of shoot thinning on 22 july on leaf chlorophyll of five-year-old ‘fuyu’ and ‘nishimurawase’ persimmon on 3 november. chlorophyll content includes chlorophylls a and b. values sharing the same letter are not significantly different from each other by duncan’s multiple range test at p≤ 0.05 (choi et al., 2003 a). µ g 166 fruit quality in vigorous persimmon by increasing light interception (mowat, 1987; ullio, 2003; george et al., 2005; yamada, 2008). however, there was no consistent effect of the pruning on fruit characteristics in many studies. table 3 shows that thinning out some water sprouts in late july increased skin color with no reduction in fruit size. this kind of positive result might be possible in vigorous ‘fuyu’ trees in densely-planted orchards, and the leaf-fruit ratio after the pruning was sufficiently high. kim (2010) also found larger size and higher coloration of fruits in vigorous ‘fuyu’ trees after thinning out some water sprouts in late july. lower water consumption and thus improved water status during the growing season after summer pruning could benefit fruit growth and relieve the potential detriment due to carbohydrate shortage in apple (li et al., 2003) and peach (lopez et al., 2006). fruit firmness was not significantly changed by the differences in pruning severity (tables 4 and 5). size, soluble solids, and skin color of persimmon fruits decreased when the leaf-fruit ratio was low (choi et al., 2010). the appropriate leaf-fruit ratio for quality ‘fuyu’ fruits has been set at about 20 in korea (choi et al., 2010) and japan (kishimoto, 1975; yamamura et al., 1989). loss of leaf area by summer pruning results in poor fruit quality table 3 effect of removing water sprouts on 28 july on light penetration and fruit characteristics in vigorous ‘fuyu’ persimmon orchard (choi et al., 2005) treatment light penetration (%) fruits average weight (g) skin color (hunter a) soluble solids (°brix) non-pruning 15 243 27.9 15.0 summer pruning 33 257 29.8 15.2 significance ** ns * ns by summer pruning of 1-year-old trees, water sprouts were removed to maintain leaf-fruit ratio 20 in an orchard planted at 6 x 3 m. ns= not significant; * = significant at p≤ 0.05; ** = significant at p≤0.01. table 4 effect of summer pruning severity on 28 july on fruit characteristics of ‘fuyu’ persimmon harvested on 31 october (choi et al., 2003 b) shoot-thinned (%) average weight (g) skin color (hunter a) fruit firmness (n) soluble solids (°brix) skin damage (%) 0 234 a 27.6 a 20.7 a 15.2 a 13.2 a 10 244 a 28.4 a 21.9 a 15.7 a 9.3 cb 20 249 a 28.3 a 20.1 a 15.1 a 7.8 c 30 233 a 29.5 a 20.3 a 15.3 a 9.8 b after summer pruning of seven-year-old trees, the leaf-fruit ratio was changed from 39 of 0% thinning to 28 of 30% thinning in an orchard planted at 6 x 3 m. skin damage: blemish or stains on fruit skin. mean values in each column with the same letter are not significantly different by duncan’s multiple range test at p≤0.05. table 5 effect of summer pruning severity on 29 july on fruit characteristics of ‘nishimurawase’ persimmon harvested on 26 september (choi et al., 2003 b) shoot-thinned (%) average weight (g) skin color (hunter a) fruit firmness (n) soluble solids (°brix) skin damage (%) 0 144 a 32.2 a 34.3 a 14.5 a 8.8 a 25 139 a 32.2 a 33.4 a 14.0 ab 3.7 ab 33 137 a 31.2 ab 32.5 a 13.6 ab 4.6 ab 50 141 a 27.7 a 31.9 a 13.0 b 1.6 b after summer pruning of seven-year-old trees on 29 july, the leaf-fruit ratio was changed from 20 of 0% thinning to 10 of 50% thinning in an orchard planted at 3.5 x 2 m. skin damage: blemish or stains on fruit skin. mean values in each column with the same letter are not significantly different by duncan’s multiple range test at p≤0.05. table 2 effect of summer pruning severity on 29 july on concentrations of carbohydrates and inorganic elements in dormant shoots of ‘nishimurawase’ persimmon on 17 february (choi et al., 2003 b) shoot-thinned (%) carbohydrates (% dw) inorganic elements (% dw) soluble sugars starch n p k ca mg 0 12.2 a 5.4 a 0.60 a 0.21 b 0.60 a 0.36 b 0.17 a 25 12.0 a 4.4 ab 0.62 a 0.24 ab 0.73 a 0.39 ab 0.17 a 33 10.3 a 3.5 ab 0.60 a 0.24 ab 0.74 a 0.43 a 0.17 a 50 9.7 a 3.2 b 0.62 a 0.28 a 0.75 a 0.44 a 0.18 a after summer pruning of seven-year-old trees, the leaf-fruit ratio was changed from 20 of 0% thinning to 10 of 50% thinning in an orchard planted at 3.5 x 2 m. mean values in each column with the same letter are not significantly different by duncan’s multiple range test at p≤ 0.05. 167 if the pruning significantly reduces the ratio. when leaffruit ratio decreased to less than 13 with severe removal of the shoots, fruit size, skin color or soluble solids were significantly reduced (table 5) (choi et al., 2003 a). on the other hand, strong winds result in blemish of fruit skin due to rubbing of shoots and leaves against fruits (george et al., 1997 a). skin staining of persimmon fruits is often caused by humid conditions in autumn (george et al., 1997 a), which become aggravated by the shoots growing vigorously. thinning out some of the shoots in summer reduces these damages by improving the micro-environment within the tree canopy (tables 4 and 5). therefore, summer pruning has been recommended as a means to reduce the occurrence of skin staining (george et al., 1997 a, 2005), especially in newly-released japanese cultivars like a crack-susceptible ‘taishu’ (yakushiji and nakatsuka, 2007). the influence of summer pruning on fruit quality varies by the degree of canopy crowding and the timing and its severity, as has been documented in other fruit crops (marini and barden, 1987; forshey et al., 1992; zamani et al., 2006). 4. water sprout management removing all water sprouts of vigorous trees too early in the season may induce severe regrowth (fujimura, 1932), which disturbs light penetration and reserve accumulation in perennial organs (kappel et al., 1983). therefore, instead of removing all water sprouts, thinning some of them combined with bending or twisting the remaining ones down to horizontal in june may be recommended (park and choi, 2000; huh et al., 2003; george et al., 2005). securing leaf area even in water sprouts helps prevent the regrowth that may be related to the action of hormones produced in old leaves (forshey et al., 1992). some of the water sprouts may have some value as mother branches the next season. water sprouts of ‘fuyu’ could form flower buds as long as their terminal buds are set by early august in south korea (choi et al., 2011): they could serve as fruiting branches for the following year. in vigorous trees with poor fruiting, using the sprouts as mother branches to set fruits the next season is extremely important to make up for the yield reduction as well as to control tree vigor. when the sprouts were twisted and bent down under horizontal in midto late june after thinning out some, flower buds were formed in 84 to 97% of them the following year (park and choi, 2000). changes in endogenous hormones might be closely involved in this treatment, particularly low gibberellin and high cytokinin, which play a role in flower bud formation (banno et al., 1985). heading-back pruning to four buds is also practiced in new zealand to ensure future fruiting site from water sprouts (mowat, 1987). 5. secondary-shoot pruning, pinching and topping secondary growth from shoot terminal occurs from mid-june in vigorous persimmon trees (nii, 1980; park et al., 2003). secondary growth often induced fruit drop (george et al., 1997 b). park et al. (2003) studied the heading-back effect of secondary shoots leaving two to three basal leaves of the secondary growth on ‘fuyu’ persimmon. when the heading-back was done on fruiting shoot, they found that the shoots had a lower dry weight which was accompanied by an increase in fruit weight. the result indicated the necessity of such cuts for fruit growth. compared with the heading-backs before 10 july or after 10 august, those on 25 july produced the largest fruits inseason and exhibited the highest percentage of shoots that bore flowers and fruits the following year, indicating that pruning the secondary shoots on 25 july helps to direct the assimilates to fruit growth and flower bud development, not to vegetative growth. ‘nishimurawase’ persimmons tend to bear staminate, not pistillate, flowers when the trees are old and not vigorous enough. chijiwa and hayashi (2007) reported a way to use water shoots to serve as fruiting mother branches for the next season by pinching at 15 cm from the base between may and june. shoots left after the pinching or summer shoots regrown from the cut bore more pistillate and fewer staminate flowers the following year than the un-treated water shoots. takano (1994) also reported a better mother branch formation by pinching adventitious buds of persimmon. the size of trees and yield efficiency are among the most important considerations in high density orchards. when the vigorous shoots of young ‘uenishiwase’ persimmon were topped to a 30cm length in early june, followed by the removal of the regrowth, trunk growth decreased and fruit set increased the following year (song et al., 2001). the occurrence of secondary growth after heading was dependent on the treatment date. topping the shoots at the fourth bud from the terminal on 20 may promoted secondary growth of ‘hiratanenashi’ persimmon, but the same treatment after 22 june did not (hasegawa and nakajima, 1984). the summer topping of long shoots in summer was effective in forming fruiting shoots the following year in their lower parts. pinching and topping the shoot would promote flower bud formation of that shoot if regrowth is not severe. 6. conclusions severe summer pruning could suppress vegetative and fruit growth due to the lowered photosynthetic capacity, reducing the carbohydrate reserves in persimmon tree. however, summer pruning which is appropriate to tree vigor and environmental conditions would increase production of quality fruits by improving light distribution in the tree canopy or restricting vegetative growth. since tree vigor is a result of the complex interactive effects with cultural practices, the pruning alone should not be used as a tool for controlling vegetative growth. it is necessary to incorporate the pruning into a comprehensive program 168 such as tree training, fruiting, irrigation, and fertilization to reduce vigorous growth. growers should employ summer pruning methods while carefully monitoring for potentially negative effects on tree growth and fruit quality. references banno k., hayashi s., tanabe k., 1985 effects of sadh and shoot-bending on flower bud formation, nutrient components and endogenous growth regulators in japanese pear (pyrus serotina rehd.). j. japan. soc. hort. sci., 53: 365-376. chijiwa h., hayashi k., 2007 the formation of fruiting mother shoot from water shoot by pinching in japanese persimmon. hort. res., 6: 265-270. choi s.t., kang s.m., park d.s., song w.d., ahn g.h., 2003 a effect of different severities of summer pruning on fruit characteristics and tree growth in young ‘fuyu’ and ‘nishimurawase’ persimmon. j. korean soc. hort. sci., 44: 569-574. choi s.t., park d.s., ahn g.h., 2003 b studies on efficient production and quality improvement of sweet persimmon effect of summer pruning on fruit qualities of sweet persimmon. rural development administration report. suwon, korea, pp. 5-34. (in korean with english summary). choi s.t., park d.s., kang s.m., 2011 nutrient accumulation and flower bud formation affected by the time of terminal bud set on water sprouts of persimmon. hortscience, 46: 523-526. choi s.t., park d.s., kang s.m., cho y.c., 2010 effect of fruit-load on the growth, absorption, and partitioning of inorganic nutrients in young ‘fuyu’ persimmon trees. sci. hort., 126: 408-412. choi s.t., park d.s., kim s.c., 2005 summer pruning and management for vigorous persimmon trees. 2005 farming technology. rural development administration. suwon, korea. (in korean) ferree d.c., myers s.c., rom c.r., taylor b.h., 1984 physiological aspects of summer pruning. acta horticulturae, 146: 243-252. forshey c.g., elfving d.c., stebbins r.s., 1992 training and pruning apple and pear trees. amer. soc. hort. sci., pp. 60-63. fujimura j., 1932 effects of summer pruning on the secondary growth of one-year-old persimmon and japanese pear trees. j. japan. soc. hort. sci., 3: 9-20. george a.p., mowat a.d., collins r.j., 1997 a factors affecting blemishing of persimmon in new zealand and australia. acta horticulturae, 436: 171-178. george a.p., mowat a.d., collins r.j., morleybunker m., 1997 b the pattern and control of reproductive development in non-astringent persimmon (diospyros kaki l.): a review. sci. hort., 70: 93-122. george a.p., nissen b., broadley r., collins r.j., rigden p., jeffers s., isaacson b., ledger s., 2005 sweet persimmons grower’s handbook. department of primary industries and fisheries, brisbane, queensland, australia. hasegawa k., nakajima y., 1984 effects of topping on the growth and fruit quality of the japanese persimmon. res. rpt. kochi univ. (agr.), 33: 43-53. huh m.s., song i.k., cho d.h., jeong y.s., 2003 bending effect of vigorous shoot on persimmon tree. 2003 annual research report. gyeongsangbuk-do agr. res. ext. serv. daegu, korea. (in korean) kappel f., flore j.a., layne r.e.c., 1983 characterization of the light microclimate in four peach hedgerow canopies. j. amer. soc. hort. sci., 108: 102-105. kim b.s., 2010 development of cultivation techniques for the stable production of high quality sweet persimmon fruits to export a study on quality enhancement of sweet persimmon for export by stable tree vigor. rural development administration, suwon, korea. (in korean). kishimoto o., 1975 estimations of optimum range of degree of fruit thinning and desirable fruit weight in japanese pears and persimmon tree. j. japan. soc. hort. sci., 43: 368-376. li k., lakso a.n., piccioni r., robinson t., 2003 summer pruning reduces whole-canopy carbon fixation and transpiration in apple trees. j. hort. sci. biotechol., 78: 749-754. lopez g., mata m., arbones a., solans j.r., girona j., marsal j., 2006 mitigation of effects of extreme drought during stage iii of peach fruit development by summer pruning and fruit thinning. tree physiol., 26: 469-477. marini r.p., barden j.a., 1982 net photosynthesis, dark respiration, transpiration, and stomatal resistance of young and mature apple trees as influenced by summer or dormant pruning. j. amer. soc. hort. sci., 107: 170-174. marini r.p., barden j.a., 1987 summer pruning of apple and peach trees. hort. rev., 9: 351-375. mediene s., jordan m.o., pagès l., lebot j., adamowicz s., 2002 the influence of severe shoot pruning on growth, carbon and nitrogen status in young peach trees (prunus persica). tree physiol., 22: 1289-1296. miller s.s., 1982 regrowth, flowering, and fruit quality of ‘delicious’ apple trees as influenced by summer pruning. j. amer. soc. hort. sci., 107: 975-978. mowat a., 1987 pruning persimmons. hortresearch publication. new zealand. march 28, 2011. http://www.hortnet. co.nz/publications/science. myers s.c., ferree d.c., 1983 influence of summer pruning and tree orientation on net photosynthesis, transpiration, shoot growth, and dry-weight distribution in young apple trees. j. amer. soc. hort. sci., 108: 4-9. nii n., 1980 current shoot and growth in japanese persimmon, diospyros kaki cv. fuyu, in relation to the development of the tissue system in the leaf. j. japan. soc. hort. sci., 49: 149-159. park d.s., choi s.t., 2000 development of technology for production of high quality fruits in sweet persimmon studies on the method of fruit-load adjustment for high quality of fruit production. rural development administration report, suwon, korea. (in korean with english summary). park d.s., kang s.m., choi s.t., lim c.a., song w.d., 2003 effect of secondary-shoot prunings on fruit growth and following year’s fruit set of ‘fuyu’ persimmon. j. korean soc. hort. sci., 44: 678-682. rom c.r., ferree d.c., 1985 time and severity of summer pruning influences on young peach tree net photosynthesis, 169 transpiration, and dry weight distribution. j. amer. soc. hort. sci., 110: 455-461. song w.d., choi s.t., kim s.c., park d.s., kang s.m., 2001 effect of pinching on fruit set of young sweet persimmon trees during the following years. 2001 annual research report. gyeongsangnam-do agr. res. ext. serv. jinju, korea. (in korean) takano s., 1994 better branch formation by pinching adventive buds of japanese persimmon. bull. nara agr. expt. sta. j., 25: 36-37. taylor b.h., ferree d.c., 1981 the influence of summer pruning on photosynthesis, transpiration, leaf abscission, and dry weight accumulation of young apple trees. j. amer. soc. hort. sci., 106: 389-393. taylor b.h., ferree d.c., 1984 the influence of summer pruning and cropping on growth and fruiting of apple. j. amer. hort. sci., 109: 19-24. ullio l., 2003 persimmon growing in new south wales. agfact h3.1.17, 3rd edition. elizabeth macarthur agricultural research institute, camden, australia. yakushiji h., nakatsuka a., 2007 recent persimmon research in japan. japan. j. plant sci., 1(2): 42-62. yamada m., 2008 persimmon propagation, orchard planting, training and pruning in japan. adv. hort. sci., 22: 269-273. yamamura h., matsui k., matsumoto t., 1989 effects of gibberellins on fruit set and flower-bud formation in unpollinated persimmon (diospyros kaki). hortscience, 38: 77-86. zamani z., saie a., talaie a.-r., fatahi r., 2006 effects of summer pruning on growth indices of two important iranian apple cultivars ‘golab’ and ‘shafi-abadi’. acta horticulturae, 707: 269-274. 148 1. introduction italian production of propagated certified grapevines by nurseries is regulated by laws (dm 8 february 2005; dm 7 july 2006) that define the procedures to obtain a certification for propagative material in order to handle healthy plants. these regulations provide detailed guidance for the registration of the primary source to be maintained by the conservative breeder and for the production of basic material or mother plants, the latter grown in nurseries and used to produce certified materials delivered to the growers. in 2009, the “working group arnadia grapevine viruses” was established, within the italian ministry of agriculture finalized project “arnadia”, with the purpose of producing validated reference diagnostic protocols for the control and monitoring of plant pathogens of phytosanitary interest. in 2011, the methods proposed to perform phytosanitary testing were reassessed (dm 13 december 2011) and it was established that mother plants grown in italian nurseries (cultivars or rootstocks) have to be checked for virus infections 10 years after transplanting. the first deadline was set for 30 june 2012 to consider mother plants transplanted in 2001 or earlier. obviously, these mother plants were checked and produced according to older regulations (starting with dpr 24 december 1969) that define pathogens (absence of grapevine leafroll and fanleaf degeneration disease) and methods for their assay (biological indexing) differently compared to the most recent regulations. presently, plants are tested for the following viruses: grapevine leafroll associated virus -1 (glrav-1) and -3 (glrav-3), grapevine fanleaf virus (gflv), arabis mosaic virus (armv) and grapevine virus a (gva). the significant presence of these viruses was recently reported in tuscany after assays carried out in sanitary selection programs. from 1997 to 2004, health tests conducted on 172 uncertified plants selected from the tuscan costal area (elba island, lucca and maremma) showed a critical phytovirologic condition, with 97.1% infected plants (materazzi et al., 2006 a). in the period 20002004, health tests conducted on 318 uncertified grapevine plants selected in d.o.c. or d.o.c.g. areas of tuscany (chianti classico, montalcino and montepulciano) revealed that 58.8% vines were virus-infected (materazzi et al., 2006 b). these findings cannot be transferred to nurseries, considering their use of certified materials. in any case, the virologic status of uncertified grapevine in tuscany indicates the presence of grapevine viruses and related vectors, underlining the importance of periodic verifications in grapevine nurseries to guarantee the highest health standards of plant production and to help reduce the sustainability of old grapevine mother plants in relation to new mandatory diagnostic tests for virus control d. rizzo*, l. stefani*, m. paoli*, e. triolo**, a. panattoni**, a. luvisi**(1) * servizio fitosanitario regionale, servizi agro ambientale di vigilanza e controllo, regione toscana, via dei fiori, 8, 51010 pescia (pt), italy. ** dipartimento di scienze agrarie, alimentari e agro-ambientali, università di pisa, via del borghetto, 80, 56124 pisa, italy. key words: arabis mosaic virus, grapevine fanleaf virus, grapevine leafroll, grapevine virus a, phytosanitary test. abstract: in 2011, the methods to perform phytosanitary tests to check for viruses in grapevine nurseries were reassessed and new regulations were defined (dm 13 december 2011). the mandatory tests require serological assays for the diagnosis of five viruses in grapevine mother plants transplanted in tuscany in 2001 or before. the aim of the present paper is to report the impact of certification programs applied before 2001 in tuscany and the sustainability of older mother plants with relation to the new mandatory diagnostic tests. among the cultivars, virus infection was reported in 19.2% of pool samples, whereas 2.4% of rootstock pool samples showed a compromised health status. glrav-3 is the most frequently found virus (10.4% and 1.3% of cultivar and rootstock pools, respectively), and it is also included in the most frequent multiple infections. multiple infections represent about 25% of infected cultivar pools and almost 50% of infected rootstock pools. adv. hort. sci., 2012 26(3-4): 148-150 (1) corresponding author: aluvisi@agr.unipi.it. received for publication 6 november 2012 accepted for publication 26 february 2013 149 the spread of grapevine pathogens, as determined by the updated regulations. in this paper, we report the results obtained from serological (elisa) tests for the diagnosis of five viruses in grapevine mother plants (cultivars or rootstock) transplanted in 2001 or before, as dictated by dm 13 december 2011. the aim is to report the impact of certification programs applied before 2001 in tuscany and the sustainability of older mother plants as stated by the new mandatory diagnostic tests. 2. materials and methods plant sampling and elisa tests plant sampling and elisa tests were carried out following procedures defined by the working group arnadia grapevine viruses, included in dm 13 december 2011. in accordance with legislation, the following steps were undertaken. sampling was performed beginning in november 2011 in 33 nurseries, collecting sample pools composed by homogenous material from five plants. phloem tissue (2 g) was collected from pools and mechanically ground (tissue lyzer with 10 ml-grinding jar, qiagen, venlo, netherlands) with extraction buffer. elisa test was performed using commercial polyclonal antibodies as well as negative and positive controls (agritest, bari, italy). absorbance at od 405 nm was recorded by photometry (titertek multiskan, titertek instruments inc., huntsville, usa). readings were normalized as r value (od-treated explant/od-hc), identifying the r= 2 threshold which distinguishes the positive versus the negative response (monette, 1983). 3. results the elisa test (table 1) showed that 19.2% of cultivar pools were infected with at least one of the viruses. all five viruses were found in cultivar samples, but glrav-3 was considerably more frequent than the others, followed by gva. the combination of these two viruses also represent the most frequent multiple infection. multiple infections represent about 25% of infected pools and they are characterized by 13 different virus combinations. the least frequent virus was armv. with regard to rootstock pools, there was a low rate of infection (2.3%), even if glrav-3 was still the most frequent virus detected. this virus was also included in the most frequent multiple infections that, for rootstocks, represent almost 50% of infected pools. multiple infections were reported in ten different virus combinations. also in rootstocks, armv was the least frequent virus. 4. discussion and conclusions monitoring revealed glrav-3 as the most frequent virus in cultivar or rootstock mother plants, as reported in sanitary selection research previously carried out in tuscany (triolo and materazzi, 2004; materazzi et al., 2006 a) and other italian areas (digiaro et al., 2000; bica et al., 2002; martelli, 2002). similarly, the low frequency of armv is in agreement with other health checks performed in tuscany on uncertified plants (borgo et al., 2000; materazzi et al., 2006 a). viruses were frequently detected in multiple infections, in particular in rootstocks, with a wide range of combinations. table 1 rates of virus infection for cultivar or rootstock pools detected by elisa test mother plant no of checked pool no of infected pool % of virus infection cultivar 712 137 19.2 rootstock 1523 36 2.4 infected pool out of total (%) glrav-1 glrav-3 gflv armv gva cultivar 2.2 10.4 1.8 1.4 5.1 rootstock 0.9 1.3 0.9 0.6 0.7 pool infected by multiple viruses out of total (%) cultivar rootstock glrav-3/gva 2.11 glrav-1/glrav-3 0.33 glrav-1/glrav-3/gva 0.56 glrav-3/gva 0.13 glrav-1/glrav-3/gva/gflv 0.42 glrav-3/gflv 0.13 glrav-1/glrav-3 0.28 armv/gva 0.13 glrav-1/gflv 0.28 others 0.46 glrav-3/gva/armv 0.28 others 0.98 total multiple infections 4.91 total multiple infections 1.18 150 considering that these findings represent the first application of dm 13 december 2011, it is not possible to evaluate the health status of mother plants grown in tuscan nurseries. moreover, comparison to other italian areas is not relevant because no homogeneous data are available. in any case, these finding can be a starting point to evaluate the health trend of plants in tuscan nurseries. the current health status of mother plants may be due to re-infection events as all tested viruses are known to be vector-transmitted (golino et al., 2002; andret-link et al., 2005; zorloni et al., 2006; demangeat et al., 2010; tsai et al., 2010) and relative vectors have been found in tuscany. however, the significant improvement in diagnostic tests over the last 30 years do not seem to exclude that the primary source or basic material were originally infected. even if these categories are considered in dm 13 december 2011, there is no updated health information available that can reconstruct the propagation links. in this case, the application of traceability tools such as electronic identification (bandinelli et al., 2009; luvisi et al., 2012 a, b) could support retrieval of health information. moreover, the activity of local conservative breeders, such as the associazione toscana costitutori viticoli (tos.co.vit.) set up in tuscany in 2003 (triolo, 2011), can promote the use of certified plants selected according to the most recent regulations. considering that the rate of infected cultivars and rootstocks was found to be very low during verifications carried out during tuscan sanitary selections, these findings confirm that the use of certified plants helps reduce the spread of grapevine viruses. references andret-link p., fuchs m., 2005 transmission specificity of plant viruses by vectors. j. plant pathol., 87: 153-165. bandinelli r., triolo e., luvisi a., pagano m., gini b., rinaldelli e., 2009 employment of radiofrequency technology (rfid) in grapevine nursery traceability. adv. hort. sci., 23(2): 75-80. bica d., nicolosi e., costa a., colombo a., buonocore e., 2002 indagine sulla presenza dei principali virus e nematodi della vite in sicilia. informatore fitopatologico, 52(1): 64-67. borgo m., ferroni g., salvi g., scalabrelli g., 2000 clonal selection of “vermentino” grapevine in tuscany. acta horticulturae, 528: 731-738. demangeat g., komar v., van ghelder c., voisin r., lemaire o., esmenjaud d., fuchs m., 2010 transmission competency of single-female xiphinema index lines for grapevine fanleaf virus. phytopathology, 100(4): 384-389. digiaro m., simeone v., boscia d., savino v., 2000 stato sanitario delle varietà ad uva da tavola di recente introduzione in puglia. informatore fitopatologico, 50(7): 54-58. golino d.a., sim s.t., gill r., rowhani a., 2002 california mealybugs can spread grapevine leafroll disease. calif. agr., 56(6): 196-201. luvisi a., panattoni a., bandinelli r., rinaldelli e., pagano m., triolo e., 2012 b propagative material of grapevine: rfid technology for supporting traceability of “basic” and “certified” material along the wine production chain. adv. hort. sci., 26(1): 39-43. luvisi a., panattoni a., triolo e., 2012 a radio-frequency identification could help reduce the spread of plant pathogens. calif. agr., 66(3): 97-101. martelli g.p., 2002 le principali virosi della vite oggi. informatore fitopatologico, 52(4): 18-27. materazzi a., luvisi a., triolo e., 2006 b diffusione e peculiarità di agenti virali su ceppi di sangiovese selezionati in toscana. in: il sangiovese vitigno tipico e internazionale: identità e peculiarità, arsia, italy, pp. 339-343. materazzi a., triolo e., scalabrelli g., d’onofrio c., luvisi a., ferroni g., 2006 a clonal selection of cv. aleatico (vitis vinifera l.) along tuscan costal area. proc. international symposium on environment identities and mediterranean area, pp. 531-535. monette p.l., 1983 virus eradication through in vitro techniques. the international plant propagatoris society, 33: 90-100. triolo e., 2011 storia ed attualità di tos.co.vit. e del suo nucleo di premoltiplicazione. in: vivaismo viticolo: nuove performance di tracciabilità e sviluppi per il mercato vivaistico. debatte, italy, pp. 44-50. triolo e., materazzi a., 2004 malattie virali e simil-virali della vite in toscana: diffusione, pecularietà ed analogie in 5 vitigni. quaderno arsia, 1: 103-116. tsai c.w., rowhani a., golino d.a., daane k.m., almeida r.p.p., 2010 mealybug transmission of grapevine leafroll viruses: an analysis of virus–vector specificity. phytopathology, 100(8): 830-834. zorloni a., prati s., bianco p.a., belli g., 2006 transmission of grapevine virus a and grapevine leafrollassociated virus 3 by heliococcus bohemicus. j. plant pathol., 88(3): 325-328. 176 1. introduction harvesting is one of the most important operations of the whole cultivating cycle in olive production, both in order to obtain high quality oils and to reduce costs (tombesi, 1990; famiani et al., 1998; cicek, et al., 2010; ferguson et al., 2010). the aim of this trial was to evaluate the efficiency of a trunk shaker with a reversed umbrella interceptor for the mechanical harvesting (visco et al., 2008; farinelli et al., 2012 a, b) of two autochthonous cultivars, ‘ortice’ and ‘ortolana’, in southern italy. 2. materials and methods the experiment was carried out in 2006 in a commercial olive grove, belonging to the “uliveto” farm, located in southern italy (41°15’ n, 14°38’ e) (province of benevento). adult trees of two autochthonous cultivars, ‘ortice’ and ‘ortolana’ (di vaio et al., 2013), trained to the vase system and planted at a spacing of 6 x 6 m were studied. the olive grove had a slope of less than 3% and was drip irrigated. pruning was carried out annually and fertilization and pest management were carried out according to local standard practices. for the mechanical harvesting, carried out on 30 november, a self-propelled machine, “f3” model with three traction wheels and a reversed umbrella interceptor manufactured by sicma (catanzaro, italy), was used (fig. 1). the main characteristics of the trunk shaker were: an engine power of 77 kw (105 cv), a very-highfrequency vibrating head (1800-2000 vibrations/min), a self-braking system and a 6-m diameter umbrella opening. the work force consisted of two workers, one for maneuvering the harvesting machine, the other for handling the olives. during drupe maturation, periodically on three samples of 100 olives per date, the following parameters were determined: olive detachment force, coloration (jaén index between 0 and 7), fresh weight, pulp firmness (with a manual penetrometer with a 1.0 mm diameter plunger on the equatorial zone of fruit) and oil content (determined using a soxlhet extractor). on four trees per cultivar, at the mechanical harvesting of oil olives by trunk shaker with a reversed umbrella interceptor c. di vaio*, n. marallo*, s. nocerino*, f. famiani** * dipartimento di arboricoltura, botanica e patologia vegetale, università di napoli federico ii, via università, 100, 80055 portici (na), italy. ** dipartimento di scienze agrarie e ambientali, università degli studi di perugia, borgo xx giugno, 74, 06121 perugia, italy. key words: mechanical harvesting, olea europaea l., ‘ortice’, ‘ortolana’, oil quality, trunk shaker with reversed umbrella. abstract: trunk shakers are primarily used for the mechanical harvesting of oil olives in intensive orchards. the objective of this trial was to determine the efficiency of mechanical harvesting of olives with a self-propelled trunk shaker with a reversed umbrella interceptor (model f3, sicma, catanzaro, italy), from adult trees of two autochthonous cultivars, ‘ortice’ and ‘ortolana’, growing in southern italy with 6 × 6 m spacing and trained to the vase system. the main characteristics of the trunk shaker were: an engine power of 77 kw (105 cv), a very-high-frequency vibrating head (1800-2000 vibrations/min), a self-braking system and a 6-meter diameter umbrella opening. the worksite consisted of two workers one for maneuvering the harvesting machine and the other for handling the olives. mechanical harvesting was carried on 30 november 2006 when the fruits of ‘ortice’ and ‘ortolana’ had a weight and detachment force around 2.8 g and 3.1 n and 3.8 g and 4.6 n, respectively, and the fruit drop was around 14% and 10%, respectively. both cultivars had a good production (26.06 and 21.18 kg/tree). the mechanical harvesting yield (percentage of mechanically harvested olives) was very high, reaching values around 97% in both cultivars. moreover, the low number of workers, the reduced time for the operation (2.5 min/tree), the good yield/tree and the high quantity of harvested fruit allowed a very high work productivity to be obtained: around 302 kg/h/worker for ‘ortice’ and 246 kg/h/worker for ‘ortolana’. the quality of the oils extracted from the harvested olives met the requirements set by european law for extra virgin olive oils. the results indicate that the use of a trunk shaker with a reversed umbrella can be an efficient solution for mechanical harvesting of the ‘ortice’ and ‘ortolana’ cultivars in southern italy. adv. hort. sci., 2012 26(3-4): 176-179 received for publication 6 february 2013 accepted for publication 14 february 2013 177 beginning of ripening, eight small branches were labeled (two per cardinal point) and the fruit was periodically counted up to harvesting time in order to estimate fruit drop. mechanical harvesting was carried out on 10 trees/ cultivar and the drupes were weighed. after harvesting, undetached olives were harvested by hand and weighed. the ratio between mechanically harvested olives/total olives on the canopy, expressed as percentage, was used to determine the mechanical harvesting yield (%). the trunk cross-sectional area (at about 0.5 m above the ground) and canopy width (w), height (h) and volume [volume = ((w/2)2 x 3.14 x h) x 2/3] were measured/calculated on each of the harvested trees. work productivity was calculated and expressed as the amount of harvested olives/h/ worker. in both cultivars, after mechanical harvesting, 100 kg of olives were collected and micro-milled to obtain two samples of mono-variety oils, on which free acidity, peroxide number, spectrophotometric indices and sensorial characteristics (by panel test) were determined. data were submitted to analysis of variance (anova) using msta-c software and mean separation was performed by the multiple range duncan test at the 5% significance level. moreover, the standard errors (se) of the means were also calculated. 3. results and discussion during maturation, the cv. ortice was resistant to drupe detachment, which decreased at the beginning of november until reaching 309.17 g at harvest time. the cv ‘ortolana’, instead, followed a rather constant course, and at harvest time reached 455.83 g (fig. 2 and table 1). pulp firmness decreased constantly during maturation, reaching fig. 1 mechanical harvesting of olives with a trunk shaker with a reversed umbrella interceptor. fig. 2 evolution of olive detachment force during maturation (mean ± standard error). table 1 characteristics of trees and fruit at harvest, machine efficiency and work productivity (mean ± standard error) cv. ortice cv. ortolana olive weight (g) 2.79 a±0.06 3.77 b±0.01 olive detachment force (g) 309.17 a±19.30 455.83 b±18.20 olive detachment force/olive weight (n/g) 1.11 a±0.08 1.21 a±0.09 pulp firmness (g/mm2) 267.00 a±5.11 231.58 b±0.45 drupe coloration jaèn index (0-7) 2.10 a±0.05 4.35 b±0.17 fruit drop (%) 13.95 a±2.27 10.36 a±2.49 canopy volume (m3) 47.34 a±2.35 56.31 b±1.56 trunk cross section area (cm2) 1587.31 a±337.96 613.43 b±172.04 total olive yield per tree (kg) 26.06 a±7.72 21.18 a±5.02 olives mechanically harvested per tree (kg) 25.17 a±7.21 20.49 a±5.03 productive efficiency of tree (kg/m3) 0.37 a±0.17 0.25 a±0.13 mechanical harvesting yield (%) 96.58 a±1.15 96.74 a±0.88 work productivity (kg/h/worker) 302.04 a±18.01 245.88 a±14.10 in each row, means with the same letter are not significantly different by duncan multiple range test (p<0.05). 178 values of 267.00 g/mm2 and 231.58 g/mm2 for ‘ortice’ and ‘ortolana’, respectively (fig. 3 and table 1). at harvest, the accumulation of oil in the drupe was 26.09% in ‘ortice’ and 16.51% in ‘ortolana’ (fig. 4). olive pigmentation, scored according to the jaén index, differed for the two cultivars: 2.10 in ‘ortice’ and 4.35 in ‘ortolana’ (table 1). both cultivars had good production (26.06 and 21.18 kg/ tree) (table 1), and the productive efficiency was 0.37 and 0.25 kg of olives/m3, respectively (table 1). the canopy volume of the trees was 47.34 m3 for ‘ortice’ and 56.31 m3 for ‘ortolana’. pre-harvest fruit drop increased during the entire period, reaching values of 13.95% for ‘ortice’ and 10.36% for ‘ortolana’ at harvest time (fig. 5 and table 1). at harvest fruit weight was 2.79 g for ‘ortice’ and 3.77 g for ‘ortolana’ (table 1). the ratio between the olive detachment force and weight decreased throughout the ripening period and was 1.11 and 1.21 n/g at harvest (fig. 6 and table 1). in general, fruit characteristics and ripening patterns were similar to those reported by di vaio et al. (2013) for the same cultivars. despite the high canopy volume of the trees, the mechanical harvesting yield (percentage of mechanically harvested olives) was very high: 96.58% and 96.74% for the ‘ortice’ and ‘ortolana’ cultivars, respectively (table 1). these high values are likely due to the relatively low olive detachment/weight ratios at the time of harvesting, which were close to one. indeed, farinelli et al. (2012 a) reported a significant negative relationship between the olive detachment/weight ratio and the mechanical harvesting yield obtained with a trunk shaker. the same authors observed that high mechanical harvesting yields are obtained when the ratio is less than 2. in this regard, it can be noted that in the present work, for both cultivars, the olive detachment/weight ratio, as a result of the medium weight and the medium/low detachment force of the olives, was less than 2 for the entire ripening period. this indicates that mechanical harvesting could be carried out efficiently fig. 3 evolution of pulp firmness during maturation (mean ± standard error). fig. 6 evolution of the ratio between olive detachment force and drupe weight during maturation (mean ± standard error). fig. 5 evolution of pre-harvest fruit drop during maturation. fig. 4 evolution of olive oil content (% f.w.) during maturation. 1.8 1.6 1.4 1.2 1.0 0.8 179 during the entire ripening period. at the farm level, this allows for flexibility, also considering that oil quality changes during olive ripening and so oils with different characteristics can be obtained by modulating harvesting time (inglese et al., 2011). moreover, the low labour requirement (only two workers), the reduced time for operation (about 2.50 min/tree, which included the approach to the tree and attachment of the shaker to the trunk, opening of the reversed umbrella, shaking and closing the reversed umbrella), the good yield/tree and high quantity of harvested fruit allowed very high values of work productivity to be obtained: about 302.04 kg/h/worker for ‘ortice’ and 245.88 kg/h/worker for ‘ortolana’ (table 1). this result highlights the importance of a good yield/tree in determining high work productivity and therefore the economic convenience of using machines for olive harvesting (famiani et al., 1998). the oil quality indices (acidity, peroxide number, spectrophotometric indices and sensorial characteristics by panel test) reported in table 2 show that all analytical and sensory evaluations of the oils from the two cultivars met the requirements set by law for extra virgin olive oils (ec regulation n. 2568, 1991). therefore, the use of the trunk shaker with interceptor allowed high quality oils to be obtained. in conclusion, the results of the present study show that the trunk shaker with the reversed umbrella interceptor, which allows for high harvesting yields and high labour productivity, can be considered an interesting solution for mechanical harvesting of the autochthonous cultivars ‘ortice’ and ‘ortolana’ in southern italy. acknowledgements this study was financed by the mi.p.a.a.f. within the riom project “research and innovation for the olive growing of southern italy”. the authors wish to thank dr. mark walters for editing the manuscript. references cicek g., sumer s.k., kocabiyik h., 2010 effect of different harvest methods on olive yield and work capacity. african journal of agricultural research, 5(23): 3246-3250. di vaio c., nocerino, s., paduano a., sacchi r., 2013 characterization and evaluation of olive germplasm in southern italy. journal of the science of food and agriculture. doi: 10.1002/jsfa.6057 ec regulation 2568, 1991 on the characteristics of olive oil and olive residue oil and on the relevant methods of analysis. off. j. eur. commun, nl 248/1 famiani f., proietti p., palliotti a., guelfi p., nottiani g. 1998 possibilità di meccanizzazione della raccolta delle olive in diverse tipologie di oliveto. rivista di frutticoltura, 7/8: 33-39. farinelli d., ruffolo m., boco m., tombesi a., 2012 a yield efficiency and mechanical harvesting with trunk shaker of some international olive cultivars. acta horticulturae, 949: 379-384. farinelli d., tombesi s., famiani f., tombesi a., 2012 b the fruit detachment force/fruit weight ratio can be used to predict the harvesting yield and efficiency of trunk shakers on mechanically harvested olives. acta horticulturae, 965: 61-64. ferguson l., rosa u., castro-garcia s., lee s.m., guinard j.x., burns j., krueger w.h., o’connell n.v., glozer k., 2010 mechanical harvesting of california table and oil olives. adv. hort. sci., 24(1): 53-63. inglese p., famiani f., galvano f., servili m., esposto s., urbani s., 2011 factors affecting extra-virgin olive oil composition. horticultural reviews, 38: 83-147. tombesi a., 1990 physiological and mechanical advances in olive harvesting. acta horticulturae, 286: 399-412. visco t., molfese m., cipolletti m., corradetti r., tombesi a., 2008 the influence of training system, variety and fruit ripening on the efficiency of mechanical harvesting of young olive trees in abruzzo, italy. acta horticulturae, 791: 425-429. table 2 quality indices of oils obtained from cv. ortice and ortolana olives mechanically harvested with a trunk shaker (mean±standard error) cultivar acidity % oleic acid peroxide value meq o 2 kg-1 uv defects at panel 0-5k232 k270 ∆k ortice 0.52±0.04 8.2 b±0.13 1.967±0.065 0.145±0.014 -0.003±0.000 0±0.00 ortolana 0.46±0.02 6.5 b±0.14 1.824±0.032 0.155±0.036 -0.005±0.000 0±0.00 impaginato 33 adv. hort. sci., 2023 37(1): 33­40 doi: 10.36253/ahsc­13908 effect of microwave mild heat treat­ ment on postharvest quality of table grapes m.l.v. de chiara (*), n. de simone, g. spano, m.l. amodio, g. colelli department of sciences of agriculture, food, natural resources and engineering (dafne), university of foggia, via napoli, 25, 71122 foggia, italy. key words: botrytis cinerea, external appearance, microbiological quality, microwave energy, mold development, ready­to­eat grape. abstract: table grapes are characterized by high susceptibility to mold develop­ ment during post­harvest, mostly due to botrytis cinerea. microwave applica­ tion on ready­to­eat product can represent an alternative to antifungal treat­ ment. with the aim of identifying the maximum energy that can be applied on grape without detrimental effects a central composite design was developed testing the application of 10 combinations of treatment time (seconds) and microwave power (watt). as a result, energies above 8000 kj negatively affect­ ed the sensorial quality of fresh product, both in the presence and absence of b. cinerea inoculum (106 log cfu g­1). the physico­chemical parameters did not show significant differences, but two time/power combinations improved sen­ sory quality of table grape, being selected for the subsequent packaging trial: 14 s/100 w and 80 s/100 w. treatments were applied before or after packaging in polypropylene bags. at the end of storage period, 100 w applied for 80 sec­ onds before packaging led to a better external appearance of the product than the other treatments, maintaining an intermediate level of mesophilic bacterial load and no significant differences in terms of nutritional quality. 80 seconds at 100 watt combined with packaging can represent a valuable starting point for further experiments. 1. introduction table grapes are characterized by high susceptibility to mold and rot development during prolonged postharvest storage and commercializa­ tion, often leading to a general decrease in overall bunch quality. among other factors, fungal decay, mainly caused by botrytis cinerea, is the prin­ cipal responsible for deterioration with grey mold development (williamson et al., 2007; ahmed et al., 2018). after harvest, a favorable environment for the germination of fungal spores is created on berries surface, mainly for damaged fruits (de simone et al., 2020). for this rea­ son, during post­harvest life of fruits and vegetables, processing technolo­ gies and biotechnologies aimed to provide physical, chemical, and biologi­ cal hurdles to limit the development of undesired microorganisms (*) corresponding author: maria.dechiara@unifg.it citation: de chiara m.l.v., de simone n., spano g., amodio m.l., colelli g., 2023 ­ effect of microwave mild heat treatment on postharvest quality of table grapes. ­ adv. hort. sci., 37(1): 33­40. copyright: © 2023 de chiara m.l.v., de simone n., spano g., amodio m.l., colelli g. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 31 october 2022 accepted for publication 29 december 2022 ahs advances in horticultural science https://doi.org/10.36253/ahsc-13908 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2023 37(1): 33­40 34 (capozzi et al., 2009). conventional thermal process­ es can result in the reduction of nutritional and sen­ sory quality of the product, due to slow heat trans­ mission within the plant tissue. application of mild thermal treatments aimed to control postharvest dis­ ease by means of microwave heating (dar et al., 2020), could allow to avoid the use of chemical com­ pounds and therefore residues in the treated product and ensuring at the same time minimal environmen­ tal impact, thus representing a valuable alternative to traditional thermal processing. microwaving ensures instead a fast and effective heat treatment reducing risk of injuries and decrease of nutritional com­ pounds. however, oversized intensity of the treat­ ment can induce an excessive temperature increase, resulting in a damage to the fresh plant tissue. moreover, high temperatures could affect grape bio­ chemical characteristics, for example losses of aroma­related compounds and development of oxidative processes (modesti et al., 2020). to date, a s m a l l n u m b e r o f s t u d i e s d e a l s w i t h t h e u s e o f microwave treatment on fresh produce (karabulut and baykal, 2002; zhang et al., 2004, 2006; sisquella et al., 2013), showing its efficiency in prolonging postharvest life of peaches, in which microwave inhibited growth of inoculated pathogens after 2 minutes, being also effective in controlling endoge­ nous microflora with a very low decay incidence. as for nectarines, brown rot incidence was significantly reduced by microwaving to less than 14% versus 45% of untreated product. treatment caused a delay of softening and internal damage. zhang et al. (2006) o b s e r v e d t h a t i n p e a r s t r e a t e d f o r 2 o r 3 m i n penicillium expansum population was significantly lower than control samples without impairing quality of the fruits. fresh­cut carrots, apples, and minimally p r o c e s s e d b o k c h o y w e r e s u b j e c t e d t o h i g h power/short time treatments showing promising results. microwave treatments maintained physical, chemical and sensory quality of fresh­cut carrots over storage period, reducing surface whitening and avoiding firmness modification, also enhancing bioac­ tive compounds concentration. however, microbial growth was greater than control samples during s h e l f ­ l i f e ( m a r tí n e z ­ h e r n á n d e z e t a l . , 2 0 1 6 ) . application of 454 w for 5 s on bok­choy decreased respiration rate, decay occurrence and etiolation, while improving integrity of cell membrane with a final better quality of product (song et al., 2018). as for fresh cut apples, a significant microbial reduction was observed for treatment at 300 w for 35 s, with no detrimental effect on nutritional parameters and a slight decrease of visual quality (colelli et al., 2021). however, there is the need to deeply study this tech­ nology and its effect on the species of interest with the aim to keep intact the fresh­like characteristics of the product. moreover, microwave application as a part of a hurdle technology and its application in combination with packaging could be recommended to avoid recontamination, thus the objective of this paper is to provide preliminary information concern­ ing the effect of microwave heating on table grape quality, in terms of efficacy in maintaining physical and microbiological quality. 2. materials and methods a preliminary test to select maximum microwave energy output to be applied on fresh table grape was carried out by means of a central composite design (ccd). treatment time (seconds) and microwave power levels (watt) were considered as ccd factors, ten combinations were identified using the software statgraphics centurion xvi.i (statpoint technologies, inc., usa) (table 1). table grape was divided into 100 g­batches and treated according to the experimental design, using a solid­state microwave oven at a labo­ ratory scale (2450 mhz, maximum power 1000 w). processed products were stored in air at 5±1°c and 95% rh up to 14 days after treatment. each combina­ tion was performed once, being the statistical vari­ ability already considered during designing of the experimental ccd plan. the experiment was also conducted on samples previously inoculated by table 1 ­ treatment time (seconds) and microwave power levels (watt) according to an experimental plan based on central composite design 22+ star with two central points run treatment time (seconds) microwave power levels (watt) 1 14 100 2 47 32 3 94 265 4 47 265 5 47 498 6 0 265 7 14 430 8 47 265 9 80 430 10 80 100 de chiara et al. ‐ microwave effect on table grape quality 35 immersion in a 1x106 spores/ml solution of botrytis cinerea cect 20973 purchased from the spanish type culture collection (cect, paterna, spain), and stored at low temperature for 7 days+7 days of room temperature shelf life. for not inoculated samples, immediately after the treatment and after 7 and 14 days, the main physicochemical and microbiological parameters were evaluated, while inoculated sam­ ples were evaluated only for external aspect due to the massive presence of b. cinerea that could affect in a non­realistic way the quality of these latter sam­ ples. obtained results were subjected to the specific s t a ti s ti c a l a n a l y s i s f o r c c d u s i n g t h e s o ft w a r e statgraphics, to create estimated response surface plots. the second trial was subsequently performed on uninoculated samples using the two most effec­ tive combinations to understand the best moment for microwave heating application throughout the minimal processing. ready­to­eat table grape bunch­ es (100 g) were subjected to treatments before or after packaging within polypropylene (pp) bags (10x15 cm), and subsequently stored at 5°c up to 14 days. during storage (at initial time and after 7 and 14 days), physicochemical, microbiological, and organoleptic evaluations were carried out and results were subjected to statistical analysis. the treatments were as follows: ctrl: samples not treated; lowmw: microwave treatment at 14 s/100 w and subsequent application of the packaging; highmw: microwave treatment at 80 s/100 w and subsequent application of the packaging; lowmw pp: microwave treatment at 14 s/100 w on packed samples; highmw pp: microwave treatment at 80 s/100 w on packed sam­ ples. all the treatments were performed in triplicate. ranking test first of all, ranking test was performed on samples treated as described for ccd to select the maximum microwave output energy to be applied on the prod­ ucts without detrimental effect. seven trained pan­ elists were asked to rank the samples according to their preference, evaluating the fresh­like appear­ ance of the product and its organoleptic properties and the presence of typical fresh­like flavor, by assigning a score (from 1 to 10 considering each sam­ ple as a whole, based on the following characteris­ tics: fresh­like appearance and organoleptic proper­ ties and the presence of typical fresh­like flavor). the sum of the score given by each panelist was com­ pared with preset values to statistically evaluate the differences between the samples in terms of the test­ ed parameters. fungal strain and growth condition botrytis cinerea cect 20973, purchased from the spanish type culture collection (cect, paterna, spain), was used to inoculate samples. cryopreserved cultures were plated on potato dextrose agar (pda, oxoid), and incubated at 25°c for 5 days. fungal spore suspension was prepared by brushing the plates surface with saline solution (8.6 g l­1 nacl) sup­ plemented with 0.01% tween 80 using a sterile swab and stored at 4°c for short­term uses. fungal spores concentration was determined by plating serial dilu­ tion on pda plates and adjusted to approximately 1 x 106 spores/ml. microbial load determination grape berries from each replicate were diluted (1:10) with nacl 8.6 g l­1 solution, and homogenized in a blender (bag mixer, interscience, saint­nom­la­ bretéche, france) for 2 min. then, samples were sub­ mitted to tenfold serial dilution. mesophilic microor­ ganisms were enumerated by plate counting on plate count agar (pca) and incubated at 25 for 48 h. yeasts and molds were plated on potato dextrose agar (pda) (oxoid) added with chloramphenicol (100 mg l­1) and incubated at 30°c for 48 h. ascorbic acid, dehydroascorbic acid and total vitamin c determination ascorbic acid, dehydroascorbic acid and total vita­ min c amounts were assessed homogenizing 5 grams o f f r u i t ti s s u e w i t h 5 m l o f e x t r a c ti o n m e d i u m (meoh: h2o (5:95) plus citric acid (21 g l ­1) with edta (0.5 g l­1) and naf (0.168 g l­1). the homogenate was filtered, centrifuged, and the supernatant was recov­ ered. ascorbic acid (aa) and dehydroascorbic acid (dhaa) contents were determined through hplc analysis (agilent technologies 1200 series; agilent, waldbronn, germany) as described by zapata and dufour (1992) with some modifications. aa and dhaa contents were expressed as mg of ascorbic or dehydroascorbic acid per 100 g of fresh weight. vitamin c content corresponds to the sum aa+dhaa and was expressed as mg 100 g fw­1. color analysis and determination of the berries’ tem‐ perature color of berries surface was measured on 5 berries per each replicate using a spectrophotometer (cm 2600d, konica minolta, japan) in the reflectance mode with the cie l*a*b* color scale. immediately after the treatment, berries surface temperature was acquired using thermal imaging camera flir c5 (teledyne technologies, wilsonville, oregon, usa). adv. hort. sci., 2023 37(1): 33­40 36 texture berries firmness evaluation on 5 berries from each replicate was performed using a ta­xt2 texture analyzer (stable micro systems ltd., england, uk) by measuring the maximum compression force at a rate of 1.5 mm s­1. sensorial quality a panel of six trained people evaluated external appearance and overall rating of bunches, berries, and rachis of the stored product from each replicate at each sampling day. it was used a hedonic scale associated to a brief description corresponding to a score from 1 to 5, where 1= really poor and 5=excel­ lent, being 3 the limit of marketability and 2 the limit of edibilit. gas analysis oxygen (o2) and carbon dioxide (co2) concentra­ tions, expressed as kpa, inside plastic bags containing table grapes were monitored over storage time by using a hand­held gas analyzer (checkpoint, pbi dansensor) to measure gases concentration in 15 ml of headspace. statistical analysis data were subjected to a two­way anova (for treatment and sampling time), and means were sep­ arated by tukey’s test at p<0.05 (5% significance level) using stat graphics centurion xvi.i software. mean values within each sampling were separated applying tukey’s test with significant difference when p≤0.05. 3. results and discussion first of all, as from ranking test results, it was observed that samples from treatment 1 and 9, treat­ ed with 14 s/100 w and 80 s/430 w, respectively, were significantly different from the other, showing better and worse characteristics than the other treat­ ments, respectively. specifically, sample number 9 reached, after treatment, a maximum temperature of 81°c, with an average of 44.7°c, consequently the panelists evaluated this sample negatively, highlight­ ing the significant presence of cooked flavor. in gen­ eral, grape temperature during treatment increased very heterogeneously among all samples (fig. 1, table 2). in fact, as observed by many researchers, microwave heating often leads to the creation of hot spots in several product zones, depending on its geometry, thickness and dielectric properties, which are in turn dependent on the moisture content, and starting temperature of the food (ho and yam, 1992; buffler, 1992; zhou et al., 1995; campanone and zaritzky, 2005; vadivambal and jayas, 2010). the increase in temperature was progressive, even if not proportional, to the increase in the total energy sup­ plied to the product as expected by the treatment. consequently, the results of the ccd showed that the more the microwave energy, the more the damage to the fresh product, leading to a worsening of the organoleptic quality of ready­to­eat table grapes for energies above 8000 kj, both in the presence and absence of b. cinerea inoculum (106 log cfu g­1). however, the chemical and physical parameters of table 2 ­ minimum, maximum, and average temperature for samples treated as described by the experimental plan of the ccd fig. 1 ­ example of temperature distribution on the bunch after microwave heating. run maximum temperature (°c) minimum temperature (°c) average temperature (°c) initial t 20.3 15.2 17.5 1 29.7 20.4 22.6 2 47.3 20.9 23.3 3 65.9 22.9 37.7 4 68.7 21.7 30.9 5 68.0 23.1 37.7 6 22.2 17.4 19.1 7 42.5 19.9 24.3 8 70.8 21.3 29.5 9 81.1 23.2 44.7 10 60.5 21.1 27.3 de chiara et al. ‐ microwave effect on table grape quality 37 uninoculated samples, did not show significant differ­ ences (data not shown), for this reason the following best treatments from a sensory point of view, as reported in figure 2, were selected for the subse­ quent packaging trials: 14 s/100 w and 80 s/100 w, respectively, treatment 1 and 10. moreover, this lat­ ter evidence was associated with a slight reduction of the total mesophilic bacterial load of the selected samples treated with combination 1 and 10 (14/100 and 80/100), equal to 0.2 log and 0.6 log respectively, compared to the untreated sample, even if not statis­ tically significant (data not shown). the selected treatments allowed to maintain a high visual quality score, above the limit of marketability, up to 14 days of storage. it is possible to observe that treatments 3, 5 and 9 caused instead a severe deterioration of table grape appearance, resulting not to be suitably applied on fresh product. there is a great lack of existing literature regarding the application of microwaves on grapes intended for fresh consump­ tion, therefore these preliminary data were used as starting point for the next experiment, and it is diffi­ cult to compare them with other data for the same product. however, similar results were observed for different products such as fresh cut carrots and apples as reported by martínez­hernández et al. (2016) and colelli et al. (2021). authors stated that high energy treatments could detrimentally affect quality of fresh product. for this reason, it became crucial to carry out the reported preliminary screen­ ing, preferably based on a statistical approach such as that of the ccd which allows to test a large num­ ber of treatments at different energies to obtain a complete picture of the effects of microwaving on the product. as for the second trial, table 3 shows the results of the two­way analysis of variance, pointing out how storage time significantly influenced all the evaluated parameters while microwave intensity, combined or not with packaging, affected the sensory aspects described as visual appearance of bunches, berries and rachis, and some of the physico­chemical para­ meters. moreover, the interaction between the two factors influenced several quality aspects of table grape subjected to microwave heating and stored up to two weeks. on the other hand, however, as described below, the significant effects of the treat­ ment on the qualitative aspects of the product are lost during storage and, for most of the evaluated table 3 ­ effect of microwave treatment, storage time and their interaction on physico­chemical, sensory and microbiological attributes of table grape stored up to 14 days mean values ± standard deviations of 9 samples are reported (3 replicates x 3 storage times). ****= p≤0.0001; *** = p≤0.001; **= p≤0.01; *= p≤0.05; ns, not significant. lowmw = 14 s/ 100 w; highmw = 80 s/100 w; pp= polypropylene bags. fig. 2 ­ visual quality score evolution during storage time for uninoculated table grape bunches treated as described in table 1. control lowmw lowmw pp highmw highmw pp treatment (a) storage time (b) interaction (a x b) total mesophilic load (logcfu g-1) 3.05±0.57 3.02±0.48 3.02±0.56 3.16±0.30 2.97±0.29 ns **** ns yeasts and molds (logcfu g-1) 3.25±0.36 b 3.32±0.23 b 3.40±0.35 ab 3.55±0.24a 3.23±0.41 b ** **** * ascorbic acid (mg 100g-1) 0.62±0.39 0.58±0.22 0.48±0.35 0.62±0.43 0.72±0.39 ns **** ns dehydroascorbic acid (mg 100 g-1) 2.04±0.32 1.95±0.17 1.74±0.24 1.98±0.20 1.75±0.41 * * * vitamin c (mg 100g-1) 2.66±0.64 2.54±0.32 2.23±0.47 2.60±0.50 2.48±0.73 ns **** ns o 2 (kpa) 11.02±8.38 10.14±8.31 10.40±8.52 10.62±7.95 10.83±7.98 ns **** *** co 2 (kpa) 6.00±4.79 bc 6.72±5.07 a 6.65±5.39 ab 6.60±5.08 abc 5.95±4.56c ** **** *** bunch appearance (score) 4.17±0.48 b 4.50±0.43 a 4.50±0.41 a 4.50±0.28 a 4.33±0.57 ab ** **** *** berries appearance (score) 4.08±0.61 b 4.58±0.47 a 4.58±0.33 a 4.58±0.35 a 4.25±0.48 b *** **** ** rachis appearance (score) 4.42±0.56 ab 4.50±0.43 a 4.42±0.35 ab 4.50±0.43 a 4.33±0.66 b ** **** **** firmness (n) 0.47±0.11 ab 0.57±0.17 a 0.47±0.09 b 0.50±0.12 ab 0.45±0.05 b * **** ** 38 adv. hort. sci., 2023 37(1): 33­40 parameters, the differences resulted to be not signifi­ cant after 14 days. the most interesting results were reached in terms of maintenance of the sensorial quality of the fresh product. specifically, all the samples, including not treated one, were characterized by a very high sensorial quality even after two weeks of storage within passive modified atmosphere packaging. however, highmw sample, treated at 100 w for 80 sec and then packed, showed the highest rating due to the very fresh­like appearance of its berries and globally, its bunches, at the end of storage time. it is widely recognized that ready­to­eat and fresh­cut products should be visually free from defects, clean, with no presence of soil or off odor up to the end of the storage time, moreover, the entire bag content should be edible without no further requirement b e f o r e c o n s u m p ti o n ( b a r r e tt e t a l . , 2 0 1 0 ) . consequently, visual appearance of fresh table grapes and fresh product in general, represents the first aspect influencing the consumers decision, and size, color, visual quality, and external appearance in general are used to describe it (musacchi and serra, 2018). in this context, it was observed in the present work that microwave treatment was able to better maintain the visual appearance of table grape during storage when packed and stored as ready­to­eat product, representing a starting point for subsequent applications and optimization of this technique on fresh produce. from a microbiological point of view, no statistically differences were observed between samples at the end of storage time, neither due to the different intensity of the treatments nor to the presence/absence of the packaging film during microwave treatment. in figure 3 it is possible to observe how, concerning mesophilic microorganisms, a lower load, even if not significant, was maintained after 14 days of storage for all the treated samples, compared to untreated, reaching the latter the high­ est value equal to 3.59 log cfu g­1. final values for lowmw, lowmw pp, highmw, highmw pp were 3.29, 3.42, 3.34 and 3.06, respectively, with highmw pp sample showing the highest difference compared to control (0.54 log cfu g­1). low efficacy of the treat­ ment in terms of reduction of the microbial content can be related to non­uniform heating process, thus leading to the incomplete action on to the microor­ ganisms due to uneven distribution of temperature (vadivambal and jayas, 2010). little literature exist­ ing on ready­to­eat samples in order to compare results. colelli et al. (2021) observed that 35 s/300 w on fresh­cut apples allowed to reach a 2­log reduc­ tion in mesophilic load at the end of storage time, however, the higher microwave intensity resulted in the appearance of side effects on nutritional quality. otherwise, as reported by martínez­hernández et al. (2016) 60 s/900 w microwave treatment applied on fresh­cut carrots led to an initial microbial reduction, followed by an increment during storage mainly due to the detrimental effect on plant tissue caused by an excessive temperature increase. as demonstrated by the present experiment, using a well­modulated microwave energy, allowed at least to maintain a good visual quality of ready­to­eat table grape (fig. 4), without significant reduction of nutritive com­ pounds and firmness. moreover, it was not possible to correlate the differences in the visual appearance with the different gaseous concentration within the plastic bags, in fact about 3 kpa of oxygen and 11 kpa of co2 were reached at the end of storage time for all the samples. this probably contribute to the mainte­ nance of grape quality, also cefola and pace (2016) reported the beneficial effect of high co2 concentra­ fig. 3 ­ total mesophilic and yeasts and moulds loads evolution over time on table grape samples untreated (ctrl) and subjected to low (14 s/100 w) and high (80 s/100 w) microwave treatment before (lowmw, highmw) and after polypropylene packaging application (lowmw pp, highmw pp). means with different letters at the same time of storage are significantly different according to tukey’s test (p≤0.05). de chiara et al. ‐ microwave effect on table grape quality 39 tion during storage on ‘italia’ table grapes, both in terms of sensory quality preservation and decay con­ trol. it is therefore possible to state that the treat­ ments, at the applied intensities, did not influence the respiratory rate of table grape and consequently there are no differences concerning metabolic activi­ ty and physiological aging. as for ascorbic and dehy­ droascorbic acid, it was observed a physiologically slightly decreasing trend, without however differ­ ences due to the intensity of the treatment (data not shown). similar results were also observed for differ­ ent table grape varieties when stored as minimally processed products (nicolosi et al., 2018). 4. conclusions at the end of the storage period, sample subject­ ed to 100 w microwave power for a treatment time of 80 seconds and subsequently packed, showed a better external appearance than the other treatment and the control samples, however maintaining an intermediate level of mesophilic bacterial load. no significant differences in terms of nutritional quality were observed. the time/power combination identi­ fied with this preliminary experiment and its combi­ nation as hurdle technology with packaging can rep­ resent a valuable starting point for further experi­ ments aiming at identifying a mild microwave treat­ ment to be applied to improve table grapes quality and safety. their combination with other treatments aimed to maximize the antifungal activity should be better investigated. acknowledgements regional project “research for innovation”, fun­ ded by “por puglia fesr fse 2014­ 2020 ­ azione 10.4 ­ interventi volti a promuovere la ricerca e per l’istruzione universitaria”. references ahmed s., roberto s., domingues a., shahab m., junior o., sumida c., de souza, r., 2018 ­ effects of different sulfur dioxide pads on botrytis mold in ‘italia’ table grapes under cold storage. ­ horticulturae, 4: 29. barrett d.m., beaulieu j.c., shewfelt r., 2010 ­ color, flavor, texture, and nutritional quality of fresh‐cut fruits and vegetables: desirable levels, instrumental and sen‐ sory measurement, and the effects of processing. ­ crit. rev. food sci. nutr., 50(5): 369­389. buffler c.r., 1992 ­ microwave cooking and processing. engineering fundamentals for the food scientist. ­ springer new york, ny, usa, pp. 14­83. campanone l.a., zaritzky n.e., 2005 ­ mathematical analysis of microwave heating process. ­ j. food eng., 69(3): 359­368. capozzi v., fiocco d., amodio m.l., gallone a., spano g., 2009 ­ bacterial stressors in minimally pro‐ cessed food. ­ int. j. mol. sci., 10: 3076­3105. cefola m., pace b., 2016 ­ high co2‐modified atmosphere to preserve sensory and nutritional quality of organic table grape (cv. ‘italia’) during storage and shelf‐life. ­ eur. j. hortic. sci., 81: 197­203. colelli g., amodio m.l., de chiara m.l.v., 2021 ­ o p e r a ti n g c o n d i ti o n s f o r m i c r o w a v e a p p l i c a ti o n throughout production process to reduce microbial load of fresh‐cut apples. ­ acta horticulturae, 1319: 223­ 230. dar a.h., shams r., rizvi q.u.e.h., majid i., 2020 ­ microwave and ohmic heating of fresh cut fruits and vegetable products, pp. 295­337. ­ in: siddiqui m.w. (ed.) in fresh‐cut fruits and vegetables: technologies fig. 4 ­ bunches and berries sensorially evaluated score over storage time for table grape samples untreated (ctrl) and subjected to low (14 s/100 w) and high (80 s/100 w) microwave treatment before (lowmw, highmw) and after polypropylene packaging application (lowmw pp, highmw pp). means with different letters at the same time of storage are significantly different according to tukey’s test (p≤0.05). adv. hort. sci., 2023 37(1): 33­40 40 and mechanisms for safety control. academic press, london, uk, pp. 396. de simone n., pace b., grieco f., chimienti m., tyibili­ ka v., santoro v., capozzi v., colelli g., spano g., russo p., 2020 ­ botrytis cinerea and table grapes: a review of the main physical, chemical, and bio‐based control treatments in post‐harvest. ­ foods, 9: 1139. ho y.c., yam k.l., 1992 ­ effect of metal shielding on microwave heating uniformity of a cylindrical food model. ­ j. food process. preserv., 16(5): 337­359. karabulut o.a., baykal n., 2002 ­ evaluation of the use of microwave power for the control of postharvest dis‐ eases of peaches. ­ postharvest biol. technol., 26(2): 237­240. martínez­hernández g.b., amodio m.l., colelli g., 2016 ­ potential use of microwave treatment on fresh‐ cut carrots: physical, chemical and microbiological aspects. ­ j. sci. food agric., 96(6): 2063­2072. modesti m., shmulevitz r., brizzolara s., tonutti p., 2020 ­ short‐term low temperature treatments of har‐ vested wine grapes (cv. vermentino) affect the volatile organic ompound profile of the berries. ­ adv. hort. sci., 34(15): 27­33. m u s a c c h i s . , s e r r a s . , 2 0 1 8 ­ a p p l e f r u i t q u a l i t y : overview on pre‐harvest factors. ­ sci. hortic., 234: 409­430. nicolosi e., ferlito f., amenta m., russo t., rapisar­ da p., 2018 ­ changes in the quality and antioxidant components of minimally processed table grapes dur‐ ing storage. ‐ sci. hortic., 232: 175­183. sisquella m., viñas i., teixidó n., picouet p., usall j., 2013 ­ continuous microwave treatment to control postharvest brown rot in stone fruit. ­ postharvest biol. technol., 86: 1­7 song l., luo h., cheng x., yan f., yang z., yu z. 2018 ­ effects of microwave treatment on physiology and quality of minimally processed bok choy (brassica campestris l.) during storage at 5°c. ­ j. food meas. charact., 12(2): 913­922. vadivambal r., jayas d.s., 2010 ­ non‐uniform tempera‐ ture distribution during microwave heating of food materials ‐ a review. ­ food bioproc. tech., 3(2): 161­ 171. williamson b., tudzynski b., tudzynski p., van kan j.a.l., 2007 ­ botrytis cinerea: the cause of grey mould disease. ­ mol. plant pathol., 8: 561­580. zapata s., dufour j., 1992 ­ ascorbic, dehydroascorbic and isoascorbic acid simultaneous determinations by reverse phase ion interaction hplc. ­ j. food sci., 57(2): 506­511. zhang h.y., fu c.x., zhen x.d., xi y.f., jiang w., wang y.f., 2004 ­ control of postharvest rhizopus rot of peach by microwave treatment and yeast antagonist. ­ eur. food res. technol., 218(6): 568­572. zhang h.y., zheng x.d., su d.m., 2006 ­ postharvest con‐ trol of blue mold rot of pear by microwave treatment and cryptococcus laurentii. ‐ j. food eng., 77(3): 539­ 544. zhou l., puri v.m., anantheswaran r.c., 1995 ­ finite element modeling of heat and mass transfer in food materials during microwave heating ‐ model develop‐ ment and validation. ­ j. food eng., 25(4): 509­529. impaginato 90 adv. hort. sci., 2011 25(2): 90-98 received for publication 8 november 2011. accepted for publication 28 march 2011. the status of chestnut cultivation and utilization in the canary islands d. ríos-mesa*, s. pereira-lorenzo**, a.j. gonzález-díaz***, j.z. hernández-gonzález*, e. gonzález-díaz****, v. galán saúco**** * centro de conservación de la biodiversidad agrícola de tenerife (ccbat), servicio técnico de agricultura y desarrollo rural, cabildo, insular de tenerife. ** escuela politécnica superior, universidad de santiago de compostela. *** servizio de agricultura, cabildo insular de la palma. **** instituto canario de investigaciones agrarias (icia). key words: castanea sativa, food consumption, timber. abstract: chestnut was introduced to the canary islands at the beginning of the 16th century during the time of the spanish conquest. it was utilised by the conquerors as a means of claiming property for communal lands. from that time until today, chestnut has been an important crop in the canary islands. it is important both as a source of food and timber and has contributed to the subsistence of the population, particularly at times when both resources were scarce. nowadays it is mainly cultivated on the islands of tenerife (around 1300 ha) and la palma. on other islands, such as gran canaria, la gomera and el hierro, semi-wild chestnut forests and small plots where farmers collect fruit still exist. morphological and molecular marker (ssrs) studies have shown a great variability within the local population of chestnut trees in the canaries. the main use for chestnut is fruit consumption, but it was also utilised in the past as an exchange commodity to obtain fish and other food. the fruit is consumed in many different ways, mainly toasted or roasted, but also cooked in soups, fish or meat dishes and even as an ingredient for the typical canary islands’ sweet ‘morcillas’ (a type of sausage). the wood of the tree has been used for furniture, with some shoots being utilised for basket making, and also as cattle food. the trunk of the chestnut has also been used to obtain cork or as a bee hive. recent efforts to add value to chestnut cultivation in the canary islands have included the creation of a chestnut farmers association in tenerife that commercializes their products under a brand name. 1. historical background according to recent studies, chestnut was first introduced to the canary islands on el hierro and gomera and then on gran canaria, la palma and finally the island of tenerife, shortly after the process of conquest and colonization of the islands. in the case of la palma, it was most likely introduced around 1493 and quickly expanded and used both for its fruit and wood (ríos mesa, 2004). in fact, there is evidence that 5 ‘arrobas’ (a measure of weight, 1 arroba = 11.502 kg) of fruits were sent from la palma to the island of el hierro in 1546 (hernández-martín, 1999), which seems to indicate the presence of numerous chestnut trees to allow for surplus production. by 1590 frutuoso (2004) had mentioned the existence of chestnuts in the region of puntallana, and there are also many other reports about the presence of chestnut early after the conquest of the islands (hernández-rodríguez, 1983; browne, 2005). many ancient trees are still alive on la palma at present. two are especially important: one located in puntallana with circumference of more than 7 m, and another even larger one with a perimeter of 10 m in the municipality of breña alta. according to the information gathered from the local population, both of them seem to be of the denomination known as “temprano”, one of the few varietal denominations identical to those of spain’s mainland cultivars, which might be a clear indication of its early introduction. the great relevance of the chestnut tree on la palma is reflected in the information given by bandini (1816) in 1813, indicating that 46% of the total production of chestnuts in the canary islands (ci) came from that island. information provided by von fritsch (2006) on exports of chestnut fruits in 1862 from different areas of la palma also confirms the importance of this crop for the island. the role of this tree as part of the landscape of la palma was also highlighted in school texts used at the end of the 19th century (de las casas pestana, 1894). it is also important to mention that the first reference to grafting of chestnuts on la palma was 91 made during the last quarter of the 18th century (rodríguez-benítez, 2004). there are also historical references to the presence of chestnut trees on tenerife as early as 1517 (pereiralorenzo et al., 2007), soon after the conquest. many historical chestnut trees reported previously still survive on this island, such as the ‘castaño de las siete pernadas’, in the area of aguamansa (fig. 1), la orotava county (mendez pérez, 2002), which may be around 500 years old. there is an interesting description of this tree given by rodríguez (2001) in which he indicates that the trunk had a circumference of more than 12 m and that the seven branches of its trunk (siete pernadas) had been reduced to five by wind action. according to ríos-mesa (2004) it is possible that this tree was one of the chestnut trees planted by el adelantado (name given to the first governor of the island), d. alonso fernández de lugo, in the valley of la orotava in the first decades of the 16th century. many well known visitors and naturalists, among them mac-gregor (2005), verneau (2003), von humboldt (2005), du cane (1993), glass (1982), von fritsch (2006) and berthelot (2005), mentioned the presence of chestnut forests as part of the natural landscape of tenerife in their canary island trip reports during 18th and 19th centuries. on gran canaria: galdar, moya, san mateo, telde, teror. on la palma: barlovento, punta gorda, punta llana, san andres y sauces, santa cruz de la palma. on lanzarote: tinajo. on la gomera: vallehermoso. there are no updated figures on the economic importance of chestnut cultivation at a regional level at present. a thorough sampling done by pereira-lorenzo et al. (2001) indicated that chestnut could be introduced in the archipelago as a crop with a double use (fruit and wood) following the example of the spanish mainland from where it was introduced. this study also revealed the presence of isolated, testimonial chestnut trees on the drier eastern islands of lanzarote and fuerteventura. chestnut fruit is nowadays almost exclusively commercialized in a few areas on tenerife and la palma. recent efforts were made to increase the value of chestnut in the canary islands by the creation of a chestnut farmers association on tenerife to market all their products through a brand name. they even differentiate chestnuts harvested in the south one month earlier than those in the north due to microclimate differences, and which usually obtain the highest prices of the year. elorrieta (1949) indicated the presence of chestnut on all the western islands of the canaries but he did not report it as a crop. a recent detailed study carried out in 2006 and 2007 by hernández et al. (2010) localised chestnut trees in 28 counties on the island of tenerife, 23 of them with chestnut orchards. according to this study, the cultivated area of chestnut on tenerife is 1374 ha, with the biggest cultivated area (280 ha) located in la orotava county. in addition 2,567 isolated trees were also found on the island of tenerife. chestnut trees on tenerife are found between 400 and 1,800 m altitude in the southern part of the island (granadilla county). most of the area (647 ha) is located between 400 and 1000 m above sea level, and diminishes progressively after this altitude. only some isolated chestnut trees can be found below 400 m above sea level in the northern county of la laguna. chestnut distribution is different in north and south portions of tenerife. the higher rainfall and humidity on the north slopes due to trade winds, in contrast with a lack of the beneficial influence of them on the south side, explains why chestnut cultivation has been developed at higher altitudes in this latter area than on the north side (ríos-mesa, 2004; hernández et al., 2010). in northern tenerife the area covered by chestnut totals 1,121.72 ha (81.6% of total) and increases constantly from 400 m to 1,000 m above sea level, with a minimum area between 1,000 and 1,200 m. chestnut orchards are located mainly in the orotava valley and tacoronte-acentejo county. most of the 112 ha cultivated in the south are located in the guimar valley, more favoured by trade winds than other southern fig. 1 ancient chestnut tree with 7 main branches named castaño del las 7 pernada. 2. economic importance, cultivated area and geographical distribution chestnut seems to have been an important crop in the middle of the 19th century according to the great number of localities in the canary islands producing chestnuts by 1852 (de leon y falcón, 2005). on tenerife: arafo, candelaria, la matanza, la orotava, el rosario, santa ursula. 92 counties, between 800 and 1,000 m and only 5.6 ha are cultivated between 600 and 800 m. another 118 ha are located between 1,000 and 1,400 m altitude, most of them also in the guimar valley, being a merely testimonial presence of chestnuts at higher altitude (fig. 2). cultivation of chestnut on gran canaria was important years ago but is now only testimonial with isolated plots in the counties of arucas, firgas, valleseco and teror (naranjo-rodríguez and escobio-garcía, 2002). most chestnut trees on the island of el hierro are located in the most humid and cooler areas of el golfo, tiñor, asofa and honduras. chestnut trees are also found on la gomera in the higher parts of the valleys of the north where they are cultivated on mountain slopes. scarce, isolated trees on lanzarote are found in the area affected by the timanfaya volcanic eruptions (1730-1736) which may suggest a later introduction on this island and, as in the case of the other dry island of fuerteventura, a lack of appropriate climatic conditions for chestnut development (pereira-lorenzo et al., 2007). 3. horticultural aspects around 1852, de león y falcón (2005) indicated that chestnuts showed excellent growth on the ci at middle and higher altitudes in soils with a predominant clay, propagated either by seeds or from shoots emerging from their roots, with grafting seldom being practiced. in the case of tenerife, chestnuts are located in highly fertile acid soils, in most cases andisoles and alfisoles, and cultivated, whenever orography and planting density allow, together with other crops such as potato, rye, corn or others (ríos-mesa, 2004). only around 430 ha (31%) of the total surface covered by chestnut on the island of tenerife can be considered to be under good conditions of cultivation and 138 of these, distributed in different counties of the island, can be considered well managed. about 670 ha (48.7% of the total), concentrated mostly on the south side, are in a clear state of abandonment (hernández et al., 2010). in tenerife, extensive areas previously devoted to chestnut cultivation have been abandoned in recent years, although in many cases new trees have grown from seeds among the old grafted trees. these new plants are called chestnut machos or ladrones (males or thieves). the only cultivation practice carried out in these places consists of possible cleaning of the soil surface to facilitate harvesting of early chestnut fruits, perhaps being the only regular horticultural practice undertaken for chestnut cultivation in the different cultivated areas of the islands. plant spacing differs greatly from place to place but it is possible to consider three main systems (ríosmesa, 2004; hernandez et al., 2010). 1 trees planted at high density (30-50 trees/ha) in places where chestnuts were planted for fruit and that correspond with the areas now in a state of abandonment. 2 planted at lower density (15-30 trees/ha) allowfig. 2 chestnut trees in the south of tenerife. three different systems of chestnut cultivation can be found on tenerife. there is a first band at lower altitudes with low density plantings where chestnuts are associated with mixed plantings of vegetables and other fruit trees; a second band with more dense plantings where the lack of light penetration does not allow association with other crops; and the last band occurs at higher altitudes where chestnuts are largely associated with endemic fayal-brezal (myrica faya and erica arborea) or even pinus (pinus canariensis) populations that, in many instances, constitute a part of the native forest and cannot be considered cultivated trees (ríosmesa, 2004). chestnut is more uniformly distributed on the island of la palma with the maximum concentration of chestnut trees being on the west side of the cumbre nueva area in santa cruz de la palma and breña alta counties, where there is more humidity, also in this case, due to an influence of trade wind; on the north and east sides of the island chestnut is more important at middle altitudes. on the drier south and west sides of the island, chestnut as well as other temperate fruit crops is less present and located at higher altitudes. as in the case of tenerife, chestnut is associated with the native vegetation at higher elevations, with laurisilva and fayal-brezal in the north and east and pine forest in the west. frequent fires, the most serious of which occurred in august 2009, have caused the death of many chestnut trees or, on occasion, destruction of the grafted portion of the tree and regrowth of the rootstock (pereira-lorenzo et al., 2007). 93 ing intercropping with associated crops. 3 chestnut trees (10-15 trees/ha) planted only along the edges of plots used for cultivation of other crops such as potatoes, grapes, corn, cabbage, lupines or other vegetables (figs. 3 and. 4). all chestnuts are cultivated without irrigation and no fertilisers are applied, but in cases 2 and 3 above they benefit from the fertilisers applied to the main associated crop. grafting on tenerife (fig. 5) was done in a similar way to that on the spanish mainland, where seedling rootstocks were grafted with two scions placed at 0.5 m height. however peculiarly shaped shrubby chestnuts found in the volcanic south of the island with large pendulous branches touching the soil indicate that this practice was not always carried out. maintenance pruning is seldom practiced except in the case of planting system 3 indicated above. even in this case pruning is scarce and rudimentary, mostly limited to the removal of dead wood or wood that fig. 3 chestnut-corn association. fig. 4 chestnut-grape association. fig. 5 scionwood for grafting. makes the cultivation of the rest of the plot more difficult. there is, however, an important exception in the case of the trees cultivated on volcanic lapilli soils in the counties of arafo (tenerife) and el paso (la palma). in both places, grafting is practiced closer to the soil with a training process orientated at keeping branches as close to the soil as possible to facilitate harvest and to obtain wind protection. trees are also protected against wind on the island of el hierro where a circular wall of rocks, locally called gorona, is built to protect the tree from wind and animals. chestnuts on the ci are in good phytosanitary condition, with no reports of the serious diseases which usually affect this plant on the spanish mainland, such as phytophthora cambivora and phytophthora cinnamomi. this is most likely due to the isolation of the islands from other chestnut areas and no introduction of new plant material for new plantings. several arthropods, such as cydia splendana and balaninus elephas that attack chestnut fruits in the archipelago, are being studied (pereira-lorenzo et al., 2007). 4. plant material efforts made to localise and identify the plant material of chestnuts on the canary islands have only 94 recently been made through extensive samplings and collecting of material during field visits to interview farmers in the producing areas: on tenerife this was carried out between 2000 and 2004; on la palma between 2001 and 2004; on el hierro in 2001; and on la gomera and gran canaria in 2003. the main objectives of these surveys were to finalise the chestnut cultivar spanish inventory, realise the morphological and molecular characterization of ci chestnut plant material, to propose the material to be kept in a germplasm bank and to define the most interesting cultivars for commercial cultivation (pereira-lorenzo et al., 2007). a total of 47 different varietal nominations, according to the names given by farmers, with one or several accessions, were found on the ci (21 on tenerife, 17 on la palma, six on gran canaria, two on el hierro and one on la gomera) coinciding on many occasions with the same names on different islands like ‘blanco’ (white) on la palma and el hierro, ‘mollar’ (freestone) on tenerife, la palma, gran canaria, gomera and el hierro, and ‘negro’ (white), ‘picudo’ (pointed), ‘manso’ (tame) and ‘temprano’ (early) on tenerife and la palma. some new plant materials have been found in recent investigations carried out by the same authors (unpublished data): ‘piñuda’ (with the shape of a pine-cone), ‘menuda’ (small) and ‘merina’ (as the sheep race) on tenerife; ‘colorado’ (red) and ‘arrancado’ (pulled out) on la palma; and ‘chabetudo’ (unknown meaning) on gran canaria. the 39 accessions found on tenerife correspond to 21 varietal nominations (fig. 6), ‘mulato’ (mulatto) with seven accessions being the most common. the 34 accessions of la palma correspond to 17 varietal nominations, ‘jabudo’ (unknown meaning) with six accessions being the most common. from all the varietal nominations of these two islands, only ‘redondo’ (round) and ‘temprano’ coincided with names of cultivars from the spanish mainland, the former in galicia and the latter in andalucía and extremadura. only the plant material from tenerife and la palma has been characterised morphologically and phonologically, using the same methodology as for previous studies of the cultivars of galicia on mainland spain and allowing a comparison among them (pereiralorenzo et al., 1996 a, b, c; pereira-lorenzo and fernández-lópez, 1997 b, c; pereira-lorenzo et al., 2006). analogous molecular characterizations of plant material from tenerife and la palma have also been made following the same methodology as for the cultivars of galicia, comprising the results obtained from 10 microsatellites being utilised in the research project “evaluation, analyses and biodiversity management of castanea sativa mill. (european chestnut) in the atlantic regions (castaneareg)”, interreg iiib, espacio atlantico, feder, 2004-2006. to further allow comparison among cultivars, the four most discriminating morphological characteristics previously studied for the cultivars of galicia (pereiralorenzo, 1994 and pereira-lorenzo et al., 1996 a), favourably tested later for cultivars of different origin (pereira-lorenzo et al., 2006 and 2007) and also utilised under the framework of this same project, have been employed: i) fruit size; ii) fruit shape; iii) type of male flowering; and iv) length of burr spines. from the results obtained through this molecular characterization, it has been possible to group the 74 accessions of tenerife and la palma into 57 different genotypes. it is worth mentioning that, as in the case of the cultivars of galicia (pereira-lorenzo et al., 1996 b), andalucia (pereira-lorenzo and ramos-cabrer, 2003), león (ramos-cabrer et al., 2003) and asturias (pereira-lorenzo et al., 2005; 2006), the cultivars from the ci are all polyclones. the few cases in which more that one genotype was found under the same name seem to indicate that more than one clone of a cultivar might have been propagated. most of the genotypes found were singulars and only 10 groups of coincidence by microsatellites have been detected. when the ci genotypes were compared with those of the spanish mainland (data unpublished), no synonyms were found. all the cases of varietal denominations with only one accession found in the ci have been found to be of a single genotype different from all the others (pereira-lorenzo et al., 2007). 5. tradition and uses chestnuts were traditionally used as an important source of food in times of scarcity and also for wood of important value (pereira-lorenzo et al., 2007). among the different uses of this plant on tenerife, ríos-mesa (2004) specifies the following: the fruits larger ones used for fresh food, for family consumption, exchange or sale, the smaller ones as cattlefig. 6 fruits of castagrande cultivar. 95 feed. chestnut fruits are important for traditional cooking on the islands. they are consumed either boiled or roasted, or even fried in oil alone or accompanying salty fish (a very traditional dish on the islands), in purees or, more recently, in desserts. the wood used for house building, furniture, wine presses, staves for wine barrels and for the bottom of boats, it is also used for making boxes to store various local units of weight, cuartillas, and sowing quantities, almudes (fig. 7). the wood has also served to build small tools such as those used to blow cereals or to fan the grain. ungrafted trees have been preferred, generally, for wood purposes as they are straighter and present fewer nodes than grafted plants. it should be mentioned that the wood has always been cut when the moon is in “its last quarter”. despite early introduction of the tree to the ci chestnut wood was not used, at least on tenerife, for building wine barrels until the middle of the 17th century, as there are reports of imports of wood for that purpose from galicia and portugal (méndez pérez, 2002). chestnut suckers were also utilised to make baskets (fig. 8) of different sizes, such as hand baskets or bigger ones for transporting rocks and even those known as raposas placed on the sides of donkeys to carry various items. suckers also served to make horquetas, long wood sticks ending in a ‘v’ shape which first served to raise and hold the grapevines away from the soil and later in the 20th century for banana cultivation, both to keep the bunch separated from the pseudostem (small horquetas) or to give support to the whole plant in case of wind (bigger horquetas called horquetones). the dry leaves have been used as cattle beds and the green ones as cattle food. ríos-mesa (2004) indicates that chestnut is closely related to cultural aspects of ci life. chestnuts are an important element of the gastronomy linked to popular celebrations. special mention should be given to: the “all the saints feast” when the early chestnut fruits are roasted and consumed together with the “aguapié” (the first liquid extracted from the grapes after the men performed the traditional stomping of the grapes). the “saint andrews feast”, when chestnuts are eaten together with the new wine of the year when the cellars are first opened. at christmas time when chestnuts are considered important christmas symbols, like the spanish turrones (hard almond cakes). it is tradition in some villages to give a small basket full of chestnuts to children as a present. during carnival in some localities in the north of tenerife dry chestnuts are also consumed accompanying wine. for the “saint anthony feast” in the county of acentejo, in the north of tenerife, it is customary to use chestnuts as rosaries to be placed over domestic animals. chestnut wood has been used on the island of la palma for wine barrels and also for making receptacles to export fruits preserved in syrup, made with the sugar coming from the sugar cane industry, or to keep salty meat. they were also used as support poles for grape cultivation (pereira-lorenzo et al., 2007). peeled chestnut wood skin bands were used for building the structure and handles of baskets. several types of baskets, including bread baskets are also made, like those on tenerife (santos-cabrera, 2002). the wood is also appreciated for smoking cheese, a very traditional and delicious dish of the gastronomy of this island. the tender young shoots coming from the main trunks have been traditionally used as cattle feed. unifloral chestnut honey is produced in tenerife and la palma where sometimes bee hives are made of chestnut wood. in the past chestnuts were preserved fig. 7 box for sowing the grain made of chestnut. fig. 8 traditional harvesting basket made with chestnut wood. 96 buried in volcanic lapilli, but it was more common to cover them with sand or to dry them in the sun (pereiralorenzo et al., 2007). it is worth mentioning the historical use of chestnut trees as a means of claiming property for communal lands. it is indicated by rodríguez-brito (1982) that the planting of a chestnut tree in a deforested plot constituted the first step toward claiming it. the “feast of saint martín” (11 november) is the day chosen to drink the new wine and on tenerife it is accompanied by the consumption of chestnut fruits. the night before this feast, children dragged empty oil tins, “cacharros”, in the air which are open at the top and filled with chestnuts and coals. with an evident sense of humour, the old people’s association in the village of san juan de puntallana organise a contest a few days after “saint martín”, coinciding with the time of the chestnut harvest, to nominate the queen of chestnuts, the big chestnut, the small chestnut and the nice chestnut. women participate in the contest by bringing chestnuts collected by themselves or by their husbands. the woman that presents the biggest one is nominated as the queen of chestnuts, and similarly for the smaller or nicer fruits. in addition, the chestnut has erotic relevance on this island as it is used to refer to the female sexual organ (pereira-lorenzo et al., 2007). on the island of el hierro chestnuts have lost their traditional importance as food during autumn and winter except at the “feast of all saints”, when chestnut fruits are used as tafeñas (local chestnut grill) and consumed with new wine. in the past chestnuts were dried in the sun and used as ingredients for the preparation of “morcillas” (blood sausages), a local product still prepared on other islands. these morcillas were used by the inhabitants of the “el golfo” county as exchange for dry figs or potatoes coming from other counties of the island (gil-gonzález 1998). the use of chestnuts on the island of la gomera must have been similar to that of other islands, but in addition chestnut wood is used for making chácaras, a musical percussion instrument similar to the spanish castañuelas (perera-lópez, 2005) (note that chestnut in spanish is castaño.) as indicated above, the consumption of chestnut fruits is very common on the ci where they have been an important source of calories since soon after the spanish conquest of the islands. they are not normally consumed raw, except occasionally when they are very soft, but generally boiled or roasted (fig. 9). boiling is generally done after making a cut in the external shell or after eliminating it. they are cooked for half an hour in plenty of water to which anise, vanilla sticks or cinnamon is added, particularly when used for desserts (iglesias garcía, 2004). they are usually roasted in homes by putting them (after making a cut in the external shell) in a perforated biscuit tin which is then put directly on the kitchen fire. chestnuts are traditionally cooked in small metal boilers placed above charcoal fires on many street corners. in both cases, coarse salt is added during cooking. while roasted chestnuts are directly consumed, boiled chestnuts may be incorporated into typical dishes of the canary island gastronomy, such as roasted sardines, meat compositions or salty fish dressed with oil and vinegar or with the typical “mojo” (a sauce made with capsicum annun) to which they give a sweeter taste. they are also smashed into a puree, either to accompany a dish or as a filling for dishes prepared in the oven mixed with dry fruits (crushed pine nuts or almonds), spices and herbs or even previously fried giblets, adding a special flavour. in addition, they are a typical side dish for wild game (iglesias garcía, 2004), or as an ingredient in sweet ci bloody sausages. chestnuts may be consumed in a broth in the typical “caldo de castañas” (chestnut soup), cooked with potatoes or sweet potatoes, offering consistency and nutritive value which is much appreciated on cold winter days (ríos-mesa, 2004; pereira-lorenzo et al., 2007). fig. 9 traditional chestnut roaster. 97 chestnuts boiled with vanilla sticks are mixed, after removing all the skin, with syrup and eaten or ground as a filling for various sweets and cakes. one of the older recipes for these fillings includes ground chestnuts in addition to sugar, milk and beaten egg yolks. the mixture is cooked on a very slow fire for a few minutes, without allowing it boil. chestnuts are also used as ingredients in stewed fruit preserves. for this use chestnuts are boiled in abundant water, well cleaned to eliminate any remaining skin and then plenty of sugar is added along with anise, mint or hortelan (menta spitaca) and a glass of orange liquor or brandy. when the chestnuts become soft and thick, a syrup is formed; they are kept well covered in the syrup in sealed jars in a dry place (iglesias, 2004). 6. future prospects technical meetings have been organised since 2003 at la matanza municipality on the island of tenerife with the assistance of the centre for conservation of crop biodiversity of tenerife (ccbat) and the extension service. lectures by national and international experts have been given to include horticultural aspects such as pruning and training, marketing and preparation of chestnut products as well as economical and structural aspects and many other subjects dealing with chestnut cultivation on tenerife. special mention should be made of the process of recovery and adding of value to chestnut cultivation initiated in 2004 by the cabildo (local government) of the island of tenerife. this project was developed by the extension service and the ccbat have initiated “ex situ” and “in situ” conservation programmes and aims to create, in connection with the chestnut farmers association of tenerife, a quality label for chestnuts produced on tenerife. this initiative, if continued and extended to other islands, may make a great contribution toward the recovery of commercial cultivation of chestnuts on the canary islands. references 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canarias. 6ª edición. edición j.a. delgado luis y excmo. ayuntamiento de la orotava. la orotava. santa cruz de tenerife, spain. viera y clavijo j., 2004 diccionario de historia natural de las islas canarias. nivaria ediciones. tenerife, spain. von fritsch k., 2006 las islas canarias, cuadros de viaje. taller de historia, 40. von humboldt a., 2005 viaje a las regiones equinocciales del nuevo mundo y otros escritos. nivaria ediciones, santa cruz de tenerife, spain. impaginato 123 adv. hort. sci., 2023 37(1): 123­132 doi: 10.36253/ahsc­13899 sustaining low­impact practices in hor­ ticulture through non­destructive approach to provide more information on fresh produce history and quality: the sus&low project m. amodio 1, g. attolico 2, l. bonelli 3, m. cefola 4, h. fazayeli 1, f.f. montesano 3, 5, b. pace 4, m. palumbo 1, 4, f. serio 3, a. stasi 1, g. colelli 1 (*) 1 department of science of agriculture, food and environment, university of foggia, via napoli, 25, 71122 foggia, italy. 2 institute on intelligent industrial systems and technologies for advanced manufacturing, national research council (cnr), via g. amendola, 122/o, 70126 bari, italy. 3 institute of sciences of food production, national research council (cnr), via giovanni amendola, 122/o, 70125 bari, italy. 4 institute of sciences of food production, national research council of italy (cnr), c/o cs‐dat, via michele protano, 71121 foggia, italy. 5 department of soil, plant and food sciences, university of bari, via aldo moro, 70126 bari, italy. key words: marketing strategies sustainability, non­destructive assessment, quality, shelf­life. abstract: the general aim of the project sus&low is to increase the sustainabil­ ity of fresh produce by testing and implementing low­input agricultural practices (lip) with positive impact on product quality with the support of non­destructive (nd) tools for real­time quality assessment and for product discrimination. additionally, new marketing strategies are generated to better support the added value of the products and to satisfy the final consumers’ preferences. the sus&low project consists of three work packages (wp) and the adopted methodology used two model crops: rocket salad and tomato. the wp1, focused on the reduction of agricultural inputs, showed that sensor­based fertigation management might improve sustainability of soilless cultivation. results coming from wp2, aimed to the evaluation of nd techniques, outlined the high poten­ tiality of hyperspectral imaging (hsi) and fourier transformed­near infrared (ft­ nir) techniques for the authentication of sustainable growing methods. moreover, project activities’ proved computer vision system (cvs) as an effec­ tive tool for evaluating the product quality also through the bag. the wp3, deal­ ing with marketing strategies, indicated a positive approach of consumers com­ pared to lip products certified through a visual storytelling platform. 1. introduction production of vegetable crops under controlled environments (i.e. greenhouses) has expanded considerably over recent decades in (*) corresponding author: giancarlo.colelli@unifg.it citation: amodio m., attolico g., bonelli l., cefola m., fazayeli h., montesano f.f., pace b., palumbo m., serio f., stasi a., colelli g., 2023 ­ sustaining low‐impact practices in horticul‐ ture through non‐destructive approach to provide more information on fresh produce history and quality: the sus&low project. ­ adv. hort. sci., 37(1): 123­132. copyright: © 2023 amodio m., attolico g., bonelli l., cefola m., fazayeli h., montesano f.f., pace b., palumbo m., serio f., stasi a., colelli g. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 26 october 2022 accepted for publication 10 january 2023 ahs advances in horticultural science https://doi.org/10.36253/ahsc-13899 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2023 37(1): 123­132 124 mediterranean areas (fao, 2013). initially, research efforts and the related introduction of technical inno­ vations focused on high­quality, healthy products. however, concern with environmentally­sustainable production has risen in the last decade as industrial greenhouse crops are usually seen as entailing high environmental impact (torrellas et al., 2012). on the other hand, there is also plenty of evidence that greenhouse vegetable production may decrease the environmental impact compared to the field cultiva­ tion (stanghellini, 2014). the efficient use of resources (water and fertiliz­ ers), in irrigated greenhouse agriculture, is a promis­ ing and increasingly adopted strategy to achieve bet­ ter crop performance, improved nutritional and sen­ sorial quality (montesano et al., 2015; montesano et al., 2018). with respect to traditional systems, soil­ less cultivation and, particularly, closed­cycle with recycling of nutrient solution (ns) produce a number of benefits, including the possibility to standardize the production process, to improve plant growth and yield, and to obtain higher efficiency in water and nutrients use. in addition, it is also possible to modu­ late the regulation of the secondary metabolism of plants through an optimal control of the nutrient solution composition, or by imposing controlled stresses, or through biofortification treatments, gen­ erally leading to an improvement in the nutritional value of products (rouphael and kyriacon, 2018; renna et al., 2022). innovative technologies based on the use of sensor networks for fertigation manage­ ment may considerably reduce water and fertilizers consumption and increase the overall use efficiency of those inputs, and may lead to qualitative and quantitative improvements while preventing both under­ and over­irrigation. the most used instrumental techniques to mea­ sure quality attributes of fruits and vegetables are destructive and involve a considerable amount of manual work, primarily due to sample preparation. in addition, most of these analytical techniques are time consuming and sometimes may require sophisti­ cated equipment. finally, they can be performed only on a limited number of specimens (samples) and therefore their statistical relevance may be limited (amodio et al., 2017 a). research has been focused on developing non­contact, rapid, environmental­ friendly, and accurate methods for non­invasive eval­ uation of quality in fruits and vegetables. nowadays, there are a few emerging non­destructive analytical instruments and approaches for this task, including spectroscopy, hyperspectral imaging, and computer vision (liu et al., 2017). near infrared spectroscopy has gained wide atten­ tion in the food sector due to its capacity of providing fingerprints of different products on the base of the interaction between their molecular structure and the incident light (workman and shenk, 2004) which is the result of different pre­harvest factors that also affect the final composition and quality. the feasibili­ t y o f n i r s ­ b a s e d a n a l y s i s t o e v a l u a t e q u a l i t y attributes of fresh fruits for commercial application have been reported by numerous authors (arendse et al., 2018; palumbo et al., 2022 a). hyperspectral imaging (hsi) combines the princi­ ples of spectroscopy and conventional imaging or computer vision. it is mainly used for internal bruise and defect detection in fruits and vegetables (ariana and lu, 2010; babellahi et al., 2020; tsouvaltzis et al., 2020) but also to predict the internal composition (piazzolla et al., 2013; yang et al., 2015; liu et al., 2017; piazzolla et al., 2017). amodio et al. (2017 a) showed the potentiality of hyperspectral imaging in the vis­nir spectral range to predict internal content of soluble solids, phenols, and antioxidant activity of fennel heads. in addition, this technique provided important information about the maturity of fennel heads which may be used to determine the optimal harvest time. some studies successfully applied these methods for the discrimination of production origin and agricultural practices, as revised in amodio et al. (2020). nir and his were in fact used for the classifi­ cation of apples (guo et al., 2013), persimmon (khanmohammadi et al., 2014), and arabica coffee (bona et al., 2017) from different origins. as for pro­ duction systems (sánchez et al., 2013) investigated the potentiality of nirs technologies to discriminate green asparagus grown under organic and conven­ tional methods. more recently, amodio et al. (2017 b) successfully discriminated conventionally and organically grown strawberries, being also able to identify two different types of organic production systems applied to the same genetic material on the same site, soil, unheated tunnel. all these studies have suggested multispectral and hyperspectral systems as valid tools to evaluate qual­ ity of different agricultural products and, more inter­ estingly, as tools for product authentication. finally, computer vision systems (cvs) may be applied to extend quality prediction and discrimina­ tion along the whole supply chain from harvesting up to consumers. cvs combine mechanics, optical amodio et al. ‐ the sus&low project 125 instrumentation, electromagnetic sensing, and digital image processing technology (patel et al., 2012). recently, cvss have been used to assess quality and marketability of tomatoes (arias et al., 2000), arti­ chokes (amodio et al., 2011), fresh­cut nectarines (pace et al., 2011), fresh­cut lettuce (pace et al., 2014), fresh­cut radicchio (pace et al., 2015), and rocket leaves (cavallo et al., 2017). moreover, they have been applied for the prediction of internal qual­ ity of colored carrots (pace et al., 2013). even more interesting is the application of these systems during the post­packaging phase and along the whole distri­ bution chain. despite the relevance of quality evalua­ tion of packaged products, few investigations were reported in literature. multi­spectral reflective image analysis has been applied to monitor the evolution and spoilage of leafy spinach covered by plastic materials (lara et al., 2013); more recently, cavallo et al. (2018) have proposed an application of image analysis by cvs for non­destructive and contactless evaluation of quality of packaged fresh­cut lettuce. therefore, the interest of investigating the applica­ tion of cvs to detect quality and shelf­life of pack­ aged products. finally, the possibility of using non­destructive technique for increasing the information on product history (e.g. growing location and agricultural prac­ tices) may be considered as baseline to develop mar­ keting tools to promote the diffusion of sustainable production system. cost barrier is an obstacle for choosing low input products instead of the conven­ tional, even if environment is mentioned as a strong commitment (krystallis and chryssohoidis, 2005). therefore, the knowledge about consumer prefer­ ences for the adoption of lip is still matter of debate. the general aim of the project is to increase the amount of sustainably­produced food by testing and implementing low­input agricultural practices with positive impact on product quality with the support of non­destructive tools for real­time quality assess­ ment and product discrimination, which may inspire new marketing strategies to better support the added value of the products and increase incomes of potential users. 2. project activities and main results the sus&low project structure consists of three work packages (wp). wp1 focused on research activi­ ties aimed to reduce agricultural inputs (water and fertilizers) in greenhouse cultivation, chosen as a strategic high­value sector for mediterranean agricul­ ture. this wp was also in charge of making available to the project team vegetables products (rocket and tomato) different for the level of sustainability char­ acterizing the cropping system adopted, to be used in other wps for the related investigations. then, wp2 was aimed to the quality assessment and to the implementation of new tools to acquire information about quality and history of fresh produce obtained with lip (wp1). non­destructive methods (including nir, hyperspectral imaging and image analysis by cvs) have been used for food authentication, show­ ing interesting and promising results. finally, wp3 realized ad hoc survey to analyze the consumer behaviour with respect to the possibility of purchas­ ing fruit and vegetables lip certified (wp1) and iden­ tified by nd technologies (wp2) with the aim to implement adequate marketing strategies. in this section, an overview of the research strategies and approaches adopted in the three wps is provided. the main results are reported and discussed. wp1: quality crops through low‐impact practices based on the overall project structure, this wp was focused on soilless cultivation, since it has the potentiality to achieve extremely high water and fer­ tilizers use efficiency, beside high yield and quality, in intensive cropping systems. however, the adoption of free­drain open cycle with empiric fertigation schedule management operated by timers (the pre­ dominant case in mediterranean area), may compro­ mise the sustainability of soilless culture. therefore, the adoption of strategies aimed to rational use of water and fertilizers and excess leaching prevention is a key­factor for increased sustainability and reduced environmental impact of soilless culture (massa et al., 2020). in this context, substrate mois­ ture/ec (electrical conductivity) sensor­based irriga­ tion is a promising and increasingly adopted strategy to reduce water and fertilizers consumption and loss­ es, and to improve the overall crop performance, product quality and production process sustainability in soilless greenhouse cultivation (palumbo et al., 2021 a). several experiments were carried out at the experimental farm la noria (mola di bari, ba) of the cnr­ispa (bari), with the common approach to com­ pare treatments providing traditionally adopted empirical fertigation management techniques with treatments in which advanced sensor­based fertiga­ adv. hort. sci., 2023 37(1): 123­132 126 tion management was implemented. the main results of selected experiments carried out during the project are reported hereafter. the research activities focused on two model species [rocket salad (diplotaxis tenuifolia l.) and tomato (solanum lycopersicum l.) selected for their relevance in mediterranean greenhouse vegetable production. in particular, rocket is reported as an e m e r g i n g l e a f v e g e t a b l e w h i c h c u l ti v a ti o n i s widespread and in further expansion (schiattone et al., 2017), while tomato is the most important green­ house crop grown in soilless cultivation systems (montesano et al., 2015). a study was carried out to test two irrigation scheduling approaches (timer­ or sensor­based) and two fertilization levels (high or low, with reference to the standard dosage range recommended for the specific fertilizers used) of open­cycle soilless rocket in mediterranean autumn­winter unheated green­ house conditions (montesano et al., 2021). rocket plants (cv. dallas, isi sementi) were grown in a peat:perlite (3:1) mixture in 4.5 l plastic pots. four treatments were compared: timer with high or low fertilization (t­hf, t­lf), and sensor­based with high or low fertilization (s­hf, s­lf). in timer­based treat­ ments, irrigation schedule was periodically adjusted based on leaching fraction measurements (≈35% was set as a target, according to common practice). in sensor­based treatments, on­demand irrigation was operated based on substrate ec/temperature/mois­ ture sensors (gs3, decagon devices). these were connected to a cr1000 datalogger programmed to automatically open irrigation valves and supply water enough to constantly maintain volumetric water con­ tent to a pre­defined set­point (0.35 m3 m­3, close to maximum water holding capacity), with no leaching. slow­release fertilizers (osmocote exact and calmag, icl) were mixed with the substrate at high (3.75 and 1 g l­1, respectively) or low dosage (2.25 and 0.6 g l­ 1). yield, quality, water use and substrate parameters trends were evaluated. sensors improved water use efficiency compared to timer (34.4 vs 21.4 g fw l­1, on average) matching water supply with plant needs, and preventing leaching (fig. 1) (no interactive effects of fertilization treatments were observed on those parameters). sensor­based irrigation also pro­ vided the best plant growth conditions, with interest­ ing interactive effects with fertilization rate. in partic­ ular, the highest and the lowest cumulative (three harvests) yield values were obtained in s­hf and t­lf respectively (144.8 and 102.2 g fw pot­1), while simi­ lar values were observed in s­lf and t­hf (131.4 g fw pot­1, on average) (fig. 1). the partial fertilizer factor productivity (g product fresh weight / g fertiliz­ ers applied) was higher at low dosage, and, with the same dosage, when the sensors were used (fig. 1). after each harvest time the fresh­cut rocket leaves were immediately transported in refrigerate condi­ tions to the postharvest laboratory (see wp2 section below) (palumbo et al., 2021 b). in another set of studies, we aimed to apply approaches for the sustainable fertigation manage­ ment of soilless tomato (semi­closed cycle recircula­ tion; sensor­based nutrient solution supply manage­ ment) in comparison with a traditional open cycle free­drain nutrient solution management providing the use timer for fertigation schedule. experiments were conducted with different tomato types (cherry ­ cv. carminio, seminis­bayer, and intermediate type ­ cv. mose, syngenta), and in different environmental conditions typical of mediterranean areas (including the use of brackish water for nutrient solution prepa­ ration). in general, both approaches (semiclosed­ cycle cultivation and open cycle with sensor­based fertigation management) reduced the environmental impact of the production process (reduced water/fer­ fig. 1 ­ water use efficiency (wue), leaching rate, total yield, and partial fertilizer factor productivity of rocket (diplotaxis tenuifolia) grown in open free drain soilless system with timer­ (t) or sensor­based (s) irrigation man­ agement, and subjected to high (hf) or low (lf) fertiliza­ tion rate. amodio et al. ‐ the sus&low project 127 tilizers usage; less nutrient solution released into the environment, increased water use efficiency) and positively affected tomato quality traits, compared to empirically management open­free drain cultivation. wp2: non‐destructive discrimination for low‐impact practices and non‐destructive quality assessment nir spectroscopy and hiperspectral imaging. in this wp, the objective of the tasks was to assess the potentiality of fourier transformed­near infrared (ft­ nir) spectrometry and hyperspectral imaging (hsi) to discriminate tomatoes and rocket leaves produced with different level of input as described in wp1, tak­ ing also into account the degree of efficiency in water and fertilizers used efficiency (wue and fue indexes). a hyperspectral line­scan scanner (version 1.4, dv srl, padova, italy) equipped with two spectrographs, one in the vis­nir range, and the second in the nir range, was used to obtain the hs images. the vis­nir spec­ trograph (400­1000 nm) has a spatial resolution of 1000 × 2000 pixels with a spectral resolution of 5 nm and was connected to a ccd camera. as for the nir spectrograph (900­1700 nm), the spatial resolution was 600 × 320 pixels with a spectral resolution of 5 nm; and a cmos (specim spectral imaging ltd., oulu, finland) with 50 frames per second equipped with c­ mount lenses was used. as for ft­nir spectrometry an mpa multi­purpose (ft­nir analyzer, bruker optics, ettlingen, germany), was used during spectral acquisition over the range of 800­2777 nm (sphere macrosample re­solution 1.71 nm, scanner velocity 10 khz, sample scan time 64 scans, background scan time 64 scans). after image processing and spectra extraction for the hsi, all spectra belonging to hsi and ft­nir were tested in discrimination using the agronomic treatments as discriminant classes and partial least squares­discriminant analysis (pls­da) as classification technique. as for rocket leaves, pls­ da was conducted with the 4 classes (t­hf; t­lf; s­ hf, s­lf) described in the paragraph related to wp1, using 70 percent of samples for calibration purpose and the remaining 30% for the external validation. the model performance was evaluated based on the accuracy, which is an average of the sensitivity calcu­ lated over the various classes, and gives an overall idea of the goodness of the classification. results indicated hsi as a promising technique for the dis­ crimination of rocket produced with different cultural techniques, with an accuracy of classification in the prediction phase of 97.2% in vis­nir and 99.5% in nir range. in figure 2, the results of the discrimination models can be observed. regarding tomato, where 2 experiments with 2 different varieties were conducted (wp1), for each trial a first pls­da was aimed to discriminate the three treatments of cultivation and a second discrimi­ nation was performed for different levels of wue and fue. according to the efficiency of use of water and fertilizers we could individuate 2 levels (high and low) in each experiment and 3 levels (high, medium, and l o w ) m e r g i n g t h e d a t a o f b o t h e x p e r i m e n t s . therefore, a pls­da with 3 levels of wue (and fue) was also generated with the full dataset. among the different non­destructive techniques, ft­nir and his in the vis­nir range gave comparable performances in discriminating tomato according to cultural prac­ tices and different use of sources. discrimination for wue for each variety improved the classification results, respect to the individual treatments, but the highest accuracy was obtained when the discrimina­ tion was based on 3 levels of wue merging the 2 datasets, reaching 92.1%. in literature there are no studies aimed to discriminate crops for wue or fue, while we may find the application of hsi for the clas­ sification of water­stressed plants, as for the case of tomatoes (rinaldi et al., 2015). in comparison to this study, reporting a mean accuracy of around 77% for discrimination of the two differently irrigated areas, our findings showed higher accuracy, exploring new area of the application for these techniques. application of cvs for non‐destructive quality evalua‐ tion on packaged products a research activity was carried out to develop and validate an innovative cvs integrating a random forest model for classification: this model automati­ cally selects from the image the most relevant colour features for the task of interest. the developed cvs was applied to digital images of fresh­cut rocket fig. 2 ­ estimated class index values in the calibration and in the prediction process for the classification based on pls­da modes shown on table 2 in a) vnir range (left) b) nir range (right). f d d ld h 128 adv. hort. sci., 2023 37(1): 123­132 leaves cultivated with lip (wp1) to objectively esti­ mate the evolution of their quality levels (ql) during storage and to discriminate the cultivation approach applied on field. at harvest, rocket leaves were stored at 10°c in open polypropylene (pp) bags for a number of days required to reach the lowest ql, according to the rating scale from 5 (very good) to 1 (very poor), as reported in figure 3. at each ql, all the samples were subjected to postharvest quality evaluation, detecting colour parameters by a traditional colorimeter (cr400, konica minolta, osaka, japan) and physical and chem­ ical parameters, in detail respiration rate (kader, 2002), electrolyte leakage (kim et al., 2005) and total chlorophyll content (cefola and pace, 2015). then, images of the same samples were acquired by the cvs for non­destructive quality assessment and for recognizing traits related to the sustainability of the cultivation management used on the field, with spe­ cific reference to water and nutrients use (wp1). image pre­processing was applied: to separate the product from the background; to identify the colour­ chart placed in the scene to estimate the effects of lights and of the sensors and to correct colours to minimize these effects. three colour correction meth­ ods (white balance, linear correction, and polynomial correction) with increasing level of complexity were evaluated and compared in terms of consistency of colour measurements and of classification perfor­ mance. linear colour correction proved to be the best trade­off between efficacy and efficiency providing a slightly lower performance than polynomial correc­ tion with significantly simpler computation. finally, a random forest model was used to train classifiers to assess the ql of rocket leaves and to identify the treatments used during the cultivation. all the postharvest quality parameters measured by traditional destructive methods were significant in ql assessment of fresh­cut rocket leaves. the pro­ posed classifier based on the random forest model was able to identify and select the most relevant colour traits for both the tasks (ql assessment and treatment identification) without human interven­ tion. the accuracy achieved in evaluating qls of rock­ et leaves during storage was high (about 95%), while the performance in discriminating the cultivation approach was lower and not sufficient for practical applications (about 65­70%). indeed, the different cultivation approaches did not significantly affect the visual characteristics of the product and the destruc­ tive measures: this task needs further investigations. another research activity was carried out to develop and validate the capability of the non­destructive and contactless cvs to assess the visual quality changes during the cold storage of fresh­cut rocket leaves coming from soil and soilless growing systems (wp1) and to estimate some internal quality attributes (chlorophyll and ammonia content) also through the packaging material. evaluating quality through the package is critical to identify the regions of the bag where the product is visible without shadows or highlights created by illumination: this is mandatory to measure colour properties in a reliable and mean­ ingful way. at harvest, rocket leaves, cultivated on soil or soilless system (wp1), were packed in open pp bags and stored at 10°c for about 18 d. during stor­ age, all samples were observed to attribute the ql according to the rating scale reported in figure 3 and the postharvest quality traits were evaluated by destructive conventional methods [colour parame­ ters, chlorophyll content, ammonia content (fadda et al., 2016) and electrolyte leakage]. then, images of unpackaged and packaged samples were acquired by the cvs. during image acquisition, no constraints were imposed on the position of the product in the bag, on the position of the bag in the scene or on the highlights created by the illumination on the surface of the bag: this was necessary to demonstrate the applicability of this technology into a real industrial line. colour correction was performed by the linear model, identified as the best trade­off between effec­ tiveness and computational complexity in the previ­ ous research activity. packed and unpacked products were processed using exactly the same phases apart from the artefacts’ elimination step applied to the images of packaged products to select the regions where the colour information was meaningful, with­ out interference from light artefacts and reflections. at last, the random forest model was used to solve fig. 3 ­ changes in the sensory quality level (ql) of fresh­cut rocket leaves during the storage at 10°c according to the 5 to 1 rating scale reported by palumbo et al. (2021 b). in detail, ql5= very good; ql4= good; ql3= fair; ql2= poor; ql1= very poor. amodio et al. ‐ the sus&low project 129 both the classification problem (assessment of the qls) and the regression problems (estimation of quality marker parameters such as chlorophyll and ammonia contents). the same architecture was used for all the tasks, by simply changing the training data. the histogram of the image, evaluated in the a­b plane of the cielab colour space, was used as the set of features. the random forest model was able to automatically select the subset of values more suit­ able for solving each task. all the postharvest quality parameters detected by conventional analysis during the storage of fresh­ cut rocket leaves were significant in the ql assess­ ment and, among them, chlorophyll and ammonia contents proved to be useful marker parameters for the objective separation of each ql considered, both on soil and soilless cultivation approach. the cvs was able to operate without relevant dif­ ferences on unpackaged and packaged products. the test was done joining all the samples, regardless of the cultivation approach: the results showed a not significant performance loss on packaged leaves (pearson’s linear correlation coefficient of 0.84 for chlorophyll and 0.91 for ammonia) with respect to unpackaged ones (0.86 for chlorophyll and 0.92 for ammonia) (fig. 4). finally, three partial least square (pls) models were performed to predict the ql using as predictors chlorophyll and ammonia contents obtained by destructive methods (model i), by cvs on packaged products (model ii) and by cvs on unpackaged ones (model iii) (table 1). the results showed high performances in terms of r2 and the model obtained by predictors estimated non­destructively by the cvs (model ii and iii) provid­ ed better performances in the ql prediction than one obtained by destructive analysis, in both calibration and validation. wp3: marketing strategies to support the added value of the products lip and nd certified implementing a marketing strategy, based on often intangible characteristics to consumers such as lip and nd, it is not an easy task. low impact prac­ tices do not have a highly distinctive impact on prod­ uct characteristics nor determine unique taste, flavour, or look elements to consumers. however, certifications could be used to signal quality through the application of standards of quality and practices. whether certifications could be effective in terms of marketing in the case of products lip and nd, or for signalling quality in general is matter of discussion. vecchio and annunziata (2011), for instance, in their work question the possibility of effective understand­ ing of certification by consumers. at this purpose the research team of wp3 decided to implement a differ­ ent strategy and test it on the market. visual story­ telling certifying lip and nd has been then hypothe­ sized to better communicate the importance and the impact of those practices on food. the research activity, therefore, has been orga­ nized in three steps: identifying the communication fig. 4 ­ values estimated by the cvs (abscissa) vs. values mea­ sured in the laboratory (ordinate) for ammonia content on unpackaged (a) and packaged (b) rocket leaves and for total chlorophyll content on unpackaged (c) and packaged (d) samples (palumbo et al., 2022 b). table 1 ­ root mean square error (rmse) and the coefficient of determination (r2) in calibration (c) or validation (v) of the partial least square (pls) models predicting visual quality of rocket leaves (palumbo et al., 2022 b) pls models predictors rmsec r 2 c rmsev r 2 v i total chlorophyll and ammonia obtained by destructive methods 0.45 0.9 0.86 0.70 ii total chlorophyll and ammonia obtained by cvs on packaged rocket leaves 0.46 0.89 0.75 0.77 iii total chlorophyll and ammonia obtained by cvs on unpackaged rocket leaves 0.46 0.89 0.7 0.8 adv. hort. sci., 2023 37(1): 123­132 130 strategy and set­up; testing through focus­groups the opportunity conditions for farms and companies; testing though a survey and an econometric analysis the consumers’ preference and their willingness to pay for products with lip and nd. therefore, a draft platform has been developed containing basic com­ munication rules in order to highlight sustainability attributes of products through storytelling. workflow has been established and a simulation has been con­ ducted (fig. 5). focus group with producers has allowed verifying the general appreciation for the marketing approach and allowed a better set­up of the strategy. finally, a picture­based simulation has been produced for the final test and the survey to consumers (fig. 6). as last activity, a questionnaire based survey has been prepared and administered to 467 consumers and an econometric model to estimate willingness to pay and consumers orientation has been set up and then estimated. the whole set of activties within the research project allowed understanding how impor­ tant is a correct communication of products and how different could be the perception of a product based o n h o w y o u c e r ti f y o r n a r r a t e t h e p r o d u c ti o n method. result allow understanding that older con­ umsers are more aware of sustainability and are more willing to pay for lip products. psicological pro­ file such as traditionalism and benevolence identify the consumer that, more than other profiles, would be willing to pay a higher price. 3. conclusions sensor­based fertigation management applied to rocket leaves and tomato confirmed to be a feasible approach to improve sustainability of soilless cultiva­ tion, also in cases where the complete and rapid switch to closed cycle recirculation systems is still impaired by economic, social, and environmental fac­ tors such as in mediterranean area. the results of this project related to non­destruc­ tive discrimination of tomatoes and rocket leaves, according to cultural practices using different levels of inputs (water and fertilizers), indicated the high potentiality of hsi and ft­nir techniques for the authentication of sustainable growing methods. moreover, project activities’ proved cvs as an effec­ tive tool for evaluating the product quality also through the bag, even working only on the regions of the image that provide meaningful colour information about the product’s surface. the integration of machine learning modules inside the cvs confirmed to be useful to simplify the design and tuning, done mostly automatically without human intervention. moreover, the flexibility introduced by machine learning makes the resulting architecture more flexi­ ble in adapting to different products and applications. as regards the marketing approach, consumers resulted willing to pay a higher price for lip products certified through a visual storytelling platform. in the next future, there could be a good chance that sus­ tainability­oriented practices coupled with a visual storytelling certification style could gain shares on food markets. acknowledgements this research was funded by the project prin 2017 “sus&low­sustaining low­impact practices in horti­ culture through non­destructive approach to provide more information on fresh produce history and quali­ ty” (grant number: 201785z5h9) from the italian ministry of education university. references amodio m.l., cabezas­serrano a.b., peri g., colelli g., 2011 ­ post‐cutting quality changes of fresh‐cut arti‐ chokes treated with different anti‐browning agents as evaluated by image analysis. ­ postharvest biol. technol., 62: 213­220. fig. 5 ­ workflow for products lip and nd certified platform. fig. 6 ­ picture based simulation of visual storytelling certifica­ tion for lip producs and nd. amodio et al. ‐ the sus&low project 131 amodio m.l., capotorto i., chaudhry m.m.a., colel­ li g., 2017 a ­ the use of hyperspectral imaging to pre‐ dict the distribution of internal constituents and to clas‐ sify edible fennel heads based on the harvest time. ­ comput. electron. agric., 134: 1­10. amodio m.l., chaudhry m.m.a., colelli g., 2017 b ­ the use of non‐destructive techniques to assess the nutritional content of fruits and vegetable, pp. 763­780. ­ i n : y a h i a e . m . 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annunziata a., 2011 ­ the role of pdo/pgi labelling in italian consumers’ food choices. ­ agric. econ. res. rev., 12(2): 80­98. workman j., shenk j., 2004 ­ agronomy monograph. ­ near­infrared spectroscopy in agriculture, 44:3­10. yang y.c., sun d.w., pu h., wang n.n., zhu z., 2015 ­ rapid detection of anthocyanin content in lychee peri‐ carp during storage using hyperspectral imagine cou‐ pled with model fusion. ­ postharvest biol. technol., 103: 55­65. 25 1. introduction tomato is the leading vegetable crop grown throughout the world and it is also the number one vegetable crop in jordan. the statistics of the ministry of agriculture (moa, 2012) reveal that the total tomato area cultivated is 12344.5 ha producing an average of 73.4 tons per hectare. in addition, the statistics indicate that the total number of plastic houses is extensively increasing. in jordan, the use of various types of mulches has become a well-established practice over recent years. it is well known that mulching helps to maintain healthy vegetable crops. the benefits of using several types of mulches have been extensively studied and recognized. however, with the global call for organic agricultural production and reduction of pesticide and fertilizer use, mulches will continue to be used. peet (1992) summarized the environmental, cultural, and anatomical factors that can increase the incidence of cracking as: irregular watering, high temperatures and light, fruit anatomy, excessive rapid fruit growth, genetical differences among cultivars, high differences between day and night temperatures, and high humidity. peet and willits (1995) concluded that growers should reduce watering tomato cultivars that are crack-prone, particularly when yielding from the upper clusters. the cuticular membrane in the outer epidermal periclinal walls of both resistant and intermediate cultivars was thicker when compared to cultivars that exhibit cracking (matas et al., 2004). cracking is mainly caused by extreme changes in fruit growth rate caused by moisture fluctuations (swiader et al., 1992). thus, the growth rate is affected by mulching material by means of manipulating the microclimate (bender et al., 2008; abubaker, 2013). differences in the microclimate, depending on the mulch material, will influence the growth rate and hence could affect fruit cracking incidence. therefore, the objective of this study was to investigate the effect of different types of mulches on ‘newton’ tomato yield and fruit cracking incidence under plastic house conditions. 2. materials and methods the research was conducted at al-balqa’ applied university research station during the 2011/2012 growing season under a green plastic house (25 m long, 9 m wide, and with a height of 2.7 m) covered with a single glaze of 200 micron clear polyethylene film. during the summer season 2011, soil solarization was applied against soil borne pests. soil was then disked and prepared for laying the types of mulches used in the study. seven types of mulches were used: black plastic, tuff gravel (thickness of covering 6-7 cm), clear plastic, compost (thickness of covering 6-7 cm), crushed stone (thickness of covering 6-7 cm), shredded wood (thickness of covering 6-7 cm), and the control (no effect of different types of mulches on ‘newton’ tomato yields and fruit cracking under plastic greenhouse conditions s. abubaker (1), i. qrunfleh, m. hasan department of plant production and protection, faculty of agricultural technology, al-balqa’ applied university, al-salt 19117, jordan. key words: lycopersicon esculentum m., mulching, tomato cracking. abstract: this study was conducted at al-balqa` applied university research station to investigate the effect of different types of mulches on ‘newton’ tomato yields and fruit cracking incidence under greenhouse conditions. the experiment consisted of seven treatments (black plastic, tuff gravel, clear plastic, compost, crushed stone, shredded wood, and the control); a randomized complete block design with three replicates was used. different mulch types showed significant effects on early, medium, late, and total yields/ha of the tomato fruits. higher early and medium yields were obtained using black and clear plastic. compost resulted with the highest total yield. results of this study clearly showed that mulching improves total tomato yields under greenhouse conditions. in addition, larger fruits were obtained by applying mulching. tomato cracking was also slightly affected by the mulch types used in this study. adv. hort. sci., 2014 28(1): 25-28 (1) corresponding author: samih_abubaker@yahoo.com received for publication 26 march 2014 accepted for publication 12 may 2014 26 mulch). these were arranged in a randomized complete block design with three replicates for each mulch type. five to six-week-old seedlings of the commercial indeterminate tomato (lycopersicon esculentum m. var. ‘newton’), with an average of 12 cm height, were transplanted in december 2011. each experimental unit consisted of a 3 m row length, with a 1 m space between every two rows. seedling distances were 30 cm between plants with ten plants per plot. the soil surface was covered with the designated type of mulch just before transplanting and it remained covered until the end of the growing season. tomato plants were then trained to one stem by continuous removal of auxiliary shoots. soil moisture content was monitored using a mpkit-160 soil moisture meter. a drip irrigation system was employed to irrigate and fertilize the plants according to local commercial tomato, plastic-house grower practices. weeds were removed by hand and pest management control practices were applied throughout the study. growth parameters (plant height, stem diameter, total number of leaves, and dry matter) were recorded (abubaker, 2013). total yield of ripe fruits was determined by recording the consecutive weights of 24 hand-harvested fruits from 6 march to 10 june 2012. total yield was subdivided into three categories: early, medium, and late yields consisting of eight harvests each. the total number of cracked fruits and the percentages were recorded for the three harvest dates. all statistical analyses were performed using sas/stat version 9.2 and analysis of variance was conducted by the proc glimmix procedure. means were separated following the fisher’s protected least significant difference (lsd) test. 3. results and discussion the different mulch types showed significant effects on early, medium, late, and total yields (t/ha) of the tomato fruits (table 1). tomato plants grown under black, clear plastic, and shredded wood mulches resulted in the highest early yields, which were significantly higher than that of the control (table 1). however, tuff gravel, crushed stone, and the plants grown using compost resulted in higher early yields compared to the control but they were not significantly different. swiader et al. (1992) mentioned that black, gray, and transparent mulches raise soil temperatures. thus, the rate of growth for plants grown under those mulches will increase. the mean yields showed fewer differences between the various types of mulches (table 1). black and clear plastic resulted in higher mean yields, however they were not significantly different from that of the control. compost also resulted in higher mean yields that were not significantly different from the control and the slow release of nutrients from the organic material early in the season may be a possible explanation. tomato plants that were grown using shredded wood and tuff gravel as a mulches recorded the lowest mean yields: 56.1 and 57.4 t/ha, respectively (table 1). however, these yields were not significantly different compared to the control. late yields showed less difference among the various types of mulches (table 1), although the highest were recorded with tuff gravel (60. 2 t/ha) and compost (59.7 t/ha). these yields were significantly different compared to the yields of plants growing without mulch. shredded wood also resulted in higher late yields that were significantly compared to the control. in addition, the control yields (48.1 t/ha) showed higher late yields but they were not significantly different compared to the clear plastic (46.4 t/ha), black plastic (43.5 t/ha), and crushed stone (43.3 t/ha). the highest overall yields were obtained using compost (table 1). the descending order from highest to lowest total yields was found to be: compost, black plastic, clear plastic, tuff gravel, shredded wood, control, and finally crushed stones. the total yields of plants grown under compost, black plastic, clear plastic, and tuff gravel were significantly different compared to the control but were not significantly different compared to the shredded wood mulch. crushed stone mulch gave the lowest total yields (144.2 t/ha), which were not significantly different compared to the control and shredded wood much. the soil temperature-increasing effect of black mulch and the late season release of organic material from the compost explain the ability of such mulches to give superior production compared to the other types. our results coincide with kayum et al. (2008) and bay (2011). both researchers showed that table 1 yield (early, medium, late, and total) and average fruit weight of greenhouse tomato ‘newton’ under different types of mulches mulch type early yield (t/ha) medium yield (t/ha) late yield (t/ha) total yield (t/ha) fruit weight (g) black plastic 46.5 a 70.1 a 43.5 b 160.1 a 173.4 ab tuff gravel 38.7 bc 57.4 b 60.2 a 156.3 a 169.5 abc clear plastic 45.2 a 66.3 a 46.4 b 157.9 a 174.7 a compost 36.7 c 66.0 a 59.7 a 162.4 a 175.1 a crushed stone 37.7 bc 62.2 ab 43.3 b 144.2 b 150.5 d shredded wood 43.3 ab 56.1 b 56.2 a 155.6 ab 160.8 bcd control 33.2 c 63.3 ab 48.1 b 144.6 b 160.2 cd lsd 0.05 6.29 8.11 7.45 11.48 13.15 different letters in a column indicate significant differences at p≤ 0.05 according to fisher’s protected lsd. 27 mulching significantly affected yield components, increased yield, and improved fruit quality. regarding fruit weight, the largest fruits were obtained using compost, clear and black plastic with average weights of 175.1, 174.7, and 173.4 g/fruit (table 1). these weights were not significantly different compared to tuff gravel. the lowest fruit weights were achieved when crushed stone was used, followed by the control and shredded wood. there were no significant differences with regard to the lowest fruit weights. dry matter contents of leaves and stems were significantly affected by mulch types (table 2). tomato dry matter was highest when grown using the compost as a mulching material (19.4%); differences with crushed stone (18.8%) and black plastic (18.3%) were not significant. abubaker (2013) attributed similar findings to the higher amounts of available minerals released from the compost which also manifested favorable effects on available water content during the growing season, directly affecting vegetative growth. regarding fruit cracking, average values are presented in figure 1 and the average percentage of cracked fruits are presented in figure 2. the results clearly indicate no significant differences between the seven mulch types used in our study. however, the average number of cracked fruits was highest when compost was used, and it is worth mentioning again that the highest overall yields were obtained using this type of mulch. however, slight differences were observed in the average percentage of cracked fruits with regard to the total number of fruits, particularly between the clear plastic and control treatments but these differences were not significantly different. our results are in agreement with suwwan et al. (1988) who indicated that seasonal cracking was not affected by the five mulch types they studied (i.e. silver plastic, black plastic, paper, white/black plastic and black/white plastic). 4. conclusions mulching improved growth parameters and yields of ‘newton’ tomato grown under plastic house conditions. the highest overall yields were obtained using compost followed by black plastic. no significant differences among types of mulches were observed regarding the number of cracked fruits. however slight, though not significant, differences were found when considering the average number of cracked fruits compared to the total number of fruits. references: abubaker s., 2013 effect of different types of mulch on performance of tomato (lycopersicon esculentum m.) under plastic house conditions. journal of food, agriculture & environment, 11(2): 132-134. bay b., 2011 effect of mulching and amount of water on the yield of tomato under drip irrigation. journal of horticulture and forestry, 3(7): 200-206. bender i., raudseping m., vabrit s., 2008 effect of organic mulches on the growth of tomato plants and quality of fruits in organic cultivation. acta horticulturae, 779: 341-346. kayum m.a., asaduzzaman m., haque m., 2008 eftable 2 dry matter percentages of ’newton’ tomato leaves and stems grown under different types of mulches mulch type dry matter of leaves and stems (%) black plastic 18.3 ab tuff gravel 17.8 b clear plastic 17.6 b compost 19.4 a crushed stone 18.8 ab shredded wood 17.7 b control 17.6 b lsd 0.05 1.30 different letters in a column indicate significant differences at p≤ 0.05 according to fisher’s protected lsd. 0 5 10 15 20 25 bp g cp c no s w n u m b e r o f c ra c k e d f ru it s mulch type fig. 1 the average number of cracked `newton` tomato fruits for the three harvest dates for each mulch type. bp= black plastic, g= gravel, cp= clear plastic, c= compost, no= no mulch (control), s= stone, and w= wood. 0 5 10 15 20 25 bp g cp c no s w p e rc e n ta g e o f c ra c k e d f ru it s mulch type fig. 2 the percentage of cracked `newton` tomato fruits for the three harvest dates for each mulch type. bp= black plastic, g= gravel, cp= clear plastic, c= compost, no= no mulch (control), s= stone, and w= wood. 28 fects of indigenous mulches on growth and yield of tomato. journal of agriculture & rural development, 6(1&2): 1-6. matas a.j., cobb e., paolillo d., niklas k., 2004 crack resistance in cherry tomato fruit correlates with cuticular membrane thickness. hortscience, 39(6): 1354-1358. moa, 2012 annual report. ministry of agriculture, amman, jordan. peet m.m., 1992 fruit cracking in tomato. horttechnology, 2(2): 216-223. peet m.m., willits d., 1995 role of excess water in tomato fruit cracking. hortscience, 30(1): 65-68. suwwan m., akkawi m., al-musa a.m., mansour a., 1988 tomato performance and incidence of tomato yellow leaf curl (tylc) virus as affected by type of mulch. scientia horticulturae, 37(1-2): 39-45. swiader j., ware g., mccollum j., 1992 producing vegetable crops. fourth edition, interstate publishers, inc., danville, illinois, usa 223 1. introduction celosia, a c3 plant, belongs to the amaranthaceae family and is of tropical origin. in ghana, celosias are not only grown as a cut flower crop, but also as bedding plants, pot plants, and vegetable crops (norman, 2004). in this study, disbudding entailed the removal of axillary buds or bud breaks on a single stem, leaving the terminal flower bud intact and able to develop into a large flower head. this practice is reported to be a standard operation in the cultivation of roses, carnations, chrysanthemums and celosias (machin and scopes, 1978; janick, 1986; dole and wilkins, 1999; norman, 2004). celosia requires one stem as a cut flower; here developing flower buds on a flowering shoot must be disbudded in order to improve the quality of the terminal flower (norman, 2004). reports indicate that disbudding increases plant height in dahlias (parshall, 2007) and in celosias reduces the number and size of undesirable side (or axillary) shoots on the flower stem and thus resulting in increased plant height, flower stem length and flower head size (norman et al., 2009). also, disbudding induces early harvesting and a more concentrated harvesting period (norman, 2004). growth analysis has been widely used to study yieldinfluencing factors and plant development as net photosynthates accumulation over time (gardner et al., 1985). this approach uses simple primary data in the form of weights, areas, volumes and contents of plant components to investigate processes within and involving the whole plant (hunt, 1990). the leaf area index (lai) of a crop at a particular growth stage indicates its photosynthetic potential or the level of its dry matter accumulation. the greater the lai, the higher the dry matter accumulation potential of the crop and vice versa (rasheed et al., 2003). its value can vary with environmental and cultivation conditions (board and harville, 1992). the leaf area and its duration (lad) are measurements of growth of plants and plant physiological processes (miralles et al., 1997). lai and lad control the total production of dry matter and subsequently yield and yield attributes (jirali et al., 1994). crop growth rate (cgr) is a prime factor in determining crop yield because it reflects the capacity of assimilates production and affects dry matter accumulation. there is a close association between maximum dry matter production and maximum cgr (ball et al., 2000). the analysis of cgr has been shown to be important in evaluating treatment differences among crop species or cultivars with species in relation to yield (fageria et al., 2006). relative growth rate (rgr) is described as the rate of increase of total dry weight per plant (hunt, 2003). relative growth rate curves of crops are in opposition to dm accumulation during the life cycle of crops (fageria et al., disbudding effects on growth analysis of celosia (celosia cristata) p. adjei-frimpong, j. ofosu-anim, j.c. norman department of crop science, university of ghana, po lg 44, legon, rupublic of ghana. key words: celosia cristata, disbudding, growth analysis. abstract: experiments to investigate the effects of disbudding on growth analysis of two celosia cultivars, ‘carmine’ and ‘chief gold’, were carried out on the field in 2009 and 2010 at the sinna garden of department of crop science, university of ghana, legon, accra, ghana. the treatments consisted of disbudding once, disbudding twice, and no disbudding, as control, and were arranged in a 3x2 split plot in a randomized complete block design with four replications in 2009 (experiment 1) and three in 2010 (experiment 2). the two cultivars were harvested weekly during the growing period and separated into the various plant parts and oven-dried for dry weights, using appropriate formulae to calculate the various growth parameters. analysis of variance (anova) was used to analyse the data and a correlation coefficient matrix showed relationships among growth parameters. disbudding resulted in increased leaf area index, leaf area ratio, leaf area duration, relative growth rate, and harvest index, but reduced crop growth rate and net assimilation rate. ‘chief gold’ had a higher harvest index than ‘carmine’. disbudding plants once gave the best flower head size and weight result. ‘carmine’ gave the best flower yield and quality results in experiment 1 and ‘chief gold’ in experiment 2. adv. hort. sci., 2011 25(4): 223-231 received for publication 7 june 2011 accepted for publication 12 october 2011 224 2006). results from the studies of medhet et al. (2000) on growth analysis of sunflower, helianthus annuus, under drought conditions indicated a reduction of rgr value from early growth stages to final stages. however, recent reports by fageria et al. (2006) indicate that values of rgr are generally higher during early growth stages of the crop and decrease with age. measurement of net assimilation rate (nar) is important to detertmine the efficiency of plant leaves for dm production. nar values decrease with crop growth due to both the shedding of leaves and reduced photosynthetic efficiency of older leaves (fageria et al., 2006). similarly, law-ogbomo and egharevba (2008) reported that abscission with plant growth of the lower leaves in tomato causes a decrease in nar. however, there is no detailed information on the quantitative growth aspects and growth analysis of celosia cristata grown for cut flower production. the only previous reference to growth characteristics is that of celosia argentea (grown as a leafy vegetable crop) by ojo (2001) who reported a positive relationship between yield and leaf area, which was enhanced by increasing population density and cutting height. the present experiment was therefore undertaken to investigate the effects of disbudding on the growth indices of two cultivars (carmine and chief gold) of celosia cristata and to identify relationships between these indices (parameters) and flower yield. 2. materials and methods experimental site the study was conducted at the sinna garden, department of crop science, university of ghana, legon, between july and september 2009 for experiment 1 and december 2009 to february 2010 for experiment 2. the soil at the experimental site is of the adenta series (brammer, 1960) and classified as ferric acrisol (fao/unesco, 1990). the soil is sandy loam and moderately well drained with moderate levels of organic matter. climatological data during the experimental period are shown in table 1. experimental design a randomized complete block design with split plot arrangement and cultivars as the main plots and disbudding as the subplots were used for the experiment. there were four replications in experiment 1 and three in experiment 2. the disbudding treatments were: disbudding once; disbudding twice; and no disbudding (as control). the cultivars used were ‘carmine’ and ‘chief gold’. the plants were established at a spacing of 15 x 9 cm. there were 90 plants (experiment 1) and 96 plants (experiment 2) per sub-plot in which five plants were sampled weekly for dry weights and 10 plants were tagged for field data collection. cultivation practices in experiment 1, seeds were first sown in seed boxes using sandy soil on 17 july 2009 and the germinated seedlings were planted in the field on 7 august 2009. before planting, each sub-plot (0.9 x 1.5 m) received an application of 15-15-15 npk fertilizer at the rate of 674 kg/ha on 6 august 2009. in experiment 2, cow dung was incorporated into the plots at 25 t/ha on 8 december 2009. seeds were sown in plastic seed trays on 16 december 2009 using peat as the soil mix and the germinated seedlings were pricked out into plastic seed trays on 30 december 2009. the seedlings were planted in the field on 13 january 2010. a day before planting, each sub-plot (0.99 x 1.65 m) received an application of 15-15-15 npk fertilizer at the rate of 600 kg/ha. in each experiment, hand watering was done twice a day. routine weed control was carried out either by handpicking of weeds or by hoeing when necessary. diseases and insect pests were controlled by spraying of insecticide and fungicide. dithane m45 was sprayed on 19 august 2009 and 11 february 2010, in both experiments respectively, to control leaf spot diseases. on 2 september 2009, 28 january and 3 february 2010, in both experiments respectively, cydim super was also sprayed to control grasshoppers and whiteflies. in experiment 1, plants were sidedressed four weeks after planting with potassium nitrate at table 1 climatological data during experimental period month mean maximum temperature (°c) mean minimum temperature (°c) total rainfall (mm) mean maximum relative humidity (%) mean minimum relative humidity (%) experiment 1 2009 july 28.3 23.3 91.5 94 77 august 28.3 23.0 11.5 92 74 september 30.5 23.2 6.3 91 69 experiment 2 2009 december 2010 33.4 24.8 10.3 92 64 january 33.3 25.0 49.6 94 65 february 33.7 25.4 57.2 94 66 source: meteorological services of ghana, mempeasem, accra, ghana. 225 a rate of 100 kg/ha while in experiment 2, side-dressing was done at three weeks after planting at the same rate. disbudding disbudding was carried out as follows: 1. disbudding once: axillary flower heads and side shoots were removed on all the plants in the field except the control plants at 22 days after planting (dap) on 29 august 2009 and at 18 dap on 1 february 2010. 2. disbudding twice: the removal of axillary flower heads and side shoots was undertaken on only the plants designated for this treatment at 27 dap on 3 september 2009 and at 25 dap on 8 february 2010. sampling sampling started two weeks after planting and every week thereafter until the sixth week in experiment 1; in experiment 2 it started a week after planting and every week thereafter until the fifth week. five plants were randomly sampled from each sub-plot, carefully dug up and the roots washed of soil particles. the leaf area was calculated using a leaf area meter (model am 100 by analytical development company limited, england). the plant parts (leaves, flower heads, flower stems, side shoots, axillary flowers and roots) were separated and chopped into pieces and put in different sampling envelopes and ovendried to a constant weight of 80°c for 48 hr to determine their dry matter. two types of measurements are needed for growth analysis: the plant weight, usually the oven dry weight (g); and the size of the assimilating system, usually in terms of leaf area (cm2). the crop growth rate, net assimilation rate (nar), relative growth rate (rgr), leaf area index, leaf area ratio (lar), leaf area duration and harvest index were calculated as follows. leaf area index (lai) leaf area index is defined as leaf area per unit area of land. it is a dimensionless ratio (watson, 1947) and calculated with the formula: leaf area index = total leaf area land area crop growth rate (cgr) crop growth rate is defined as the increase in plant dry matter per unit of time per land area unit (radford, 1967) with the formula: cgr (gm-2 day-1) = w2 – w1 t2 – t1 relative growth rate (rgr) relative growth rate is the increase of plant material per time unit. it was calculated for each interval between sampling with the formula given by radford (1967). the rgr of the first harvest could not be calculated because there was no dry weight before the first harvest. rgr (mgg-1 day-1). = ln w2 – ln w1 t2 – t1 net assimilation rate (nar) the net assimilation (nar) is the increase of plant material per unit of the assimilating material per unit of time. it was calculated for each interval between two samplings with the formula described by watson (1947) and radford (1967). the nar of the first harvest could not be calculated because there was no leaf area value before the first harvest. nar (gm-2 day-1) = (w1)(w2) lad leaf area duration (lad) leaf area duration is the photosynthetic potential of a plant, i.e. a measurement of the entire opportunity for assimilation a plant possesses during a growth period (watson, 1947). this was calculated using the formula: lad = (lai1 + lai2) x (t2 – t1) 2 leaf area ratio (lar) leaf area ratio of a plant at an instant in time (t) is the ratio of the assimilatory material per unit of plant material present. the lar was calculated with the following formula: lar (cm2/g) = (lai1) – (lai2) (w1)(w2) harvest index (hi) the harvest index is the ratio of economic yield (flower head and stem) to biological yield (donald and hamblin, 1976). its computation uses the following formula: harvest index (hi) = economic yield (flower head and stem) biological yield (total dry weight) x 100 where w2 and w1 = dry weight at second and first harvest, t2 and t1= time corresponding to second and first harvest. leaf chlorophyll content leaf chlorophyll content was measured using a chlorophyll meter (model spad, minolta, japan). flower head size index this was calculated as the product of the vertical and horizontal lengths of the flower head divided by 2. number of side shoots this was obtained by stripping off side shoots on the flower stem and counting. 226 harvesting harvesting of the 10 tagged plants of each plot started 60 days after sowing for experiment 1 and 63 days after sowing for experiment 2. ‘carmine’ was harvested two days earlier than ‘chief gold’. statistical analysis the data collected were analysed using analysis of variance (anova) (genstat, ver. 9). significant differences among treatment means were determined using the least significant difference (lsd) test at p = 0.05. correlation analysis correlation analysis for flower quality parameters and other measured growth variables was also determined using spearman’s rank correlation coefficient. 3. results and discussion flower head dry weight and size tables 2 and 3 show the effects of disbudding and cultivar on flower head dry weight and size. disbudding significantly influenced flower head size production. in experiment 1, disbudding twice produced the heaviest flower heads with the control producing the lightest. plants subjected to disbudding once produced the heaviest flower heads with the control producing the lightest in experiment 2. larger flower heads were also produced by disbudding-once plants followed by disbudding-twice, with the control producing the smallest flower heads. ‘carmine’ produced significantly larger flower heads than ‘chief gold’ in experiment 1. however, the opposite was true in experiment 2. flower head size has the potential to increase when the sink-source ratio is reduced, i.e. when the number of competing sinks for assimilates is reduced or the source activity is increased (cockshull, 1982; lee et al., 2001). in the present study, disbudding increased flower head size significantly. similar observations were made by carvalho et al. (2006) in chrysanthemums and norman et al. (2009) in celosia. in a celosia plant, the axillary flower heads, roots, leaves and side shoots compete with the flower stem and head for assimilates. as the number of flower heads per plant increases, the flower head size tends to decrease. reducing the number of flower heads on a flower stem allows the plant to distribute assimilates to the terminal flower that then attains a larger size. competition among the terminal flowers as well as between the flower and the vegetative plant parts for available assimilates explains the smaller and lighter flower heads produced by the control plants. these experienced a high intra-plant competition for photosynthetic radiation, thus influencing the assimilate allocation to the terminal flower. an increased in the number of small flowers has also been reported in chrysanthemum by carvalho et al. (2006) as a result of removal of the terminal flower bud. crop growth rate (cgr) figure 1 shows the effects of disbudding and cultivar on cgr of celosia plants. significant interactions were observed but showed no differences among treatments at 3 wap in experiment 1. however, in experiment 2, disbudding significantly affected cgr with the control plants recording the highest cgr and this was significantly different from the other treatments. significant table 2 effects of disbudding on flower head dry weight and size of two celosia varieties at harvest. experiment 1 treatment flower head dry weight (g) flower head size index (cm) ‘carmine’ ‘chief gold’ mean ‘carmine’ ‘chief gold’ mean disbudding once 2.08 2.20 2.34 21.7 19.7 20.7 disbudding twice 2.48 1.81 1.94 18.9 15.0 17.0 control 1.09 0.84 0.97 12.2 9.5 10.9 mean 1.89 1.62 17.6 14.7 lsd(5%):cultivar ns ns lsd(5%):disb 0.56 7.20 lsd(5%):cultivar x disb 1.22 9.65 table 3 effects of disbudding on flower head dry weight and size of two celosia varieties at harvest. experiment 2 treatment flower head dry weight (g) flower head size index (cm) ‘carmine’ ‘chief gold’ mean ‘carmine’ ‘chief gold’ mean disbudding once 2.21 2.47 2.34 16.9 23.9 20.4 disbudding twice 2.11 2.13 2.12 18.1 20.2 19.1 control 1.43 1.02 1.08 11.9 10.2 11.1 mean 1.82 1.87 15.7 18.1 lsd(5%):cultivar ns ns lsd(5%):disb 0.36 3.50 lsd(5%):cultivar x disb 0.41 4.30 227 interactions were observed with ‘chief gold’ having significantly higher cgr than ‘carmine’ at the 3 wap. crop growth rate was observed to increase with plant development (fig. 1a and b). an increase in cgr was recorded for both experiments at the final sampling (5-6 wap). crop growth rate is a prime factor in determining crop yield because it reflects the capacity of assimilates production and affects dry matter accumulation. there is a close association between maximum dry matter production and maximum cgr (ball et al., 2000). the observed significant and positive correlation between total aboveground biomass and cgr (r = 0.241*) and (r = 0.245*) in both experiments, respectively, supports this hypothesis. celosia plants produced a lot of side shoots and axillary flower heads during growth. therefore, it can be speculated that the dm accumulated in these organs, in addition to the other plant organs, accounted for the higher cgr and also enhanced nar in the control plants (fig. 2a and b). crop growth rate was significantly and positively correlated with flower stem dry weight (r = 0.3*). leaf area index (lai) disbudding did not significantly affect lai at the initial growth stages. leaf area index was significantly different among the various treatments (fig. 3a and b) and at harvesting (5-6 wap), disbudding significantly affected lai in both experiments. in experiment 1, plants disbudded twice had the highest lai (2.47) followed by those disbudded once (2.43); the control plants produced the lowest (1.55). in experiment 2, plants disbudded once had the highest lai (2.58) followed by those disbudded twice (2.52), while the control plants had the lowest lai (1.83). all disbudded treatments had a significantly higher lai than the control treatments. ‘carmine’ produced a lot of leaves in experiment 1, and they were broader than the ones of ‘chief gold’, hence ‘carmine’ had a higher lai. ‘chief gold’ responded earlier to disbudding than ‘carmine’ in lai as the control plants recorded lower values right from the initial stages. fig. 1 crop growth rate as affected by disbudding and cultivar: ‘carmine’ (a) and ‘chief’ gold (b). experiment one experiment two fig. 2 effects of disbudding and cultivar on side shoot production: ‘carmine’ (a) and chief gold’ (b) cultivars of celosia over the growing period in both experiments. experiment one experiment two fig. 3 leaf area index as affected by disbudding and cultivar: ‘carmine’ (a) and ‘chief gold’ (b). experiment one experiment two disbudding once disbudding twice control c ro p gr ow th r at e (m g2 da y -1 ) c ro p gr ow th r at e (m g2 da y -1 ) n um be r of s id e sh oo ts n um be r of s id e sh oo ts l ea f ar ea i nd ex l ea f ar ea i nd ex l ea f ar ea i nd ex l ea f ar ea i nd ex n um be r of s id e sh oo ts n um be r of s id e sh oo ts disbudding once disbudding twice control disbudding once disbudding twice control disbudding once disbudding twice control disbudding once disbudding twice control disbudding once disbudding twice control disbudding once disbudding twice control disbudding once disbudding twice control disbudding once disbudding twice controldisbudding once disbudding twice control disbudding once disbudding twice control weeks after planting c ro p gr ow th r at e (m g2 da y -1 ) c ro p gr ow th r at e (m g2 da y -1 ) weeks after planting weeks after planting weeks after planting weeks after planting weeks after planting weeks after planting weeks after planting weeks after planting weeks after planting weeks after planting weeks after planting disbudding once disbudding twice control 228 the lai determines the photosynthetic capacity of a crop. the higher lai of the disbudded treatments means that there were more (expanded) leaves per plant for higher radiant energy interception for photosynthesis and, therefore, more dry matter partitioning into the economic yield. this assertion is supported by the significant positive correlation between chlorophyll content and lai in experiment 1 (r = 0.040*). a significantly higher number of leaves per plant was produced by ‘carmine’ (17.44) than ‘chief gold’ (12.82) (fig. 4). the lower lai induced by the control treatments might be due to a lower leaf number and area which might have resulted from the competition among the various plant parts for assimilate partitioning. maximum dm production is achieved at an optimal lai. the optimal lai obtained for disbudded plants in celosia is between 2 and 2.5. although the control plants had a lower lai, they had the highest cgr. previous reference to growth characteristics of celosia argentia was made by ojo (2001) who reported a positive relationship between yield and leaf area. the results of experiment 2 confirm what reported above this. a linear relationship was observed between total aboveground biomass and lai (r = 0.040*). however, total aboveground biomass had a significantly negative relationship with lai (r = -0.343*) in experiment 1. lai had a positive and significant association with flower stem dry weight (r = 0.04*) and flower head dry weight (r = 0.07*). relative growth rate (rgr) in both experiments, rgr decreased linearly during the early growth and increased towards maturity (fig. 5a and b). in experiment 1, plants disbudded twice exhibited a higher rgr than control and disbudded-once plants. however, in experiment 2, plants disbudded once had a higher rgr than the other treatments. ‘carmine’ exhibited a higher rgr in experiment 1 than ‘chief gold’ in experiment 2. the observed decrease in rgr may be attributed to the decreasing trend in leaf area ratio (lar) with plant growth as indicated by the linear relationship between lar and rgr (r = 0.343*), (r = 0.168*) in both experiments, respectively. relative growth rate had a positive and significant association with flower stem dry weight (r = 0.12*) and flower head dry weight (r = 0.39*). increased rgr due to disbudding also resulted in increased flower yield. relative growth rate also had a significant negative relationship with cgr (r = -0.04*). net assimilation rate (nar) net assimilation rate showed no significant differences among treatments at 3 wap in experiment 2 (fig. 6a and b). in experiment 1, disbudding did not affect nar significantly; in experiment 2 disbudding lowered nar significantly. correlation analysis shows that nar had a negative and significant correlation with flower head dry weight (r = -0.05*) and flower stem dry weight (r = -0.02*) in experiment 1 but correlated positively and significantly with flower head dry weight (r = 0.13*) in experiment 2. thus, the lower nar observed in experiment 2 was compensated for bigger flower head production. generally, ‘chief gold’ had a higher nar than ‘carmine’. the decline in nar with plant growth observed in experiment 2 after disbudding might be due to both the shedding of leaves and reduced photosynthetic efficiency of older leaves (fafig. 4 leaf growth as influenced by disbudding and cultivar: ‘carmine’ (a) and ‘chief gold’ (b) cultivars of celosia. experiment one experiment two fig. 5 relative growth rate as affected by disbudding and cultivar: ‘carmine’ (a) and ‘chief gold’ (b). experiment one experiment two n um be r of l ea ve s n um be r of l ea ve s n um be r of l ea ve s n um be r of l ea ve s disbudding once disbudding twice control disbudding once disbudding twice control disbudding once disbudding twice control disbudding once disbudding twice control disbudding once disbudding twice control disbudding once disbudding twice control disbudding once disbudding twice control disbudding once disbudding twice control weeks after planting weeks after planting weeks after planting weeks after planting weeks after planting weeks after planting weeks after planting weeks after planting r el at iv e gr ow th r at e (m g2 da y -1 ) r el at iv e gr ow th r at e (m g2 d ay -1 ) r el at iv e gr ow th r at e (m g2 da y -1 ) r el at iv e gr ow th r at e (m g2 da y -1 ) 229 geria et al., 2006). similarly, law-ogbomo and egharevba (2008) reported that the abscission of the lower leaves with plant growth in tomato causes a decline in nar. leaf area ratio (lar) leaf area ratio decreased for both cultivars in both experiments with plant age. from 3-6 wap, disbudding significantly affected lar (fig. 7a and b). the disbudded plants had a significantly higher lar than the control plants. since lar indicates how much leaf area a plant produces per gram of dry matter, a high lar indicates that a plant is efficient at producing leaf area. since leaf area determines light interception, which is also an important parameter affecting plant growth, a high lar would be expected to result in a high growth rate (kang and van iersel, 2004). this further explains the linear relationship between lar and rgr (r = 0.343*), (r = 0.168*) in both experiments, respectively. leaf area ratio correlated negatively and significantly with flower stem dry weight (r = -0.05*) and flower head dry weight (r = -0.02*). leaf area duration (lad) the effect of disbudding and cultivar on lad is shown in figure 8a and b. disbudding affected lad but this was significant. however at harvesting (5-6 wap), significant differences were observed among disbudded treatments. significant interactions were also observed with all disbudded plants of ‘carmine’ producing a higher lad than that of ‘chief gold’ (fig. 8a and b). according to gifford and evans (1981), lad is more important for determining the final yield. however, in the current study, the higher lad of ‘carmine’ in both experiments did not lead to a yield advantage since ‘chief gold’ had a higher yield in terms of both the flower head and flower stem (economic sinks). fig. 6 net assimilation rate as affected by disbudding and cultivar: ‘carmine’ (a) and ‘chief gold’ (b). experiment one experiment two fig. 7 leaf area ratio as affected by disbudding and cultivar: ‘carmine’ (a) and ‘chief gold’ (b). experiment one experiment two fig. 8 leaf area duration as affected by disbudding and cultivar: ‘carmine’ (a) and ‘chief gold’ (b). experiment one experiment two disbudding once disbudding twice control disbudding once disbudding twice control disbudding once disbudding twice control disbudding once disbudding twice control disbudding once disbudding twice control disbudding once disbudding twice control disbudding once disbudding twice control disbudding once disbudding twice control disbudding once disbudding twice control disbudding once disbudding twice control disbudding once disbudding twice control disbudding once disbudding twice control weeks after planting weeks after planting weeks after planting weeks after planting weeks after planting weeks after planting weeks after planting weeks after planting weeks after planting weeks after planting weeks after planting weeks after planting n et a ss im ila ti on r at e (m g2 d ay 1 ) n et a ss im ila ti on r at e (m g2 d ay 1 ) l ea f ar ea r at io ( cm 2 / g) l ea f ar ea d u ra ti on l ea f ar ea d u ra ti on l ea f ar ea d u ra ti on l ea f ar ea d u ra ti on l ea f a re a ra ti o (c m 2 / g) l ea f ar ea r at io ( cm 2 / g) l ea f ar ea r at io ( cm 2 / g) n et a ss im ila ti on r at e (m g2 d ay 1 ) n et a ss im ila ti on r at e (m g2 d ay -1 ) 230 the most likely explanation for this disagreement is the inefficiency of ‘carmine’ to use its entire lad for dm production even though flower heads were harvested before they were fully matured (market requirement) for both cultivars. a positive linear association was observed between lad and flower head dry weight (r = 0.02*) and flower stem dry weight (r = 0.04*). harvest index (hi) the effects of cultivar and disbudding on mean harvest index are presented in table 4. disbudding significantly influenced hi. all disbudded plants had a higher hi than the control plants (table 4). cultivars did not differ significantly in mean hi. significant disbudding and cultivar interactions were also observed. the hi for the disbudded ‘chief gold’ plants was relatively higher than that of ‘carmine’, indicating that ‘chief gold’ had a more efficient translocation system compared to ‘carmine’. differences in hi may be related to differences in the pattern of allocation of photosynthate (gent and kiyomoto, 1989). ‘chief gold’ had higher hi than ‘carmine’, which indicates that ‘carmine’ is less efficient in converting dm to flower stem and head yield (flower yield). hi showed linear associations with lai (r = 0.131*), lar (r = 0.154), nar (r = 0.019*) and rgr (r = 0.010*). 4. conclusions the overall result of the present study shows the effect of variations in disbudding on growth and development of the two considered cultivars. disbudding increased leaf area index, leaf area ratio, leaf area duration, relative growth rate, and harvest index, but reduced crop growth rate and net assimilation rate. ‘chief gold’ had a higher harvest index than ‘carmine’. disbudding plants once gave the best flower yield and quality in terms of flower head size and weight. in addition, ‘carmine’ gave the best flower yield and quality results in experiment 1 and ‘chief gold’ in experiment 2. disbudding once is therefore a highly recommended technique for celosia cut flower growers. references ball r.a., purcell l.c., vories e.d., 2000 yield compensation in response to plant population and water regime for short-season soybean production. crop sci., 40: 757-764. board j.e., harville b.g., 1992 explanations for greater light interception in narrowvs. wide-row soybean. crop sci., 32: 198-202. brammer h., 1960 soils of the accra plains. memoir no. 3, soil research institute, kumasi, republic of ghana. carvalho m.p.s., heuvelink e.p., harbinson j., van kooten o., 2006 role of sink-source relationships in chrysanthemum flower size and total biomass production. physiologia plant., 128: 263-273. cockshull k.e., 1982 disbudding and its effect on dry matter distribution in chrysanthemum morifolium. j. hort. sci., 57: 205-207. dole j.m., wilkins h.f., 1999 floriculture: principles and species. prentice hall, new jersey, usa. donald c.m., hamblin j., 1976 the biological yield and harvest index of cereals as agronomic and plant breeding criteria. advances in agronomy, 28: 361-405. fageria n.k., baligar v.c., clark r.b., 2006 physiology of crop production: physiology of growth and yield components. the haworth press, inc., pp. 61-82. fao/unesco, 1990 soil map of the world. revised legend. fao, pp. 119. gardner f., pearce r.b., mitchell r.l., 1985 physiology of crop plants. iowa state university press, ames, iowa, usa, pp. 327. gent m.p.n., kiyomoto r.k., 1989 assimilation and distribution of photosynthate in winter wheat cultivars differing in harvest index. crop sci., 29: 120-125. gifford r.m., evans l.t., 1981 photosynthesis, carbon partitioning and yield. annual review of plant physiology, 32: 485-509. hunt r., 1990 basic growth analysis: plant growth analysis for beginners. unwin hyman, london, uk, pp. 112. hunt r., 2003 growth analysis, individual plants. in: thomas b., d.j. murphy, and b.g. murray (eds.) encyclopedia of applied plant sciences. academic press, london, uk, vol. 3, pp. 2000. janick j., 1986 horticultural science. w.h. freeman and company, san francisco, california, usa, pp. 325-326. jirali d.i., panchai y.c., janagoudar b.s., patil b.c., 1994 studies on the growth pattern and yield in chicktable 4 the effects of cultivar and disbudding on mean harvest index of two celosia cultivars in both experiments treatment mean harvest index ‘carmine’ ‘chief gold’ mean epx 1 exp 2 epx 1 exp 2 epx 1 exp 2 disbudding once 36.99 29.34 39.26 29.34 38.12 29.34 disbudding twice 37.41 37.03 42.90 28.49 40.15 27.76 control 21.35 15.58 20.10 15.47 20.73 15.53 mean 31.92 23.99 34.09 24.44 lsd(5%):cultivar ns ns lsd(5%):disb 4.35 4.04 lsd(5%):cultivar x disb 5.48 4.87 231 pea (cicer arietinum l.) genotypes under receding soil moisture conditions. indian j. of plant physiol., 37: 275-276. kang j.g., van iersel m.w., 2004 nutrient solution concentration affects shoot:root ratio, leaf area ratio, and growth of sub irrigated salvia (salvia splendens). hortscience, 39: 49-54. law-ogbomo k.e., egharevba r.k.a., 2008 effects of planting density and npk fertilizer on growth and fruit yield of tomato (lycospersicon esculentus mill). res. j. agric. and biol. sci., 4: 265-272. lee b.j., won m.k., lee d.h., shin d.g., 2001 effects of source sink on flowering and growth in chrysanthemum (dendranthema grandiflora tzvelev). journal of korean soc. hort. sci., 42: 625-630. machin b., scopes n., 1978 chrysanthemum year-round growing. blandford press ltd., dorset, uk, pp. 233. medhet k.h., ejaz r., syed k.a., 2000 growth analysis of sunflower (helianthus annuus l.) under drought conditions. int. j. agric. and biol., 2: 136-140. miralles o.b., de juan valero j.a., martin de santa olalla f., 1997 growth, development and yield of five sunflower hybrids. european journal of agronomy, 6: 47-59. norman j.c., 2004 tropical floriculture. national science and technoolgy press, accra, ghana, pp. 179-182. norman j.c., ofosu-anim j., bassaw e.n., adjeifrimpong p., boateng b.o., 2009 response of field grown celosia (celosia cristata var. carmine) to disbudding. adv. hort. sci., 23(3): 171-174. ojo o.d., 2001 density and cutting height affect the production of celosia (celosia argentea l.). journal of vegetable crop production, 7: 45-55. parshall r., 2007 how to disbud and disbranch. dparshal@ scdahlias. org. radford p.j., 1967 growth analysis formula their use and abuse. grop science, 7: 171-175. rasheed m., hussain a., mahmood t., 2003 growth analysis of hybrid maize as influenced by planting techniques and nutrient management. int. j. agric. and biol., 2: 169-171. watson d.j., 1947 comparative physiological studies on the growth of the field crops. i. variation in net assimilation rate and leaf area between species and varieties and within and between years. annals of botany, 11: 41-76. 158 1. introduction in almost all fruits including mango, the stage of maturity at harvest is very important as it markedly influences not only ripening and storage but also taste and palatability of the fruit. consumers seek a mango with a bright, fully developed skin colour without any blemishes, uniformly softened flesh and a fruit with small stone, more pulp and also good flavour and taste with appreciable storage life. mango fruits when matured on the tree develop most of the above characteristics but such fruits have been associated with environmental hazards such as insect attack, sunburn, wind abrasion, sap spurt at harvest, damage due to hail and pre-harvest disease infection (oosthuyse, 1997). bagging may be useful as a means of preventing such problems in mango and can reduce disease and physical damage as well as improve colour at harvest in a number of fruits (bentley and viveros, 1992; byers and carbaugh, 1995). this approach has been tested to produce high quality unblemished mango fruits in queensland (hofman et al., 1997), south africa (oosthuyse, 1997) and the philippines (bugante et al., 1997). however, different bagging materials behave in different ways, as has been reported by ann et al. (1998). according to these authors fruit bagging at an early stage was the most effective method to control mango anthracnose disease. the use of different bagging materials did not affect disease controls, although it did affect fruit maturation, colour and °brix. while some benefits (e.g. reduction in physical damage) could be expected, there may also be negative effects on quality as different days of bagging can delay the development of ripening characteristics of fruits. the present paper describes the results of experiments on bagging in fruit quality and mineral element content of mango fruit cv. himsagar, an important commercial cultivar of west bengal, india. influence of bagging on fruit quality and mineral composition of himsagar mango grown in new alluvial zones of west bengal p. dutta*, d. majumder** * department of fruits and orchard management, faculty of horticulture, bidhan chandra krishi viswavidyalaya, p.o. mohanpur, 741252 west bengal, india. ** department of agricultural statistics, bidhan chandra krishi viswavidyalaya, p.o. mohanpur, 741252 west bengal, india. key words: fruit bagging, fruit quality mineral composition, himsagar mango. abstract: the effect of polybagging of mango (mangifera indica l.) fruits was evaluated at different stages of fruit development (35, 45, 55 and 65 days after fruit set). fruits were harvested at different stages of maturity (75, 85 and 90 days after fruit set) and allowed to ripen at room temperature (34-36°c, rh 85-90%). the use of bagging at different stages of fruit development improved the appearance of fruit, fruit weight and size through other effects such as increased relative humidity and a consequently reduced fruit water loss. the maturity of fruits, at all stages of fruit harvest, was delayed with increasing bagging duration. early bagging of fruit (35 days after fruit set) delayed the development of ripening characteristics in comparison to delayed bagging and unbagged control fruit, which ripened earliest. this was clearly evident from the carotene content in the mango flesh, at the different stages of harvest and of ripening fruit, which was the result of higher temperature inside the bags. in bagged fruits usually day/night temperature fluctuations were reduced and there was a cut off in the temperature curve inside the bag. the total soluble solids and sugar content were higher and titratable acid content was always less in unbagged fruit at all stages of fruit harvest and fruit ripening. mineral elements were also affected by the number of days of bagging. the reduced ca concentration in long-duration bagging (early bagging) might be due to increased rh around the fruits. fruits bagged for 55 days recorded an increased content of n, p, zn, mn and fe while fruit calcium concentration was reduced by bagging for 55 days. anthracnose and stemend-rot (ser) caused by colletotrichum and diplodia spp. respectively were reduced by bagging in both years through a reduction in contact between disease propagules and fruits. these results indicate that bagging can improve fruit quality by reducing disease, lead to a better appearance of fruit and increase fruit weight and size. adv. hort. sci., 2012 26(3-4): 158-162 received for publication 16 january 2012 accepted for publication 24 september 2012 159 2. materials and methods the experiment was conducted at the mondouri horticultural research station and the post harvest technology laboratory of the faculty of horticulture, bidhan chandra krishi viswavidyalay, nadia, west bengal, india during 2006-2008. study trees of mango cv. himsager were selected from the orchard at mondouri (23.5° n latitude and 89° e longitude). the trees were healthy, uniform in size and more than 15 years old. about 250 developing mangoes were tagged and bagged with transparent polyethylene bags at different stages of fruit development: 35, 45, 55 and 65 days after fruit set and a control without any bagging. then on each sampling date (75, 85, 90 days after fruit set), 10 mangoes were harvested at random from each bagging-date lot of polyethylene-bagged fruits. the fruits were washed and dried at ambient temperature (32°c±1°c), and kept in the laboratory for ripening. ripe fruits were analyzed for physico-chemical characteristics; bagged fruits were analysed 55 days after fruit set for mineral elements and disease incidence. physico-chemical analysis a. physical characters. weight, length and diameter. the weight of ripe fruit was determined using a digital balance and expressed in grams. length of fruit was measured from the base to the apex and diameter at its widest part, near the shoulder of the fruit, with the help of a vernier caliper. both were expressed in centimeters. b. bio-chemical constituents. total soluble sugars, total soluble solids (tss), titratable acidity. total soluble sugar content was analysed using fehlings’ a and b solution, according to the methods of the aoac (1996) and expressed as percentage. in this method, for inversion at room temperature an aliquot of clarified and diluted solution was transferred to a flask. 10 ml of hcl (1:1) was added and allowed to stand at room temperature for 24 h. the solution was then neutralized with concentrated naoh solution and made to volume. an aliquot was taken and the total soluble sugars were determined as invert sugars using fehling’s a and b solution. in determining reducing sugar, acid hydrolysis was not done. total soluble solids (tss) content of juice was determined using a hand refractometer and expressed as °brix at 20°c. titratable acidity (% malic acid) was estimated by titrating fruit juice (5 ml) to ph 8 against 0.1 m naoh using phenolphthale as an indicator. total carotenoids total carotenoids were estimated by the method of ranganna (1977). five grams of fresh sample were taken, a few crystals of anhydrous sodium sulphate were added, and then crushed in 10 ml acetone with the help of a pestle and mortar. the supernatant was decanted into a beaker. the process was repeated twice or thrice and the combined supernatant was transferred to a separating funnel out on standing. petroleum ether (10 to 15 ml) was added in the separating funnel and rinsed; the pigment was then transferred to the petroleum ether phase by diluting the acetone with water or water containing 5% sodium sulphate. the extraction of the acetone phase with a small volume of petroleum ether was repeated, if necessary, until no more colour was extracted. the lower layer was discarded and the upper layer was collected in a 100 ml volumetric flask. the petroleum ether extract was filtered through anhydrous na 2 so 4 and the volume was made up to 100 ml with petroleum ether. the optical density was recorded at 452 nm using petroleum ether as blank containing 3 ml acetone per 100 ml and expressed as µg 100 g-1 pulp. as carotenoids are light sensitive, all steps were performed under subdued light. flesh minerals dried flesh samples from the ripe fruit (unbagged and bagged 55 days after fruit set) were collected and ground with a mortar and pestle, further dried at 70°c for 2 days, then finely ground in a shatter box. a 0.3-0.5 g sub-sample was digested in 15 ml nitric acid/perchloric acid and analysed by atomic absorption spectroscopy against standards prepared in the same matrix. nitrogen was determined using kjeldahl digestion. disease incidence disease incidence was measured 10 days after harvest. anthracnose lesions (caused by colletotrichum spp.) on the side of the fruit, and stem end rot lesions (ser : caused by diplodia spp.) at the stem end of the fruit were rated for incidence (percentage of the fruit affected). statistical analyses data regarding observed characters were statistically analysed by complete randomised design and test of significance was carried out following the method described by panse and sukhatme (1967). 3. results and discussion fruit weight, length and diameter fruit bagging with polyethylene bags significantly increased the weight of fruit as compared with the control at all stages of fruit harvest (75, 85 and 90 days after fruit set). early bagging (35 days after fruit set) proved most effective in increasing fruit weight compared to those bagged later (table 1). it is evident from table 2 that fruit length table 1 effect of bagging on fruit weight (g) at different stages of harvest bagging (days after fruit set) stages of fruit harvest (days after fruit set) 75 85 90 35 303.50 322.75 345.00 45 287.50 296.75 319.60 55 244.75 291.20 310.17 65 229.72 259.86 301.87 control 205.25 229.50 276.02 sem ± 21.22 28.52 24.12 lsd (p= 0.05) 63.62 85.11 72.42 160 increased along with the increase in duration of bagging and also with delayed harvesting. fruits bagged early (35 and 45 days after fruit set) were almost always longer than those bagged later (55 days after fruit set). however, the increase in fruit length due to polybagging was not significant for fruits sampled 85 days after fruit set. like fruit length, the diameter of fruit also increased due to polybagging; the increase in diameter was significant at all stages of fruit harvest (table 3). total soluble solids (tss), total soluble sugar and titratable acidity (ta) total soluble solids and sugar contents of fruit were significantly affected by treatment with polybagging (tables 4 and 5). tss content of ripened fruit was always higher in those bagged later or not bagged at all. at fruit harvest 90 days after fruit set, tss content was higher in fruits that were bagged later (at or after 55 days of fruit set) than the rest of bagged or not bagged fruits, however tss content was greatest in fruits bagged earliest (35 day after fruit set). as tss, total sugar content of fruits was always greater than those that were bagged later (65 days after fruit set) or those that were not bagged at all (control). fruit acidity decreased with a decrease in bagging period (table 6). however titratable acid content of ripened fruits did not show any significant variations due to bagging at different stages of fruit development. carotenoid contents analysis at the eating ripe stage showed that the carotenoid content of fruit increased as harvesting was delayed from 75 to 90 days (table 7). early bagged fruits usually developed less carotenoid upon ripening as compared to those bagged later or to the control. the unbagged fruits table 2 effect of fruit bagging with polyethylene on length (cm) of fruit at different stages of harvest bagging (days after fruit set) stages of fruit harvest (days after fruit set) 75 85 90 35 10.75 12.75 13.91 45 10.30 11.50 12.28 55 10.06 11.35 11.94 65 9.95 11.12 11.82 control 9.40 10.92 11.32 sem ± 0.425 0.414 0.380 lsd (p = 0.05) 1.241 ns 1.144 table 3 effect of fruit bagging with polyethylene on diameter (cm) of fruit at different stages of harvest bagging (days after fruit set) stages of fruit harvest (days after fruit set) 75 85 90 35 7.77 8.00 8.43 45 7.33 7.71 8.25 55 7.19 7.70 7.95 65 7.05 7.44 7.94 control 6.91 7.25 7.68 sem ± 0.172 0.210 0.211 lsd (p = 0.05) 0.521 0.591 0.610 table 4 effect of fruit bagging on total soluble solids (°brix) content of ripe mango fruit bagging (days after fruit set) stages of fruit harvest (days after fruit set) 75 85 90 45 9.90 12.20 15.40 55 10.05 13.50 17.30 65 10.40 14.30 17.85 control 11.90 15.30 16.25 sem ± 0.161 0.072 0.051 lsd (p = 0.05) 0.483 0.219 0.153 table 5 effect of fruit bagging on total soluble sugar (% of fresh weight) content of ripe mango fruit bagging (days after fruit set) stages of fruit harvest (days after fruit set) 75 85 90 35 4.42 6.85 11.81 45 4.77 6.85 12.57 55 5.02 7.52 13.69 65 5.50 7.81 13.82 control 6.20 7.97 12.83 sem ± 0.129 0.061 0.051 lsd (p = 0.05) 0.391 0.180 0.158 table 6 effect of fruit bagging on titratable acid (% malic acid) content of ripe mango fruit bagging (days after fruit set) stages of fruit harvest (days after fruit set) 75 85 90 35 0.250 0.210 0.200 45 0.240 0.220 0.190 55 0.210 0.190 0.180 65 0.180 0.180 0.150 control 0.170 0.160 0.140 sem ± 0.012 0.014 0.015 lsd (p = 0.05) ns ns ns table 7 effect of fruit bagging on carotene (µg/100 g pulp) content of ripe mango fruit bagging (days after fruit set) stages of fruit harvest (days after fruit set) 75 85 90 35 2247.50 4345.50 7782.50 45 2380.00 4773.50 8276.00 55 3565.00 6566.00 8958.50 65 3955.00 7318.50 9168.00 control 4354.00 8485.50 9768.00 sem ± 12.95 17.25 19.00 lsd (p = 0.05) 36.79 52.12 57.12 161 (control) at all stages of harvest developed maximum content of carotene while early bagged fruits (35 days after fruit set) contained minimum carotene upon ripening at all different stages. fruit minerals and disease incidence mineral element contents of fruits varied due to different days of bagging. table 8 shows that n, p, zn, mn and fe content of fruits were higher in bagged fruit (55 days bagged) while k and ca contents were higher in control fruit (0 day bagged) as compared to 55 days of bagging. bagging did not significantly reduce the incidence of anthracnose and stem-rot (ser) (table 9) of ripe fruit. reductions of only 5.5 and 6.0% of anthracnose and stemend-rot, respectively, were noted in fruits bagged 55 days after fruit set. table 8 effect of fruit bagging on mineral elements contents of ripe mango fruit mineral elements days bagged significance 0 55 n (%) dry weight 0.57 0.74 * p (%) dry weight 0.12 0.14 * k (%) dry weight 0.71 0.64 * ca (%) dry weight 0.07 0.03 * zn (ppm) 22.50 23.33 ns mn (ppm) 11.75 12.18 ns fe (ppm) 49.00 49.75 ns assessment were made at eating soft stage. ns indicates p>0.1. * represent effects significant at p<0.05. the results of the present investigation clearly demonstrate the benefits of polybagging on the development of mango fruit and fruit quality. bagging of fruits improved fruit weight and size of litchi (tyas et al., 1998), banana (johns and scott, 1989) and pomegranate (hussein et al. 1994). bagging can affect fruit size through other effects such as increased relative humidity and therefore reduced fruit water loss (tombesi et al., 1993). according to hofman et al. (1997), in the present study fruit mass and size may be due to the use of polybag instead of paper bag. the polybagging of fruits delayed the development of ripening characteristics of fruits. however, the extent of the fruit surface coloured yellow was greater with polybagged fruits, which was due to maintenance of a higher temperature inside the bags. in bagged fruits usually day/night temperature fluctuations were reduced, hence the range of minimum temperatures was between 18 and 27°c, which is very close to the optimal temperature range (24-30°c) for the development of himsagar mango (mukherjee, 1953; whiley et al., 1989). the total soluble solids and total sugar content of ripe mango were almost always greater in control fruits and those with a shorter bagging period as compared to early-bagged fruits. titratable acid content of ripe fruits was greater in early-bagged fruits and acid content of ripe fruits declined as the period of bagging was reduced; minimum acid levels were noted in the control. this is only due to bagging which delayed the development of ripening characteristics of fruits. similarly, the carotene content in the pulp of ripe mango fruit was at maximum in control and minimum in early-bagged fruits. this is mainly due to fact that polybagging of mango fruits delayed the development of ripening characteristics and therefore there was less development of carotene in fruits that were bagged early in comparison to control. these results are in close conformity with the previous findings of singha (2002). however, hofman (1997) opined that fruit quality of mango was not affected by bagging. fruit mineral (n, p, k and ca) contents were significantly influenced by bagging. bagged fruit in the 55-day group had a amount of calcium in comparison to unbagged fruits. calcium is transported mainly in the transpiration stream (grange and hand, 1987), thus tissues with greater transpiration generally have higher tissue ca concentrations (witney et al., 1990 b). increased rh around the fruit can reduce fruit ca accumulation (grange and hand, 1987, combrink et al., 1995) and bagging of apple fruit has been associated with reducing fruit ca concentrations and increasing bitter pit incidence (witney et al., 1991). similar effects were observed in mangoes bagged for 55 days, although the higher fruit ca concentration with longer bagging times may suggest a capacity of the fruit to import ca by a mechanism other than transpiration. longer bagging times may have caused changes in the surface of fruits that allowed higher transpiration rates and thereby the higher ca accumulation noted with increased bagging times as described by hofman et al. (1997). reduction in fruit disease as a consequence of bagging in a number of different fruits has been noted by kitagawa et al. (1992). in mango, colletotrichum infection occurs during fruit development and remains quiescent until fruit ripening (dodd and jeffries, 1989). the reduction in anthracnose incidence with bagging (55 days) could be partly due to a reduction in contact between disease propagules and fruit as mentioned by hofman et al. (1997). in conclusion, bagging of mango can have important commercial benefits as a means to prevent problems like table 9 effect of bagging on fruit disease incidence (percent of fruit affected) parameter incidence (%) days bagged anthracnose stem-end-rot 0 82.5 77.0 55 56.5 50.5 significance ns ns all assessment were made at 10 days after harvest. ns indicates p>0.1. 162 insect attack, sunburn, sap spurt at harvest and damage due to small hail storms by reducing physical damage of fruit, as well as delaying the development of ripening characteristics of fruits. acknowledgements we thank the university grant commission, india for financial support through minor research project. references ann p.j., lu l.s., chuang t.y., kao c.w., 1998 effect of fruit bagging and mulching on control of mango fruit anthracnose disease. plant pathology bull., 7: 19-26. aoac, 1996 aoac methods. association of official analytical chemists, washington, dc., usa. bentley w.j., viveros m., 1992 brown bagging 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journal of horticultural science, 64: 753-765. witney g.w., hofman p.j., wolstenholme b.n., 1990 b mineral distribution in avocado trees with reference to calcium cycling and fruit quality. scientia horticulturae, 44: 279-291. witney g.w., kushad m.m., barden j.a., 1991 induction of bitterpit in apple. j. of amer. soc. of hortic. sci., 47: 173-176. 187 1. introduction ampelography is a traditional morphological method used for the identification and discrimination of varieties of grapevine (vitis vinifera l.). it has also been found to be very useful in detecting and describing variability among chosen potential clone candidates (poljuha et al., 2006). nowadays, a link between historic descriptions and the molecular fingerprints is also very important, especially in cases of possible homonymy and synonymy of autochthonous varieties (cervera et al., 2001). official descriptors have been published to be used together with the analysis of germplasm material (dettweiler, 1993; ortiz et al., 2004). all the organs used in ampelography (leaves, grapes, shoots etc.) usually change their aspect during phenologic phases, hence it is important to find the characteristics able to discriminate between varieties according to these phases: shoots in the early stage, leaves in the moment when they are mature, and later also the grapes and mature berries (cancellier, 2007). the use of fractal-based measurements of digitallyacquired images eliminates problems with different phenologic phases as well as problems with subjectivity. this allows defining the good shape measure that can be effectively applied to leaf shapes, so they can be compared and analysed by meaningful and objective criteria (mancuso, 1999). using fractal parameters and phyllometric outputs, an artificial neural network can be constructed and effectively used to differentiate varieties and accessions. it is an easy method that requires low-cost equipment such as an optical scanner, personal computer and free software (mugnai et al., 2008). st. claire’s vineyard is an historical area located close to the centre of prague, and where a long tradition of grapevine cultivation dates back to the 13th century. following a period of decline, the vineyard is today regaining its former importance. new varieties are being planted and no ampelographic observations have yet been carried out. the main aim of the present work was to discriminate the grapevine varieties cultivated under the climatic conditions of prague (czech republic) using either the subjective method according to the international descriptors published by ipgri (1997), or an objective computing method which consists of analysis using an artificial neural network. these two approaches are also compared in this paper to assess differences between subjective and objective analysis. discrimination of grapevine varieties cultivated in the czech republic by artificial neural networks e. svobodová*(1), c. pandolfi**, p. hlásná čepková*, s. mancuso** * department of crop sciences and agroforestry in tropics and subtropics, faculty of tropical agriscience, czech university of life sciences prague, czech republic. ** dipartimento di scienze delle produzioni agroalimentari e dell’ambiente, università degli studi di firenze, viale delle idee, 30, 50019 sesto fiorentino (fi), italy. key words: ampelography, phyllometry, vitis vinifera, variety identification. abstract: an artificial neural network approach, based on fractal leaf parameters, and classical ampelography were used to identify nine grapevine varieties cultivated at the st. claire’s vineyard, prague botanic garden. fifty healthy, fully-expanded leaves were collected for each variety, scanned using an optical scanner and then elaborated by computer programs. fourteen phyllometric parameters were qualitatively and quantitatively analysed by the digital image analysis. comparative frames were constructed for each variety and the relationships among varieties were assessed using artificial neural networks. results were then compared with the outcome from traditional ampelographic analysis. the artificial neural network technique appears to be a complementary approach to the traditional ampelography methods commonly used for cultivar discrimination, since the equipment necessary for this analysis is very inexpensive and available. application of the technique led to the distinction of nine selected varieties of vitis vinifera. adv. hort. sci., 2012 26(3-4): 187-192 (1) corresponding author: evasvobodova@its.czu.cz. received for publication 3 september 2012 accepted for publication 6 march 2013 188 2. materials and methods plant material leaf samples for the analysis of nine varieties were collected at the st. claire’s vineyard, prague botanic garden. nine varieties (table 1), cultivated in a sufficient quantity to provide a sufficient source of samples, were chosen for analysis. berry and grape samples were collected at the time of the harvest, which was done according to the further utilization of the grapes. table 1 list of selected varieties variety 1 ‘müller thurgau’ 2 ‘gewürztraminer’ 3 ‘rhine riesling’ 4 ‘italian riesling’ 5 ‘moravian muscat’ 6 ‘sauvignon’ 7 ‘blue portugal’ 8 ‘white chasselas’ 9 ‘red chasselas’ morphological and phenological characterization for the classical ampelography, the following method was applied. in the period 2006-2008, 22 morphological and six phenological traits were evaluated in 10 randomly chosen plants for each variety, according to the descriptors for grapevine (iprgi, 1997). all parameters chosen for the study are listed in table 2 with the codes of updated descriptors (oiv, 2009). digital image analysis of leaves, phyllometric parameters and fractal analysis for each variety, 50 healthy, fully-expanded leaves were collected from ten randomly selected plants in late spring 2008. leaf images were acquired (200 dpi, 256 greyscale) using an optical scanner. based on the method described by mancuso and nicese (1999), 14 phyllometric parameters (table 3) were determined for each leaf using image-analysis software (uthscsa image tool program 3.0). table 2 list of characteristics selected for the description of observed cultivars. the codes are in line with oiv guidelines (oiv, 2009) character code description 001 young shoot: aperture of tip 003 young shoot: intensity of anthocyanin colouration on prostrate hairs of tip 006 shoot: attitude 007 shoot: colour of dorsal side of internode 008 shoot: colour of ventral side of internode 051 young leaf: colour of the upper side of blade (4th leaf) 065 mature leaf: size of blade 067 mature leaf: shape of blade 068 mature leaf: number of lobes 070 mature leaf: area of anthocyanin colouration of main veins on the upper side of blade 076 mature leaf: shape of teeth 079 mature leaf: degree of opening/ overlapping of petiole sinus 102 woody shoot: structure of surface 103 woody shoot: main colour 202 bunch: length (peduncle excluded) 204 bunch: density 220 berry: length 223 berry: shape 241 berry: formation of seeds 225 berry: colour of skin 236 berry: particularity of flavour 303 time of beginning of berry ripening (véraison) 233 berry: must yield 502 bunch: weight of a single bunch 503 single berry weight 505 sugar content of must the codes in bold letters indicate the characteristics which were evaluated by anova. table 3 fourteen phyllometric parameters measured by image analysis software parameter definition 1 area the area of the leaf 2 perimeter the perimeter of the leaf 3 major axis length the length of the longest line that can be drawn through the leaf 4 minor axis length the length of the longest line that can be drawn through the leaf perpendicular to the major axis 5 roundness computed as: (4*π*area) / perimeter2 6 elongation the ratio of the length of the major axis to the length of the minor axis 7 feret diameter the diameter of a circle having the same area as the leaf 8 compactness computed as: sqrt(4*area/π) / major axis length 9 integrated density computed as the product of the mean grey level and the number of pixels in the image of the leaf 10 minimum grey level minimum grey level of the leaf 11 mean grey level mean grey level of the leaf 12 median grey level median grey level of the leaf 13 mode grey level mode grey level of the leaf 14 maximum grey level maximum grey level of the leaf 189 the fractal spectrum of the leaves was obtained using fractal image analysis software (harfa, harmonic and fractal image analyzer 4.9.1,), according to the method described by mancuso (2002). briefly, greyscale image of each leaf was thresholded for a grey value between 0 and 255 and the fractal dimension for each grey value was then assessed using the box counting method. the implementation of these methods has been described in detail by mancuso et al. (1999). after drawing the baseline (fractal dimension = 1) which separates the fractal (>1) from the non-fractal (<1) zone of the spectrum, five fractal parameters (first x, peak x, last x, peak y and total peak area) were calculated (fig.1 a). fig. 1 fractal spectrum of one leaf and identification of five parameters to be implemented in the ann. graphical representation of fractal parameters: theoretical situation (a); and a real value (b). artificial neural network analysis an artificial neural network (ann) was constructed as previously described in pandolfi et al. (2009 a). fourteen phyllometric parameters and five fractal parameters were used as input layers, and the nine grapevine accessions represented the output. to optimize the neural network activity, the number of hidden neurons and the number of iterations was modified. with regard to the hidden layer, many factors (such as learning scheme, numbers of nodes of the output and input and connections between them) play an important role for the determination of the best configuration (zurada and malinowsji, 1994). the ann outputs are represented by a xy-graph for each accession, with the accession names on the x-axis, and the yaxis representing the output. each graph aims to show how the ann was able to discriminate the selected accession in comparison with the others. the level of similarity is expressed by number, which ranges between 0 (false) and 1 (true) (fig. 1 b). due to the natural variability among leaves, the output of the expected class tends to report a value close to 1, but less than 1, while the others should be close to 0 (mugnai et al., 2008). statistical analysis statistical analysis of the selected characteristics was performed by analysis of variance (anova) and a comparision was done using the turkey hsd test (program statistica 7.0 cz). the graphical presentation of variability of the tested varieties was carried out using principal component analysis (pca) (software statistica 7.0 cz). ntsys 2.1 was used to investigate neural network outputs performing a cluster analysis by unweighted pair group method analysis (upgma) based on the similarity matrix calculated using the cosine function (eq. 1). equation 1: 3. results morphological data seven characteristics (table 2), as evaluated by anova, showed differences between the varieties during the three consecutive years of sampling, as well as variability within the variety. the number of lobes observed in the mature leaves varied during the study period in three varieties (‘müller thurgau’, ‘rhine riesling’, and ‘blue portugal’). a significant variation was observed both in the weigh of a bunch and the weight of berries, as well as in the size of berries within one variety. with regard to must yield and its sugar content, there was no significant variance among the varieties, however all the varieties demonstrated a significant correlation between the sugar content of the must and the time of berry ripening (r=0.74). the highest sugar content was measured in 2006, when the mean temperatures remained quite high during whole vegetative period (in july as high as 25˚c), whilst the total precipitation was about 5 mm in july. for most of the 19 characteristics that were not included in the anova analysis, there was considerable stability over the course of the years. the greatest variability was shown in the size of a blade of a mature leaf; it remained stable only in ‘rhine riesling’. in the other eight varieties, the size of the leaves varied significantly. correlations between morphological and phenological characters were also assessed. the output obtained from both morphologic and phenological characteristics after three years of observations (fig. 2) was a score plot crefig. 2 pca representation. 190 ated by pca analysis. according to the graph, factor 1 explained 29.02% of the total variation, whereas factor 2 expressed 22.67% of the total. evaluating both approaches (morphological and phenological), ‘red chasselas’ and ‘white chasselas’ were identified as being quite close, which indicated their relationship to one another. the second group was formed in the very centre of the graph and includes ‘blue portugal’, ‘italian riesling’ and ‘sauvignon’. more distant from the centre group and also from each other were ‘müller thurgau’ and ‘rhine riesling’. the only variety which remained completely separate was ‘gewürztraminer’. neural network analysis in all eight cases, the artificial neural network was able to recognise all the accessions presented in the learning phase. throughout there was a clearly defined peak for the selected accession; therefore the varieties were well-separated one from another. the highest level of similarity was present in ‘müller thurgau’ (0.810). however, all other accessions showed quite high average output in a range from 0.541 (‘rhine riesling’) to 0.657 (‘moravian muscat’) (fig. 3). fig. 3 neural networks of eight chosen varieties. ‘müller thurgau’ also showed the lowest degree of similarity with the other varieties and this was confirmed by the upgma diagram, where ‘müller thurgau’ remained completely separated from all other varieties. another interesting result was the position of both ‘chasselas’ in the neural network diagram and the final dendrogram. in the dendrogram, ‘red chasselas’ was more closely related to ‘italian riesling’ than to ‘white chasselas’. using the data from the neural network, euclidean distances were calculated and a dendrogram based on the distance matrix data, by applying an unweighted pair group method with average mean (upgma) cluster analysis, was constructed (fig. 4). fig. 4 upgma dendrogram. the dendrogram formed two clusters, and left two varieties more (‘müller thurgau’) or less (‘moravian muscat’) separated from the others. two other varieties, ‘rhine riesling’ and ‘sauvignon’, were indicated at the same level. even though they were the two most related samples, they still remained quite distant, with the similarity coefficient at 4.43. the results showed a closer relationship between ‘italian riesling’ and ‘red chasselas’ than the relationship between both ‘chasselas’, even though they are considered to be colour variations of the same variety. 4. discussion and conclusions in the present investigation all general characteristics which are common to vitis vinifera, without regarding the differences between the varieties (ipgri, 1997), were confirmed with regard to morphology for the varieties under study. there was only a slight variation in the number of lobes observed in the mature leaves, which is due to the genotype fixation of this trait, as proposed by poljuha et al. (2006). in three varieties (‘müller thurgau’, ‘rhine riesling’ and ‘blue portugal’) the number of lobes varied during the observation period. this way have occurred due to the unisindentification of the depth of the status. (cancellier, 2007). the size of a berry was strongly correlated either with the weight of a berry, or with the weight of a bunch. the smallest berries were observed in an important must variety 191 ‘rhine riesling’. this result corresponded with the findings of fregoni (2005), who indicated that varieties which produce wines of excellent quality generally have smaller berries with higher amount of skin and lower amount of pulp, in comparision with the table varieties. ojeda et al. (2001) pointed out that the size of a berry is an environment-dependent trait. water deficit that occurs in the early phase from flowering to véraison causes an irreversible decrease in cell volume. due to changes in water import by xylem, this may lead to a decrease of mesocarp cell turgor (thomas et al., 2006). under water stress, especially in the early growth stages, the division and elongation of cells decreases (williams, 2000), thus berries remain smaller. in 2008, when precipitation events were regularly distributed throughout the year, the berries of all nine varieties were larger compared to other observation years. sugar content, and thus the quality of the wine, is significantly influenced by the weather, as proven by grifoni et al. (2006) from observations of italian wines. a surprisingly low sugar content was found in ‘moravian muscat’, despite it being a variety bred in former czechoslovakia (pavloušek 1999), and thus adapted to its climatic conditions. however, in this case the sugar content was influenced by the early harvest date, since the grapes were used for the production of federweisser (a freshly fermented must with low alcohol content). the must yield was also strongly correlated to the size of the berry. according to fregoni (2005) table varieties have lower must yields than must varieties. however, ‘white chasselas’, despite being a table variety, gave the highest must yield during the three study years, while the must varieties, like for instance ‘blue portugal’, produced significantly lower quantities the must (57.00±6.25 ml). this may be the influence of rains right before harvest. in all eight cases, the artificial neural network was able to recognise all the accessions presented in the learning phase, as published for example by mancuso (2002) or mugnai et al. (2008). an interesting result was the position of ‘müller thurgau’, which remained separated from all other accessions. dettweiller et al. (2000) confirmed that ‘müller thurgau’ is the descendent of a crossing between ‘madeleine royal’ and ‘rhine riesling’. nevertheless, in our results ‘rhine riesling’ remained distant from ‘müller thurgau’, and was assessed as close to ‘sauvignon’, corresponding with the data published by moravcovà et al. (2004). as reported in their work (moravcová et al., 2004), ‘red chasselas’ is only a mutation of berry colour of ‘white chasselas’. in the dendrogram, however, ‘red chasselas’ was more closely related to ‘italian riesling’ than to ‘white chasselas’. comparing the dendrogram with the pca score plot, the results were slightly different. both ‘chasselas’ varieties were the closest, which corresponded with the results of moravcovà et al. (2004). but also in this case, there was no evident relationship between ‘rhine riesling’ and ‘müller thurgau’, since they occurred in different quadrants. unfortunately, the position of ‘gewürztraminer’ in the pca graph could not be compared with the ann output. however, the score plot put this variety completely separate from other varieties, e.g. ‘rhine riesling’, as already described by imazio et al. (2002). the great distance of ‘gewürztraminer’ from other varieties might also be due to its closer relationship to vitis silvestris (regner et al., 2000; lacombe et al., 2003). when using the ann approach it is very important to choose well-developed, healthy leaves (mugnai et al., 2008). an ann needs a suitable set of sample data to achieve excellent quality in the training set, as this operation can be usually defined as the key point of all the ann building process (pandolfi et al., 2009 a). unfortunately, the euclidean distance did not permit a clear clustering and thus in our case a pca graph could be considered a more precise tool, even though the data were mostly obtained from subjective observations. however, the possibility of a primary misidentification while still in the vineyard must always be considered: in early spring, when the plants do not have mature leaves or even grapes, an identification error may occur, especially when the varieties are not clearly divided. both approaches ampelography or artificial neural networks – are important methods based on morphological traits, which complement each other to give clear classification of the varieties. the ann technique represents an more economic alternative to genetic methods commonly used for cultivar discrimination since the equipment necessary for this analysis is inexpensive and commonly available (pandolfi et al., 2009 a). in this study, application of anns led to the distinction of nine selected varieties of vitis vinifera. for the further uses, the ann profiles obtained from our analysis may be kept in databases for breeding programs, as well as for the description of new cultivars. the data obtained might also be helpful for rapid and reliable identification and description of still unknown varieties, as shown by pandolfi et al. (2009 b). acknowledgements the authors would like to thank st. clair’s vineyard (prague botanic garden), especially martin beránek, ph.d., for his support and assistance during the experiment. we would also like to acknowledge an unknown reviewer for his/her kind and useful suggestions. references cancellier s., 2007 proposta di una guida per il riconoscimento dei vitigni. esempio: i vitigni di veneto. rivista di viticoltura e di enologia, 60(1): 3-16. cervera m.t., rodriguez i., cabezas j.a., chavez j., martínez-zapater j.m., cabello f., 2001 morphological and molecular characterization of grapevine accessions known as ‘albillo’. amer. j. of enol. and vitic., 52(2): 127-135. 192 dettweiler e., 1993 evaluation of breeding characteristics in vitis.vitis, 32: 249-253. dettweiler e., jung a., zyprian e., töpfer r., 2000 grapevine variety ‘müller-thurgau’ and its true-to-type descent. vitis, 39(2): 63-65. fregoni m., 2005 viticoltura di qualità. 2nd edition, phytoline s.r.l., affi, pp. 819. grifoni d., mancini m., maracchi g., orlandini s., zipoli g., 2006 analysis of italian wine quality using freely available meteorological information. amer. j. of enology and viticulture, 57(3): 339-346. imazio s., labra m., grassi f., winfield m., bardini m., scienza a., 2002 molecular tools for clone identification: the case study of the grapevine variety ‘traminer’. plant breeding, 121: 531-535. ipgri, 1997 descriptors for grapevine (vitis spp.). international union for the protection of new varieties of plants, geneva, switzerland; office international de la vigne et du vin, paris, france and international plant genetic resources institute, rome, italy. lacombe t., laucou v., di vecchi m., bordenave l., bourse t., siret r., david j., boursiquot j.m., bronner a., merdonoglu d., this p., 2003 contribution à la caractérisation et à la protection in situ des populations de vitis vinifera l. ssp. silvestris (gmelin) hegi, en france. les actes du brg, 4: 383-404. mancuso s., 1999 fractal geometry-based image analysis of grapevine leaves using the box-counting algorithm. vitis, 38(3): 97-100. mancuso s., 2002 discrimination of grapevine (vitis vinifera l.) leaf shape by fractal spectrum. vitis, 41(3): 137-142. mancuso s., nicese f.p., 1999 identifying olive (olea europaea) varieties using artificial neural networks. j. amer. soc. hort. sci., 124(5): 527-531. moravcová k., baránek m., pidra m., 2004 the use of rapd markers for differentiation of grapevine varieties registered in the czech republic. horticultural science (prague), 31 (3): 96-101. mugnai s., pandolfi c., azzarello e., masi e., mancuso s., 2008 camelia japonica l. genotypes identified by an artificial neural network based on phyllometric and fractal parameters. plant systematics and evolution, 270: 95-108. oiv, 2009 the 2nd edition of the oiv descriptors list for grape varieties and vitis species. office international de la vigne et du vin, paris, france. ojeda h., deloire a., carbonneau a., 2001 influence of water deficits on grape berry growth. vitis, 40(3): 141-145. ortiz j.m., martín j.p., borrego j., chávez j., rodríguez i., muñoz g., cabello f., 2004 molecular and morphological characterization of a vitis gene bank for the establishment of a base collection. genetic resources and crop evolution, 51: 403-409. pandolfi c., messina g., mugnai s., azzarello e., masi e., dixon k, mancuso s., 2009 b discrimination and identification of morphotypes of banksia integrifolia (proteaceae) by an artificial neural network (ann), based on morphological and fractal parameters of leaves and flowers. taxon, 58(3): 925-933. pandolfi c., mugnai s., azzarello e., bergamasco s., masi e., mancuso s., 2009 a artificial neural networks as a tool for plant identification: a case study on vietnamese tea accessions. euphytica, 166: 411-421. pavloušek p., 1999 vinohradnictví-odrůdy révy vinné. 1. vydání. mendelova lesnická a zemědělská univerzita v brně, brno, pp. 122. poljuha d., sladonja b., peršurić d., 2006 evaluation of intravarietal variability of cv. ‘istrian malvasia’ by the ampelographic parameters. rivista di viticoltura e di enologia, 2(3): 13-18. regner f., stadlbauer a., eisenheld c., kaserer h., 2000 considerations about the evolution of grapevine and the role of traminer. acta horticulturae, 528: 179-184. thomas t.r., matthews m.a., shackel k.a., 2006 direct in situ measurement of cell turgor in grape (vitis vinifera l.) berries during development and in response to plant water deficits. plant, cell and environment, 29: 993-1001. williams l.e., 2000 grapevine water relations, pp. 121126. in: christensen l.p. (ed.), raisin production manual. university of california, division of agriculture and natural resources, publication 3393, oakland, ca. zurada j.m., malinowsji a., 1994 multilayer perceptron networks: selected aspects of training optimization. international journal of applied mathematics and computer science, 4: 281-307. 143 1. introduction summer pruning is a cultural techniques which drives vine vigour to ensure fruit quality and plant vegetative balance. while summer pruning is the most expensive cultural operation 44.2% of total management costs (crescimanno et al., 2011) it helps to improve the microclimate in the canopy, promotes good ripening of the grapes and creates less suitable conditions for the development of pathogens. good results depend on the vegetative-productive behaviour of the vineyard, intensity and age of cultural operation (crescimanno et al., 1986). summer pruning defines the final productivity of plants by modifying the number of shoots per plant with shoot thinning, the number of clusters per shoot with cluster thinning, and the number of berries per bunch with berry thinning. other summer operations include leaf removal, shoot trimming and girdling (di lorenzo, 2003). general indications about summer pruning techniques to enhance quality of production are very difficult to formulate because cultivar behaviour, vigour of the vineyard and environmental conditions must all be considered. 2. leaf removal leaf removal causes a reduction of vine leaf area. if it occurs at or before bloom, it may cause berry drop, a reduction in fruit set or a reduction in bud fertility in the following season (candolfi-vasconceloset and koblet, 1990). the intensity of leaf removal should be based on canopy density and light penetration into the fruit zone. the removal of basal leaves around the clusters is widely adopted to improve grape quality and to reduce the incidence of fungal infection (gubler and marois, 1987; caspari et al., 1998). leaf removal should be performed near berry set or after fruit softening (dokoozlian et al., 2000 a). the leaves immediately above the cluster are the main source for photosynthates translocated to the cluster, particularly during the early stages of its development (hunter and visser, 1988). also at pea-size stage the loss of basal leaves increases fruit abscission, reduces berry size and decreases bud fertility; it has no effect when applied at veraison (caspari et al., 1998). after berry setting, usually all primary leaves and lateral shoots beginning from the base of the shoot to the node opposite the top cluster on each shoot are removed. elimination of apparently superfluous sinks, such as lateral shoots, reduces canopy density and °brix, but it has minor impact on ta and ph (reynolds and wardle, 1989; barbagallo et al., 2007 a). the leaves left on the vines after defoliation increase photosynthetic activity to recover the reduction on total leaf area activity and to supply the photoassimilates demand of sinks (poni et al., 2006; scafidi et al., 2010). on the other hand, candolfi-vasconcelos and co-workers (1994) found that defoliated plants had similar or even slightly lower photosynthetic rates compared to control plants, not only during the stress period but also in the following season. a photosynthesis response to leaf removal may be apparent only if the source-sink ratio is sufficiently limited. under conditions of source deficiency due to leaf removal in the fruit zone, plants promote the activity of apical meristems to replace the missing leaf area (barbagallo et al., 2007 b). basal leaves should not be removed before veraison, especially in varieties susceptible to heat damage or sunburn like ‘red globe’, ‘thompson seedless’. during fruit ripening leaves opposite the clusters have limited importance compared to the younger leaves at the top of the canopy (candolfi-vasconcelos et al., 1994; hunter et al., 1995). younger leaves show a higher transpisummer pruning in table grape r. di lorenzo, c. gambino, p. scafidi dipartimento demetra, università degli studi di palermo, viale delle scienze, 11, 90128 palermo, italy. key words: girdling, leaf removal, thinning, trimming, vitis vinifera l. abstract: this paper reviews cultural practices to improve fruit quality in table grape during vegetative and reproductive seasons. summer pruning in table grape (vitis vinifera l.) has more effects than winter pruning, above all with regard to plant productivity and final number of bunches for harvesting. thinning is one of the most cultural technique and it consists in the elimination of vegetative or reproductive organs in excess. other summer canopy management techniques include leaf removal, fruit shoots positioning, shoot trimming and girdling. adv. hort. sci., 2011 25(3): 143-150 received for publication 9 june 2011 accepted for publication 8 september 2011 144 ration rate, but also higher water use efficiency than those opposite the clusters (candolfi-vasconcelos et al., 1994). some weeks before harvest random defoliation is usually undertaken to fully develop the colour of white, red and some black grape varieties. 3. thinning thinning consists in the elimination of vegetative or reproductive organs in excess. it is very rarely performed before bloom since negative climatic events can lead to the loss of many shoots or irregular fruit set; in some areas and for some cultivars thinning performed before bloom can lead to excessive fruit set and tight bunches. shoot thinning shoot thinning is the elimination of double, weaker and sterile shoots and it is very important to aerate the canopy, improve the growth of remaining shoots and adjust cluster numbers. there may be an advantage with shoot thinning in vigorous vines to reduce shoot crowding and thus increase light exposure of the remaining shoots. shoot thinning should be performed when shoot length reaches 25-30 cm. (dokoozlian et al., 2000 b) when it is possible to define which shoots have bunches in good position and which are well located as pruning material for the next year. on spur-pruned vines two shoots per spur are retained and latent shoots are removed from older wood, arms and cordons, while cane-pruned vines are sometimes shoot thinned, especially when several canes are wrapped together on a single wire. cluster thinning cluster thinning is usually performed after fruit set in order to adjust the crop load, distribute clusters evenly on the vine and canes, select the best clusters (shape, size and position) and eliminate those that are misshaped and weak (figs. 1 and 2). generally the aim is to have an equal number of cluster and shoots on the plant, leaving two clusters on the distal shoots. the number of flowers per inflorescence, berry per cluster and cluster weight (table 1) is positively affected by the node position long the cane (sottile et al., 1996). a cluster/shoot ratio of less than 0.8 usually determines a reduction in terms of yield (tables 2 and 3) without any significant improvement in terms of quality (di lorenzo, 2003). several studies demonstrated that crop removal significantly increases soluble solids (fig. 3) and berry colour (dokoozlian et al., 1995). in a trial conducted on ‘flame seedless’ in fresno california, berry weight, size and fruit composition varied little among vines thinned one week prior to bloom and those thinned four weeks following fruit set (dokoozlian et al., 1995). berry thinning berry thinning is a widely performed technique and involves the removal of a few berries from the cluster (di lorenzo, 2003). this operation is necessary to decrease the compactness of bunches and to give them a more attractive shape with large, uniform-size berries (fig. 4 table 4). berry thinning is performed when berries are at pea-size in order to give more uniform clusters in terms of weight and shape, satisfying packaging and marketing needs. in some cases for some cultivars, the partial removal of inflorescence or flowers with small scissors or small combs is performed in order to avoid an excessive clusters weight and/or closeness (di lorenzo, 2003). how the berries are removed depends on the cultivar. the best results are obfig. 1 effect of cluster thinning on evolution of berry weight (dotted line) and volume (continuous line) in treated (square) and control (triangle) plants. fig. 2 cluster thinning. table 1 influence of node position on number of flowers per inflorescence, number of berries per cluster, and berry and cluster weight (sottile et al., 1996) node position flower (no.) berry (no.) berry weight (g) cluster weight (g) 1-4 258 101 7.0 779 5-9 517 128 7.3 1015 10-12 744 148 6.9 1090 145 tained with the “helicoidal” method, which consists of eliminating shoulders arranged in a spiral around the axis of the rachis. another method is the “fish spine” system where two parallel cuts are made on each side of the axis of the rachis, but without injuring it. the resulting bunch is very flat, but when the berries grow, the respective ramification occupies the space around the rachis. in ‘thompson seedless’ the most common method is to clip the cluster leaving only the upper four to six shoulders (dookolzian et al., 1995); in ‘red globe’ and ‘flame seedless’ usually the upper six to eight shoulders are kept (dookolzian and hirschfelt, 1995); in superior seedless® one-third of the bottom part of the cluster is removed. in cultivars such as ‘italia’, instead, berry thinning requires plucking small seedless or irregularly developed berries by hand, a very expensive operation which may take up to 50-80 labour days/hectare. in seedless varieties the use of giberelic acid (ga3) is widespread; dose and time of application is highly dependent on the variety. the success of treatment is extremely variable, and is greatly influenced by climate during flowtable 2 influence of cluster:bud ratio on production of grape cv. italia. score of grapes from different vineyards and different thesis (crescimanno et al., 1986) vineyards different time i ii iii 0.6 0.8 0.6 0.8 0.6 0.8 1 627.45 509.15 592.20 565.40 581.17 499.62 2 680.17 ab 539.25 b 724.47 a 708.97 a 731.80 a 704.95 a 3 336.27 abab 305.20 abcd 355.67 aab 230.77 bc 393.22 aa 303.65 abbc 4 948.55 816.72 960.07 842.97 846.05 860.92 2a 919.72 890.85 945.70 845.75 851.62 894.35 3a 821.75 ab 871.47 ab 698.35 b 782.85 ab 946.00 a 787.62 ab 4a 832.05 a 633.78 b 714.30 ab 756.52 ab 818.62 ab 774.40 ab values sharing the same letter are not significantly different from each other by duncan’s multiple range test at p≤ 0.01 and p≤ 0.05. table 3 influence of cluster:bud ratio on production of grape cv. italia. score of grapes from different vineyards and different thesis (crescimanno et al., 1986) different time i ii iii vineyards 0.6 0.8 0.6 0.8 0.6 0.8 1 2 9 8 9 8 8 8 3 4 9 8 8 8 7 8 2a 10 9 8 8 9 8 3a 10 9 9 9 9 8 4a 10 7 10 10 8 8 thesis average 9.6 8.4 8.8 8.6 8.2 8 time average 9 8.7 8.1 = poor quality of the product (not packable). fig. 3 effect of cluster thinning on evolution of °brix content (dotted line) and total acidity (continuous line) in berries of treated (square) and control (triangle) plants. fig. 4 berry thinning. 146 ering (especially air temperature). one of the goals of breeding programs is to obtain varieties that do not require berry thinning. 4. girdling and cane-scoring girdling is the removal of a ring of bark (only phloem) around the trunk or bases of the individual canes, while scoring is a simple knife-cut encircling the branch (fig. 5 a and b). the phenological stage at which girdling is carried out is the greatest factor determining the nature and magnitude of the obtained effects (di lorenzo, 2003). both operations stop movement through the phloem, modifying the hormonal balance of the vine after girdling (kriedemann and lenz, 1972) and consequently producing an increase of carbohydrates above the girdle (weaver and mccune, 1959); carbon exchange between the shoot and the rest of the vine is thus eliminated. girdling reduces net co2 assimilation rate and stomatal conductance of leaves until the girdle heals (kriedemann and lenz, 1972; williams and ayars, 2005). water use efficiency decreases following girdling without an application of ga3 at berry set. once the girdle heals, vine water use increases up to harvest (bucks et al., 1985; williams and ayars, 2005). the reduction in stomatal conductance, and concomitant reduction in vine water use in response to girdling is probably due to an accumulation of abscisic acid (aba) in the leaves (loveys and kriedemann, 1974; during, 1978; williams et al., 2000; williams and ayars, 2005). girdling has negative effects on some berry characteristics, such as a decrease of malic acid concentration in the must (orth et al., 1994). the effect of girdling is reduced by leaf removal and declines while the number of leaves decreases (caspari et al., 1998). cane girdling at 12°brix sugar content on cv. vittoria determines a qualitative improvement of grapes (tables 5 and 6): particularly, single girdling increases ratio sugar: acidity, double girdling (first time performed at pea-size stage, second time at veraison) increases the berry weight (fig. 6) (di lorenzo and gambino, 2010). canescoring increases the average berry size of ‘emperatriz’ seedless grape and bunch weight compared to unscored vines, but has no effect in ‘aledo’ seeded grape (casanova et al., 2009). the author supposes that in seeded fruits the availability of carbohydrates is guaranteed by the seed’s ability to synthesize plant growth hormones leading to powerful sink capacity, while seedless fruit has an insufficient sink capacity to grow. trunk girdling is a more rapid technique than cane girdling and all clusters are subjected to treatment. with cane girdling or scoring there may be a few clusters, located below the cut, that remain unaffected. the bark ring removed has to be complete; incomplete cuts result ineffective (jensen et al., 1979). usually the girdle cut heals in approximately four weeks through callus formation that recovers the vascular connections (williams et al., 2000). girdling and cane scoring are carried out seven to 10 days before flowering to improve berry-set, at berry set to increase berry size, and at veraison to advance sugar and colour development in red varieties. table 4 effect of two different thinnings on qualitative traits (di lorenzo, 2003) thinnings cluster weight (g) berry weight (g) berry weight variation coefficient (%) closeness index thinning intensity more than 40% of traditional 626 10.6 23.1 2.59 “traditional” berry thinning 934 8.9 28.1 3.39 fig. 5 cane girdling and trunk girdling. a 147 in a trial of trunk girdles applied at fruit set on ‘crimson seedless’, vines girdled at fruit set produced larger berries compared to vines girdled at berry softening and ungirdled vines. trunk girdles applied at fruit set increased berry weight 38%, berry length 12% and berry diameter 10% compared to the fruit of ungirdled vines. the berry weight and diameter of vines girdled at veraison were significantly lower than those of ungirdled vines, while the berry length of these treatments was similar. in addition, berry firmness of vines girdled at fruit set was significantly greater compared to vines girdled at berry softening and ungirdled vines. due primarily to their larger berry size, the total yield of vines girdled at fruit set was approximately 45% greater than vines girdled at berry softening and ungirdled vines. a fruit quality defect among the girdling treatments was poor colour, and so only a portion of this increase in total yield was packable fruit (dokoozlian et al., 1995; dokoozlian et al., 2000 a). in contrast, in the same variety, brar and coworkers (2008) indicated that girdling at berry set was an effective practice to stimulate berry colour development. in ‘autumn royal’ berry weight can be increased 10 to 15% by girdling at berry set, but also in this variety girdling delays colour development and harvest (dokoozlian et al., 2000 a). trunk girdling at berry set and bunch thinning, in an early-season black seedless table grape variety (sugrathirteen® or midnight beauty®) improved berry size, sugar content and berry firmness (gentilesco et al., 2011). girdling increases the risk of skin burn, and should never be done on the same vine more than once a year. repeated girdling over a number of years may reduce bunch size and the life expectancy of the plant. 5. shoot trimming intensive growth of vines in warm climates requires measures to control vigour in order to ensure fruit quality and vegetative balance of the plants. the main control measure, besides the careful use of water and fertilizers, is shoot trimming which is usually performed after flowering; the exact moment depends on the cultivar and the objective of the culture (camargo, 2005). shoot trimming carried out just before bloom may improve fruit set: in fact in this stage it stops trophic competition of top shoot. in “t”, “y” or open gable trellis, shoot trimming or hedging can be performed to improve cluster exposure to sunlight and to reduce humidity within the fruit zone. early hedging may stimulate lateral shoot growth. hedging should be performed after berry softening to avoid potential problems with fruit sunburn. both sides of the canopy should be trimmed to allow the uniform penetration of sunlight into the canopy interior. care must be taken not to remove too much foliage when hedging as excessive foliage removal may slow fruit maturation and significantly retard fruit colour development (dokoozlian et al., 2000 a). table 5 effect of early girdling (1), girdling at 12°brix sugar content (2), and double girdling (3) on parameters of berries at ripening on cv. vittoria (di lorenzo et al., 2010) average berry weight ±se (g) weight range average p.d. ±se (mm) average e.d ±se (mm) average berry form (dp/de)<6 6-8 > 8 1 8.3 c ±0.20 15 42 43 27.3 bc ±0.39 21.1 b ±0.30 1.30 ±0.02 2 7.4 b ±0.16 21 44 35 26.8 b ±0.23 19.7 a ±0.20 1.47 ±0.01 3 8.7 c ±0.19 10 35 55 27.8 c ±0.33 21.0 b ±0.17 1.32 ±0.01 control 6.8 a ±0.10 38 43 19 25.0 a ±0.21 21.1 b ±0.14 1.18 ±0.01 table 6 effect of early girdling (1), girdling at 12°brix sugar content (2), and double girdling (3) on harvest parameters (cv. vittoria) (di lorenzo et al., 2010) soluble solids °brix total acidity (g/l) 05-07 15-07 05-07 15-07 1 13.2 b 13.3 a 5.8 a 5.5 a 2 12.0 a 14.2 b 6.2 b 5.8 b 3 13.2 b 13.5 a 5.8 a 5.9 b control 12.0 a 13.5 a 6.2 b 5.8 b fig. 6 effect of early girdling (1), girdling at 12°brix sugar content (2), and double girdling (3) on growth rate of the shoot and berry (di lorenzo and gambino, 2010). 148 6. plant growth regulators plant growth regulators play a notable role in current worldwide table grape cultivation. some of these can be included among summer management techniques, in order to reduce berry set, increase berry size and accelerate or improve fruit ripening. before discussing their effects and possible uses, it must be pointed out that in each country there are different rules and regulations for their use (e.g. forchlorfenuron and ethephon are forbidden in many countries). gibberellic acid (ga3) gibberellic acid (ga3) is commonly used to reduce fruit set and increase berry size of seedless table grape cultivars. ga3 rates and timing applications are quite specific and depend on the cultivar, region, and desired effects on berry growth and fruit quality (dokoozlian et al., 1995). ga3 sprays are generally carried out: several weeks before bloom to elongate the cluster rachis. while many studies have reported that pre-bloom ga3 application has no effect on cluster length or compactness at harvest (dokoozlian, 2000), commercially it is still used (about 10 ppm rate). it could have a negative influence on bud fruitfulness in the following year. between 30 and 100% bloom to improve berry thinning. the mechanism by which gibberelic acid works as fruit thinner is still not understood. an initial hypothesis was that ga3 acts as a pollenicide, interfering with pollen germination, however many studies have shown that the ga3 concentration normally applied for thinning does not reduce pollen germination. some authors suggest, instead, that ga3 applied at bloom alters the endogenous hormone balance causing flower or fruit abscission. the most reliable hypothesis is that ga3 induces nutrient competition between flowers and shoots, and among flowers/small fruits within the cluster. in the latter case ga3 stimulates nutrient competition among berries, and so physiologically advanced berries become strong sinks, while weaker berries are unable to compete for nutrients and drop (dokoozlian, 2000). the ga3 rate is closely related to variety and climate conditions, and it can vary from 1 to 20 ppm. a higher rate of ga3 applied at bloom generally does not improve thinning, but can significantly increase the number of shot berries per cluster. single or multiple applications usually result in similar levels of fruit thinning, however it seems that multiple applications produce larger berries at harvest compared to single applications (dokoozlian, 2000). ga3 spray at bloom often produces inadequate levels of berry thinning, which results in a need for manual berry thinning. after fruit set to increase berry size. gibberellic acid applied to growing berries increases cell division and elongation. also in this case the rate depends on the cultivar and prefixed quality target. the timing of application has a big influence on the efficacy of treatment; usually berry size should be in the range 4-6 mm, to a maximum of 10 mm. ga3 treatments can increase berry size at harvest 50% or more, but they delay fruit maturity and reduce berry colour in red varieties (dokoozlian, 2000). also in this stage, high rates might cause a decrease in bud fruitfulness in the following year. ga3 molecules enter in plant tissues better if applied in low ph solution (ph ≈ 4) since at low ph ga3 molecules are neutral and are able to move easily through plant tissues. forchlorfenuron (cppu) forchlorfenuron (cppu) is a synthetic cytokinin that increases cell division and elongation. usually, cppu can be sprayed on grape: immediately before bloom to increase fruit set (≈ 10 20 g/ha) (dokoozlian, 2000); after fruit set to increase berry size (≈ 5 40 g/ha). in different varieties (‘thompson seedless’, ‘ruby seedless’, ‘redglobe’ and ‘melissa’) cppu applied at fruit set increased berry weight, diameter and length, while cppu applied at fruit softening had no significant effect on berry growth. a two-week delay in harvest of most cultivars was obtained when 9-12 mg/l cppu was applied at berry set, while pigment accumulation was either delayed or significantly reduced (dokoolzian, 2001). cppu does not reduce the fruitfulness of either seedless or seeded table grape cultivars, while it increases the rachis size and the force required to remove berry from the capstem (dokoolzian et al., 1995). ethephon ethephon (trade name ethrel®) is commonly applied to red-pigmented table grape cultivars at the beginning of fruit ripening to enhance berry colour. the active ingredient in ethephon, [(2-chloroethyl) phosphonic acid], produces ethylene upon its degradation. ethylene is an endogenous plant hormone that accelerates the ripening of many fruits, including grapes. ethephon, applied on ‘crimson seedless’ when approximately 5 to 10% of the berries were showing red colour, had no effect on fruit soluble solids content, however vines treated with ethephon had lower titratable acidity compared to untreated vines (dokoozlian et al., 1995). ethephon had no significant effect on berry weight, length or diameter, while it significantly improved fruit colour, increasing packable yield (+38%), but significantly reduced berry firmness compared to untreated vines (dokoozlian et al., 1995). abscisic acid (aba) the plant hormone abscisic acid (aba) appears to be one of the factors for anthocyanin accumulation. exogenous applications of aba increased the anthocyanin content of grape skins (peppi et al., 2006; peppi et al., 2007). 149 application of abscisic acid (aba) may improve colour more effectively than ethephon, but it may potentially influence postharvest quality, though in a trial carried out on ‘crimson seedless’ the aba and ethephon treatments did not affect berry firmness or predispose the fruit to postharvest shatter (cantína et al., 2007). in that trial grapes treated with 300 μl l−1 aba coloured quickly and thus were harvestable about 30 days earlier than untreated grapes, and 10 days earlier than grapes treated with ethephon. on average, grapes treated with 150 μl l−1 aba were harvestable at about the same time as grapes treated with 300 μl l−1 aba or ethephon, and grapes treated with either 150 μl l−1 aba or ethephon were harvestable about 15 day before non-treated grapes. however, tss, ta, and the ratio of tss to ta differed among treatments. grapes treated with 300 μl l−1 aba were harvested at the lowest tss, followed by grapes treated with 150 μl l−1 aba, and grapes treated with ethephon or not treated. grapes treated with 300 μl l−1 aba or ethephon had the highest acidity (≈ 5.0 g l−1) and the lowest tss:ta ratio (cantína et al., 2007). in ‘flame seedless’ 300 ml l−1 aba applied at veraison was superior to the other aba concentrations and to ethephon applied at any of the tested times. moreover, any concentration of aba between 75 and 300 mg l−1 applied after veraison improved colour better than ethephon applied at the same time (peppi et al., 2006), although the same rate in ‘red globe’ increased pigmentation and improved colour, it also caused fruit softening (peppi et al., 2007). references barbagallo m.g., grippi f., scafidi p., lino t., 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of thompson seedless, red malaga and ribier grapes. hilgardia, 28: 421456. williams l.e., ayars j.e., 2005 -water use of thompson seedless grapevines as affected by the application of gibberellic acid (ga 3) and trunk girdling practices to increase berry size. agricultural and forest meteorology, 129: 85-94. williams l.e., retzlaff w.a., yang w., biscay p.j., ebisuda n., 2000 effect of girdling on leaf gas exchange, water status, and non-structural carbohydrates of fieldgrown vitis vinifera l. (cv. flame seedless). am. j. enol. vitic., 51: 49-54. 9 1. introduction temperature is a limiting element for distribution of plants worldwide (sakai and larcher, 1987). cold season turfgrasses such as festuca in temperate regions have a good adaptability to low temperature, but under severe winter conditions may suffer considerable damage (levitt, 1980). although fescues are cultivated in transition zones, warm season turfgrasses such as bermudagrass are preferred (carrow, 1994). plant responses to cold stress and subsequent adaptation occurs at physiological and biochemical levels, as well as cellular and molecular extents (gulzar et al., 2011). harsh low temperatures result in oxidative stress and a change in proline and sugar content of the cells. the vital means for interaction of plants to these stresses is a balance between antioxidant enzymes and reactive oxygen species (ros). the damaging ros responsible for oxidative stress consists of free radicals: superoxide (o 2 .-), hydroxyl (oh.), hydroperoxyl (ho 2 .) and other molecules such as hydrogen peroxide (h 2 o 2 ) and singlet oxygen (1o 2 ) (gill and tuteja, 2010). the precise role of antioxidant enzymes which give tolerance to cold stress in turfgrasses has not yet been investigated, but their relieving effect to other oxidative stresses has been reported by other researchers (jiang et al., 2005). rogers et al. (1975) examined the proline amount of zoysia japonica steud. ‘meyer’ during the months from october to march and found that there is an increase in proline from october to december. it has been shown that for the period of adaptation to cold, sod and cat activity in agrostis stolonifera l., poa pratensis l. and lolium perenne l. significantly increased (sarkar and bhowmik, 2009). the main objective of the present study was to investigate the effects of low temperature stress on biochemical and physiological responses of tall fescue and common bermudagrass. to the best of our knowledge, this is the first report on how these turfgrasses counter cold stress. 2. materials and methods plant materials and experimental conditions the experiment was conducted in a growth chamber (gallenkamp, germany) at the department of horticultural sciences at the college of agriculture, shiraz university (29°36’ n and 52°32’ e, elevation 1810 m). seeds of common bermudagrass (cynodon dactylon [l.] pers. ‘california origin’) and tall fescue (festuca arundinacea schreb. ‘starlett’) were cultivated in 5 l plastic pots containing a biochemical and physiological adjustments in common bermudagrass (cynodon dactylon [l.] pers.) and tall fescue (festuca arundinacea schreb.) under low temperature stress r. manuchehri, h. salehi(1), a. jowkar department of horticultural science, college of agriculture, shiraz university, shiraz, iran. key words: antioxidant enzymes activity, common bermudagrass, low temperature stress, tall fescue, turfgrasses. abstract: low temperature is a restrictive factor for turfgrass growth and development in temperate regions. a study was conducted with the purpose of examining the physiological and antioxidant response of two turf species, festuca arundinacea schreb. ‘starlett’ and cynodon dactylon [l.] pers. ‘california origin’ to cold stress in a growth chamber at the college of agriculture, shiraz university. five temperatures (25, 15, 7.5, 0 and -7.5°c) in four replicates were examined in a completely randomized design experiment. it was revealed that under low temperature stress, soluble sugar contents, proline, malondialdehyde (mda) and hydrogen peroxide (h 2 o 2 ) were increased in both turfgrasses. antioxidant enzyme activity, particularly catalase (cat, ec 1.11.1.6) and superoxide dismutase (sod, ec 1.15.1.1), was increased as a result of temperature reduction from 25°c to 0°c. tall fescue is thought to be better adapted to cold stress than common bermudagrass due to higher soluble sugar contents, proline, malondialdehyde and antioxidant enzyme activity. the results show that scavenging enzymes have a direct effect in cold season tolerance of turfgrass and improve the defense mechanism of plants, but their exact role merits further investigation. adv. hort. sci., 2014 28(1): 9-13 (1) corresponding author: hsalehi@shirazu.ac.ir received for publication 6 december 2013 accepted for publication 18 march 2014 short note 10 mixture of 1:2 (v/v) of loamy soil/decomposed farmyard manure. irrigation was carried out on a daily schedule. established turfs were clipped from 3 cm above ground by a hand mower and were transferred to the growth chambers prior to the application of treatments. all treatments received a constant light intensity of 3000 lux, relative humidity of 65±5% and a 12 h photoperiod. low temperatures were maintained at 25, 15, 7.5, 0 and -7.5°c for 48 h. experimental design and data analysis experiment factors were arranged in a completely randomized design with four replications. data were analyzed using sas software (ver. 9.1.3) and means were compared using the least significant difference (lsd) test at p<0.05. reducing sugars and proline content phenol-sulfuric acid reactions were used to determine the reducing sugar content. shoot samples were oven dried at 60°c for 48 h and then ground to a fine powder using an electric mill. samples (0.2 g) were diluted with 80% ethanol and centrifuged at 13500 rpm. supernatant was further diluted to 25 ml by 80% ethanol. then, 1 ml of extract was mixed with 1 ml of 5% phenol. five ml of concentrated sulfuric acid were added to tubes and immediately stirred. light absorption was measured by a spectrophotometer (biochrome, uk) at 490 nm wavelength (dubois et al., 1956). proline was determined according to the method used by bates et al. (1973) using a spectrophotometer at 520 nm wavelength. measurement of antioxidant enzyme activity to extract antioxidant enzymes, fresh leaf samples (0.5 g) were collected and ground to a fine powder in a mortar by adding liquid nitrogen and then homogenized with an ice cold enzyme extraction buffer containing 0.5% polyvinylpyrrolidone (pvp), 3 mm edta, and 0.1 m potassium phosphate buffer (ph=7.5). the extracted samples were centrifuged for 10 min at 13500 rpm and 2-4°c and stored on ice until used. the resulting supernatants were used for enzyme analysis. cat activity was determined according to the procedure used by dhindsa et al. (1981) and sod activity was determined as described by beauchamp and fridovich (1971). malondialdehyde (mda) as for h 2 o 2 , 0.25 g of leaf samples were ground in a mortar containing 5 ml tca (0.1%). leaf extracts were centrifuged at 10000 rpm for 5 min. supernatants (250 µl) were mixed with 1 ml mda solution containing 20% tca and 0.5% thiobarbituric acid. the mixtures were warmed at 95°c for 30 min and then immediately cooled on ice. sample tubes were centrifuged at 10000 rpm for 10 min. absorption of light was measured by a spectrophotometer at 532 nm wavelength according to heath and packer (1969). hydrogen peroxide (h 2 o 2 ) leaf samples (0.25 g) were ground in a mortar containing 5 ml trichloroacetic acid (tca) (0.1%). extracts were centrifuged at 10000 rpm for 5 min. supernatants (250 µl) were mixed with 250 µl phosphate buffer (100 mm) and 500 µl potassium iodide (1 m). absorption of light was measured by a spectrophotometer at 390 nm wavelength according to alexieva et al. (2001). 3. results with a decrease of temperature from 25°c to -7.5°c, reducing sugars increased considerably, with tall fescue showing a greater increase than common bermudagrass. the highest soluble sugar content in tall fescue was formed at -7.5°c and the highest reducing sugar content produced in common bermudagrass was detected at 0°c (table 1). there was no significant difference between the turfgrasses for proline content. the highest proline content was observed at 0°c and the lowest proline content was seen at 25°c. the interaction of temperature and turf species showed that tall fescue at 25°c had the lowest proline content, while tall fescue at -7.5°c had the highest (table 1). it was found that as temperature decreased from 25°c to 7.5°c, cat activity increased. the greatest cat activity was observed in tall fescue at 7.5°c and the least was seen in bermudagrass at -7.5°c (table 1). comparison of the means showed that sod activity in tall fescue is greater, but not significantly different from common bermudagrass. maximum sod activity in bermudagrass was detected at 0°c, while the minimum was found at 25°c in tall fescue. as the temperature diminished from 25°c to -7.5°c, mda amassed continuously in the plants. mda accumulated significantly more in common bermudagrass with the highest amount built up at -7.5°c (table 2). h 2 o 2 increased in plants as the temperature lowered to 0°c. the most h 2 o 2 was produced in common bermudagrass at 0°c, whilst the lowest was observed in tall fescue at 25°c (table 2). 4. discussion and conclusions as the temperature decreased from 25°c to -7.5°c, soluble sugars and proline content increased, which tall fescue had higher amounts at -7.5°c (table 1). a similar behavior was found in saltgrass (distichlis spicata l.), centipedegrass (eremochloa ophiuroides [munro]), annual bluegrass (poa annua l.) and buffalograss (bouteloua dactyloides [nutt.]) (fry, 1993; shahba et al., 2003). generally, one of the first reactions by these plants to counter the chilling stress of winter is a buildup of sugar (fry, 1993; ball et al., 2002), whilst amino acids help adapt the plants to low temperature (guy, 1990). proline and reducing sugars serve as cryoprotectants through increasing the concentration of cell content and reducing the water potential (ball et al., 2002). comparable results were observed in zoysiagrass (zoysia japonica steud.) and annual bluegrass (dionne et al., 2001). 11 the increase in cat and sod activity found in this study is assumed to protect the cells from oxidative damage caused by cold stress as seen in other plants (matsumura et al., 2002; larkindale and huang, 2004; jiang et al., 2011). sod converts superoxide (o 2 .-) to h 2 o 2 and cat detoxifies the latter to water and oxygen (fuchs et al., 1997; polidoros and scandalios, 1999). antioxidant enzymes help maintain cell homeostasis under severe low temperatures by scavenging as well as signaling, although their definite function should be further elucidated (polle, 1997). higher antioxidant enzyme activity in tall fescue could be attributed to better cold tolerance compared to common bermudagrass. mda and h 2 o 2 increase in the turfgrasses in this research is dependent on the cold stress received (table 2). mda and h 2 o 2 are produced by lipid peroxidation of plants under chilling stress (leshem, 1987; wise and naylor, 1987). these two sensitive indicators are considered to point toward the extent of low temperature stress and damage inflicted to the plant (xu et al., 2006). greater amounts of these two substances in common bermudagrass compared to tall fescue could be interpreted as a greater sensitivity to and injury from low temperatures (table 2), which is consistent with the reports in manila grass (zoysia matrella l.) (wang et al., 2009). overall, cold stress produces large amounts of ros which causes oxidative damage to plants through vast destruction of proteins, carbohydrates, lipids, cellular membranes, dna and major decline of atp reserve, and finally cell death (dionne et al., 2001; gill and tuteja, 2010). since ros has multifunctional roles, it is essential for the cells to control the level of ros tightly to avoid any oxidative injury and not to eliminate them table 1 effects of cold stress on biochemical changes [reducing sugar, proline content, catalase (cat) and superoxide dismutase (sod) activity] in the two turfgrasses used in this study turfgrass temperature (°c) mean -7.5 0 +7.5 +15 +25 reducing sugar (mg·g-¹ d.w.) tall fescue 176.4±60.1 a 160.8±16.3 ab 130.2±27.3 bcd 114.8±13.2 cd 104.3±22.4 dz 137.4 a bermudagrass 121.9±11.5 bcd 158.9±21.0 abc 140.9±18.7 a-d 117.3±19.8 bcd 96.5±14.7 d 127.1 a mean 149.2 a 159.8 a 135.5 ab 116.1 bc 100.4 c proline content (µg·g-¹ d.w.) tall fescue 35.8±3.7 a 35.1±3.4 a 27.2±2.7 b 13.7±3.1 cd 9.2±0.9 d 24.2 a bermudagrass 26.4±1.7 b 31.8±1.5 ab 27.3±5.1 b 17.3±4.5 c 12.7±1.3 cd 23.1 a mean 31.1 a 33.5 a 27.2 b 15.5 c 11.0 d cat (u g·g-¹ d.w.) tall fescue 36.3±5.4 c 46.7±6.1 ab 52.8±9.8 a 37.5±3.6 c 32.8±5.7 c 41.2 a bermudagrass 31.5±4.5 c 39.5±4.4 bc 47.5±2.9 ab 41.5±3.9 bc 36.9±5.8 c 39.3 a mean 35.4 c 43.1 b 50.1 a 39.5 bc 34.8 c sod (u g·g-¹ d.w.) tall fescue 179.6±17.6 abc 161.6±33.1 a-d 186.6±30.5 ab 126.0±19.0 de 106.6±17.0 e 152.1 a bermudagrass 127.3±23.1 cde 193.3±25.1 a 145.0±42.7 a-e 137.6±58.6 b-e 120.6±11.7 de 144.8 a mean 153.5 ab 177.5 a 165.8 ab 131.8 bc 113.6 c z in each variable, data followed by the same letters±sd (small letters for interactions and capital letters for means) are not significantly different at 5% level of probability using lsd test. table 2 effects of cold stress on malondialdehyde (mda) and hydrogen peroxide (h 2 o 2 ) in the two turfgrasses used in this study turfgrass temperature (°c) mean -7.5 0 +7.5 +15 +25 mda (µmol g-1 f.w.) tall fescue 7.5±1.6 cd 7.8±1.3 bc 6.8±0.8 cde 4.4±0.7 fg 3.9±0.7 gz 6.1 b bermudagrass 11.6±0.7 a 9.5±0.7 b 7.7±0.8 c 5.9±1.0 def 5.1±0.7 efg 8.0 a mean 9.6 a 8.6 a 7.3 b 5.1 c 4.5 c h 2 o 2 (µmol g-1 f.w.) tall fescue 5.1±0.9 a 4.7±0.7 a 3.5±0.6 b 2.9±0.4 b 2.7±0.4 b 3.8 b bermudagrass 5.0±0.3 a 5.3±0.4 a 4.9±0.7 a 3.5±0.5 b 3.1±0.7 b 4.3 a mean 5.0 a 5.0 a 4.2 b 3.2 c 2.9 c (z) in each variable, data followed by the same letter ± sd (small letters for interactions and capital letters for means) are not significantly different at 5% level of probability using lsd test. 12 entirely (sharma et al., 2012). it is concluded that both turfgrasses increase reducing sugars, proline, cat, sod, mda and h 2 o 2 in response to lower temperatures, but tall fescue has a better defense mechanism than common bermudagrass and is more tolerant to cold stress. the results show that scavenging enzymes have a direct effect in cold season tolerance of turfgrass and improve the defense mechanism of plants, but their exact role merits further investigation. acknowledgements the authors wish to thank the administration of the students’ scientific association of shiraz university for their financial support. references alexieva v., sergiev i., mapelli s., karanov e., 2001 the effect of drought and ultraviolet radiation on growth and stress markers in pea and wheat. plant cell and environment, 24: 1337-1344. ball s., qian y.l., stushnoff c., 2002 soluble carbohydrates in two buffalograss cultivars with contrasting freezing tolerance. j. amer. soc. hortic. sci., 127(1): 45-49. bates l.s., waldern r.p., teave i.d., 1973 rapid determination of free proline for water stress standies. plant and soil, 39: 107-205. beauchamp c., fridovich i., 1971 superoxide dismutases: improved assays and an assay predictable to acrylamide gels. analytical bioch., 44: 276-287. carrow r., 1994 zoysiagrass cultivar performance as affected by traffic and n-level. agronomy abstracts, asa, madison, wi, usa, p. 183. dhindsa r.s., plumb-dhindsa p., thorpe t.a., 1981 leaf senescence: correlated with increased levels of membrane permeability and lipid peroxidation, and decreased levels of superoxide dismutase and catalase. j. exp. bot., 32(1): 93-101. dionne j., castonguay y., nadeau p., desjardins y., 2001 freezing tolerance and carbohydrate changes during cold acclimation of green-type annual bluegrass (poa annua. l) ecotypes.crop science, 41: 443-451. dubois m., gilles k.a., hamilton j.k., rebers p.a., smith f., 1956 colorimetric method for determination of suger and related substances. analytical bioch., 28: 350-356. fry j.d., 1993 freezing tolerance and carbohydrate content of low-temperature-acclimated and nonacclimated centipedegrass. crop science, 33: 1051-1055. fuchs d., bitterlich g.b., wede i., wachter h., 1997 reactive oxygen and apoptosis, pp. 139-167. in: scandalios j.g. 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doi: 10.1155/2012/217037. wang y., yang z.m., zhang q.f., li j.l., 2009 enhanced 13 chilling tolerance in zoysia matrella by pre-treatment with salicylic acid, calcium chloride, hydrogen peroxide or 6-benzylaminopurine. biologia plantarum, 53: 179-182. wise r.r., naylor a.w., 1987 chilling-enhanced photooxidation, evidence for the role of singlet oxygen and superoxide in the breakdown of pigments and endogenous antioxidants. plant physiology, 83: 278-282. xu s., li j.l., zhang x.q., wei h., cui l.j., 2006 effects of heat acclimation pretreatment on changes of membrane lipid peroxidation, antioxidant metabolites, and ultrastructure of chloroplasts in two cool-season turf grass species under heat stress. environ. exp. bot., 56: 274-285. impaginato 3 adv. hort. sci., 2011 25(1): 3-13 received for publication 21 september 2010. accepted for publication 28 december 2010. pollen characteristics, pollination behaviour and pollinizer compatibility of some exotic and indigenous almond [prunus dulcis (miller) d.a. webb] genotypes g. sharma, n. sharma department of fruit science, dr. y.s. parmar university of horticulture and forestry, nauni, solan, hp 173230, himachal pradesh, india. key words: almond, compatibility, pollen, pollination behaviour, pollinizer, abstract: pollination is the most critical and complex part of fruit production, particularly in cross pollinated crops like almond, and it is affected by pollen characteristics. in the present study 100% pollen viability was observed when stained by acetocarmine for all considered genotypes, except gp-17 (99%). optimum stigma receptivity was observed for two days before anthesis, on the day of anthesis and one day afterwards all the stigmas remained receptive. in open pollination, maximum fruit set was noted in primorskij (32.37%) with minimum in makhdoom (17.96%). no fruit set was observed in any of the genotypes by self-pollination, confirming the self-incompatible nature of all tested genotypes. in cross pollination, fruit set was found between 0.00 and 42.84% for different cross combinations. makhdoom, pranyaj, shalimar, waris, nonpareil, waris, waris and shalimar yielded maximum fruits when cross-pollinated with ixl, merced, drake, primorskij, pranyaj, nonpareil, shalimar, makhdoom and waris, respectively. no pollen tube growth was observed in the style when genotypes were crossed with their own pollen. in crosses between ixl and nonpareil, pollen tube growth was arrested in the styles. in compatible crosses, the pollen tube reached the base of the style after different times following pollination. 1. introduction almond [prunus dulcis (miller) d.a. webb], which belongs to the family rosaceae, is an important edible nut with widespread popularity. nuts are a rich source of energy and contain high amounts of fat, protein, minerals and vitamins. presently, the united states of america is the leading producer of almonds and india ranks third as an importer of almonds from the usa after spain and germany. in india, almond cultivation is confined mainly to northern areas including the regions of jammu and kashmir and high hills of himachal pradesh. during 2006-2007, the area under almond cultivation was 16,404 ha with annual production of 15207 mt in jammu and kashmir (directorate of horticulture, personal communication), whereas in himachal pradesh the area was 5766 ha and the production was 1303 mt (directorate of horticulture, personal communication). pollination is the most critical and complex part of fruit production, particularly in cross pollinated crops like almond. it involves a complex and sensitive sequence of events and interactions on a morphological, physiological and biochemical level. climatic conditions and genetic factors of the cultivars have an important impact on pollination. the number of flowers which develop, their interaction and their position within the inflorescence as well as pollen compatibility or incompatibility are some of the determinants for fruiting. the self-incompatibility system in almond limits in-breeding and therefore fails to produce an adequate crop, as a consequence, almond requires pollen from some other compatible cultivar(s) for cross-pollination. trees do produce abundant bloom but fail to set adequately due to lack of pollination and unfavourable weather conditions. sometimes cultivars are self unfruitful, and require pollen from other compatible cultivars for fruit production. according to socias i company (1992) most almond cultivars are self incompatible and nearly 30% pollinizers are required to have an economic crop (kester and griggs, 1959) which necessitates the planting of more than one 4 cultivar with sufficient overlapping of flowering periods to ensure adequate cross pollination and fruit set. some cultivar combinations also exhibit cross incompatibility (talaie and imani, 1998). knowledge of the pollen characteristics and pollination behaviour of different cultivars is therefore an important prerequisite for the successful cultivation of almonds. therefore, studies on pollen characters and pollination behaviour of almond genotypes were undertaken to find the compatibility groups between some exotic and indigenous selections of almond. 2. materials and methods the study of pollen characteristics, pollination behaviour and pollinizer compatibility of almond genotypes was conducted on four introductions from the usa (ixl, merced, drake and nonpareil), two from ukraine (primorskij and pranyaj) and seven indigenous selections from india (shalimar, makhdoom, waris, gp 10, gp 14, gp 17 and gp 19) at the central institute of temperate horticulture, srinagar, jammu and kashmir during 2005-06 and 2006-07. the experimental site was located at a latitude of 34o 05’ north and longitude 74o 50’ east, and at an altitude of 1640 m above mean sea level. the plants were five to six years old and were laid out at a spacing of 4 × 4 m. meteorological data are reported in appendix 1. pollen studies pollen viability was studied by staining with acetocarmine (2% solution) as per the method suggested by das (1995). in vitro pollen germination in sucrose solution was studied in 5, 10, 12.5, 15 and 20% sucrose concentrations. pollen tube growth was assessed for each genotype under a microscope after 24 hr of incubation at 22±2°c. the pollen grains with a pollen tube at least two times longer than pollen size were considered germinated. pollination studies stigma receptivity was studied in unopened, aboutto-open buds and opened flowers with the help of a magnifying lens to visualize the presence of exudates (watery fluid) on the stigmatic surface indicating the stigma to be receptive. to monitor fruit set under open pollination, four shoots in all the directions were selected for each genotype and the number of flowers on each shoot was counted. shoots were left open for natural pollination to occur. percent fruit set was calculated at harvest. under natural self pollination (by bagging) branches with flower buds were selected and all the opened flowers and shrivelled buds were removed. numbers of buds at popcorn stage left on each shoot were counted. these shoots were covered with muslin cloth bags, tied at the lower end and properly labelled. after 35-40 days the bags were removed and percent fruit set was determined at harvest. for hand self-pollination flowers were emasculated at balloon or popcorn stage by removing the entire calyx and corolla, leaving only the pistil. whole branches with emasculated buds were then covered with muslin cloth bags and properly tied and labelled. pollination of the emasculated buds was done on the following or next day with the freshly collected pollen of the same genotype. the pollen was applied to the receptive stigma with the help of a camel hair brush. after pollination the bags were again placed onto the branches. for cross pollination studies, crosses were made in the nine genotypes (exotic and indigenous cultivars) in all the possible combinations. the procedure followed to determine the extent of cross pollination was the same as discussed for self pollination studies, except that the pollen used for crossing belonged to different cultivars. fruit set was counted one month after pollination and percent fruit set was calculated as fruit harvested/flower pollinated × 100. in-vivo pollen tube growth was studied according to the procedure given by ortega et al. (2004). a sample of five pistils was collected at 24, 48, 72, 96 and 120 hr after hand self-pollination and immediately fixed in faa: ethanol 500 ml; formaldehyde 100 ml; acetic acid 50 ml; distilled water 350 ml. pollen tube growth in the style was viewed under fluorescent microscope (olympus bx-40 model) and the extent of pollen tube growth penetration into the style at different intervals of time after pollination was recorded. the data recorded were analysed statistically as per the methods described by panse and sukhatme (1978). 3. results and discussion almond has very low chilling requirements, so trees blossom in early spring, hence pollination and fertilization are negatively affected by the low temperature and rain which may prevail at that time. low temperatures during the flowering period can prevent germination of pollen on the stigma or prevent development of pollen tubes in style. further pollen can be washed away by rains and bees are not active at low temperatures. these factors can create some hindrance in the pollination and fertilization of almond, and subsequently affect fruit set percentage and final yield. therefore, fruit set under open pollination is influenced by a number of factors such as genetic makeup of cultivars, nearness or distance from a compatible pollen source, prevailing weather conditions, bee activity, stigma receptivity, pollen germination, pollen tube growth and fertilization process. pollen viability pollen viability tested with acetocarmine (2%) in different almond genotypes revealed that all the genotypes had 100% pollen viability, except for gp 17 in which 99.00% pollen viability was recorded differing 5 significantly from the others. similar higher values for pollen viability accessed through acetocarmine (2%) were observed previously by das (1995). under invitro pollen germination the percent germination varied significantly in different concentrations of sucrose (table 1). a significant increase in pollen germination percentage was recorded up to 15% sucrose solution and thereafter it decreased significantly in 20% solution. peak pollen germination was recorded in either 12.5 or 15% solution for most of the genotypes. in 5% sucrose solution pollen germination ranged from 12.67% in gp-19 to 76.90% in waris. in 10% solution it varied from 40.40 to 87.83% for gp-14 and ‘nonpareil’, respectively. this latter genotype gave values of 94.05, 96.45 and 90.05% germination in 12.5, 15 and 20% sucrose solution, respectively, whereas, gp 19 demonstrated low pollen germination of 12.67, table 1 in vitro pollen germination of different cultivars of prunus dulcis (miller) d.a. webb genotypes in different sucrose concentrations. values are expressed in percentage ixl merced drake primorskij pranyaj nonpareil shalimar makhdoom waris gp-10 gp-17 gp-19 gp-14 cd0.05 genotypes 37.88 e 19.84 d 18.18 e 41.13 e 45.19 d 34.68 e 28.80 d 16.90 e 76.90 c 39.92 e 31.92 e 12.67 d 23.98 d sucrose solution (%) 5 10 12.5 15 20 55.91 d 65.72 b 52.86 d 72.77 d 64.73 b 87.83 d 77.62 c 55.41 d 78.13 c 48.49 d 53.21 d 47.59 c 40.40 c 86.03 a 83.89 a 85.72 b 85.95 c 88.01 a 94.05 b 86.85 a 81.05 b 91.30 a 81.44 b 80.05 b 72.54 b 75.81 b 83.10 b 85.88 a 92.44 a 96.44 a 87.17 a 96.45 a 80.47 b 84.00 a 92.71 a 85.99 a 82.66 a 79.80 a 79.75 a 77.31 c 56.99 c 80.57 b 93.50 c 59.10 c 90.05 c 78.52 c 64.53 c 86.08 b 73.98 c 70.99 c 72.30 b 75.32 b genotypes (g) concentration (c) genotypes x concentration (gxc) 3.27 2.03 7.33 47.59, 72.54, 79.80 and 72.30% respectively at similar levels of sucrose concentrations. the variation in pollen germination in different genotypes under the same sucrose concentration may be attributed to their varied genetic constitution. dhillon et al. (1982) in ‘california papershell’ almond found the highest (80.36%) pollen germination in 20% sucrose solution. eti (1994) found 10 to 15% sucrose concentrations quite suitable for almond pollen germination. stigma receptivity in almond, the importance of the length of the period of flower receptivity to obtain good yield was first described by griggs and iwakiri (1964). the percent of flowers showing stigma receptivity at different durations before and after anthesis in various genotypes is presented in table 2. two days prior to anthesis stigma table 2 stigma receptivity in different almond genotypes ixl merced drake primorskij pranyaj nonpareil shalimar makhdoom waris gp 10 gp 17 gp 19 gp 14 genotypes 52 68 72 32 44 56 36 32 44 36 40 28 36 sucrose solution (%) -2 days -1 day 0 day +1 day +2 day 76 72 88 64 84 92 60 56 84 60 84 72 76 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 82 76 68 76 80 72 88 76 80 72 76 72 72 (-) before anthesis, (0) on anthesis, (+) after anthesis. 6 receptivity varied from 28 to 72%; maximum receptivity was observed in drake (72%) and the minimum value (28%) was observed for seedling selection gp19. one day before anthesis, stigma receptivity varied from 60% in shalimar and gp-10 to 92% in nonpareil. in addition, it was observed that all the stigmas were receptive on the day of anthesis and remained so for the second day after anthesis; receptivity decreased thereafter for all genotypes. the work of ortega et al. (2007) is in accordance with the present findings. pollination studies fruit set data under open pollination and self pollination for the years 2006 and 2007 is presented in table 3 and revealed that it varied according to the genotype and the year. the analysis of variance showed that there were significant differences for all the genotypes as well as for the interaction among the genotypes under open pollination. the average effect of the year was observed to be significant as average set for 2006 (26.42%) was higher than that of second year (22.41%). the principal reason for this difference is that the average temperature was higher in march 2006 than it was in march 2007: in 2006 the minimum temperature was above 1°c for all the days while it was 0°c or less in ten out of first 17 days of march 2007. low temperature along with snowfall on 12 and 13 march damaged the blossom of early flowering varieties like makhdoom, shalimar and gp-10. maximum fruit set was recorded for ‘primorskij’ (32.37%) which was at par with ‘pranyaj’ (30.96%) and ‘drake’ (30.51%) and significantly higher than ‘ixl’ (26.27%), ‘nonpareil’ (26.53%), ‘waris’ (25.17%) and ‘shalimar’ (25.02%). minimum fruit set was observed for cultivar makhdoom (17.96%). maximum fruit set in 2006 was recorded for cultivar shalimar (32.99%) and was at par with ‘pranyaj’ (32.25%) and ‘makhdoom’ (30.15%) whereas, the minimum value was observed for merced (21%). in 2007, ‘primorskij’ had the highest fruit set (36.57%) and the minimum recorded was 5.77% for ‘makhdoom’; both these values differed significantly from all other genotypes. low fruit set in early blooming cultivars, due to spring frost, was reported previously by connell (2000). for commercial fruit production fruit set in almond must range between 25 and 40% of the initial number of flowers (kester and griggs, 1959). low fruit set values for both the years of the present study can be further attributed to the non availability of supplemented pollinators (bee hives) during bloom for adequate pollination. variation in fruit setting behaviour under open pollination was reported by talaie and imani (1998), ak et al. (2001) and socias i company et al. (2005). the degree of self compatibility in almond genotypes, assessed by observing fruit set following unassisted self pollination (bagging), revealed that there was no fruit set following bagging in any of the genotypes, thus indicating total self-incompatibility. almond shows a gametophytic self incompatibility system (socias i company, 1992) controlled by a multiallelic locus, known as locus ‘s’ (gagnard, 1954). this implies that the pollen tube of a flower of the same tree, the same cultivar and sometimes of certain other cultivars, will not grow down the style (kester, 1969). in this regard, most almond breeding programmes have fostered the development of self-compatible cultivars to overcome the problems related to cross-pollination of a mostly self incompatible species such as almond table 3 fruit set of prunus dulcis (miller) d.a. webb genotypes calculated for open and self pollination ixl merced drake primorskij pranyaj nonpareil shalimar makhdoom waris gp-10 gp-17 gp-19 gp-14 mean cd(0.05) pooled cd 0.05 g y gxy genotypes fruit set (%) open pollination selfing by bagging 2006 2007 pooled 2006 2007 pooled 27.20 21.00 26.93 28.16 32.25 24.23 32.99 30.15 28.70 22.89 24.28 21.97 22.67 26.42 2.07 25.33 21.28 34.09 36.57 29.67 28.83 17.05 5.77 21.65 15.04 19.42 16.99 19.63 22.41 3.24 1.85 0.72 2.63 26.27 c 21.14 de 30.51 b 32.37 a 30.96 ab 26.53 c 25.02 c 17.96 f 25.17 c 18.97 f 21.85 d 19.48 ef 21.15 de 24.41 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 7 (social i company, 2002). our results are in accordance with a previous report of kester et al. (1994) wherein a low level of fruit set was recorded following hand pollination in otherwise self-incompatible cultivars. cross pollination cross pollination is essential in almond orchards as most of the cultivars are self-incompatible. in order to guarantee good pollination, at least two cultivars must be inter planted which not only coincide in flowering time, but are also cross-compatible. as the commercial part of the fruit is the seed, a decrease in the number of pollinated flowers often results in crop reduction (kester and griggs, 1959). thus rainy, windy or cold weather interferes with pollination by inhibiting bee foraging (socias i company et al., 1996). in the current work, nine almond cultivars were pollinated with one another in all possible combinations. the data pertaining to fruit set following cross pollination is presented in table 4. the maximum fruit set value was recorded for cross-combination ixl x makhdoom (42.84%) and was at par with nonpareil x pranyaj (39.69%), pranyaj x nonpareil (38.55 %), primorskij x waris (36.29%) and waris x shalimar (35.88%). minimum fruit set was observed when ixl was crossed with its own pollen (0.95%). no fruit set was recorded in merced, makhdoom and shalimar when they were pollinated with their own pollen. the data further revealed that when ixl was used as pollinizer, maximum fruit set was recorded in the cultivar pranyaj (34.47%) followed by waris (25.82%) and primorskij (23.87%). when merced was used as a pollinizer, maximum fruit set was observed with pranyaj (34.71%) followed by drake, waris, ixl, primorskij and ‘nonpareil’ as 28.37, 21.88, 21.29, 20.94 and 14.09%, respectively when merced was used as pollinizer. no fruit set was observed when merced was pollinated by its own pollen. fruit set ranged between 0.88 and 32.67% in different genotypes when drake was used as pollinizer: the maximum was with pranyaj (32.67%) which was statistically at par with fruit set in nonpareil (29.10%) but differed significantly from ixl (25.19%), waris (20.59%), merced (19.69%) and primorskij (18.79%); minimum fruit set (0.88%) was observed when drake was pollinated by its own pollen. in addition, when primorskij was used as a pollinizer, the maximum fruit set was observed with ixl (29.29%), at par with nonpareil (27.44%). fruit set ranged between 2.44 and 38.55% in different genotypes when nonpareil was used as pollinizer. maximum fruit set was noted in pranyaj (38.55%) followed by primorskij (28.12%), drake (27.28%), waris (21.48%) and merced (14.01%). minimum fruit set was observed when nonpareil was pollinated by its own pollen (2.44%), statistically at par with fruit set in ixl (3.05%) when pollinated with nonpareil pollen. when shalimar was used as pollinizer the highest ta bl e4 fr ui ts et (% )i n pr un us du lci s( m ill er )d .a .w eb b ge no ty pe so bt ain ed in di ffe re nt in ter -v ar iet al cr os se s fe m ale ix l m er ce d dr ak e pr im or sk ij pr an ya j no np ar eil sh ali m ar m ak hd oo m w ar is m ea n cd 0.0 5 1x l m er ce d dr ak e pr im or sk ij pr an ya j no np ar eil sh ali m ar m ak hd oo m w ar is m ea n cd 0.0 5 cd 0.0 5 ge no ty pe s( g) 0.9 5 21 .29 25 .19 29 .29 13 .72 3.0 5 21 .64 42 .84 24 .14 20 .24 2.2 2 = 16 .16 0.0 0 19 .69 22 .32 32 .24 14 .01 28 .37 23 .95 18 .76 19 .42 1.3 2 6.2 4 18 .16 28 .37 0.8 8 13 .75 12 .64 27 .28 29 .37 28 .55 20 .70 19 .97 2.3 8 23 .87 20 .94 18 .79 1.2 7 19 .37 28 .12 28 .29 31 .60 36 .29 23 .17 2.1 9 34 .47 34 .71 32 .67 21 .56 1.5 7 38 .55 28 .89 28 .44 35 .91 28 .53 5.1 6 2.8 1 14 .09 29 .10 27 .44 39 .69 2.4 4 29 .52 26 .06 31 .90 22 .56 4.9 0 x x x x x x 0.0 0 23 .33 28 .97 17 .43 2.2 9 x x x x x x 14 .23 0.0 0 15 .85 10 .03 2.0 9 25 .82 21 .88 20 .59 24 .50 24 .35 21 .48 35 .88 25 .13 0.9 1 22 .28 1.7 7 17 .46 19 .94 20 .99 20 .02 20 .50 19 .27 24 .06 25 .46 23 .71 1.7 0 2.0 5 2.7 5 2.1 1 3.3 0 3.1 1 3.1 0 2.8 1 3.5 9 m ale x = cr os se sn ot att em pt ed . 8 value of fruit set was observed in waris (35.88%), which differed significantly from other cultivars. the data also showed that no fruit set was observed in cultivar shalimar when pollinated by its own pollen. when makhdoom was used as a pollinizer, the highest fruit set was recorded in ixl (42.84%) followed by primorskij (31.60%). drake, pranyaj, nonpareil, waris, merced and shalimar had fruit set of 28.55, 28.44, 26.06, 25.13, 23.95 and 23.33%, respectively, which were at par with each other when pollinated with makhdoom. no fruit set was observed when makhdoom was pollinated by its own pollen, as was also the case when waris was pollinated by its own pollen. the mean fruit set induced by different pollinizers ranged from 17.46 to 25.46%. makhdoom, as a pollinizer, affected the highest average fruit set (25.46%), followed by shalimar (24.06%), waris (23.71%) and drake (20.99%). the minimum fruit set value was observed when ixl (17.46%) was used as pollinizer. among female parents, the maximum fruit set was observed in pranyaj (28.53%), followed by primorskij (23.17%) and waris (22.28%). cultivar makhdoom (10.03%) had the lowest fruit set value as female parent when pollinated with different pollinizers. cross-incompatibility of ixl with nonpareil had been previously established (gagnard, 1954) and the present study revealed low fruit set in ixl and nonpareil crosses, thus supporting the findings. the rest of the cultivars showed optimum fruit set with crossing (13.72 to 42.84%) thus indicating cross-compatibility between the cultivars. the differences in fruit set may be due to various factors such as genotypic differences of the cultivars under study, response of genotypes to different pollen sources, ovary degeneration, unfavourable climatic conditions during flowering, flower sterility and heterostyly. other workers (talaie and imani, 1998; dalal et al., 2004) reported similar results. in vivo pollen tube growth in temperate tree crops the rate of pollen tube growth to the base of the style is quite low (sedgley, 1982). the present studies, pertaining to in-vivo pollen tube growth, have revealed that in all the cultivars which were pollinated by their own pollen, pollen tube growth was arrested in the style. observations regarding in vivo pollen tube growth are presented in table 5 a and 5 b. the findings indicate that in all crosses where pollen of the same cultivar was used for pollination, the pollen tube failed to reach up to the base of the style. the observations revealed that in ixl the pollen tube reached the base of the style after 120 hr when crossed with pollen from merced and shalimar, whereas it took 96 hr with pranyaj and makhdoom. the pollen tube reached the style after 72 hr when ixl was pollinated by drake, primorskij and waris, while the pollen tube failed to grow in the style of ixl when nonpareil was used as pollen source. in merced it was observed that the pollen tube reached the base of the style after 72 hr when crossed with shalimar and makhdoom; with ixl, drake, nonpareil and waris it reached the same point after 96 hr. primorskij and pranyaj pollen tubes reached the base after 120 hr. likewise, the study of drake styles revealed that pollen tubes reached the base after 72 hr of pollination with merced and waris whereas with other cultivars it reached after 96 hr. the pollen tube of ixl, nonpareil and waris reached up to the base of styles of primorskij after 72 hr of pollination; with merced, drake, shalimar, makhdoom and pranyaj, it reached after 96 hr. it was also revealed that in pranyaj the pollen tube reached up to the base of the style after 72 hr when pollinated with ixl, merced, drake and shalimar while with others it took 96 hr. in vivo pollen tube growth in nonpareil revealed that pollen of drake primorskij, pranyaj and makhdoom reached the earliest (i.e. 72 hr) after pollination, whereas with other pollinzers it took 96 hr to reach the base. in cultivar shalimar it was observed that the pollen tube reached up to the base of the style after 96 hr when crossed with makhdoom and with waris it reached after 120 hr. similarly, in makhdoom the pollen tube reached up to the base after 120 hr and 96 hr when crossed with shalimar and waris, respectively. the pollen tube reached the base of the style at different durations in waris. it took 72 hr for ixl, drake, and pranyaj pollen whereas, with merced, primorskij, nonpareil, shalimar and makhdoom it took 96 hr after pollination. these findings, along with fruit set data, confirm the self incompatibility of cultivars under study. similar results were observed by ak et al. (2001). these authors found that the rejection of incompatible male gametophyte occurred on the stigma, as well as in the style. similarly in pistils of nonpareil and ixl none of the pollen tubes reached the base of the pistil when they were inter-pollinated, thus confirming the cross incompatibility between these two cultivars. however, in the compatible pollination crosses, the pollen tube reached the base of the style after different durations of pollination. the difference did not affect the compatibility relationship of the pollinations. the observed differences must be mostly attributed to the interaction of weather conditions at the time of pollination and thereafter. temperature is an important component for pollen tube growth and the most suitable temperature for pollen tube growth in almond is 12-13°c, and under these temperatures the pollen tube can reach the ovarium within three to four days (loreti and viti, 1984). moreover, pistils may react differently to different pollen sources. overall, it generally took three to five days for pollen tubes to reach the base of the pistil in otherwise compatible pollination. the present findings are in consonance with those of ak et al. (2001) and das and kumar (2004) who reported that pollen tubes reached the base of pistils after four or five days of pollination in almond. other in vivo pollen tube 9 ta bl e5 ain viv o po lle n tu be gr ow th in sty les of pr un us du lci s( m ill er )d .a .w eb b ge no ty pe so bt ain ed in di ffe re nt in ter -v ar iet al cr os se s fe m ale po lli ni ze r m er ce d dr ak e pr im or sk ij pr an ya j ix l 24 hrpo lle n tu be pe ne tra tio n (s ty le len gt h) co mp ati bil ity sta tus po lle n tu be pe ne tra tio n (s ty le len gt h) co mp ati bil ity sta tus po lle n tu be pe ne tra tio n (s ty le len gt h) co mp ati bil ity sta tus po lle n tu be pe ne tra tio n (s ty le len gt h) co mp ati bil ity sta tus po lle n tu be pe ne tra tio n (s ty le len gt h) co mp ati bil ity sta tus 48 hr 72 hr 96 hr 12 0 hr 24 hr 48 hr 72 hr 96 hr 12 0 hr 24 hr 48 hr 72 hr 96 hr 12 0 hr 24 hr 48 hr 72 hr 96 hr 12 0 hr 24 hr 48 hr 72 hr 96 hr 12 0 hr ix l m er ce d dr ak e pr im or sk ij pr an ya j no np ar eil sh ali m ar m ak hd oo m w ar is x x 1/ 4 1/ 4 1/ 4 x 1/ 4 1/ 4 1/ 4 x 1/ 4 2/ 4 3/ 4 2/ 4 1/ 4 2/ 4 1/ 4 2/ 4 x 2/ 4 at th e ba se at th e ba se 3/ 4 x 3/ 4 2/ 4 at th e ba se x 3/ 4 at th e ba se x 3/ 4 at th e ba se x at th e ba se x at th e ba se at th e ba se in co m pa tib le co m pa tib le co m pa tib le co m pa tib le co m pa tib le in co m pa tib le co m pa tib le co m pa tib le co m pa tib le 1/ 4 1/ 4 1/ 4 1/ 4 1/ 4 1/ 4 1/ 4 1/ 4 1/ 4 2/ 4 x 2/ 4 1/ 4 2/ 4 2/ 4 3/ 4 3/ 4 2/ 4 3/ 4 x 3/ 4 2/ 4 2/ 4 3/ 4 at th e ba se at th e ba se 3/ 4 at th e ba se x at th e ba se 3/ 4 3/ 4 at th e ba se at th e ba se x at th e ba se at th e ba se co m pa tib le in co m pa tib le co m pa tib le co m pa tib le co m pa tib le co m pa tib le co m pa tib le co m pa tib le co m pa tib le 1/ 4 1/ 4 1/ 4 1/ 4 1/ 4 2/ 4 1/ 4 1/ 4 2/ 4 2/ 4 3/ 4 x 2/ 4 2/ 4 3/ 4 2/ 4 2/ 4 3/ 4 3/ 4 at th e ba se x 3/ 4 3/ 4 3/ 4 3/ 4 3/ 4 at th e ba se at th e ba se x at th e ba se at th e ba se at th e ba se at th e ba se at th e ba se x co m pa tib le co m pa tib le in co m pa tib le co m pa tib le co m pa tib le co m pa tib le co m pa tib le co m pa tib le co m pa tib le 1/ 4 1/ 4 1/ 4 1/ 4 1/ 4 1/ 4 1/ 4 1/ 4 1/ 4 3/ 4 2/ 4 2/ 4 x 2/ 4 3/ 4 2/ 4 2/ 4 3/ 4 at th e ba se 3/ 4 3/ 4 x 3/ 4 at th e ba se 3/ 4 3/ 4 at th e ba se at th e ba se at th e ba se x at th e ba se at th e ba se at th e ba se x co m pa tib le co m pa tib le co m pa tib le in co m pa tib le co m pa tib le co m pa tib le co m pa tib le co m pa tib le co m pa tib le 1/ 4 1/ 4 1/ 4 1/ 4 x 1/ 4 1/ 4 1/ 4 1/ 4 2/ 4 2/ 4 2/ 4 1/ 4 x 1/ 4 3/ 4 2/ 4 2/ 4 at th e ba se at th e ba se at th e ba se 2/ 4 x 3/ 4 at th e ba se 3/ 4 3/ 4 at th e ba se x at th e ba se at th e ba se at th e ba se co m pa tib le co m pa tib le co m pa tib le co m pa tib le in co m pa tib le co m pa tib le co m pa tib le co m pa tib le co m pa tib le 10 ta bl e5 b -i n viv o po lle n tu be gr ow th in sty les of pr un us du lci s( m ill er )d .a .w eb b ge no ty pe so bt ain ed in di ffe re nt in ter -v ar iet al cr os se s fe m ale po lli ni ze r no np ar eil po lle n tu be pe ne tra tio n (s ty le len gt h) co m pa tib ili ty sta tu s 24 hr 48 hr 72 hr 96 hr 12 0 hr sh ali m ar po lle n tu be pe ne tra tio n (s ty le len gt h) co m pa tib ili ty sta tu s 24 hr 48 hr 72 hr 96 hr 12 0 hr m ak hd oo m po lle n tu be pe ne tra tio n (s ty le len gt h) co m pa tib ili ty sta tu s 24 hr 48 hr 72 hr 96 hr 12 0 hr w ar is po lle n tu be pe ne tra tio n (s ty le len gt h) co m pa tib ili ty sta tu s 24 hr 48 hr 72 hr 96 hr 12 0 hr ix l m er ce d dr ak e pr im or sk ij pr an ya j no np ar eil sh ali m ar m ak hd oo m w ari s 1/ 4 1/ 4 2/ 4 1/ 4 1/ 4 x 1/ 4 1/ 4 1/ 4 1/ 4 2/ 4 3/ 4 3/ 4 3/ 4 1/ 4 2/ 4 3/ 4 2/ 4 x 3/ 4 at th e ba se at th e ba se 3/ 4 x 3/ 4 at th e ba se 3/ 4 x at th e ba se at th e ba se x at th e ba se at th e ba se x x in co m pa tib le co m pa tib le co m pa tib le co m pa tib le co m pa tib le in co m pa tib le co m pa tib le co m pa tib le co m pa tib le x 1/ 4 1/ 4 x 2/ 4 2/ 4 x 3/ 4 3/ 4 x at th e ba se 3/ 4 x at th e ba se in co m pa tib le co m pa tib le co m pa tib le 1/ 4 x 1/ 4 1/ 4 x 2/ 4 2/ 4 x 3/ 4 3/ 4 x at th e ba se at th e ba se x co m pa tib le in co m pa tib le co m pa tib le 1/ 4 1/ 4 1/ 4 1/ 4 1/ 4 1/ 4 1/ 4 1/ 4 x 2/ 4 2/ 4 2/ 4 2/ 4 3/ 4 2/ 4 2/ 4 2/ 4 1/ 4 at th e ba se 3/ 4 at th e ba se 3/ 4 at th e ba se 3/ 4 3/ 4 3/ 4 x at th e ba se at th e ba se at th e ba se at th e ba se at th e ba se x x co m pa tib le co m pa tib le co m pa tib le co m pa tib le co m pa tib le co m pa tib le co m pa tib le co m pa tib le in co m pa tib le (-) no ob se rv ati on re co rd ed . (x )n o po lle n tu be gr ow th ob se rv ed . 11 growth studies in almond indicate that pollen tubes require two to four days or more to reach the ovule (pimienta and polito, 1983; polito et al., 1996). 4. conclusions the present study has shown that all the considered genotypes had optimum pollen viability and confirmed their self-incompatible nature. furthermore, examination of cross pollination has indicated that there is a potential to renew the declining almond industry of india by exploiting the existing diverse gene pool. exotic varieties can be used for commercial cultivation or in future breeding programs to develop varieties suited to local conditions. references ak b.e., acar i., sakar e., 2001 an investigation on the determination of pomological and morphological traits of wild almond grown at sanlurfa province. options mediterraneennes, 56: 139-144. connell j.hr., 2000 pollination of almonds: practices and problems. hort. technology, 10(1): 116-119. dalal m.a., farooqui k.d., das b., 2004 studies on varietal diversity in blooming, productivity and quality characteristics of almond germplasm in kashmir valley. acta horticulturae, 662: 151-156. das b., 1995 studies on compatibility and xenia in almond (prunus amygdalus batsch). m. sc. thesis, dr. y.s. parmar university of horticulture and forestry, solan, himachal preadesh, india. das b., kumar k., 2004 in-vivo pollen germination on stigma and pollen tube growth in relation to inter-varietal cross compatibility in almond. app. biol. res., 6: 44-47. dhillon d.s., dhatt a.s., gill r.p.s., 1982 pollination studies in almond (prunus amygdalus batsch) growing under subtropical conditions. indian j. of hort., 39: 190-195. eti s., paydas v., kuden a.b., kasks n., kurnaz s., ilgin m., 1994 investigations on the pollen viability, germination capability and the growth of pollen tubes on some selected almond types under cukurova conditions. acta horticulturae, 373: 225-233. gagnard j.m., 1954 research on systematic of almond varieties grown in algeria and on the phenomena of sterility. ann. inst. agric. alger., 8(2): 163. griggs w.h., iwakiri b., 1964 timing is critical for effective cross pollination of almond flowers. californian agric., 18(1): 6-7. kester d.e., 1969 almonds, pp. 302-314. in: richard a.j. (ed.) handbook of north american nut trees. northern nut grower association, knoxville, tennessee, usa, pp. 421. kester d.e., gradziel t.m., micke w.c., 1994 identifying pollen incompatibility groups in california almond cultivars. proc. of the amer. soc. of hort. sci., 119(1): 106109. kester d.e., griggs w.hr., 1959 fruit setting in the almond: the effect of cross pollinating various percentages of flowers. proc. of the amer. soc. of hort. sci., 74: 206-213. loreti f., viti r., 1984 recherches sur la pollinisation de certaines varieties d’amandier dans les conditions climatiques du littoral toscan. ciheam options mediterraneennes, 84(ii): 177-183. ortega e., dicenta f., egea j., 2007 rain effect on pollen stigma adhesion and fertilization in almond. scientia hort., 112(3): 345-348. ortega e., egea j., dicenta f., 2004 effective pollination period in almond cultivars. hort. science, 39(1): 19-22. panse v.g., sukhatme p.v., 1978 statistical methods for agricultural workers. indian council of agricultural research, new delhi, india, pp. 347. pimienta e., polito v.s., 1983 embryo sac development in almond [prunus dulcis (mill.) d.a. webb] as affected by cross, self and non pollination. annals of botany, 51: 469-479. polito v.s., micke w.c., kester d.e., 1996 bud development, pollination and fertilization, pp. 98-102. in: micke w.c. (ed.) almond production manual. division of agriculture and natural resources, university of california, usa. sedgley m., 1982 floral development, anthesis and pollination. acta horticulturae, 240: 177-183. socias i company r., 1992 breeding self fertile almonds. plant breeding reviews, 8: 313-338. socias i company r., 2002 latest advances in almond self compatibility. acta horticulturae, 591: 205-212. socias i company r., alonso j.m., gomez j.a., 2005 evaluation of almond selection for fruit set under field conditions. ciheam options mediterraneennes, 63: 133-139. socias i company r., felipe a.j., aparisi j.g., 1996 genetics of late blooming in almond. acta horticulturae, 484: 261-266. talaie a.r., imani a., 1998 flowering, pollination and fruit set patterns in some new iranian almond genotypes. acta horticulturae, 470: 123-130. 12 appendix i meteorological data march, 2006 days temperature maximum minimum i ii rh (%) rh (%) i ii rain (mm) weather 7:30 hr 14:30 hr i ii 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 15.00 16.00 17.50 16.00 15.80 15.60 13.00 14.00 16.00 16.00 19.00 20.00 16.00 11.00 13.00 11.00 14.00 16.50 9.00 6.00 14.00 17.50 18.50 11.50 11.40 15.00 14.50 16.50 20.00 20.50 21.60 15.20 1.00 2.50 2.00 3.00 2.60 3.80 2.50 0.50 3.00 3.30 1.50 0.80 3.00 6.40 4.40 0.50 1.20 0.40 5.20 5.00 5.00 3.40 4.60 4.50 5.50 6.50 6.50 5.60 3.00 4.00 9.50 3.57 85.00 94.00 85.00 86.00 94.00 81.00 89.00 94.00 85.00 75.00 88.00 89.00 75.00 90.00 87.00 86.00 81.00 78.00 78.00 94.00 94.00 87.00 73.00 75.00 95.00 75.00 92.00 85.00 95.00 75.00 60.00 84.51 78.00 69.00 53.00 56.00 63.00 77.00 77.00 56.00 60.00 45.00 46.00 42.00 73.00 72.00 87.00 74.00 60.00 67.00 74.00 92.00 58.00 53.00 46.00 69.00 66.00 68.00 68.00 56.00 39.00 39.00 38.00 61.96 0.00 0.00 0.00 0.00 1.00 0.00 5.80 0.00 0.00 0.00 0.00 0.00 0.00 1.80 12.20 7.40 2.20 0.00 0.00 13.00 4.80 0.00 0.00 0.00 8.20 1.60 4.20 4.00 0.00 0.00 0.00 66.20 clear clear clear clear clear cloudy cloudy clear cloudy cloudy clear clear clear cloudy p. cloudy cloudy cloudy clear cloudy rain cloudy clear clear cloudy rain cloudy cloudy cloudy clear clear cloudy clear clear clear clear clear rain cloudy cloudy clear clear clear clear rain rain rain rain clear cloudy cloudy cloudy clear clear clear rain rain cloudy cloudy clear clear clear clear year 2006 month temperature maximum minimum rh% rh% rain (mm) january february march april may june july august september october november december 4.07 12.59 15.20 20.94 28.27 27.96 31.12 28.31 25.05 22.13 14.17 7.47 -1.82 2.38 3.57 5.69 11.45 13.41 17.91 17.26 11.23 6.78 2.79 -0.54 92.19 90.71 84.52 71.30 74.26 79.43 77.35 86.80 88.86 92.16 92.00 93.06 84.06 72.75 61.97 45.17 53.06 65.00 63.32 64.35 69.53 63.94 67.67 76.23 168.10 53.20 66.20 55.50 38.60 35.80 151.60 149.20 108.00 19.00 82.50 94.90 13 march, 2007 days temperature maximum minimum i ii rh (%) rh (%) i ii rain (mm) weather 7:30 hr 14:30 hr i ii 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 11.40 12.00 10.00 10.50 13.50 14.50 16.00 17.00 13.50 15.20 9.00 2.00 7.50 9.00 11.50 11.50 10.00 11.40 9.50 5.50 6.00 11.50 15.50 16.50 18.00 19.40 22.50 22.50 23.50 25.00 25.00 13.73 2.70 -1.20 2.50 1.00 0.20 -1.00 -1.00 -0.50 1.50 3.40 4.00 0.00 0.00 -1.50 -2.00 -2.60 -0.50 3.80 4.50 4.50 3.20 3.80 4.00 3.40 1.80 2.40 3.50 3.50 5.00 6.60 8.50 2.04 88.00 100.00 73.00 97.00 94.00 96.00 90.00 87.00 72.00 82.00 71.00 93.00 100.00 93.00 90.00 69.00 85.00 92.00 90.00 97.00 92.00 94.00 89.00 75.00 73.00 62.00 79.00 61.00 65.00 73.00 80.00 83.93 59.00 43.00 64.00 75.00 47.00 35.00 35.00 39.00 53.00 46.00 78.00 90.00 77.00 64.00 50.00 55.00 54.00 76.00 87.00 91.00 97.00 66.00 52.00 53.00 46.00 49.00 45.00 42.00 38.00 42.00 38.00 57.61 5.40 0.00 0.00 3.60 3.00 0.00 0.00 0.00 0.00 0.00 0.00 35.00 165.00 5.40 0.00 0.00 0.00 2.40 0.00 25.40 29.80 6.80 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 281.8 cloudy clear cloudy cloudy cloudy clear clear clear clear cloudy cloudy snow snow cloudy clear clear cloudy cloudy cloudy rain cloudy cloudy p. cloudy clear clear clear clear clear clear clear p. cloudy cloudy clear rain cloudy clear clear clear clear cloudy clear rain snow clear clear clear clear cloudy cloudy rain rain cloudy clear clear cloudy clear clear clear clear clear clear clear year 2007 month temperature maximum minimum rh% rh% rain (mm) january february march april may june july august september 9.36 11.11 13.74 24.99 25.13 28.54 29.90 29.79 26.88 -2.95 1.8 2.04 6.87 10.52 14.55 16.86 16.53 12.35 88.8 90.03 83.94 70.90 77.32 75.80 82.00 81.84 85.43 53.80 64.43 57.61 39.23 53.29 55.27 56.58 57.39 58.97 8.90 50.50 281.8 1.40 44.50 49.70 57.60 46.40 23.20 source (sasa). 100 1. introduction ozone is a major gaseous pollutant in the troposphere and ozone concentrations have in recent years increased rapidly in developing asian countries. indeed, the emission of anthropogenic nitrogen oxides (ozone precursors) in asia under a no-further-control scenario was predicted to increase by 350% between 1990 and 2020 (aunan et al., 2000). an elevated ozone concentration will reduce the growth and yield of crop plants including rice, the most important food crop in asia (kobayashi et al., 1995; yonekura et al., 2005). many researchers have described the mechanisms responsible for visible injury on plant leaves by acute ozone exposure (reviewed by kangasjarvi et al., 2005). the primary mechanism is oxidative damage caused by an increase in levels of reactive oxygen species (ros). however, the cause for yield reductions under chronic ozone stress remains unclear. in a previous report we described how ozone sensitivity in evaluated rice cultivars, in terms of visible injury (chlorotic or necrotic lesions), did not coincide with that indicated by the grain yield reduction (sawada and kohno, 2009). in addition, conventional evaluation of chronic ozone effects relies on measurements such as growth and yield reductions, which require large-scale studies (e.g. in a field or greenhouse) and long time periods (e.g. about six months). a rapid and small-scale method for early evaluation of chronic ozone effects, such as the use of molecular markers, would make it faster, easier, and less expensive to select ozone-tolerant cultivars. kubo et al. (2011) reported that during ozone stress, sakuranetin, a flavonone in the phytoalexin family, appears to serve as a molecular marker of the stress response. sakuranetin contents in rice leaves exposed simultaneously to ozone and high temperature increased only in the three cultivars whose grain yield was unaffected by ozone stress. however, their experiment was performed under both elevated ozone and elevated temperature, making it difficult to determine the separate effect of each factor. moreover, the ozone concentration was 150 nl l-1 (ppb), much higher than ambient ozone levels. therefore, more practical markers are needed. proteomic studies are useful to reveal protein markers associated with various stress tolerance (reviewed by kosova et al., 2011). in a previous study, we conducted differential proteome analysis using three rice cultivars that showed different levels of ozone sensitivity (indicated by the reduction in grain yield) when exposed to elevated ozone during the cultivation season in open-top chambers (sawada et al., 2012). in these cultivars, we observed significant changes in the size of spot that contained three proteins: a 60-kda chaperonin (cpn-60), chloroplastic atp synthase, and enolase 1. the change in size of this spot was proportional to their ozone sensitivity, measured as the reduction in grain yield. these results suggest that these proteins are closely involved in the mechanisms that underlie the yield reduction potential marker proteins for ozone-induced yield reduction in rice h. sawada*(1), s. komatsu**, m. tamaoki***, y. kohno* * environmental science research laboratory, central research institute of electric power industry (criepi), 1646 abiko, abiko-shi, chiba 270-1194, japan. ** national institute of crop scinence, 2-1-18 kannondai, tsukuba, ibaraki 305-8518, japan. *** center for environmental biology and ecosystem studies, national institute for environmental studies, onogawa 16-2, tsukuba, ibaraki 305-8506, japan. key words: grain yield, ozone stress, protein markers, 60-kda chaperonin. abstract: three proteins a 60-kda chaperonin (cpn-60), chloroplastic atp synthase, and enolase 1 were evaluated as potential markers of ozone-induced yield responses in six rice (oryza sativa l.) cultivars (‘kirara 397’, ‘koshihikari’, ‘nipponbare’, ‘takanari’, ‘kasalath’, ‘suphanburi 90’) under ozone stress in laboratory-scale tests. the levels of all three proteins decreased after ozone exposure in cultivars identified as ozone-sensitive while they increased or remained constant after ozone exposure in tolerant cultivars, although atp synthase tended to decrease. furthermore, the protein level and grain yield in each cultivar exposed to ozone were significantly positively correlated for all three proteins. thus, cpn-60 and enolase 1 are potential markers for chronic ozone stress in rice. adv. hort. sci., 2014 28(2): 100-104 (1) corresponding author: sawada.hiroko@nies.go.jp received for publication 12 march 2014 accepted for publication 16 june 2014 101 that occurs under elevated ozone levels, and therefore they have potential as molecular markers that can predict the ozone-induced yield loss. to clarify the usefulness of these proteins for use in laboratory-scale tests, we investigated the levels of these candidate proteins in the seedlings of six rice cultivars under short-term ozone exposure in growth chambers, and tested for a significant correlation between the protein levels and relative grain yield. 2. materials and methods chronic ozone exposure six rice (oryza sativa l.) cultivars were used in this study: ‘kirara 397’, ‘koshihikari’, ‘nipponbare’ (japonica cultivars), ‘takanari’ (a hybrid indica cultivar), ‘kasalath’, ‘suphanburi 90’ (indica cultivars). seedlings (n = 40) of each cultivar were grown in seedling boxes for three weeks in a glasshouse under ambient atmospheric conditions, then transplanted into pots (at four plants per pot with a 0.05-m2 surface area and a 0.015m3 volume) in open-top chambers (otcs; 3.6 × 3.6 m) at an experimental field of the akagi testing center of the criepi (maebashi, japan) in the late spring of 2007, 2008, and 2009. fertilizer was supplied at a rate of np 2 o 5 -k 2 o=15-15-15 g m-2. the otc fumigation system has been described previously (frei et al., 2011). ozone was added in the chambers using a mass-flow controller combined with a pid controlling system to maintain the designated concentrations. three ozone-level treatments were established, from transplanting of rice plants into the pots to harvest, for three years with a regular diurnal pattern: charcoal-filtered air (cf), ambient ozone (ozone ×1), and twice ambient ozone (ozone ×2). concentrations of ozone were continuously monitored in each chamber at 3-min intervals using a uv absorption ozone analyzer (ml9810, monitor labs, englewood, co, usa). mean ozone concentration, air temperature and relative humidity in the different treatments are summarized in table 1. measurement of the yield the rice cultivars were harvested between september and november in 2007, 2008, and 2009. harvesting of each cultivar was conducted when about 80% of the grains had turned yellow. after harvesting, grains were separated from the panicles and categorized into two groups (filled and unfilled grains) using an automatic seed-sorting machine (fv-459a, fujiwara seisakusho kk, tokyo, japan). the filled grains (rough rice) were weighed to determine the grain yield. short-term ozone exposure rice seedlings were grown in indoor growth chambers at 28/23°c (day/night), photosynthetic photon-flux density of 400 μmol m-2 s-1, with a 12-h photoperiod, and a relative humidity of 60±5%. after two weeks, the ‘kirara 397’, ‘koshihikari’, and ‘takanari’ seedlings were exposed to three levels of ozone (12 h/day) for three days in three individual replicates: cf, ambient ozone (40 ppb), and twice ambient ozone (80 ppb). similarly, ‘nipponbare’, ‘kasalath’, and ‘suphanburi 90’ seedlings were exposed to cf and 40 ppb of ozone. ozone was generated with a silent electrical discharge in dry oxygen. the concentration of ozone in the chambers was monitored continuously during exposure with a uv absorption ozone detector (model 1150, dylec inc., tokyo, japan). at the end of the exposure, we removed the third leaves, immediately froze them in liquid nitrogen, and stored them in -80°c until the immunoblot analysis was performed. immunoblot analysis leaves (100 mg) were homogenized in sodium dodecyl sulfate (sds) buffer (10% (w/v) glycerol, 5% (v/v) β-mercaptoethanol, 2.3% (w/v) sds, and 62.5 mm trishcl, ph 6.8). equal amounts of protein samples were separated using 15% sds-polyacrylamide gel electrophoresis (page). after the sds-page, the protein samples were transferred onto a polyvinylidene fluoride membrane or they were stained by coomassie brilliant blue (cbb). table 1 ozone concentrations and environmental conditions in the open-top chambers during the cropping seasons of rice ozone concentration (ppb) temperature (ºc) 24 h mean relative humidity (%) 24 h mean12 h mean 24 h mean mean daily maximum 2007 cf 3.1 2.1 4.1 – – ozone x1 37.6 31.1 61.3 – – ozone x2 68.6 56.3 101.7 – – 2008 cf 4.7 3.9 6.5 21.1 83.4 ozone x1 40.4 27.5 57.6 21.3 83.5 ozone x2 82.7 57.0 118.3 21.3 81.7 2009 cf 5.1 5.0 9.7 20.6 78.7 ozone x1 35.1 27.9 56.9 20.7 78.9 ozone x2 73.5 54.7 110.2 20.9 77.4 measurements of environmental conditions and ozone concentrations were recorded at 3 and 10-minute interval throughout the experiment, respectively. average values of the two replicate chambers per treatment are shown. 12 h means were calculated for the period from 6:00 to 17:59 hours. the temperature and relative humidity were not measured in 2007. 102 the blotted membrane was blocked for 1 h in tbs-t (20 mm tris-hcl, ph 7.6, 150 mm nacl and 0.1% v/v tween-20) containing 5% (w/v) nonfat milk (skim milk; difco, sparks, md, usa). the membrane was subsequently incubated with the monoclonal antibody anti-heatshock protein 60 (acris antibodies gmbh, herford, germany), with the polyclonal antibodies anti-atp synthase β-subunit (antiprot, pullach i. isartal, germany), and antienolase (aviva system biology, san diego, ca, usa) at 1:5000 dilutions for 1 h at room temperature. as secondary antibodies, we used anti-mouse or anti-rabbit igg with conjugated hrp (bio-rad laboratories inc., hercules, ca, usa). after incubation for 1 h with the appropriate horseradish peroxidase (hrp)-conjugated secondary antibodies, we detected the immunoblot signals using the ecl plus western blotting detection kit (ge healthcare, piscataway, nj, usa) following the manufacturer’s protocols and the results were visualized using an las-3000 luminescent image analyzer (fujifilm, tokyo, japan). the relative intensities of the bands were calculated using pdquest software (version 8.0.1, bio-rad). 3. results and discussion after chronic ozone exposure during three years of growing seasons (from 2007 to 2009) each cultivar showed a similar yield response to ozone in all years of the experiment. the grain yields of ‘kirara 397’, ‘takanari’ and ‘kasalath’ decreased significantly by 15 to 36%, 10 to 21%, and 12 to 19%, respectively, under twice the ambient ozone level (about 80 ppb treatment, daily 12-h mean concentration), although the grain yields did not differ significantly from cf under ambient ozone level (about 40 ppb treatment), except for ‘kirara 397’ and ‘takanari’ in 2007 (fig. 1, p <0.05). the grain yields of ‘koshihikari’, ‘nipponbare’, and ‘suphanburi 90’ did not decrease significantly with ozone stress. on this basis, we defined ‘kirara 397’, ‘takanari’, and ‘kasalath’ as ozone-sensitive cultivars, and ‘koshihikari’, ‘nipponbare’, and ‘suphanburi 90’ as ozone-tolerant cultivars. to confirm whether cpn-60, atp synthase, and enolase 1 can be used as markers for ozone-induced rice yield loss in laboratory-scale tests, we analyzed the levels of these proteins (fig. 2a). levels of cpn-60 decreased significantly after three days of exposure to 40 (the ambient concentration) and 80 ppb (twice the ambient concentration) of ozone in ‘kirara 397’ and 80 ppb of ozone in ‘takanari’ (fig. 2b, p<0.05). levels of atp synthase and enolase 1 tended to decrease after ozone exposure, although not significantly (except for ‘takanari’ exposed to 80 ppb of ozone), in both ‘kirara 397’ and ‘takanari’. these cultivars also showed lower grain yield under ozone exposure (fig. 1). in contrast, levels of cpn-60 and enolase 1 in ‘koshihikari’ exposed to 40 ppb of ozone increased significantly and remained the same compared with the levels in cf (p <0.05). moreover, enolase 1 production also remained constant in ‘koshihikari’ at 80 ppb ozone exposure. the level of atp synthase tended to decrease after ozone exposure in ‘koshihikari’. because the levels of cpn-60 and enolase 1 decreased and increased at 40 ppb ozone exposure in ozone-sensitive and ozonetolerant cultivars, respectively, ‘kasalath’, ‘nipponbare’, and ‘suphanburi 90’ seedlings were exposed to cf and fig. 1 effects of chronic ozone exposure on the grain yields of six rice cultivars in 2007, 2008, and 2009. values are mean ± se (n= 40). asterisk indicates a significant difference compared with cf according to dunnett’s test (p<0.05). ‘kasalath’ and ‘suphanburi 90’ were not cultivated in 2007 and 2009, respectively, and yields are shown as “nc”. 103 40 ppb of ozone. the levels of all three proteins increased significantly or were maintained under 40 ppb of ozone in ‘nipponbare’ and ‘suphanburi 90’, but tended to decrease in ‘kasalath’. therefore, the levels of cpn-60 and enolase 1 differed between the ozone-sensitive and ozone-tolerant cultivars: they decreased and increased, respectively at least at 40 ppb. cpn-60 is a molecular chaperone. many molecular chaperones were originally identified as heat-shock proteins (hsps), which function in protein folding, assembly, fig. 2 (a) immunoblot analysis of cpn-60, atp synthase, and enolase 1 in leaves of rice seedlings exposed to charcoal filtered air (cf), or to 40 or 80 ppb of ozone. immunodetection was performed with antibodies specific to these proteins. (bottom panel) cbb-stained sds-page showing the quality and loading quantity of the protein samples. (b) relative intensity for each protein estimated from the immunoblot analysis in panel (a). values are mean ± se (n = 3). asterisk indicates a significant difference compared with cf according to dunnett’s test or t-test (p<0.05). nb, nipponbare; sp90, suphanburi 90. translocation, and degradation during many normal cellular processes, and can assist in protein refolding under stress (wang et al., 2004). cpn-60 (hsp60) appears to be involved in the defense response that mitigates oxidative stresses (wang et al., 2011). enolase 1 is an enzyme involved in glycolysis in the cytosol. bohler et al. (2007) suggested that the enzymes involved in glycolysis increase to produce more energy and to increase the reduction capacity for detoxification of ros and repair oxidative damage in response to ozone stress in the leaves of poplar (populus). in arabidopsis thaliana, cpn60b (at1g55490), encoding homologous protein to cpn-60 in rice, was upregulated in response to drought, uv-b, heat, wounding and oxidative (methyl viologen) stress within 30 min (winter et al., 2007). similarly, eno2 (at2g36530) in a. thaliana, encoding homologous protein to enolase 1 in rice, was upregulated in response to cold, drought, uvb, wounding and heat stress (winter et al., 2007). these studies suggest that cpn-60 and enolase 1 are induced by stresses involved in the production of ros. therefore, the alterations of these protein levels with ozone exposure might result in ozone-derived ros rather than ozone itself. however, there has been no report describing whether cpn-60 and enolase 1 influence grain production in crops under environmental stress, although these proteins might not be specific markers to ozone. further studies will be needed to clarify the relationship between the reduction in grain yield and decreased production of these proteins by ozone-sensitive cultivars. in order to compare the relative levels of each protein upon short-term ozone exposure with the relative grain yields under chronic ozone exposure we performed a linear regression analysis (fig. 3). we found significant positive correlations between the levels of cpn-60, atp synthase, and enolase 1 and the relative grain yield (i.e. yield decreased as the protein concentrations decrease). therefore, the three proteins may serve as potential markers for chronic ozone stress in rice, although further experiments will be required for atp synthase that also tended to decrease in ‘koshihikari’ at 40 ppb ozone exposure (fig. 2b). the level of cpn-60 had the highest goodness of fit (r2 = 0.786) with the grain yield. this suggests that the potential ozone-induced yield reduction can be evalu104 ated using the level of cpn-60 at the seedling stage in laboratory-scale tests. moreover, the protein markers that we identified in this study may be useful in crop breeding to quickly select ozone-tolerant rice varieties. vincent et al. (2007) indicated that the inhibition of shoot growth was best correlated with the level of cpn-60 in two wine grape cultivars exposed to salinity and water deficit stress, suggesting that the protein marker is also applicable to other plant or crops. acknowledgements this work was supported by the environmental research and technology development fund (a0806) of the ministry of the environment, japan. references aunan k., berntsen t.k., seip h.m., 2000 surface ozone in china and its possible impact on agricultural crop yields. ambio, 29:294-301. bohler s., bagard m., oufir m., planchon s., hoffmann l., jolivet y., hausman j.f., dizengremel p., renaut j., 2007 a dige analysis of developing poplar leaves subjected to ozone reveals major changes in carbon metabolism. proteomics, 7: 1584-1599. frei m., kohno y., wissuwa m., makkar h.p.s., becker k., 2011 negative effects of tropospheric ozone on the feed value of rice straw are mitigated by an ozone tolerance qtl. global change biol., 17: 2319-2329. kangasjarvi j., jaspers p., kollist h., 2005 signalling and cell death in ozone-exposed plants. plant cell environ., 28: 1021-1036. kobayashi k., okada m., nouchi i., 1995 effects of ozone on dry matter partitioning and yield of japanese cultivars of rice (oryza sativa l.). agric. ecosyst. and environ., 53: 109-122. kosova k., vitamvas p., prasil i.t., renaut j., 2011 plant proteome changes under abiotic stress contribution of proteomics studies to understanding plant stress response. j. proteomics, 74: 1301-1322. kubo a., cho k., rakwal r., kohno y., shibato j., 2011 method for evaluating influence of ozone on rice using sakuranetin. japan patent kokai, 2011-33533. sawada h., kohno y., 2009 differential ozone sensitivity of rice cultivars as indicated by visible injury and grain yield. plant biol., 11(suppl. 1): 70-75. sawada h., komatsu s., nanjo y., khan n.a., kohno y., 2012 proteomic analysis of rice response involved in reduction of grain yield under elevated ozone stress. environ. exp. bot., 77: 108-116. vincent d., ergul a., bohlman m.c., tattersall e.a.r., tillett r.l., wheatley m.d., woolsey r., quilici d.r., joets j., schlauch k., schooley d.a., cushman j.c., cramer g.r., 2007 proteomic analysis reveals differences between vitis vinifera l. cv. chardonnay and cv. cabernet sauvignon and their responses to water deficit and salinity. j. exp. bot., 58: 1873-1892. wang c.r., gu x.y., wang x.r., guo h.y., geng j.j., yu h.x., sun j.a., 2011 stress response and potential biomarkers in spinach (spinacia oleracea l.) seedlings exposed to soil lead. ecotoxicol. environ. saf., 74: 41-47. wang w., vinocur b., shoseyov o., altman a., 2004 role of plant heat-shock proteins and molecular chaperones in the abiotic stress response. trends plant sci., 9: 244-252. winter d., vinegar b., nahal h., ammar r., wilson. g.v., provert n.j., 2007 an “electronic fluorescent pictograph” browser for exploring and analyzing large-scale biological data sets. plos one, 2: e718. yonekura t., shimada t., miwa m., arzate a., ogawa k., 2005 impacts of tropospheric ozone on growth and yield of rice (oryza sativa l.). j. agric. meteorol., 60: 1045-1048. fig. 3 regression analysis for the relative levels of cpn-60, atp synthase, and enolase 1 in rice seedlings exposed to 40 and 80 ppb ozone, plotted as a function of relative grain yield. the grain yields are relative to the values for six cultivars grown in opentop chambers. significance levels: **, p< 0.01; *, p< 0.05. 92 1. introduction the spread of grapevine varieties through different countries over time has produced many of cases of synonymy that now need clarification. correct identification is very important in order to gain more precise knowledge of the varietal assortment and to better manage grapevine catalogues and germplasm repositories. the italian catalogue of grapevine varieties is one of the most copious in the world. nonetheless, it contains various duplicates or triplicates, such as ‘alicante’/‘cannonao’/‘tocai rosso’, ‘vermentino’/ ‘pigato’/‘favorita’, ‘biancame’/‘trebbiano toscano’, ‘albarola’/‘bianchetta genovese’. also ‘verdicchio’, one of the most prized white grape varieties of the marche region (central italy) and registered in the italian catalogue with code no. 254, has an officially recognized duplicate, ‘trebbiano di soave’ (code no. 239), the name used in several provinces of the veneto region (north-eastern italy). other synonyms in the same region are ‘trebbiano di lugana’ and ‘turbiana’. in the lazio region (central italy) it is known as ‘trebbiano verde’ (bruni, 1962). to our knowledge, the first citation of ‘verdicchio’ was by the botanist costanzo felici as far back as 1569 (in arbizzoni, 1986). ‘verdello’ is considered a minor grapevine variety of the umbria region (central italy). it is registered in the italian catalogue with code no. 253 and is used for the doc wines of orvieto, colli amerini, colli del trasimeno and torgiano. scalabrelli and grasselli (1988) report that ‘verdello’ is also cultivated in the south of tuscany under the synonym ‘duropersico’, mainly in the territory of pitigliano doc, where it has been present for at least two centuries and contributes to the wine of the same name. the two authors refer that in the same territory a variety known as ‘uva angiola’ is also present; they generically link it to the trebbiano group, but this variety more specifically resembles to those analyzed in the present study. furthermore, in the most northern part of tuscany, a variety morphologically similar to ‘verdicchio’ has been grown for a long time. it is called ‘verduschia’ (soderini, 1600; acerbi, 1825), ‘verdella’ or ‘verdarella’, depending on the cultivation area. the diffusion of this variety is limited to the small lunigiana territory (provinces of la spezia and massa carrara) and to a few residual specimens in old vineyards (scalabrelli and dodi, 1998). ‘verduschia’ falls into the category of minor grapevine varieties on the ‘verdello’, ‘verdicchio’ and ‘verduschia’: an example of integrated multidisciplinary study to clarify grapevine cultivar identity m. crespan*, a. armanni**, g. da rold*, b. de nardi*, m. gardiman*, d. migliaro*, s. soligo***, p. storchi** * consiglio per la ricerca e la sperimentazione in agricoltura, centro di ricerca per la viticoltura, cra-vit, viale xxviii aprile, 26, 31015 conegliano (tv), italy. ** consiglio per la ricerca e la sperimentazione in agricoltura, unità di ricerca per la viticoltura, cra-vic, via romea, 53, 52100 arezzo, italy. *** centro regionale per la viticoltura, l’enologia e la grappa, cerveg, veneto agricoltura, via zamboni, 31015 conegliano (tv), italy. key words: ampelography, ‘duropersico’, ssr markers, ‘trebbiano di soave’, ‘turbiana’, ‘uva angiola’. abstract: ‘verdello’, ‘verdicchio’ and ‘verduschia’ are registered in the official italian catalogue as three distinct grapevine varieties. twenty-five accessions of these cultivars, encompassing known or presumed synonyms, coming from cra repositories and from vineyards where they are traditionally cultivated, have been genotyped with eleven ssr markers. for morphological comparison, one accession for each variety has been described with 57 characters of oiv 2009 list; phenological and yield traits have also been recorded. in addition, the phenotypic comparison has been extended to the literature descriptions. the same dna profile has been obtained for all 25 accessions; moreover, present and historical ampelographic data showed a very high similarity. all this information leads to the conclusion that these three varieties are, in fact, the same cultivar. adv. hort. sci., 2012 26(2): 92-99 received for publication 16 february 2012. accepted for publication 31 may 2012. 93 verge of extinction; although registered in the italian catalogue since 1971 with code no. 297, it does not seem to have been propagated. dna genotyping allowed us to hypothesise an unrecognized synonymy among ‘verdicchio’, ‘verdello’ and ‘verduschia’. in fact, based on previous results obtained with molecular analysis of some accessions of ‘verdello’, interesting for the clonal selection of materials from lazio, we found that they had the same profile of the ‘verdicchio’ accession held in the centro di ricerca per la viticoltura (cra-vit) repository. given the interest aroused by this preliminary information, we extended the molecular comparison to numerous accessions of ‘verdicchio’, ‘verdello’ and their synonyms, as well as to ‘verduschia’, the tuscan variety morphologically similar to ‘verdicchio’. the samples for genotypic comparison were singled out both in the cra-vit repositories and in the cultivation areas of each variety, for a total of 25 accessions, including a commercial clone of ‘verdello’ (clone vcr1) and another of ‘verdicchio bianco’ (clone r2). this study was integrated with morphological comparison: one accession for each variety was characterized with 57 descriptors of the oiv 2009 list; phenological and yield traits were also recorded. in addition, ampelographic comparison was extended to the literature descriptions of these three varieties: most of the traits reported in literature were retrieved and harmonized according to the oiv 2009 descriptors list used for examining the actual materials. 2. materials and methods plant material twenty-five accessions were sampled, coming from cra-vit repositories in spresiano (tv) and susegana (tv), from unità di ricerca per la viticoltura (cra-vic) repositories in arezzo, from the veneto agricoltura collection and from vineyards in veneto, tuscany, umbria, marche and lazio regions (table 1). ampelographic, phenological and yield data comparison three accessions were described: one ‘verdello’ of umbrian origin (accession no. 4 in table 1) and one ‘verduschia’ (no. 15 in table 1) coming from lunigiana (province of massa carrara), both held in the cra-vic germplasm collection in arezzo, and one ‘verdicchio’ coming from marche (no. 25 in table 1), held at the azienda poggio gagliardo in montescudaio (pisa, italy). morphological descriptions of the three varieties were performed according to 57 descriptors (oiv, 2009); phyllometric analyses were also carried out on samples of 20 leaves per cultivar using superampelo software (soldavini et al., 2009). the comparison was widened to the ‘verdello’, ‘verdicchio’ and ‘verduschia’ descriptions given in the literature: in particular, the umbrian ‘verdello’ was described by cartechini and moretti (1989), the ‘verdicchio’ from marche by bruni (1962) and the tuscan ‘verduschia’ by breviglieri and casini (1965). to facilitate the comparison, all these descriptions were standardized according to the oiv 2009 descriptor list. dna extraction total genomic dna was extracted from young leaves of the 25 accessions using nucleospin® 8 plant kit (macherey-nagel gmbh, düren, germany) automated on the microlab® star liquid handling robot according to the macherey-nagel nucleospin® 8 plant protocol. dna concentration and quality were assessed with a spectrophotometer and by 1% agarose gel electrophoresis. dna samples were then diluted to 10 ng/µl prior to amplification. ssr analysis ssr analysis was performed in order to verify the varietal identity of the studied accessions. eleven microsatellite loci were analyzed: the six core loci selected within genres 081 european project (vvs2, vvmd5, vvmd7, vvmd27, vrzag62 and vrzag79) (this et al., 2004); vvmd28 (bowers et al., 1999); isv2 (vmc6e1), isv3 (vmc6f1) and isv4 (vmc6g1) (crespan, 2003); vmcng4b9 (welter et al., 2007). a multiplex pcr mixture was prepared, amplifying simultaneously all 11 ssr loci. the reaction mixture consisted of: 1 x pcr buffer (promega; pharmacia biotech), 200 µm of each dntps, 1 u of taq dna polymerase (promega; pharmacia biotech), 2.0 mm mgcl 2 and the primer concentrations ranged between 0.09 µm and 0.40 µm, according to signal  intensity; the forward primers were labelled with 6-fam, vic, pet or ned fluorescent table 1 list of the analysed accessions id accession name provenance 1 verdicchio cra-vit repository 2 verdello cra-vit repository 3 verdello clone vcr1 vivai cooperativi of rauscedo (pordenone) italy 4 verdello covio 6 orvieto (terni) italy 5 verdello sugano orvieto (terni) italy 6 verdello fausto 1 porano (terni) italy 7 verdello 553 ercolani farm, capodimonte (viterbo) italy 8 verdello 550 ercolani farm, capodimonte (viterbo) italy 9 verdello 549 ercolani farm, capodimonte (viterbo) italy 10 verdello 514 catercia farm, capodimonte (viterbo) italy 11 verdello 405 bianchi farm, bagnoreggio (viterbo) italy 12 verdello 146 ar pitigliano (grosseto) italy 13 verdello lazio 14 verdello cra-vit repository 15 verduschia aulla (massa carrara) italy 16 verdella aulla (massa carrara) italy 17 verdicchio carr 10 terranuova bracciolini (arezzo) italy 18 turbiana vicenza italy 19 trebbiano verde viterbo italy 20 trebbiano di soave cra-vit repository 21 trebbiano di soave cra-vit repository 22 trebbiano di lugana veneto 23 duropersico 165 ar pitigliano (grosseto) italy 24 uva angiola cra-vit repository 25 verdicchio clone r2 marche 94 dyes; the final volume was 12.5 µl. pcr was carried out in geneamp 9700 (applied biosystems) with the following thermal profile: 2 min at 94°c, followed by 30 cycles at 94°c for 45 sec, 55°c for 1 min and 30 sec, 72°c for 1 min and a final step at 72°c for 30 min. pcr products (1 µl) were added to 0.1 µl liz 500 size standard and 8.9 µl hi-di formamide (applied biosystems) and separated by capillary electrophoresis using an abi prism 3110xl dna analyzer (applied biosystems) and pop-7 polymer (applied biosystems). after data collection, genotyping analysis was performed with abi prism® genemappertm software version 3.0. 3. results the results of the ampelographic comparison between ‘verdello’, ‘verdicchio’ and ‘verduschia’ are reported in table 2. the descriptions from the present study show that the three grapevine varieties share 47 out of 57 traits. ten diverge (in bold in table 2): in particular, ‘verdello’ differs from ‘verdicchio’ and ‘verduschia’ by having a slight bronze tinge on the young leaf and the mature leaf with less pronounced lower lobes and shorter petiole compared to the length of the middle vein. ‘verdicchio’ showed a larger average size of the mature leaf, a longer middle vein n1 and a greater density of prostrate hairs on the lower side of the blade (descriptor no. 84), as well as longer internodes (no. 353). finally, three morphological traits differ in ‘verduschia’ with respect to ‘verdello’ and ‘verdicchio’: the dorsal side of the shoot is green (oiv codes 007 and 009) and berry size is smaller. therefore, comparison of our descriptions shows a substantial analogy among these varieties, which share 82% of the expression levels, with very minor discrepancies. their similarity is also confirmed by the elaboration of the mature leaf measurements with superampelo software: the cluster analysis performed by the program shows that the degree of similarity is even higher and above 93% (fig. 1). fig. 1 cluster analysis of ‘verdello’, ‘verdicchio’ and ‘verduschia’ phyllometric data elaborated with superampelo software. percentage of similarity values are indicated. table 2 list of ampelographic traits of the varieties ‘verdello’, ‘verdicchio’ and ‘verduschia’. there are two types of comparisons: i) among current descriptions: discordant characters observed in the present study are written in bold; ii) among descriptions from other authors: slightly discordant traits inferred from the literature are underlined; clearly discordant characters are underlined in italics. an asterisk indicates the characters defined by the software superampelo. nr: not recorded character. organ oiv code 2009 description verdello verdicchio verduschia present study cartechini and moretti, 1989 present study bruni, 1962 present study breviglieri and casini, 1965 sh oo t 002 young shoot: distribution of anthocyanin coloration on prostrate hairs of the shoot tip absent absent absent absent absent piping 003 young shoot: intensity of anthocyanin coloration on prostrate hairs of the shoot tip none or very low none or very low none or very low none or very low none or very low low 004 young shoot: density of prostrate hairs on the shoot tip medium very high medium very high medium medium or very high 006 attitude semi-erect semi-erect semi-erect semi-erect semi-erect semi-erect 007 colour of the dorsal side of internodes green and red green and red green and red green and red green green 008 colour of the ventral side of internodes green and red green and red green and red green and red green and red green and red 009 colour of the dorsal side of nodes green and red green and red green and red green and red green green 010 colour of the ventral side of nodes green and red or red green green and red or red green and red green and red green and red 013 density of prostrate hairs on nodes low none or very low low none or very low low low 015_2 intensity of anthocyanin coloration on the bud scales weak weak weak nr weak nr 016 number of consecutive tendrils 2 or less 2 or less 2 or less 2 or less 2 or less 2 or less 95 organ oiv code 2009 description verdello verdicchio verduschia present study cartechini and moretti, 1989 present study bruni, 1962 present study breviglieri and casini, 1965 yo un g le af 051 colour of upper side of blade (4th leaf) green / bronze / yellow green green / yellow green / bronze green / yellow green 053 density of prostrate hairs between main veins on lower side of blade (4th leaf) high very high high very high high very high 055 density of prostrate hairs on main veins on lower side of blade (4th leaf) low low low nr low nr m at ur e le af 065 size of blade medium-small* medium medium* medium medium-small* medium-small 067 shape of blade wedge-shaped or pentagonal* circular wedge-shaped or pentagonal* circular or pentagonal wedge-shaped or pentagonal* pentagonal 068 number of lobes five or three five five five or three five five or three 069 colour of the upper side of the blade between medium green and dark green medium green between medium green and dark green between medium green and dark green between medium green and dark green dark green 070 area of anthocyanin coloration of main veins on upper side of blade absent absent absent absent absent absent 071 area of anthocyanin coloration of main veins on lower side of blade absent absent absent absent absent absent 073 undulation of blade between main or lateral veins present present present present present present 074 profile of blade in cross section involute, v-shaped or twisted v-shaped involute, vshaped or twisted flat or twisted involute, vshaped or twisted flat or twisted 075 blistering of upper side of blade medium weak medium medium medium nr 076 shape of teeth mixture between straight and convex sides one side concave, one side convex mixture between straight and convex sides mixture between straight and convex sides mixture between straight and convex sides both sides convex 078 length of teeth compared with their width short short short short short short 079 degree of opening/overlapping petiole sinus overlapped strongly overlapped overlapped closed or overlapped overlapped overlapped 084 density of prostrate hairs between main veins on lower side of blade medium medium high very high medium very high 086 density of prostrate hairs on main veins on lower side of blade low low low high low nr 090 density of prostrate hairs on petiole low none or very low low none or very low low none or very low 093 length of petiole compared to length of middle vein (n1) shorter than n1 longer than n1 equal to n1 nr equal to n1 nr 601 length of vein n1 short* nr medium* nr short* nr 602 length of vein n2 medium* nr medium* nr medium* nr 603 length of vein n3 medium* nr medium* nr medium* nr 604 length of vein n4 very long* nr very long* nr very long* nr w oo dy sh oo t 101 cross section circular or elliptic circular circular or elliptic circular or elliptic circular or elliptic circular or elliptic 103 main colour brownish brownish brownish between brownish and grey brownish brownish in fl or es ce nc e 151 flower: sexual organs hermaphrodite hermaphrodite hermaphrodite hermaphrodite hermaphrodite hermaphrodite 152 insertion of 1st inflorescence 3rd and 4th node 3rd and 4th node 3rd and 4th node 3rd and 4th node 3rd and 4th node 3rd and 4th node 153 number of inflorescences per shoot 1.1 to 2 1.1 to 2 1.1 to 2 1.1 to 2 1.1 to 2 1.1 to 2 96 organ oiv code 2009 description verdello verdicchio verduschia present study cartechini and moretti, 1989 present study bruni, 1962 present study breviglieri and casini, 1965 bu nc h 202 length medium-long medium medium-long medium-long medium-long long 204 density dense very dense dense dense or medium dense medium 206 length of peduncle short very short short medium short short 208 shape conical / funnel shaped / cylindrical conical conical / funnel shaped / cylindrical conical or cylindrical-conical conical / funnel shaped / cylindrical conical 209 number of wings of the primary bunch 1-2/3-4 with wings 1-2/3-4 wings with wings 1-2/3-4 with wings be rr y 220 length medium medium medium medium between medium and short short 222 uniformity of size uniform uniform uniform uniform uniform uniform 223 shape globose globose globose globose globose globose 225 colour of skin yellow yellow yellow yellow yellow yellow 227 bloom medium medium medium medium medium medium 228 thickness of skin thin thick thin thin thin thick 229 hilum visible visible visible visible visible visible 241 formation of seeds complete complete complete complete complete complete ve ge ta tio n 306 autumn coloration of leaves yellow yellow yellow yellow yellow yellow 351 vigour of shoot growth medium-strong medium medium-strong medium-strong medium-strong medium-strong 352 growth of lateral shoots weak weak weak medium weak weak 353 length of internodes medium long long medium-long medium medium 354 diameter of internodes medium small medium medium medium medium with regard to the descriptions from the literature, most of the 57 oiv descriptors we used were retrieved. only the measurements from code 601 to 604 relative to the mature leaf, three traits of ‘verdicchio’ and five of ‘verduschia’ were excluded (indicated as not recorded characters in table 2) and so a total of 50 descriptors for ‘verdello’ and ‘verdicchio’ and 48 for ‘verduschia’ were taken into consideration. as expected, the literature data comparison highlighted greater differences, given that the three descriptions were made by different authors. twenty-eight descriptors, underlined in table 2, showed slightly discordant expression levels, as they are limited to a single interval or with different coexisting or intermediate expression levels. these are mainly characters whose expression may be influenced by environmental factors like the exposure to the light or by cropping factors and vegetative vigour, in addition to the subjectivity of the observer. other descriptors (7 out of 52), underlined in italics in table 2, showed markedly different expression levels. four of them regard the mature leaf and in particular the density of prostrate hairs on the lower side of the blade (no. 84 and 86) and the profile and shape of the teeth (no. 74 and 76); the other three regard the bunch and the berry (no. 204, bunch density; no. 206, peduncle length; no. 228, thickness of skin). comparing this last group of seven morphological features with the set of results from our field measurements, it appears that our descriptions match better with those of ‘verdicchio’ described by bruni (1962); instead, fewer similarities are noted with the descriptions in the literature for ‘verduschia’ and even fewer for ‘verdello’. lastly, the comparison of the phenological and yield data collected in the two environments [arezzo and montescudaio (pisa)] are reported in table 3. the data obtained from the same references for ampelographic descriptions are also shown for comparison and ‘trebbiano toscano’ is proposed as common reference variety to enhance data significance. ‘verduschia’ differs by the longer vegetative cycle and the lower berry weight. instead, there are no relevant differences between the other two varieties. molecular analysis of the 25 accessions listed in table 1 produced the same dna profile, reported in table 4 together with ‘sangiovese’, ‘pinot noir’ and ‘muscat blanc à petits grains’ profiles, proposed as reference varieties for easier data comparison. to our knowledge this is the first time that the synonymy with ‘trebbiano verde’ from lazio, ‘duropersico’, ‘verduschia’ or ‘verdella’ and ‘uva angiola’ from tuscany and ‘turbiana’ from veneto, is confirmed by molecular data. 97 table 3 phenology and yield data for ‘verdello’, ‘verdicchio’ and ‘verduschia’: average of 2007-2010 for the vineyard in arezzo, average of 2004-2008 for the vineyard in montescudaio (pisa). the data from some reference ampelographic descriptions are also shown for comparison. ‘trebbiano toscano’ is proposed as common reference variety to enhance data significance ‘verdello’ ‘verdicchio’ ‘verduschia’ ‘trebbiano toscano’ place of data collection arezzo1 orvieto (terni)2 montescudaio (pisa)3 iesi (ancona)4 arezzo1 firenze5 arezzo1 montescudaio (pisa)3 bud burst 13 april (medium) medium 7 april (medium) medium-late 12 april (medium) 10 20 april (medium) 16 april (medium) 7 april (medium) flowering 3 june (medium early) medium 2 june (medium -early) early 7 june (medium) 1 10 june (medium) 8 june (medium) 3 june (medium) veraison 11 august (medium) medium 5 august (medium) medium 13 august (medium late) 21 31 august (late) 17 august (medium late) 6 august (medium) ripening 25 september (medium) late 20 september (medium) medium-late 27 september (late) 1 10 october (late) 4 october (late) 28 september (late) average weight of the bunch (g ± sd) 320±95 269 455±43 280 352±66 245 357±51 402±60 average weight of the berry (g ± sd) 1.87±0.18 1.80 1.94 2.05 1.60±0.64 0.80 1.71±0.21 1.82±0.19 ° brix (± sd) 22.0±1.2 22.3 21.4±0.6 20.0 20.6±0.7 21.0 18.9±1.2 21.1±0.8 titratable acidity (g/l ± sd) 6.71±1.36 8.90 6.80±0.40 8.25 6.42± 0.75 6.97 7.32±0.48 5.29±0.62 1 cra-vic repository, lon: 11°49’29’’ e, lat: 43°28’30’’ n. 2 data from cartechini and moretti (1989). lon: 12° 12’ e, lat: 42° 42’ n. 3 poggio gagliardo farm, lon: 10°32’53’’ e, lat: 43°18’52’’ n. 4 data from bruni (1962), lon: 13° 11’ e, lat: 43° 38’ n. 5 data from breviglieri and casini (1965), lon: 11°19’21’’ e, lat: 43°45’11’’ n. table 4 ssr profiles of ‘verdicchio’/‘verdello’/‘verduschia’ and three cultivars proposed as reference to favour comparison with other databases ssr loci verdicchio, verdello and verduschia sangiovese pinot noir muscat blanc à petits grains vvs2 133 133 137 133 155 133 151 133 vvmd5 228 226 228 228 240 236 238 236 vvmd7 239 239 239 233 247 263 243 249 vvmd27 179 179 185 179 185 185 189 194 vvmd28 239 237 221 249 261 247 239 271 vrzag62 195 193 187 185 195 195 193 195 vrzag79 248 242 238 250 256 258 244 254 isv2 (vmc6e1) 165 143 151 141 165 165 165 143 isv3 (vmc6f1) 135 139 133 133 139 139 145 139 isv4 (vmc6g1) 169 177 169 169 197 197 177 187 vmcng4b9 164 158 158 158 166 168 162 166 98 4. discussion and conclusions the large number of accessions analyzed, most of which come from traditional cultivation areas, together with the results of the ampelographic comparisons, allow us to affirm that ‘verdicchio’, ‘verdello’ and ‘verduschia’ represent a new group of synonyms. this discovery is important because the three varieties are registered as distinct in the italian catalogue. this is further supported by the results of the molecular analysis on the commercial clone of ‘verdello’ (clone vcr1), which showed to be identical to the other ‘verdello’ accessions analyzed in this study. it emerges from present work that the extent of the cultivation area in italy and the interest in ‘verdicchio’/ ‘verdello’/‘verduschia’ are greater than previously supposed from the information available on the already known synonyms. according to the italian census of agriculture data (istat, 2004) the total area under ‘verdicchio’, ‘verdello’, ‘verduschia’ and ‘trebbiano di soave’ cultivation in italy is about 6000 ha, mainly in marche (53%) and veneto (30%) and, to a much lesser extent, in umbria (8.3%) and lazio (1.6%). cultivation is also authorized in emilia romagna, tuscany, abruzzo, molise and even in sardinia. the success with which this variety has been cultivated for centuries in central and north-eastern italy is obvious; the broad and historic diversification of the denominations, even in very close areas, supports the old age of the variety and also the interest in the locally produced wines, with recognised quality. the synonymy ‘verdicchio’/‘verdello’/‘verduschia’ is added to a growing number of redundancies found in the italian catalogue and highlights the value of molecular analysis to facilitate rapid comparison among varieties. this tool has been very useful in revealing other, never previously suspected cases of synonymy, such as ‘greco di tufo’ and ‘asprinio’ (costantini et al., 2005), ‘malvasia delle lipari’ and ‘malvasia di sardegna’ (crespan et al., 2006), or to definitively clarify long disputed synonymies, such as ‘malvasia nera di brindisi’ and ‘malvasia nera di lecce’ (crespan et al., 2008; gasparro et al., 2008). it is extremely difficult to hypothesize the centre of diffusion of ‘verdicchio’/‘verdello’/‘verduschia’, also because the pedigree of this variety is unknown. one not scientifically supported hypothesis infers that this cultivar arrived in marche from northern italy in the second half of the 15th century, with colonies of farmers from veneto and lombardy, and from there it moved to lazio, to finally turn up in tuscany (pollini, 2006). the discovery of this new group of synonyms has been greatly facilitated by the use of ssr markers, which suggest comparisons among varieties independently from preliminary ampelographic indications. given the wealth of varieties registered in the italian catalogue, molecular analysis, associated with the building of a reliable and complete molecular database of reference, has been shown to be very useful to highlight redundancies. ampelographic comparison is indispensable to confirm the preliminary data acquired via molecular fingerprinting. indeed, the definition of a cultivated variety is tied to the dus criteria (distinctness, uniformity and stability), since the somatic mutants for agronomically important characters are legally registered as separate cultivars, as are mutants for berry colour or earliness of ripening. lastly, genotyping represents a strategic tool in order to avoid the recording of duplicates in the future. acknowledgements this research was financed by the ministry of agricultural, food and forestry policies as part of the rgv-fao and aser-identivit projects, and with funds from the regional agency ‘veneto agricoltura’. the authors thank francesco anaclerio (vcr) for providing the clone of ‘verdello’ vcr1 and marina niero for the information on the clonal selection of ‘verdello’ in the lazio region. references acerbi g., 1825 delle viti italiane. silvestri, milano. arbizzoni g., 1986 costanzo felici, scritti naturalistici. dell’insalata e piante che in qualunque modo vengono per cibo del’homo. quattroventi, urbin, italy, pp. 105. bowers j.e., dangl g.s., meredith c.p., 1999 development and characterisation of additional microsatellite dna markers for grape. am. j. enol. vitic., 50: 243-246. breviglieri n., casini e., 1965 verduschia. in: i principali vitigni da vino coltivati in italia. vol. iv, ministero dell’agricoltura e delle foreste, rome, italy. bruni b., 1962 verdicchio bianco. in: i principali vitigni da vino coltivati in italia. vol. ii, ministero dell’agricoltura e delle foreste, rome, italy. cartechini a., moretti g., 1989 verdello, pp. 679690. in: ministero dell’agricoltura e delle foreste i principali vitigni da vino coltivati in italia, nuova serie. vol. i:. ministero dell’agricoltura e delle foreste, istituto sperimentale di viticoltura di conegliano, grafiche zoppelli, dosson (treviso), italy. costantini l., monaco a., vouillamoz j., forlani m., grando m.s., 2005 genetic relationships among local vitis vinifera cultivars from campania (italy). vitis, 44(1): 25-34. crespan m., 2003 the parentage of muscat of hamburg. vitis, 42(4): 193-197. crespan m., cabello f., giannetto s., ibáñez j., kontić j.k., maletić e., pejić i., rodriguez i., antonacci d., 2006 malvasia delle lipari, malvasia di sardegna, greco di gerace, malvasia de sitges and malvasia dubrovačka synonyms of an old and famous grape cultivar. vitis, 45(2): 69-73. crespan m., coletta a., crupi p., giannetto s., antonacci d., 2008 ‘malvasia nera di brindisi/lecce’ grapevine cultivar (vitis vinifera l.) originated from ‘negroamaro’ and ‘malvasia bianca lunga’. vitis, 47(4): 205-212. 99 gasparro m., caputo a.r., brini m.l., coletta a., crespan m., la penna f., antonacci d., 2008 characterization of malvasia nera accessions with molecular markers. cd-rom, section varieties: knowledge, characterization, valorization. proceedings of the 31th oiv congress held in verona, 15-20 june. istat, 2004 5° censimento generale dell’agricoltura 2000, “la coltivazione della vite in italia”. volume ii. vitigni. istat, rome, italy. oiv, 2009 oiv descriptor list for grape varieties and vitis species (2nd edition). http://www.oiv.int/oiv/info/frplubicationoiv. pollini l., 2006 viaggio attraverso i vitigni autoctoni italiani. alsaba, siena, italy, pp. 481 scalabrelli g., dodi s., 1998 caratteristiche ampelografiche di alcuni vitigni ad uva bianca reperiti in lunigiana. proceedings “il recupero del patrimonio viticolo della lunigiana”, aulla, massa-carrara, italy, pp. 77-130. scalabrelli g., grasselli a., 1988 recupero dei vitigni coltivati nella zona del “bianco di pitigliano”. l’informatore agrario, 41(5): 59-66. soderini g., 1600 trattato della coltivazione delle viti. filippo giunti, firenze. soldavini s., stefanini m., dallaserra m., policarpo m., schneider a., 2009 superampelo: a software for ampelometric and ampelographic descriptions in vitis. acta horticiculturae, 827: 253-258. this p., jung a., boccacci p., borrego j., botta r., costantini l., crespan m., dangl g.s., eisenheld c., ferreira-monteiro f., grando m.s., ibáñez j., lacombe t., laucou v., magalhães m., meredith c.p., milani n., peterlunger e., regner f., zulini l., maul e., 2004 development of a standard set of microsatellite reference alleles for identification of grape cultivars. theor. appl. genetics, 109(7): 1448-1458. welter l.j., göktürk-baydar n., akkurt m., maul e., eibach r., töpfer r., zyprian e.m., 2007 genetic mapping and localization of quantitative trait loci affecting fungal disease resistance and leaf morphology in grapevine (vitis vinifera l.). mol. breed., 20: 359-374. impaginato 53 1. introduction calla and peruvian lily are commonly cultivated in tuscany (italy) and they represent one of the main cut flower productions. calla lily [zantedeschia aethiopica (l.) spreng] is known to be susceptible to a t l e a s t 1 3 v i r u s s p e c i e s , m a i n l y b e l o n g i n g t o potyviridae, bunyaviridae, and tombusviridae (huang et al., 2007). peruvian lily (alstroemeria spp.) has become one of the most popular cut flowers worldwide. it has been reported as the natural host of various plant viruses, including members of potyviridae, betaflexiviridae or bunyaviridae (park et al., 2010). in tuscany, virus surveys were carried out on various woody plants such as grapevine (rizzo et al., 2012; 2015 a) but to our knowledge no reports are available for calla and peruvian lily in italy. in order to evaluate the health status of these plants in tuscan nurseries, various viruses were assayed, belonging to the following families: betaflexiviridae [alstroemeria carla virus (alcv), lily symptomless virus (lsv)], bromoviridae [cucumber mosaic virus (cmv)], bunyaviridae [impatiens necrotic spot virus (insv), iris yellow spot virus (iysv), tomato spotted wilt virus (tswv)], comoviridae [arabis mosaic virus (armv), broad bean wilt virus 1 (bbwv-1), broad b e a n w i l t v i r u s 2 ( b b w v 2 ) ] , p o t y v i r i d a e [alstroemeria mosaic virus (almv), bean yellow mosaic virus (bymv), dasheen mosaic virus (dsmv), konjac mosaic virus (komv), lily mottle virus (lmov), turnip mosaic virus (tumv), zantedeschia mosaic v i r u s ( z a m v ) , z a n t e d e s c h i a m i l d m o s a i c v i r u s (zammv)], tombusviridae [carnation mottle virus (carnmv)] and unassigned [tobacco rattle virus (trv)]. an additional aim of this survey was to evidence new viral records in italy for these widespread c u l t i v a t i o n s , d u e t o t h e i n t e n s e i n t e r n a t i o n a l exchanges that characterize the commercialization of lily. 2. materials and methods tests were carried out on 90 z. aethiopica plants and 48 alstroemeria spp. plants collected from 12 tuscan nurseries in two years. plants showed sympadv. hort. sci., 2016 30(1): 53-56 doi: 10.13128/ahs-18702 occurrence of viruses in calla and peruvian lily in tuscan nurseries and evidence of new viral records in italy a. luvisi 1,2 (*), d. rizzo 3, l. stefani 3, a. panattoni 2, a. materazzi 2 1 dipartimento di scienze e tecnologie biologiche ed ambientali, centro ecotekne università del salento, via provinciale lecce monteroni, 73100 lecce, italy. 2 dipartimento di scienze agrarie, alimentari e agro-ambientali, università di pisa, via del borghetto, 80, 56124 pisa, italy. 3 servizio fitosanitario regionale, regione toscana, via ciliegiole, 99, 51100 pistoia, italy. key words: alstroemeria, rt-pcr, zantedeschia. abstract: in order to evaluate the health status of calla and peruvian lily in tuscan nurseries, 18 viruses belonging to six families and one unassigned virus were assayed. tests were carried out on 90 zantedeschia aethiopica plants and 48 alstroemeria spp. plants collected from 12 tuscan nurseries in two years, via rt-pcr tests. z. aethiopica was mainly affected by viruses belonging to the potyviridae family, with the main infection caused by dasheen mosaic virus (dsmv) and zantedeschia mild mosaic virus (zammv). even if alstroemeria spp. plants were affected by potyviridae family viruses too, higher infection rates were recorded for betaflexiviridae, where lily symptomless virus infected more than half of plants. this is the first known report of lily mottle virus (lmov) in alstroemeria spp. and z. aethiopica or zammv in alstroemeria spp. in italy. (*) corresponding author: andrea.luvisi@unisalento.it received for publication 24 november 2015 accepted for publication 22 february 2016 copyright: © 2016 author(s). this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. adv. hort. sci., 2016 30(1): 53-56 54 toms such as foliar chlorosis, yellow spot and stripes. total rna was extracted from foliar tissue (2 g) using rneasy plant mini kit (qiagen, netherlands) protocol, modified according to mackenzie et al. (1997). tissues (2 g) were ground using a tissue lyser (qiagen) adding 5 ml of grinding buffer (4.0 m guanidine isothiocyanate, 0.2 m sodium acetate ph 5.0, 25 mm edta, 2.5% pvp-40 and 2.0% sodium bisulfate) just before use. the homogenate (1 ml) was transferred to a 1.5 ml tube and 100 μl of 20% sarkosyl were added. after 2 min centrifugation, 600 μl were transferred to a qiashredder spin column (qiagen) placed in a 2 ml collection tube. the subsequent steps of rna extraction were according to the manufacturer’s protocol. the extracted rna was then retro-transcribed into cdna using the iscript cdna synthesis kit (biorad, usa). for each sample, 2 µl of cdna were amplified in a total volume of 20 μl containing 1x hotmaster buffer, 0.5 μg/μl bsa and 1 u of hotmaster taq dna polymerase (eppendorf, germany). primers and rt-pcr parameters were chosen following the protocols reported in table 1. 18s rrna was used as internal control (osman and rowhani, 2006). finally, 10 μl of the amplification mix was electrophoresed in a 1.5% agarose gel in tae buffer [40 mm tris base, 20 mm sodium acetate, 1 mm edta ph (8.0)]. the amplified cdna fragments were visualized on a uv transilluminator. data were analyzed using sigma-plot software (version 11; systat software, san jose, ca). the software was used to perform analysis of variance (anova). data expressed in percent were converted to arcsin values. p <0.05 was considered to be significant. 3. results the health status of calla and peruvian lily as determined by the present survey is reported in table 2. z. aethiopica was mainly affected by viruses belonging to the potyviridae family. more than two plants out of three were infected by dsmv and 50% table 1 list of references for primers and rt-pcr conditions target rt-pcr assay comoviridae armv faggioli et al., 2005 bbwv-1 ferrer et al., 2008 bbwv-2 ferrer et al., 2008 betaflexiviridae alcv spence et al., 2000 lsv lim et al., 2009 bromoviridae cmv faggioli et al., 2005 bunyaviridae iysv kritzman et al., 2000 insv liu et al., 2009 tswv mumford et al., 1994 potyviridae almv spence et al., 2000 bymv ganesh selvaraj et al., 2009 dsmv wen-chi et al., 2010 komv wen-chi et al., 2010 lmov lim et al., 2009 tumv wen-chi et al., 2010 zamv kwon et al., 2003 zammv wen-chi et al., 2010 tombusviridae carnmv cevik et al., 2010 unassigned trv wei et al., 2009 table 2 health status of calla lily (zantedeschia aethiopica) and peruvian lily (alstroemeria spp.) expressed as percentage of infected plants target zantedeschia aethiopica alstroemeria spp. comoviridae armv bbwv-1 bbwv-2 betaflexiviridae alcv lsv 56.3 a bromoviridae cmv 12.5 b bunyaviridae iysv insv 3.3 c * tswv 3.3 c potyviridae almv dsmv 66.7 a 13.5 b komv lmov 16.7 b 12.5 b tumv zammv 50.0 a 6.3 c tombusviridae carnmv unassigned trv * values in the same column followed by the same letter do not differ significantly according to duncan’s multiple range test (p=0.05). luvisi et al. occurrence of viruses in calla and peruvian lily in tuscan nurseries 55 of tested plants were infected by zammv. lower infection rates were reported for viruses belonging to bunyaviridae. even if alstroemeria spp. plants were affected by potyviridae viruses too, higher infection rates were recorded for betaflexiviridae, where lsv infected more than half of plants. further infections were caused by bromoviridae viruses. comoviridae, tombusviridae or trv infections were not found in both plants. in z. aethiopica, mixed infections were set at 40% for dsmv/zammv, 6.7% for dsmv/lmov/zammv and 3.3% for dsmv/lmov(data not shown). in alstroemeria spp., mixed infections were set at 6.3% for lsv/zammv, lsv/lmov, dsmv/zammv, cmv/lsv or cmv/dsmv/lsv. with regard to alstroemeria spp., the sequence obtained from a zammv amplicon (genbank accession no. kf156666 (table 3) had 99% nucleotide identity with the corresponding fragment of a reference zammv isolate (genbank accession no. ay626825). further isolates of lmov were detected (genbank accessions no. kf156667, kf156668, kf156669). each further isolate had 99% nucleotide identity with the corresponding fragment of a reference zammv isolate. all four isolates are different but had 99% nucleotide identity. the sequence obtained from lmov amplicon (genbank accession no. kf156662) (table 3) had 94% nucleotide identity with the corresponding fragment of a reference lmov isolate (genbank accession no. jn703466). the same lmov amplicon was obtained from a z. aethiopica sample as well. further isolates of lmov were detected in both species with 100% nucleotide identity with kf156662 (genbank accessions no. kf156663, kf156664, kf156665). 4. conclusions various viral infections, mainly due to viruses belonging to two families, betaflexiviridae and potyviridae, seem to affect calla and peruvian lily in tuscan nurseries. most detected viruses are frequently reported for both plants and mixed infection of virus belonging to potyviridae are quite common in z. aethiopica (huang et al., 2007). however, some evidence is rarer. to our knowledge this is the first report of lmov in alstroemeria spp. and z. aethiopica or zammv in alstroemeria spp. in italy, while zammv was recently identified in taiwan (huang and chang, 2005) and in italy (rizzo et al., 2015 b). this report puts in evidence how widespread the virus is within these common plants and the need for constant monitoring of the health status for flower production. even if some of viruses that affect calla and peruvian lily may be eradicated by thermotherapy (panattoni et al., 2013) and heat treatment may help in bromoviridae control (luvisi et al., 2015), prevention represents the preferred method of virus control. references cevik b., bakir t., koca g., 2010 f i r s t r e p o r t o f carnation mottle virus in turkey. plant pathol., 59: 394. faggioli f., ferretti l., albanese g., sciarroni r., pasquini g., lumia v., barba m., 2005 distribution of olive tree viruses in italy as revealed by one-step rtpcr. j. plant pathol., 87: 49-55. table 3 sequence of isolates of zantedeschia mild mosaic virus and lily mottle virus isolate detected in italy zantedeschia mild mosaic virus isolate 2079 polyprotein gene, partial cds (genbank: kf156666) tcattgagtaccaaccccaacagtccgatctgtttaatactcgcgcgtcacaaacccaattcaataattggtatgatgcgatcaaaaatgagtatggggttgatgatagtcagatgcagagaatcatgaatggcttcatggtgtggtgtctcgagaatgggacatcaccaaacataaatggcgtgtgggttatgatggatggggatgaacaagtagaatttccactaaaaccaatggtggagaatgccaagcctacgctgcgtcaaataatgcaccacttttcagacgcagccgaggcttacattgaacttaggaatgccgctgccccatatatgcctagatatgggttgctgcggaacttaagagacagaggtctagcacgcttcgcattcgacttctatgaagtcacttcaaagacaccagatcgtgctagagaagctgtagcgcagatgaaggcagcagcgctaaacaatgtttccacaaggatgtttggattggatggaaatattgcaactgccacggagaacactgaaaggcacactgctaaggatgtaagtccgagcatgcactcgctactcgggatctcagccttgcagtaaaggagctggaaacagcccacagttattgtcttgcgatagggtttaaatagccgtactattgtcgttgctagatgttgcagtgtgggcctcccaccttaaggtttatcagtgtggctttccacctagttccttacattgcgcatagtatgtgt lily mottle virus isolate 2409 coat protein gene, partial cds (genbank: kf156662.1) tgctggggcctctagctccacacaaacgagtcgcccaacacgtccagagattgccgcggtcgatgtagcaccacaacagagctctgaggctagagtgcgtgatcgtgatgttgatgctggcaccgtgggaacataccaaatccctcgactgaaagcactggcaacaaagattaacgtacccaaggtcaaggggcgaacaatagtgaacactgggcaccttgtgaactacaacccagaccaaacagatatttcaaatacaaggtcaacccagaagcaatttgagacctggcataacgctgtgaaagatgagtatggtctcaacgacgagagtatggctctcgcaatgaatggtctgatggtttggtgcatagagaatggcacctcaccaaacataaatggcgtgtggctcatgatggacggagatcagcaagttgaatttcctttacgtcctatacttgaacacgcaaaaccgacgctgcgccaaattatggcgcatttctcaaacctcgctgaagcttatattgagaagcaaaatttggagaaaccgtacatgcctaggtacggccttcagcgaaatctcaccgatttcaatctagcacgatttgcttttgatttctatga adv. hort. sci., 2016 30(1): 53-56 56 ferrer r., escriu f., luis-arteaga m., guerri j., moreno p., rubio l., 2008 new molecular methods for identification of broad bean wilt virus 1. mol. cell. probes, 22: 223-227. ganesh selvaraj d., pokorny r., holkova l., 2009 comparative analysis of elisa, one step rt-pcr and icrt-pcr for the detection of bean yellow mosaic virus in 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korean journal of microbiology, 45: 251-256. liu h.y., sears j.l., mou b., 2009 spinach (spinacia oleracea) is a new natural host of impatiens necrotic spot virus in california. plant dis., 93: 673. luvisi a., panattoni a., materazzi a., 2015 heat treatments for sustainable control of soil viruses. agron. sustain. dev., 35: 657-666. mackenzie d.j., mclean m.a., mukerji s., green m., 1997 improved rna extraction from woody plants for the detection of viral pathogens by reverse transcriptionpolymerase chain reaction. plant dis., 81: 222-226. mumford r.a., barker i., wood k.r., 1994 the detection of tomato spotted wilt virus using the polymerase chain reaction. j. virol. methods, 46: 303-311. osman f., rowhani a., 2006 application of a spotting sample preparation technique for the detection of pathogens in woody plants by rt-pcr and real-time pcr (taqman). j. virol. methods, 133: 130-136. panattoni a., luvisi a., triolo e., 2013 elimination of viruses in plants: twenty years of progress. span. j agric. res., 11: 173-188. park t.h., han i.s., kim j.b., 2010 review on the development of virus resistant plants in alstroemeria. j. plant biotechnol., 37: 370-378. rizzo d., materazzi a., stefani l., farina p., vanarelli s., panattoni a., luvisi a., 2015 a distribution of regulated viruses in cv. sangiovese vineyards in tuscany. j. plant pathol., 97: 131-135. rizzo d., panattoni a., stefani l., paoli m., nesi b., lazzereschi s., vanarelli s., farina p., della bartola m., materazzi a., luvisi a., 2015 b first r e p o r t o f z a n t e d e s c h i a m i l d m o s a i c v i r u s o n zantedeschia aethiopica (l) spreng in italy. j. plant pathol., 92: 399. rizzo d., stefani l., paoli m., triolo e., panattoni a., luvisi a., 2012 the sustainability of old grapevine mother plants in relation to new mandatory diagnostic tests for virus control. adv. hort. sci., 26(3-4): 148150. spence n.j., mills p.r., barbara d.j., 2000 a surbey of viruses of alstroemeria in the uk and the characterization of carlaviruses infecting alstroemeria. eur. j. plant pathol., 106: 843-847. wei t., lu g., clover g.r.g., 2009 a multiplex rt-pcr for the detection of potato yellow vein virus, tobacco rattle virus and tomato infectious chlorosis virus in potato with a plant internal amplification control. plant pathol., 58: 203-209. wen-chi h., chin-hsing h., shu-chuan l., chun-i w., ya-chun c., 2010 detection of four calla potyviruses by multiplex rt-pcr using nad5 mrna as an internal control. eur. j. plant pathol., 126: 43-52. impaginato foreword when i was asked to prepare a brief preface to this special issue of the journal advances in horticultural sciences, i gladly accepted because i thought that the topic of the recovery and enhancement of indigenous germplasm of a country so far away and so problematic as afghanistan was fascinating and full of great potential in various fields of agricultural science and beyond. while i was reading the articles included in this issue, i heard news of a further attack by isis on the architectural masterpieces of the city of palmyra, syria, on 20 january. the facade of the roman theater and the spectacular tetrapylon were destroyed, irretrievably. although there is no clear link between the two events (the recovery of the fruit germplasm of afghanistan and the destruction of historical monuments) it set me thinking that these two events are actually part of a single negative picture of modern times that man, consciously or not, is perpetrating against the biological, cultural and artistic gifts that our ancestors have been handing down to us for centuries. the systematic deletion of the vestiges of the past carried out by isis is not so different from what decades of uninterrupted war have caused to a country like afghanistan, which for historical and cultural reasons, is one of the most important points of contact and exchange between cultures and the biodiversity of the east, the middle east, africa and europe. a country which through commercial and military exchanges along the silk road, has witnessed a merging of genotypes of many species of agricultural interest, as well, of course, of traditions and cultures belonging to people who had never met before. it is easy to imagine what kind of genetic variability has been able to generate through these exchanges which allowed the spread, throughout the rest of the globe, of fruit species which today are some of the most cultivated worldwide. in the temperate climates where these crops have since been established, the strong selection imposed by man in favour of genetic or phenotypes traits of production and commercial interest has meant that, over the centuries, the gene pool of these species has been increasingly shrinking. this became particularly acute when specialized vegetative propagation began to be used through grafting techniques in order to obtain almost total uniformity in the reproduced trees and the fixing of those positive characters that are essential from an economic point of view. however in an area like afghanistan, these species have survived, as witnessed by the authors of the articles in this special issue, in an environment in which the technological evolution of agricultural practices has remained almost stationary since the beginning of the last century. in this environment selective processes have taken place that are much closer to the natural processes than to those imposed by man for cultivation purposes. given that afghanistan is a country rich not only in contradictions, but also in geomorphological variability, soil and climate, the strategic importance of the conservation of its biodiversity is clear. this country, which is considered by many as ‘the land of dust’, with extreme climatic conditions, both in terms of drought and flooding, high and low temperatures, and a topography with considerable altimetric changes, has led to an evolutionary process of adaptation to these conditions on the part of both cultivated and non-cultivated species. these conditions thus suggest the presence of genes that can resist extreme climatic conditions, which may be a key to the future adaptation of fruit tree species grown in temperate climates to the progressive and unstoppable changes that climate changes are causing. unfortunately, genetists from around the world have established that the fruit tree species grown in temperate areas no longer have, and maybe have never even had, a gene pool that would lead to improvements in the resistance abiotic stress that has become increasingly frequent. hence the vital importance of participatory research programs such as those described in this special issue. and here again lies the analogy between the destruction of the archaeological sites and the gradual loss of irreplaceable biodiversity that has been handed down for generations and generations: the loss of this heritage permanently eliminates the future possibility of re-establishing a balance between us and our environment and achieving harmony between the people, nature and history of a country. it is difficult, however, to assume that the preservation of this heritage can be obtained through protective measures aimed merely at conservation, without any organized and responsible participation by the local populations. just as the statues of the bamiyan buddhas and ancient books and statues of the mosul museum were not saved from the devastating fury of the taliban and isis, so too the fragile afghan germplasm of the fruit tree species would have little chance of survival if its conservation was confined to collection orchards designed like an open air museum but subjected to some nefarious purpose in a country where the emergency of war, combined with the food and economic emergency, leaves little room for investments in an unproductive genetic heritage that cannot actually be exploited by the local people. the papers in this special issue thus represent an effective model of approach to the problem which, in my opinion, is the only way to reach such a high objective the protection of biodiversity threatened by genetic erosion. a fundamental part of this approach is also the participation of local people who can then gain a possible means of livelihood and income, providing that they are properly trained and supported both technically and culturally. this is an enormously difficult process, most importantly due to the need to recover all that knowledge that enabled the older generation to pass on, until a few decades ago, the cultivation and planting techniques of suitable plants for the particular geographical area in question. the gradual abandoning of agricultural practices for food in order to concentrate on the almost universal production of opium poppies has led to the disappearance of several generations of farmers and the almost irretrievable loss of know-how in agriculture. introducing new and effective cultivation and propagation techniques for fruit species can play a vital role in the reconstruction of an agricultural micro-entrepreneurship, which is both self-sufficient and motivated to succeed. the challenge for researchers who are attempting this enterprise is very great. we can only hope that their efforts will be rewarded by the expected results so that they can guarantee for tomorrow the survival of our agricultural production if we can keep that genetic heritage that will represent the basis for future breeding programs. for now we can only wish the greatest success to the researchers in this volume who, with great personal sacrifice, have started working on the challenge with so much enthusiasm and so much hard work. good luck and thank you! rossano massai 14 1. introduction grapevine phylloxera (daktulosphaira vitifoliae fitch) is considered the most destructive grapevine insect pest in syria where more than 70,000 ha of this crop, annually producing approximately 540,000 t of grapes, are planted (makee et al., 2010). grape phylloxera invaded syria on nursery stock from bordering countries during the 1920s where it quickly spread in syrian vineyards among the non-resistant local vitis vinifera l. grape (makee et al., 2003). in syria, the most commonly used resistant rootstocks are ru140 (v. rupestris x v. berlandieri), r99 (v. rupestris x v. berlandieri), 3309c (v. riparia michaux x v. rupestris) and 41 b (v. vinifera x v. berlandieri planchon). however, local phylloxera demonstrate an ability to develop and reproduce on all american rootstocks (makee et al., 2010). in addition, the susceptibility of ru140 rootstock was found to be higher than that of r99 and 3309c rootstocks (makee et al., 2003). the rootstocks often allow limited growth and reproduction of phylloxera, but they may also reduce the growth vigor of grafted varieties because of incompatibility phenomena. moreover, most syrian grapevines are planted in areas of 300-500 mm rainfall, thus they are unirrigated and such conditions are inappropriate for resistant rootstocks (makee et al., 2010). granett et al. (1996) reported that once a vineyard is infested with grape phylloxera it is expected to cease its production in about two to five years. generally, spring phylloxera is found primarily on the small feeder roots that proliferate during root flush. however, during summer when the climate is dry and the top of the plant is growing, phylloxera is found extensively on the infected mature older roots. in winter, phylloxera is found on mature roots due to the disappearance of feeder roots (omer et al., 1997). once the phylloxera’s proboscis is inserted into root bark cells, the parenchyma cells below the suberized outer layer, they inject saliva that, in turn, trigger the root cells to increase in size and number. these galls provide a feeding site from which stored nutrients such as sugars and amino acids can be extracted by developing phylloxera, which leads to a decrease in vine vigor and eventually destroys the roots causing vine death (omer et al., 2002). the injured grapevines are usually attacked by secondary soil-borne pathogens at the insect feeding site (omer and granett, 2000). granett et al. (1998) and lotter et al. (1999) revealed that fungal infections associated with pylloxera galls are pathogenic and cause root death. in addition, fungal invasion of the roots at grape phylloxera feeding sites cause severe infections in grapevines. fusarium is a large genus of filamentous fungi, and most fusarium species are harmless saprobes and relatively abundant members of the soil microbial community (domsch et at., 1980; nwanma et al.,1993). this ecological habitat of the fungus implies that fusarium could be a useful resource of extra cellular enzymes. the fungal isolate f. solani sy7 was the best xylanase producer among tested isolates (arabi et al., 2011; bakri et al., 2012). vine varieties susceptible to grape phylloxera were also highly susceptible to f. oxysporum (omer et al., 1995; 1999). granett et al. the relationship between grape phylloxera and fusarium root infection i. idris,(1) m.i.e. arabi department of biotechnology, atomic energy commission of syria, p.o. box 6091, damascus, syria. key words: fusarium ssp., grape phylloxera, grape root. abstract: phyloxera seems to have key role in the fungal pathogen infection ratio while the fungal spread reduces the ability of pheloxera to reproduce. intact roots of four-month-old grape plants were inoculated with phylloxera eggs in presence or absence of fungal pathogens. fusarium solani sy7 infection was detected in all plant parts when grapevine roots were infested with phylloxera. the spread ratio of fusarium solani sy7 increased from 74 to 100% of the infested plants with phylloxera. on the other hand, the phylloxera on f. solani sy7 infected roots were developed more slowly, since the nymphs and tuberosities were significantly decreased 49% and 31% respectively. the total plant biomass decreased to 29% in the presence of both f. solani sy 7 and phylloxera as compared to 9 and 17% in the presence of f. solani sy7 or phylloxera, respectively. this study sheds light on the correlation between fungi, phylloxera and grapevine and could help in the application of integrated pest management (ipm) programs against grape phylloxera. adv. hort. sci., 2014 28(1): 14-19 (1) corresponding author: ascientific@aec.org.sy received for publication 26 march 2014 accepted for publication 29 april 2014 15 (1998) also demonstrated that the two root types with v. vinifera parentage, carignane and axr#1, which are most susceptible to phylloxera feeding, are also susceptible to infection by f. oxysporum pathogen. the present study therefore aims to investigate whether the presence or absence of phylloxera could enhance f. solani sy7 fungal infection in the local balady v. vinifera variety vine roots and the consequences of the interaction between the grape phylloxera and f. solan sy7 on the vine. in this context, the interaction between root infection and the three fusarium species root and rootfeeding grape phylloxera were first investigated under controlled conditions, then the effects of fungal infection with and without the presence of phylloxera on grape vine vigor (plant biomass, roots, branches and leaves, internodes weights) were determined. the process included identification of the fusarium species that invade the roots from grape phylloxera feeding sites. 2. materials and methods establishment of the phylloxera colony grape phylloxera was originally collected from fieldinfested roots of the local grape varieties in southern parts of syria. the phylloxera colony was established according to the procedures mentioned by de benedictis and granett (1993). fresh and healthy pieces of local grape variety (makee et al., 2008) “balady” roots, 4-7 mm in diameter and 5-7 cm long, were taken and washed with tap water. each piece was wrapped with moist cotton wool at one end, and then 10 to 15 phylloxera eggs were placed on each piece. the infested root pieces were placed on a wet filter paper disk inside plastic petri dishes (12 cm diameter and about 1 cm deep, three to four root pieces per dish). for ventilation purposes, the petri dish lid was modified with a 1-1.5 cm cloth–screened hole. dish edges were sealed with parafilm, kept in plastic boxes with tightly fitting lids and incubated at 25°c in the dark with 75% relative humidity. the root pieces were replaced when they desiccated rotted or the phylloxera became crowded. potted inoculation procedure before inoculation, three-day-old eggs (n =100) were removed from the colony and placed in 1.5 ml plastic tubes for surface sterilization. one ml of formaldehyde was added to the eggs and the tubes were gently shaken for 10 min. the sterilizing solution was removed from the mix and the eggs were extracted and placed on a sterile filter paper and dried for 5 min. they were then kept in a petri dish, sealed with parafilm to prevent contamination and the escape of the phylloxera crawlers. egg sterilization was carried out under sterile conditions (makee et al., 2003). fungal isolates the fungal isolates were obtained from the plant pathology laboratory of the atomic energy commission of syria (arabi et al., 2011) (table 1). host plant root samples infested with fusarium were collected from different locations in syria. roots were sterilized in 5% sodium hypochlorite (naocl) for 5 min. after three washings with sterile distilled water, roots were dipped in 70% ethanol for 1 min and then washed once with distilled water. roots were cut into small slides under sterile conditions and transferred to petri dishes containing potato dextrose agar (38g/l) (pda, difco, detroit, mi, usa) (alazem, 2007). thirteen mg/1 kanamycin sulphyate were added after autoclaving and 10 days incubation at 23 ± 1°c in the dark to allow mycelia growth. all isolates were identified morphologically, according to nelson et al. (1993). emphasis was placed on selecting isolates that induced differential reactions on specific genotype, pathogencity and in vitro xylanase activity (alazem, 2007; arabi et al., 2011; bakri et al., 2012). the above mentioned parameters lead to select of three monosporic isolates f. culnorum sy3, f. solani sy7 and f. equisesti sy24. the fusarium isolates used in this study, their location, year of collection and xylanase production are listed in table 1 (arabi et al., 2011). the cultures were maintained on silica gel at 4°c until needed. eighty “balady” grape stem pieces were dipped in a solution of 2000 ppm iaa (indol butric acid) for 2 min, then planted in plastic pots contacting sterile moistened soil. finally, they transferred to 10-l plastics pots, after four months. the experiment was conducted in greenhouse, using a randomized complete block design with four replicates of four plants for each of the following treatments in each treatment. 1 infection with phylloxera: roots of vines were infected with phylloxera eggs (50 eggs/root). 2 infection with fusarium spp: vine roots were dipped in a fungal solution (5 x 104 spores/ml) containing an equal mix of spores (f. culnorum sy3, f. solani sy7, f. equiseti sy24) for 15 s, then dried for 30 min. 3 infection with fusarium spp and phylloxera: vine roots were dipped in a fungal solution (5 x 104 spores/ml) containing an equal mix of spores (f. culnorum sy3 , f. solani sy7, f. equiseti sy24) for 15 s, dried for 30 min, and then infected randomly by phylloxera eggs. 4 plant control (free of fusarium spp. and phylloxera): plants were transplanted into 10-l pots. each experimental unit consisted of two plants. pots were filled with sterile soil. the pots were all placed in a greenhouse at 25 ± 1°c ( day) and 23±1°c (night) with 16-h table 1 fusarium isolates, location, year of collection and extra cellular xylanase production in solid state fermentation after five days of inoculation at 30°c isolate location of syria year of collection xylanase (u/g) f. culnorum sy3 north-west 2005 163.69 f. solani sy7 middle-region 2003 908.2 f. equiseti sy24 north-east 2005 122.43 16 daylight and 85-95% relative humidity. plants were irrigated with water as needed. a year later, the following parameters were measured: plant biomass, root weight, root number, internode weight. five root pieces of each tested plant were sampled. microscopic inspection was performed to determine the number of tuberosities and feeding nymphs for each tested plant. fungal inspection plant samples (roots, leaves and branches) were collected from each tested plant, surface sterilized for 3 min in 5% naocl, and rinsed twice in distilled water. six disks to each roots, leaves and branches were transferred to petri dishes containing pda with 13 mg/l kanamycin sulphyate added after autoclaving and incubated for 4 weeks at 23 + 1°c in the dark to allow mycelia growth. mycelia edges of f. culnorum sy3, f. solani sy7, and f. equiseti sy24 were identified morphologically as describe by nelson et al. (1993). the infection percentage of fungal ratio was calculated using the formula r = f e / ft x 100, where r = the percentage of fungal ratio, f e = number of disks that contain the fungi from roots, leaves and branches, and ft = number of the total disks with or without fungi from roots, leaves and branches. statistical analysis was performed using the statistic program version 6 (statsoft, inc. 2003) at 5% level (p = 0.05). means were subjected to analysis of variance tested for significance using tukey hsd test. 3. results incidence of fusarium spp infection in different parts of plant with or without phylloxera the results demonstrated that fungal infection ratio in the whole plant was 74, 1.7 and 0% of f. solani sy7, f. equiseti sy24 and f. culmorum sy3, respectively. furthermore, the ratio of infection with f. solani sy7 in treatment 4 increased 26% to reach 100% (table 2). effect of different treatments on plant the plant biomass (fig. 1, i) decreased significantly in the presence of phylloxera and fusarium solani sy7 (9 and 17%, respectively). this effect is more evidenced in the presence of fungi and phylloxera 29% (f=2; df=1, 3; p<0.05). vine roots infection with fusarium spp. had no effect on root weight (fig. 1, ii) compared with the control, while infection with phylloxera increased 36% significantly comparing to control (f=2; df=1, 3; p<0.05). branch and leaf weight (fig. 1, iii) decreased significantly (f=2; df=1, 3; p<0.05 ) compared to the control in the presence of phylloxera and f. solani sy7 (53% and 31%, respectively). this effect is more evidenced in the presence of phylloxera with infection of f. solani sy7 58%. the average internode weight (fig.1, iv) in treated plants with both phylloxera and fungi increased considerably (50%) as compared to the control. however, no obvious correlation between phylloxera and fungi concerning the internode weights was shown in the experiment. in addition, the root number of vines exposed to the fungus was notably decreased but to a lesser degree compared to plants infested with phylloxera or both fungi and phylloxera (f=3.6; df=1, 3; p<0.05) (fig. 2). effects of f. solani sy7 infection on nymph and tuberosity numbers the numbers of nymphs and tuberosities in vines infested with phylloxera decreased significantly (f=85.3; df=1.1; p<0.05), (f20=; df=1.1; p<0.05) in comparison with vines infested with both phylloxera and fungi. in addition, nymphs and tuberosities were reduced by 49 and 31%, respectively, compared to the control plants (table 3). 4. discussion and conclusions phylloxera seems to have key a role in the fungal pathogen infection ratio and, in contrast, the fungal spread reduces the ability of pheloxera to reproduce. the high infection ratio of f. solani sy7 (74%) may attribute to its ability to spread in the phloem parenchyma through a special mechanism which allows the fungi to infect the roots (omer et al., 1999). phylloxera can serve as a vector and transport fungal propagates from infected to healthy roots (omer et al., 2000). therefore, our data suggest that the injury caused by phylloxera may give benefit to f. solani table 2 incidence of fusarium spp. infection in different parts of plant with or without phylloxera treatment f. solani sy7 % f. equiseti sy24 % f. culnorum sy3 % roots branches leaves roots branches leaves roots branches leaves phylloxera 0 0 0 0 0 0 0 0 0 fusarium mix 72 77 75 0 0 0 0 0 0 phylloxera + fusarium mix 100 100 100 5 0 0 0 0 0 control 0 0 0 0 0 0 0 0 0 data represents the infection percentage of fusarium spp. ratio in roots, branches and leaves in each treatment. 17 sy7 to spread throughout the entire plant 100%. however, the other two fungi (f. culmorum sy24 and f. equiseti sy3) species may not possess this mechanism to spread in the phloem parenchyma and eventually they are not able to infect the vine despite the presence of phylloxera which may be attributed either to the lack of proper growth condition in vine or to the presence of immune response in the plant which prevents the growing of the later fungi species (omer et al., 1999; fossen, 2002). to confirm the effect of f. solani sy7 and phylloxera on vines, we established an experiment to evaluate five parameters (the weights of plant biomass, root internodes, roots, branches and leaves and root numbers in presence or absence of phylloxera compared to control plants). from these biological parameters, we can estimate the relationship between f. solani sy7 and phylloxera. omer et al. table 3 effects of f. solani sy7 infection on numbers of nymphs and tuberosities treatments nymphs number (se) tuberosities number (se) phylloxera 25 ± 1 a 4.9 ± 0.9 a f. solani sy7 0 0 phylloxera + f. solani sy7 12.9 ± 0.9 b 3.4 ± 0.5 b control 0 0 data represents nymphs number mean in tested roots of 16 plants in each treatment. data were subjected to anova analysis and the differences between means were tested for significance using tukey hsd test. means followed by different letters (columns) are significantly different at p <0.05. b a b b 0 10 20 30 40 50 60 control phylloxera fungi phylloxera+fungi r oo ts w ei gh ts gr am treatments ii a b c d 0 20 40 60 80 100 control phylloxera fungi phylloxera + fungi p la n ts b io m a ss g ra m treatments i b a a a 0 5 10 15 20 25 control phylloxera fungi phylloxera+fungi in te rn o d es w ei g h ts g ra m treatments iv a c b c 0 10 20 30 40 50 60 control phylloxera fungi phylloxera+fungi b ra n ch es a n d le av es w ei g h ts g ra m treatments iii fig. 1 effect of different treatments on plant biomass (i), root (ii), branches + leaves weights (iii) and internode weights (iv). a b b b 0 5 10 15 20 25 30 35 40 45 50 control phylloxera fungi phylloxera+fungi r o o ts n u m b er s treatments fig . 2. effects presents of phylloxera and fungi on roots numbers fig. 2 effects presents of phylloxera and fungi on roots numbers i ii iii iv 18 (1995) demonstrated that greenhouse experiments with effects of fungal infection on grapevine vigor after pruning at week 13 showed that damage was significantly greater in vines infested by phylloxera, f. solani and pythium ultimum than the damage in vines infested with phylloxera alone. total biomass was reduced by 16% in vines infested with phylloxera and by 24% in vines infested by phylloxera and f. solani (omer et al., 1995). in our study it was found that the total biomass was reduced by 9% in vines infested with phylloxera and by 29% in vines infested with phylloxera and f. solani sy7, compared to the controls. the differences between f. solani sy7 and f. solani (omer et al., 1995) may be attributed to the disparity of aggressiveness of the isolates. therefore, the infection with phylloxera and fungi together caused a synergetic effect where the plant biomass tend to decrease in presence of phylollxera or fungi. this can be attributed to the damage in the vine roots caused by either phylloxera or fungi which cause dysfunction of the root as they should be in the normal conditions. as both phylloxera and fungi affected the roots directly therefore, more work focused on the roots. the root weight and internodes increased significantly by 36 and 63% comparing to the control, in the presence of phylloxera and fungi respectively. this increase is normal in the presence of phylloxera due to the tuberosities and nodosities formed by the phylloxera. however, fungi alone did not cause changes in the root weight (omer et al., 1995). our results demonstrated that the significantly growth of infested vines with phylloxera alone or with a combination of phylloxera and f. solani sy7 may attribute to the rapid growth observed after potting. moreover phylloxera may require more time necessary to establish feeding sites (omer et al., 1995). additionally, a reduction of 50%, 30% respectively was observed in branch and leave weight and in the number of roots in the presence of phylloxera or both phylloxera and fungi compared to control, is attributed to roots death.therefore, f. solani sy7 infection can spread radially causing necrosis in the parenchyma and phloem. ultimately the infection kills the roots and eventually the plant (omer et al., 1999). similarly fossen (2002) found that the virulence isolates in present of v. vinifera on vine roots led to proportion of the root circumference that became necrotic in a 5-week period. some isolates were highly virulent, causing up to 80% necrosis while other isolates were negligibly virulent (fossen, 2002). it has been shown in other plant-herbivore-pathogen systems that co-occurrence can, through direct interaction or changes in host’s susceptibility, affect the performance of the pest or the pathogen (karban et al., 1987; hatcher., 1995). omer et al. (2002) reported that the ability of grape phylloxera to exploit grape roots increased in the absence of fungal pathogens and indicated that phylloxera on infected roots developed more slowly, and had substantially reduced survival and reproduction rates. therefore our results revealed the presence of fungi reduced the ability of phyloxera to form tuberosities by 31%. our data also demonstrated that the total root weight decreased in presence of fungi with the phylloxera-infested vine roots which were also reflected by a decrease in the number of nymphs. therefore our assays demonstrated that phylloxera on f. solani sy7 infected roots were developed more slowly, since the nymphs and tuberosities were significantly decreased by 49% and 31% respectively. the reproduction and feeding activities of phylloxera were significantly decreased in the presence of fungal infection, consequently this result is in agreement with omer et al.( 2000). furthermore the ability of f. solani sy7 to spread within the plant parts increased when grapevine roots were infested with phylloxera grape. these results provide interesting piece of information about the relationship between f. solani sy7 pathogens and phylloxera. however these results are to be proved in the field. the present study provides preliminary information that could help in application of integrating pest management (ipm ) program against grape phylloxera. acknowledgements the authors thank the general director of aecs and the head of the molecular biology and biotechnology department for their continuous support throughout this work . we also wish to thank dr h. makee, and dr t. charbaji for helping us in this work. references alazem m., 2007 characterization of syrian fusarium species by cultural characteristics and aggressiveness. ph.d. thesis, faculty of agriculture, university of damascus, damascus, syria. arabi m.i.e., bakri y., jawhar m., 2011 extracellular xylanase production by fusarium species in solid state fermentation. polish. j.m., 60(3): 209-212. bakri y., jawhar m., arabi m.i.e., 2012 correlative analysis of fusarium species pathogenicity and in vitro xylanase activity. j. plant bio. research, 1(2): 86-92. de benedictis j., granett j., 1993 laboratory evaluation of grape roots as host of california grape phylloxera biotypes. am. j. enol. vitic., 44(3): 285-291. domsch k.h., gams w., anderson t.h., 1980 compendium of soil fungi. academic press, london. fossen m., 2002 resistance of grapevine rootstocks to rootpathogenic fungi vectored by grape phylloxera (daktulosphaira vitifoliae fitch). ms thesis, university of california, davis, pp. 28. granett j., omer. d.a., pesserequ p., walker m., 1998 fungal infections of grapevine roots in phylloxerainfested vineyards. vitis, 37: 39-42. granett j., walker m., de benedictis j.a., fong g., lin h., weber e., 1996 california grape philloxera more variable than expected. calif. agric., 50(4): 9-13. hatcher p.e., 1995 three-way interactions between plant pathogenic fungi, herbivorous insect and their host plants. 19 bio. rev. cambridge philosoph. soc., 70: 639-694 karban r., adamchak r., schnathorst w.c., 1987 induced resistance and interspecific competition between spider mites and a vascular wilt fungus. science, 235: 678-680 lotter d.w., granett j., omer a.d., 1999 differences in grape phylloxera-related grapevine damage in organically and conventionally managed vineyards in california. hort sci., 34: 1108-1111. makee h., ammounha h., idris i., 2010 development and reproduction of phylloxera on some local grape varieties in syria. adv. hort. sci., 24(3): 169-175. makee h., charbaj i.t., ayyou b.z., idris i., 2003 evaluating resistance of some rootstocks to grape phylloxera with an in vitro and excised root testing systems. in vitro cell. dev. biol. plant., 40(2): 225-229. makee h., charbaj i.t., idris i., ayyoubi z., 2008 effects of gamma irradiation on survival and reproduction of grape phylloxera. adv. hort. sci., 22(3): 1-5. nelson p.e., toussoun t.a., marasas w.f.o., 1983 fusarium species: an illustrated maual for identification. the pennsylvania state university press, university park, usa, pp. 203. nwanma b., onyike n., nelson p.e., 1993 the distribution of fusarium species in soils planted of millet and sorghum in lesotho, nigeria and zimbabwe. mycopathologia, 121: 105-114 omer d.a., granett j., 2000 relationship between grape phylloxera and fungal infections in grapevine roots. j. plant diseases and protection, 107: 285-294. omer d.a., granett j., de benedictis j.,walker m.a., 1995 effects of fungal root infections on the vigor of grapevines infested by root-feeding grape phyloxera. vitis, 34(3): 165. omer d.a., granett j., downie d.a., walker m.a., 1997 population dynamics of grape philloxera in california vineyards. vitis, 36(4): 199-205. omer d.a., granett j., walker m.a., 1999 pathogeniticy of fusarium oxysparum on different vitis rootstocks. phytopathology, 147: 433-436. omer d.a., granett j., walker m.a., 2002 influence of plant growth stage on grape phylloxera (homoptera: phylloxeridae) populations. environ. entomol., 31(1): 120-126. 131 1. introduction despite considerable advances in technology, agriculture is exposed to climate changes in all parts of the world. among climatic factors, rainfall is the most critical because 70% of the main areas under cultivation are still without irrigation (wilhite, 2001). water shortage is the main characteristic of agriculture in mediterranean regions, inducing water stress during spring and summer. fruit trees survive in this situation because they are prone to physiological or morphological changes which enable them to avoid drying damage, cast it back, or tolerate it (torrecillas et al., 1999). drought tolerance is observed with different rates in almost all plant species. understanding plant responses to the external environment is an essential component for selection stress tolerance (reddy et al., 2004). in recent years, in southern iran, low and poor distribution of rainfall has caused great damage to plants. water stress, particularly in rain-fed fig production areas (e.g. estahban) has become a big issue. in the estahban region not only the annual production has decreased, but also the highly productive trees are in danger of destruction. one strategy to confront this problem is the identification and selection of more tolerant genotypes. traditional breeding approaches for selection of tolerant plants are time consuming and complex processes. imposing water stress in field-grown crops is difficult because of the unpredictability of rainfall and the possibility of seepage from adjoining plots and drought escape of deep roots from osmotic stress. since field evaluation of drought effects is highly correlated with environmental conditions, in vitro screening techniques allow for a better control of culture conditions. tissue culture also offers the possibility of screening many plants in limited space and time, assuming that there is a correlation between cellular, tissue, organ and in vivo plant responses (mohammad et al., 2000). polyethylene glycol (peg) has been used to simulate water stress in plants. peg of high molecular weight is a non-ionic osmoticum lowering the water potential of nutrient solution without being taken up or being phytotoxic (hassan et al., 2004). it has been shown that the shoot length decreases with increasing water stress via in vitro culture in cherry (sivritepe et al., 2008) and mulberry (tewary et al., 2000). in a study on mulberry, leaf relative water content (rwc) decreased with increasing water stress (ramanjulu et al., 1998). accumulation of proline is the most common plant response to decreasing water potential (helal ragab and samir moustafa, 2008). it has been reported that with increasing peg in date palm seedling culture, the rate of proline was increased in tolerant cultivar (djibril et al., 2005). carbohydrates have an important role in osmotic regulation in different plant parts (masoudi-sadaghiani et al., 2011). simple sugars such as glucose and fructose were increased and the rate of sucrose and starch decreased under water stress (sharp and davies, 1979; munns and weir, 1981; wang and stutte, 1992; nawar and ezz, 1993; clifin vitro drought effects on morphological and physiological indices of two fig (ficus carica l.) cultivars a. shekafandeh(*), s. hojati department of horticultural science, college of agriculture, shiraz university, shiraz, iran. key words: drought, growth parameters, polyethylene glycol, proline. abstract: in vitro responses of two fig cultivars, ‘sabz’ and ‘siah’, were evaluated in ms media containing four levels of polyethylene glycol (peg) (0, 2, 4, 6%) as a simulation of water stress. the results showed that in sabz cultivar, shoot length, shoot fresh and dry weights were 43, 36 and 25%, respectively, lower than control in drought treatments caused by 4% peg, while the leaf area and specific leaf area were not significantly (p>0.05) affected. in siah cultivar, shoot length, fresh and dry weights were 57, 58 and 40%, respectively, lower in stressed media in comparison to control. in contrast to sabz cultivar, leaf area and specific leaf area of siah cultivar were significantly reduced by addition of 6% peg (56 and 21.5%, respectively). naturally, the amount of proline in ‘sabz’ was higher than in ‘siah’ (81.8 μmole g-1 versus 16.7 μmole g-1). however, in both cultivars, with addition of peg in culture media, leaf proline content was increased, in comparison to control. with increasing peg% in culture media, the amount of leaf soluble sugars content increased and the amount of starch decreased. the result show that ‘siah’ is more sensitive to drought than ‘sabz’ and that in vitro culture can be used to evaluate drought tolerance of cultivars. adv. hort. sci., 2012 26(3-4): 131-137 (*) corresponding author: shekafan@shirazu.ac.ir. received for publication 26 april 2012 accepted for publication 12 september 2012 132 ford et al., 1998; perez-perez et al., 2007; mafakheri et al., 2010). in iran, fars province with about 36,000 hectares under fig cultivation is the primary fig production area. estahban district with 20,000 hectares under fig cultivation is the center of fig cultivation in fars province, as well as in iran. as mentioned above, due to drought in recent years, highly productive trees are in danger of destruction. the cultivars sabz and siah are the most desirable cultivars in iran; there has not been report on water stress tolerance of these cultivars. selection of more drought tolerant cultivars can be used to establish of new orchards and a promising rootstock for other interested cultivars. the aim of this investigation was to evaluate the effects of drought created by polyethylene glycol (peg) on morpho-physiological changes of two important fig cultivars (‘sabz’ and ‘siah’) and leading to selection of more drought tolerant varieties using in vitro culture. 2. materials and methods plant material and explant decontamination in this research, the in vitro-derived micro-shoots of two cultivars of ficus carica l. ‘sabz’ and ‘siah’ were used. the branches (20~30 cm long) were cut from rain-fed, mature mother trees established in estanban fig research station located at 29° 07’ n latitude and 54° 02’ e , 197 km southeast of shiraz. freshly grown stems with 2~6 nodes were cut from the above branches and washed in a solution of water-detergent (three drops of detergent in 100 ml of water) for 30 min, then placed in a solution of benomyl (3%) for 1 h (torres, 1998). to prevent the secretion of phenolic compounds into the culture media, explants were treated in a solution of 2% ascorbic and citric acid for 45 min. at this point, the stems were transferred to a laminar air flow cabinet. they were first dipped in 70% (v/v) ethanol for 45 s, then treated with 15% (v/v) chlorox solution (a household bleach containing 5.25% sodium hypochlorite) for 15 min and rinsed three times with sterilized distilled water. nodal segments of 1~1.5 cm long were cut and used as explants. the basal medium was ms (murashige and skoog, 1962), containing 3% sucrose (merck, lgaa 64271darmstadt, germany) and 1mg l-1 (4.4 µm) benzyl adenine (ba) and solidified by 0.8% agar-agar (merck, lgaa 64271darmstadt, germany). the ph was adjusted to 5.7±0.05 prior to autoclaving at 1.2 atm pressure and 121˚c for 20 min. cultures were maintained in a growth chamber at 25±2˚c, 58% relative humidity and a photon flux density of 40 μmol m-2 s-1 was provided by white fluorescent tubes, with a 16 h photoperiod. peg treatments after four weeks, the micro-shoots (2 cm length) from the above cultures were transferred into 500 ml culture vessels containing 100 ml ms basal media supplemented with different levels of peg (0, 2, 4 and 6%) as selective agent and maintained under the environmental conditions described above. growth measurements seven weeks after the beginning of experiments, shoot length and shoot fresh and dry weight were evaluated. leaf area was measured by leaf area meter (deltatdevices england) and specific leaf area was calculated as follows: specific leaf area = leaf area (cm2)/leaf dry weight (g) relative water content leaf relative water content (rwc) was estimated according to the method described by whetherley (1950). twenty healthy leaf discs of 0.7 cm diameter were cut from plants, using a leaf punch and washed three times with double distilled water. leaf discs were weighed (fw), then placed into a 10 ml conical flask, immersed in 10 ml distilled water for 4 h in dark. turgid weight (tw) of leaf discs were then measured and samples were dried in an oven (80ºc) until constant weight (dw) was achieved. rwc was calculated from the following equation: relative water content % = fw dw/tw dw x 100 chlorophyll content for chlorophyll determination, prior to extraction fresh leaf samples were washed with deionized water to remove any surface contamination. one g leaf samples were ground in 80% acetone using a pestle and mortar. the mixture was centrifuged at 4800 rpm for 20 min. the optical density of the supernant was measured at 663 and 645 nm wavelengths and chlorophyll content was calculated using the following equation: mg chl g fw = [20.2 (od645 nm) + 8.02 (od663 nm)] x v/fw x 1000 where v is final volume of solution (ml) and fw, leaf fresh weight (mg). proline contents to determine proline content of the leaves, 0.5 g of plant material was homogenized in 10 ml of 3% aqueous of sulfosalcylic acid and the homogenate filtered through whatman # 2 filter paper. two ml of filterate was reacted with 2 ml acid ninhydrin and 2 ml of glacial acetic acid in a test tube for 1 h at 100°c and the reaction terminated in an ice bath. the reaction mixture was extracted with 4 ml toluene, mixed for 15~20 seconds. chromophore containing toluene was aspirated from the aqueous phase and the absorbance was read at 520 nm. the proline concentration was determined based on standard curve and calculated as follows: μ moles proline/g fw = (μg proline/ml x ml toluene) / g sample/5 115.5 μg / μmole electrolyte leakage electrolyte leakage was measured as an assessment of cell wall permeability. electrolyte leakage was measured using an electrical conductivity meter. two mature leaves per plant were taken and cut into 1 cm segments. after three washes to remove surface contamination, leaf samples were placed in individual vials containing 10 ml of 133 distilled water. the samples were incubated at room temperature on a shaker (100 rpm) for 24 h. electrical conductivity (ec) of bathing solution (ec1) was read after incubation. samples were then placed in an autoclave at 120°c for 20 min and the second reading (ec2) was determined after cooling the solution to room temperature. the electrolyte leakage was calculated as ec1/ec2 and expressed as percent. soluble sugars content soluble sugars were extracted from 0.1 g fresh leaves by heating with 5 ml of 80% ethanol in a water bath at 70°c for 30 min. the insoluble residue was removed by centrifuging at 5000 g for 10 min. one ml of the resulting extract was mixed with 1 ml of 5% phenol solution and 5 ml of sulfuric acid. the mixed solution was permitted to cool to room temperature, then vortexed. the absorbance was read at 490 nm using a spectrophotometer. the soluble sugar content of each sample was determined using standard curve for glucose and expressed as mg glucose g-1 fw (mccready et al., 1950; dubois et al., 1956). starch content the solid residue remaining in the centrifuge tube after removal of all soluble sugars in the previous section was washed, re-extracted and re-centrifuged four times using 80% (v/v) ethanol. starch content in the samples was determined colorimetrically using anthrone method (mccready et al., 1950). the absorbance was determined at 630 nm in a digital spectrophotometer (spectronic 20 d+; spectronic instruments inc., new york, usa.) as described by lópez et al. (2002). statistical analysis the experiment was arranged as a 2×4 factorial experiment in completely randomized design (crd) with 10 replicates, each consisting of three plants. thus, there were 30 plants in each treatment and a total of 240 plants in the experiment. data were subjected to analysis of variance using the spss software (ver. 13.0) spss inc. mean differences were determined by duncan’s multiple range tests at p≤0.05. 3. results and discussion water stress caused a reduction in micro-shoot growth of the two fig cultivars (table 1). in ‘sabz’, shoot fresh and dry weights and shoot length were 36, 25, and 43% lower, respectively, in drought treatments caused by 4% peg compared with the control, while in ‘siah’, these traits were 58, 40 and 57% lower respectively, in the same stressed media (4% peg), than the control. in this latter cultivar, in contrast to ‘sabz’, leaf area and specific leaf area were significantly affected by water deficit (56 and 21.5%, respectively) (table 2). the findings of this study showed that the growth rate of 'siah' in optimum conditions (control) was the same as sabz cultivar, but with increasing intensity of water stress, the growth reduction rate in ‘siah’ was more than in ‘sabz’. the main effect of cultivars showed that without respect to different levels of peg, ‘sabz’ had a significantly higher leaf area (8.2 cm) than ‘siah’ (5.51) (table 2). water stress significantly reduced vegetative growth indices of fig micro-shoots. this phenomenon has been previously reported (oukabli et al., 2008). the long-term use of peg in vitro on growth reduction and shoot regeneration in other plants has been well documented (bressan et al., 1982; handa et al., 1982; handa et al., 1983; dami and hughes, 1995; al-khayri and al-bahrany, 2004). in most cases, peg has been used to stimulate table 1 interaction of water stress and cultivar on micro-shoot length, fresh and dry weight drought stress (peg%)  0 2 4 6 cultivar average fresh weight (g) mean siah 3.80 a (z) 2.1 bc 1.6 c (58) 2.10 bc 2.4 a sabz 3.00 ab 2.3 bc 1.9 bc (36) 2.10 bc 2.3 a mean 3.41 a 2.2 b 1.8 b 2.08 b  average dry weight (g) siah 0.50 a 0.30 bc 0.3 bc (40) 0.30 bc 0.34 a sabz 0.40 ab 0.40 ab 0.3 bc (25) 0.30 bc 0.36 a mean 0.46 a 0.34 b 0.3 b 0.31 b shoot length (cm) siah 2.60 a (z) 1.4 b 1.1 b (57) 1.20 b 1.57 a sabz 2.33 a 1.4 b 1.3 b (43) 1.30 b 1.59 a mean 2.47 a 1.4 b 1.2 b 1.25 b (z) in each row and column, means with similar letters (small letters for interaction and big letters for main effects) are not significantly different using duncan’s multiple rang test p≥0.05. the percentage of reduction with respect to control is reported in parentheses. table 2 interaction of water stress and cultivar on leaf area (cm2) and specific leaf area (g cm-2) of micro shoots drought stress (peg%) 0 2 4 6 cultivar leaf area (cm2) mean siah 8.2 a (z) 6.9 ab 3.4 b 3.6 b (56) 5.51 b sabz 12.4 a 8.2 a 6.9 ab 6.6 ab 8.52 a mean 10.0 a 7.6 ab 5.4 b 5.4 b specific leaf area (g cm-2) siah 14.4 a 14.6 a 13.4 a 11.3 b (21.5) 13.4 a sabz 16.8 a 15.3 a 15.1 a 13.1 a 15.1 a mean 15.6 a 14.9 a 14.4 a 12.2 a (z) in each row and column, means with similar letters (small letters for interaction and big letters for main effects) are not significantly different using duncan’s multiple rang test p≥0.05. the percentage of reduction with respect to control is reported in parentheses. 134 water stress in plants. peg with high molecular weight is a non-penetrating inert osmoticum which lowers the water potential of nutrient solutions without being taken up or being phytotoxic (hassan et al., 2004). it has been shown that in vitro growth reduction of apple (molassiotis et al., 2006) and cherry (sivritepe et al., 2008) was due to the decrease in water potential created by peg. under water stress conditions, the reduction of mineral absorption has resulted in limited leaf growth and development and decreased plant water transpiration. therefore, producing smaller leaves can be the first plant defense mechanism against water deficit. hsiao (1973) reported that a decrease in leaf area results in lower light absorption and photosynthetic capacity; thereby reducing photosynthetate and plant growth. this can be true with plants in vivo, but probably this is less important in vitro, because in vitro plants are more heterotrophic, so decreases in their growth cannot be due to a deficiency of carbohydrates. in the present work it was clearly shown that the decrease in water availability is the main cause of decreasing growth. specific leaf area (sla) is a function of leaf area. the reduction of sla in water deficit conditions is due to the fact that leaf area development is more affected than deposition of dry matter (blum and pneul, 1990). in both cultivars with increasing peg% in culture media, leaf relative water content (rwc) decreased and an increase in ion leakage was recorded (table 3, fig. 1). these findings are in agreement with the results obtained by other researchers in vitro (sawwan et al., 2000; al-khayri and al-bahrany, 2004; chai et al., 2005). leaf yellowing and chlorosis were the consequence of structural damage to cell membranes in explants. interestingly, in 6% peg, although the rate of relative water content reduction in leaves of sabz cultivar (50%) was more than ‘siah’ (20%), the difference in their ion leakage was not significant. leaves accumulated significant quantities of proline and, in contrast, the amount of chlorophyll was decreased with increasing peg percentage in culture media (fig. 2). table 3 interaction of water stress and cultivar on leaf relative water content (%) and leaf ion leakage (%) drought stress (peg%) leaf relative water content (%) cultivar 0 2 4 6 mean siah 69.2 a (z) 68.6 a 59.3 ab 54.9 ab (20) 63.0 a sabz 73.6 a 51.4 ab 61.7 a 36.7 b (50) 55.8 a mean  71.4 a 60.6 ab 58.8 ab 45.7 b leaf ion leakage (%) siah 50.6 d 57.1 bcd 55.5 bcd 64.2 abc 56.9 b sabz 54.1 cd 57.0 bcd 68.7 a 66.2 a 61.5 a mean 52.3 c 57.0 bc 62.1 ab 65.2 a (z) in each row and column, means with similar letters are not significantly different using duncan’s multiple rang test p≥0.05. the percentage of reduction with respect to control is reported in parentheses. fig. 1 changes in leaf water content (rwc) and ion leakage in two fig cultivars: ‘sabz’ (a) and ‘siah’ (b). fig. 2 changes in proline and total chlorophyll in two fig cultivars: sabz (a) and siah (b). 135 naturally, the amount of proline in ‘sabz’ was higher than in ‘siah’ (81.8 µmole g-1 versus 16.7 µmole g-1) (table 4). in both cultivars, with increasing peg % in media, proline content increased. however, the results indicated that, in all levels of peg treatments, the amount of proline in ‘sabz’ was higher than in ‘siah’. plants produce and accumulate such compounds compatible to their metabolism to overcome adverse effects of drought stress (zhu, 2001). accumulation of such substances would cause more negative water potential in plants, a necessary condition to absorb and keep the water in plant tissues (sivritepe et al., 2008). table 4 interaction of water stress and cultivars on leaf proline content (µm g-1 fresh weight) drought stress (peg%) cultivar 0 2 4 6 mean siah 16.8 c (z) 50.5 b 73.7 b 121.0 b 65.5 b sabz 81.8 b 320.7 a 293.6 a 289.5 a 246.4 a mean 42.8 b 170.6 a 199.4 a 205.2 a (z) in each row and column, means with similar letters are not significantly different using duncan’s multiple rang test p≥0.05 with increasing the severity of water stress, proline and soluble sugars (as compatible substances) accumulated significantly in explant leaves of both cultivars. the accumulation of these substances has been well documented in field (clifford et al., 1998; zamani et al., 2002; mafakheri et al., 2010) and in vitro conditions (handa et al., 1982; brito et al., 2002; al-khayri and al-bahrany, 2004; molassiotis et al., 2006; sivritepe et al., 2008). a notable point in our results was the significant increase in proline accumulation in leaves of both cultivars. it has been reported that there is a direct positive relationship between drought tolerance and proline concentration in plant tissues (al-khayri and albahrany, 2004; sivritepe et al., 2008). the role of proline in destroying reactive oxygen species (ros) in water stress conditions has been reported in different plant species (turkan et al., 2005; verslues et al., 2006). therefore, it can be expected that plants which accumulate more proline under water stress are more tolerant. thus, it may be concluded that ‘sabz’ is more tolerant than ‘siah’, although in some plants a relationship was not found between proline accumulation and drought tolerance. the changes in leaf sugar and starch contents under different levels of peg are shown in table 5 and figure 3. in both cultivars, with increasing peg%, the amount of soluble sugar content increased and the amount of starch deceased. in drought conditions, soluble sugar can act as osmoticum and also osmoprotectant. in addition, accumulation of sugar may partly protect protein against the oxidative damage created by free radicals (bohnert and shen, 1999). in the present work, soluble sugars content in ‘sabz’ was greater than in ‘siah’, but it did not have significant effect on water absorption. accumulating soluble sugars can be due to starch hydrolysis, which is in agreement with the results obtained by shawky et al. (1997). taylor et al. (1982) also reported that carbohydrates (reducing and non-reducing sugars) are the most abundant component in osmotic adjustment in tomato seedlings. table 5 interaction of water stress and cultivar on leaf tss and starch (mg g-1 dry weight) drought stress (peg%) 0 2 4 6 cultivar tss (mg g-1 dry weight) mean siah 170.3 c 168.9 c 256.6 bc 287.0 b 220.69 b sabz 276.4 bc 344.5 b 357.5 b 522.2 a 375.2 a mean  215.8 c 256.7 bc 311.6 b 404.6 a starch (mg g-1 dry weight) siah 133.5 ab 103.8 bc 73.7 bc 65.3c 94.1 a sabz 167.0 a 124.2 abc 76.3 bc 80.3 bc 111.9 a mean  152.56 a 112.83 b 75.4 b 73.9 b (z) in each row and column, means with similar letters (small letters for interaction and big letters for main effects) are not significantly different using duncan’s multiple rang test p≥0.05 in this study, with increasing peg in culture media, the amount of total chlorophyll declined in leaves of the two fig cultivars. effect of drought stress on reduction of chlorophyll content of other plants in vitro has previously fig. 3 changes in leaf sugar and starch content in two fig cultivars: ‘sabz’ (a) and ‘siah’ (b). 136 been reported (hernández-sebastià et al., 2000; brito et al., 2002; molassiotis et al., 2006). under field conditions, also chlorophyll content was reduced with increasing drought (munne-bosch and penuelas, 2004). in the present experiment, with increasing water deficit (with addition of peg), chlorophyll content was decreased along with accumulation of leaf proline (fig. 3). this may be due to the fact that chlorophyll and proline are synthesized through glutamate pathway, causing an increase in synthesis of proline under drought conditions, which resulted in reduction of chlorophyll synthesis (aspinall and paleg, 1981). 4. conclusions the assessment of drought tolerance of two fig cultivars showed that under water stress conditions in both cultivars, leaf fresh and dry weight, total chlorophyll and starch contents decreased, but proline and soluble sugar 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al., 2013; lee et al., 2014), decreases blood pressure and pulse rate (park and mattson, 2009; park et al., 2012), and decreases cortisol concentration (park et al., 2012). studies by li et al. (2007, 2008 a, b) demonstrated that staying in a forest environment for three days and two nights improved the immune function of office workers (li et al., 2007), and this effect was sustained for approximately one month (li et al., 2008 a, b). another study reported that walking in an urban park enhances parasympathetic nervous activity and decreases heart rate (song et al., 2013). in addition, spending time in rooftop gardens enhances parasympathetic nervous activity and suppresses sympathetic nervous activity in elderly people requiring care (matsunaga et al., 2011). evidence-based medicine has been attracting attention globally, with physiological data from field tests making a significant contribution. we expect that accumulating physiological data from field experiments will continue to demonstrate the preventive medical effects of nature therapy in the future (lee et al., 2012). in modern society, many individuals spend the majority of their time in intensely stressful states, and they have no time to make contact with nature outside of their immediate surroundings. high school students, who spend most of their everyday life at school, are typical examples. previous studies have evaluated the psychological stress levels in high school students (anda et al., 2000; takakura and sakihara, 2001). moreover, many high school students have stressful relationships with friends or teachers (miura and kawada, 2008). in a document by the japanese ministry of education, the percentage of students who progressed to universities or junior colleges in 2010 was 56.9%, which was 18.5% higher than the rate in 1975 (statistics bureau, ministry of internal affairs and communications, 2012), and the pressure from entrance examinations is extremely high among high school students (equal employment, children and families bureau, ministry of health, labour and welfare, 2009). physiological and psychological relaxing effects of visual stimulation with foliage plants in high school students h. ikei*, c. song*, m. igarashi*, t. namekawa**, y. miyazaki*(1) * center for environment, health and field sciences, chiba university, 6-2-1 kashiwanoha, kashiwa, chiba 277-0882, japan. ** chiba prefectural kashiwanoha senior high school, 6-1 kashiwanoha, kashiwa, chiba 277-0882, japan. key words: heart rate variability, minors, house plant, stress reduction, visual stimuli. abstract: with lifestyles in modern society becoming increasingly stressful, there is growing interest in the physiological relaxing effects of the natural environment. particular interest has been paid to the physiological effects of indoor plants, however no studies have revealed the effects of such visual stimulation on minors. in this study 85 (41 male and 44 female; 16.5±0.9 years; mean±sd) students were exposed, or not as control, to a typical foliage plant, dracaena (dracaena deremensis; lemon lime), for 3 min. physiological indices included heart rate variability (hrv) and pulse rate, using an accelerated plethysmography at the fingertip, were collected continuously during the experiments. the results indicated that the high frequency component (hf), a general index of parasympathetic nervous activity, was significantly higher; the low frequency component [lf/(lf+hf)], a general index of sympathetic nervous activity, was significantly lower; and the pulse rate was significantly lower. after exposure, or not, the subjects completed a questionnaire as psychological evaluation. a 13-point rating scale was used for following parameters: “comfortable-uncomfortable,” “relaxed-awakening,” and “natural-artificial.” results of the study showed that subjects felt more comfortable, relaxed and natural after visualizing the dracaena plants. overall, the physiological and psychological relaxing effects of visual stimulation with foliage plants in high school students is confirmed. adv. hort. sci., 2014 28(2): 111-116 (1) corresponding author: ymiyazaki@faculty.chiba-u.jp received for publication 31 march 2014 accepted for publication 29 july 2014 112 flowers and foliage plants are common natural surroundings that can be incorporated into the school and home. we previously conducted surveys to evaluate the physiological effects of visual stimulation with fresh rose flowers in high school students (ikei et al., 2013), middle-aged and elderly medical staff (komatsu et al., 2013), and office workers (ikei et al., 2014). the results showed enhanced parasympathetic nervous activity (ikei et al., 2013; komatsu et al., 2013; ikei et al., 2014), suppressed sympathetic nervous activity (ikei et al., 2013), and decreased pulse rates (komatsu et al., 2013) during visual stimulation. it was reported in a previous study that natural views from hospital windows or the presence of indoor plants hasten the recovery of patients after surgery and decrease systolic blood pressure (park and mattson, 2009). these effects have also been studied in a classroom, demonstrating that the ambience of indoor space can be improved by including foliage plants (doxey et al., 2009), as reflected by enhanced feelings of comfort among the students (han, 2009). however, there have been no reports on the influence of visual stimulation with foliage plants on heart rate variability (hrv) and subjective feelings in minors. therefore, this study was conducted to examine the effects of exposure to the foliage plant dracaena (ministry of agriculture, forestry and fisheries, 2008) on physiological and psychological variables (hrv, pulse rate, and subjective responses) in high school students. 2. materials and methods the experiments were conducted in a classroom of the chiba prefectural kashiwanoha senior high school in october 2012. the room temperature was approximately 25.9°c, relative humidity approximately 52.6%, and illumination approximately 900  lux. eighty-five high school students (41 male and 44 female; 16.5±0.9 years; mean±sd) participated in the experiment. the study was conducted with the approval of the ethics committee of the center for environment, health and field sciences, chiba university. all subjects provided written informed consent. three dracaena plants (dracaena deremensis, lemon lime), 55-60 cm high, were placed at intervals of 8 cm on a desk in front of each subject (test situation). the distance from the subject’s eyes to the plants was approximately 55 cm, and they were adjusted according to the height of the subjects. no exposure to foliage plants was used as the control condition. before visual stimulation, the plants and the control were covered by a corrugated cardboard box (rest condition). figure 1 shows the study protocol, figure 2 the rest condition, and figure 3 the visual stimuli (the dracaena fig. 1 study protocol for testing the physiological and psychological relaxing effects of visual stimulation with foliage plants in high school students. fig. 2 the rest condition. fig. 3 -the visual stimulation condition. 113 plants or control). after viewing the cardboard box at rest in a sitting posture for 1 min (fig. 2), the subject was exposed to the plants or control for 3 min (fig. 3). after the experiments, each subject completed a questionnaire. the order of stimuli was counterbalanced among subjects. hrv and pulse rate were measured as physiological indices. hrv was calculated by spectral analysis of the coefficient of variation of the a-a interval on an accelerated plethysmograph (apg; artett, u-medica inc., osaka, japan). previous studies have reported that the a-a interval on an apg and r-r interval on an electrocardiogram are strongly correlated (takada and okino, 2004; takada et al., 2008). the sampling frequency was set at 1000 hz. the maximum entropy method was used for frequency analysis, and variance of the low frequency (lf; 0.04-0.15 hz) and high frequency (hf; 0.15-0.40 hz) components were calculated. the lf/(lf+hf) ratio for r-r interval variability was also assessed. the hf component was used as an index of parasympathetic nervous activity and the lf/(lf+hf) ratio was used as an index of sympathetic nervous activity (weise and heydenreich, 1989; cacioppo et al., 1994; sawada et al., 1997). generally, parasympathetic nervous activity is enhanced during relaxation and sympathetic nervous activity is enhanced at the time of awakening and stress (ackerknecht, 1974). therefore, the pulse rate was converted by dividing 60 by the a-a interval on apg. the hrv and pulse rate data were collected continuously during the 3-min experiments and averaged. in addition, the subjects subjectively evaluated the emotional effects of the dracaena plants and control using the modified semantic differential (sd) method (osgood et al., 1957), which uses three pairs of adjectives on 13 scales, including “comfortable-uncomfortable,” “relaxedawakening,” and “natural-artificial.” the statistical package for social sciences software (v20.0, ibm corp., armonk, ny, usa) was used for all statistical analyses. a paired t-test was used to compare the physiological responses to visual stimulation with dracaena plants or control, followed by holm correction of the changes in each 1-min average, while the wilcoxon signed rank test was used to compare the psychological responses to visual stimulation with dracaena plants or control. for both conditions, one-sided tests were used because of the hypothesis that humans are relaxed by visual stimulation with foliage plants. statistical differences were considered significant at p<0.05. 3. results significant differences were found in the values of the hf component and the lf/(lf+hf) ratio of hrv between dracaena and the control. figure 4 shows the hf component of hrv, an estimate of parasympathetic nervous activity. hf between 0 and 1 min was 1210.7±120.3 (mean±se) ms2 during the test condition and 1032.9±95.2 ms2 during the control condition, showing a significant increase of 17.2% (p<0.05) (fig. 4a) during the test condition. similarly, hf between 1 and 2 min was 1052.0±109.3 ms2 during the test condition and 893.1±87.1 ms2 during the control condition, showing a significant increase of 17.8% (p<0.05) (fig. 4a) during the test condition. there was no significant difference in hf between 2 and 3 min. the overall hf during the 3-min experiment was 1083.9 ± 101.5 ms2 in the test condition and 954.8±83.5 ms2 in the control condition, showing a significant increase of 13.5% (p<0.01) (fig. 4b) with the test condition, indicating that parasympathetic nervous activity was significantly higher during dracaena plant exposure. the results of the lf/(lf+hf) ratio, a marker of sympathetic nervous activity, are shown in figure 5. for 1-min segment analysis, the lf/(lf+hf) ratio between 0 and 1 min was 0.47±0.02 during the test condition and 0.52±0.02 during the control condition, showing a significant decrease of 9.6% (p<0.05) (fig. 5a) with the former. fig. 4 the 1-min averages and overall mean high frequency component (hf) of heart rate variability (hrv) during visual stimulation with dracaena plants and control. (a) changes in each 1-min average hf value over 3 min. (b) overall mean hf values. n=85. mean±se. *p < 0.05, **p < 0.01 as determined by the paired t-test, holm correction. fig. 5 the 1-min averages and the overall mean lf/(lf+hf) ratio of heart rate variability. (hrv) during visual stimulation with dracaena plants and control. (a) changes in each 1-min average lf/(lf+hf) value over the 3 min. (b) overall mean lf/(lf+hf) values. n=85. mean±se. *p<0.05 as determined by the paired t-test, holm correction. 114 similarly, between 1 and 2 min, the ratio was 0.48±0.02 during the test condition and 0.54±0.02 during the control condition, showing a significant decrease of 11.1% (p<0.05) (fig. 5a) during the test. no significant difference was observed between 2 and 3 min. for the entire 3-min duration, lf/(lf+hf) was 5.6% lower during the test condition than during the control condition (dracaena: 0.51±0.02, control: 0.54±0.02; p<0.05) (fig. 5b), indicating that sympathetic nervous activity was significantly lower during dracaena plant exposure. clear differences in pulse rate were observed between dracaena and control exposure. the pulse rate between 0 and 1 min was 71.9±1.2 beats/min during the test condition and 72.7±1.2 beats/min during the control condition, showing a significant decrease of 1.1% (p<0.05) (fig. 6a) during the test condition. similarly, between 1 and 2 min, the pulse rate was 72.6±1.2 beats/min during the test condition and 73.3±1.2 beats/min during the control condition, showing a significant decrease of 1.0% (p<0.05) (fig. 6a) during dracaena plant exposure. no significant difference was observed between 2 and 3 min. the mean pulse rate was 0.1% lower during the test condition than during the control condition (dracaena: 72.4±1.2 beats/min, control: 73.0±1.2 beats/min; p<0.05) (fig. 6b). the subjective evaluation data clearly showed the effect of the two different visual stimuli on the psychological states of participants. participants felt significantly more comfortable (dracaena: “slightly comfortable”; control: “indifferent”; p<0.01) (fig. 7, left), relaxed (dracaena: “slightly relaxed”; control: “indifferent”; p<0.01) (fig. 7, center), and natural (dracaena: “slightly natural”; control: “indifferent”; p<0.01) (fig. 7, right) in the dracaena condition than in the control condition after stimuli. 4. discussion and conclusions an improved ambience resulting from the placement of foliage plants in a classroom may significantly enhance physiological relaxation and mental health in high school students, as shown in the present investigation. relatively brief (3 min) visualization of foliage plants resulted in significantly enhanced parasympathetic nervous activity (13.5%), suppressed sympathetic nervous activity (-5.6%), and decreased pulse rate (-0.8%), results which are consistent with previous studies involving the visualization of a forest scene (park et al., 2010; tsunetsugu et al., 2010; park et al., 2011). furthermore, our findings are consistent with those of our previous report on the calming effects of roses in high school students (ikei et al., 2013), where similar physiological responses were found. also in line with our findings, visual stimulation with fresh roses enhanced parasympathetic nervous activity and significantly decreased the heart rate in middle-aged and elderly medical staff (komatsu et al., 2013), while it enhanced parasympathetic nervous activity in office workers (ikei et al., 2014). according to our analysis of the three questionnaires, the subjects in the present study felt more comfortable, relaxed, and natural after visualizing the dracaena plants. this result is consistent with that of our previous report on the calming effects of roses (ikei et al., 2013; komatsu et al., 2013; ikei et al., 2014). the results of this study support the hypothesis that placement of foliage plants in classrooms can induce a relaxing effect, improve physiological activity, and improve the psychological state in high school students. because of the growing interest in mental health in modern times (murray and lopez, 1996), the psychological benefits of indoor plants are expected to play an important role in the promotion of mental health in the future. however this study had limitations. first, we only evaluated hrv. thus, the results cannot be interpreted in terms of a complete physiological evaluation. other experimental indices such as brain activity and stress hormone levels should be assessed to determine the effects of visual stimulation with natural objects, such as foliage plants on human response. second, only dracaena plants were used. fig. 6 the 1-min averages and overall mean pulse rate during visual stimulation with dracaena plants and control. (a) changes in each 1-min average pulse rate over the 3 min. (b) overall mean pulse rates. n=85. mean±se. *p < 0.05 as determined by the paired t-test, holm correction. fig. 7 changes in subjective evaluation for “comfortable–uncomfortable,” “relaxed–awakening,” and “natural–artificial” with dracaena plant exposure and control exposure. n=85. mean±se. **p < 0.01 as determined by the wilcoxon signed-rank test. 115 in future experiments, we will examine human responses to exposure to multiple types of foliage plants. we predict that the physiological data will support the physiological and psychological relaxing effects of foliage plants, which may subsequently lead to their increased use in educational establishments in attempts to decrease stress among students. a brief visual stimulation of foliage plants shifted the sympathetic/parasympathetic balance and improved mood, suggesting a simple method to decrease stress and improve the health of high school students. acknowledgements this work was supported by a grant from the policy research institute, ministry of agriculture, forestry and fisheries (extramural research program for agricultural, forestry and fishery policy research). we would like to express our gratitude to ms. misako komatsu and ms. mariko aga for their valuable contributions to data collection. references ackerknecht e.h., 1974 the history of the discovery of the vegetative (autonomic) nervous system. med. hist., 18: 1-8. anda d., baroni s., boskin l., buchwald l., morgan j., ow j., gold j.s., weiss r., 2000 stress, stressors and coping among high school students. child youth serv rev., 22: 441-463. cacioppo j.t., berntson g.g., binkley p.f., quigley k.s., uchino b.n., fieldstone a., 1994 autonomic cardiac control. ii. noninvasive indices and basal response as revealed by autonomic blockades. psychophysiology. 31: 586–598. doxey j.s., waliczek t.m., zajicek j.m., 2009 the impact of interior plants in university classrooms on student course performance and on student perceptions of the course and instructor. hortscience, 44: 384-391. equal employment, children and families bureau, ministry of health, labour and welfare, 2009 2-6-1. anxiety and worries of current. nationwide survey on families and children 2009. japan ministry of health, labour and welfare, tokyo, japan. han k.t., 2009 influence of limitedly visible leafy indoor plants on the psychology, behavior, and health of students at a junior high school in taiwan. environ. behav., 41: 658692. ikei h., komatsu m., song c., himoro e., miyazaki y., 2014 the physiological and psychological relaxing effects of viewing rose flowers in office workers. j. physiol. anthropol., 33: 6. ikei h., lee j., song c., komatsu m., himoro e., miyazaki y., 2013 physiological relaxation of viewing rose flowers in high school students. jpn. j. physiol. anthropol. 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linse et al., 2001; busey, 2003; argenti and ferrari, 2009) especially in areas modified by human intervention. recently, the need for larger spaces for individual, family environments and common areas for socialization has increased the concept that green spaces represent a higher standard of living in urban areas (hull, 1990; roberts, 1990). this awareness has increased interest in turf, as a surface capable of fulfilling technical, aesthetic and recreational goals, ensuring high quality standards, practicability and durability of the turf (volterrani and magni, 2007). in turn, this has led to the development of precise management practices (gaetani et al., 2013) through which to minimize the negative impact on the environment. the main problem connected to turfgrass is the amount of input necessary to obtain a high quality surface (easton and petrovic, 2005). the predominant challenge is the utilization of water for irrigation (youngner et al., 1981; sevostianova and leinauer, 2014). this aspect may reach critical levels especially in mediterranean regions, characterized by low rainfall and hot summer temperatures. these climatic characteristics suggest the use of a low water rate and drought tolerant species such as warm-season turfgrasses (marchione, 2008) that can provide high quality turf with suitable water consumption and low fertilizer and pesticide input (turgeon, 2002; schiavon et al., 2013). the main objection to the use of these grasses is their lack of green color during the winter period, when they enter in dormancy and loose chlorophyll (bernardini, 2007). this occurs especially in central italy, an area characterized by high summer and cold winter temperatures, where cold tolerance and rapidity of recovery from winter dormancy are also important qualities (magni et al., 2014). the objective of this investigation was to assess performance and adaptation, especially in relation to cold temperature, of three species (seven cultivars) of c4 warm-season turfgrass species and a cool-season grass as a comparison, in an internal area of the tuscany region. 2. materials and methods the experimental site was located in a borderline area of mediterranean climate in the centre of tuscany, characterized by hot summer and low winter temperatures. the experimental trial started in the late spring of 2011 in antria (tuscany, central italy) on natural soil, containperformance of warm-season turfgrasses in an area of central italy i. seppoloni (1) *, n. staglianò (2), s. cecchi (2), g. argenti (2) (1) c.n.r., ibimet, via giovanni caproni 8, 50145 firenze, italy. (2) dispaa, università degli studi di firenze, piazzale delle cascine, 18, 50144 firenze, italy. key words: bermudagrass, ground cover, growing season, turf quality, weeds. abstract: traditionally, in italy, the c3 cool-season grasses have been the dominant species used for turfs, even though they do not appear to be the most suitable for the mediterranean climate. however, recent limited water availability and the need to reduce energy inputs have placed drought tolerant warm-season turfgrasses under the spotlight. these species combine aesthetics with performance advantages in terms of water consumption, and with regard to the reduction of fertilizer and pesticides use. the present research was aimed to test the performance of warm-season turfgrass species (three cultivars of cynodon dactylon, two cultivars of paspalum vaginatum and two of zoysia japonica) in a climatic transition zone in tuscany, to evaluate their potential for use in this environment. the assessment of several parameters, which were estimated periodically, permitted performance evaluation of each species/cultivar, thereby enhancing the existing knowledge of these species and their potentiality in this environment. results showed that the species with the best adaptation to the environment was cynodon dactylon, which had higher performances compared to the other species. paspalum vaginatum reported good quality in terms of color and density, but was damaged by low temperatures during winter. zoysia japonica displayed a poor performance during the first year, but quality increased during the second year, yielding satisfactory results. adv. hort. sci., 2015 29(1): 53-58 * corresponding author: i.seppoloni@ibimet.cnr.it received for publication 28 january 2015 accepted for publication 1 may 2015 54 adv. hort. sci., 2015 29(1): 53-58 ing 16.4% sand, 54.1% silt and 29.5% clay. the study was conducted over two growing seasons (2011 and 2012), starting after full establishment of the turf, up to the period of dormancy. due to the presence of a meteorological station nearby, weather parameters were recorded in detail for the entire experimental period. the species under comparison included cynodon dactylon (‘black jack’, ‘casino royale’ and ‘la paloma’), paspalum vaginatum (‘marina’ and ‘sea spray’), and zoysia japonica (‘compadre’ and ‘zenith’) and a cool-season grass as control (lolium perenne ‘kokomo’). the eight different accessions tested were replicated three times for a total of 24 plots (2x3 m with a surface area of 6 m2) according to a complete randomized block design. sowing took place on the 10 june 2011. seed rate was 25 g m-2, as an average of the suggested dose for warm-season species (panella et al., 2000). seeding was performed manually and followed by rolling. cultural practices for the growing season included four or five applications of standard fertilizers (yearly total amount 318-85-81 kg ha-1 npk). irrigation was performed to restore 100% of crop evapotranspiration (etc) and it was applied with a sprinkler system. periodically, mowing was carried out with a mowing height of 30 mm performed by a rotary mower. a glyphosate treatment was performed during the dormancy period to reduce the presence of weeds (1 l ha-1). the following parameters were monitored during the trial and evaluated every two weeks (pardini et al., 2002; reyneri and bruno, 2008): aesthetic quality of the turf: scores were visually assigned on a scale ranging from 1 to 9 (1= poorest quality, 9= highest quality) (piano, 2005; bigelow and walker, 2008). quality was also compared to additional parameters measured during the trial (color, ground cover and weeds) by multiple regression analysis to establish which aspects are the most related to quality; turf color: scores were visually assigned on a scale ranging from 1 to 9 (1= light green, 9= dark green) (bullitta et al., 2005; kir et al., 2010); ground cover: estimated visually as a percentage of soil cover; weed infestation: estimated visually as percentage of soil cover. all the data were grouped to obtain seasonal aggregation, where s1 was the first season of trials corresponding to summer 2011, s2 was autumn 2011, s3 spring 2012, s4 summer 2012 and s5 was the end of the study in autumn 2012. the mean length of winter dormancy was estimated for each species, and plants were considered dormant when color scores means reached a value of 2. statistical analysis was carried out by means of anova and tukey test to discriminate differences among averages species values. moreover, through multiple regression analysis, it was possible to evaluate which of the tested parameters (color, ground cover and presence of weeds) was strongly related to the quality on a global scale. all analysis were performed utilizing ibm spss statistics software (release 20). 3. results meteorological trends during experimental period both years were characterized by very hot and dry summer periods. in particular, 2011 was exceptionally dry until late autumn and had a total rainfall of 500 mm compared to the average of 800 mm (2002-2010). the highest recorded temperature occurred in august (38°c). during winter the coldest month was february 2012 with severe temperatures below 0°c (-9°c minimum recorded) and, even in full dormancy, warm-season grasses can be damage by the cold. damage is also proportional to the duration of low temperatures. in 2012 there was a total rainfall of approximately 900 mm but it was concentrated at the end of the year. for this reason, during the summer, there was a severe water deficit replaced by irrigation. spring was exceptionally warm, whereas the summer was one of the driest in recent decades. turf quality figure 1 shows the interaction between quality and season (s1= summer 2011, s5= autumn 2012) for the eight species or cultivars. the species that showed the best performance over time was cynodon dactylon, with good values and a constant quality trend. all three cultivars belonging to this species performed well: in particular, ‘black jack’, obtained higher scores at the beginning of the trial. the highest value (7.1) was attained during summer 2012 (s4), even though the presence of thatch probably influenced the estimation. paspalum vaginatum, after a good start and an improvement in autumn, proved to be the most sensitive species to low winter temperatures, having slower vegetative growth during the spring 2012 (s3) and the worst score, due to damage that occurred during winter. ‘sea spray’ showed better results in comparison to ‘marina’. zoysia japonica performed badly during the year of establishment until the second growing season. thereafter, fig. 1 global quality of the species/cultivars under investigation during the trial period (assessment with 1-9 scale). bars = standard error. l.p. = lolium perenne, c.d. = cynodon dactylon, p.v. = paspalum vaginatum, z.j. = zoysia japonica. 55 seppoloni et al., performance of warm-season turfgrasses in an area of central italy it gradually improved and gave very good scores at the end of the second year. ‘zenith’ performed better than ‘compadre’ on an overall scale. lolium perenne was the most unsuitable species, showing a rapid initial establishment, after which the quality began to decrease from s2, attaining the worst score (2) in s4. color the best performance regarding color was obtained with lolium perenne: it was clearly superior with a darker and brighter color in each period (fig. 2). only p. vaginatum, in s3, showed equivalent scores and it resulted to be the species with the best color among the tested warmseason grasses. cynodon dactylon gave unsatisfactory results, showing a decrease in quality in both years (in late summer and in the beginning of autumn), due to a rapid entrance into dormancy. moreover, the light green color, typical of these seeded cultivars of cynodon and the high production of thatch, influenced the scores. the trend of the two cultivars of zoysia japonica appears to be very similar to that of cynodon. ground cover and weed infestation table 1 shows the average ground cover values for each species during the different seasons of the trial. it is clear that the warm-season grasses increased the soil cover over time, while the percentage of l. perenne remained almost unchanged compared to the initial one (approximately 5560%). p. vaginatum, due to the winter cold, had a poor cover percentage in the spring of the second year but it was able to regenerate an efficient soil cover thanks to its strong stolons, and this trend is evident for both cultivars. c. dactylon demonstrated good and constant soil coverage and it was more satisfactory than other species, whereas z. japonica displayed the typical behavior of this species, attaining optimal values at the end of the growing season of the second year (s5). as regards the presence of weeds (table 1), infestation spread rapidly during the first year on l. perenne and z. japonica. the high presence of weeds on lolium is also due to the fact that this species is characterized by a bunch type table 1 average value (±se) of ground cover and infestation of the tested cultivars over the 5 seasons species/ cultivar ground cover (%) s1 s2 s3 s4 s5 lp 59.0±7.5 a 58.3±2.1 bcd 58.3±4.6 ab 51.7±4.8 d 61.7±2.5 c cd1 73.7±5.2 ab 86.3±5.1 a 76.7±8.8 a 92.8±1.5 a 95.0±0.1 a cd2 63.7±5.8 ab 81.3±4.4 a 78.3±2.2 a 92.2±1.1 a 94.4±0.6 a cd3 57.0±5.6 ab 79.6±4.2 ab 73.3±3.6 a 92.2±1.1 a 93.9±1.1 a pv1 67.0±3.2 ab 78.3±2.1 ab 40.0±6.3 b 58.1±9.3 cd 83.3±5.9 b pv2 61.7±2.6 ab 76.7±6.9 ab 42.5±14.4 b 71.9±12.3 bc 88.9±5.3 ab zj1 24.7±2.4 c 40.8±3.4 d 52.5±6.3 b 78.9±1.5 ab 87.2±2.0 ab zj2 39.7±7.6 bc 56.3±6.3 cd 60.0±3.8 ab 83.3±2.5 ab 89.4±0.6 ab species/ cultivar infestation (%) s1 s2 s3 s4 s5 lp 4.0±2.3 ab 16.7±4.0 bc 30.0±5.8 c 23.3±4.4 b 30.6±4.0 b cd1 0.3±0.3 a 5.8±0.8 ab 8.3±2.2 a 6.7±1.0 a 16.1±2.9 ab cd2 1.7±0.9 a 10.8±1.1 abc 8.3±2.2 a 9.4±2.8 a 18.9±5.3 ab cd3 1.7±1.2 a 5.4±1.1 a 7.5±2.5 a 9.4±4.4 a 13.9±5.6 a pv1 1.3±0.9 a 3.3±1.1 a 19.2±5.8 bc 14.7±6.8 b 20.0±3.3 ab pv2 1.0±1.0 a 4.2±0.4 a 15.0±2.5 ab 12.5±2.7 ab 19.4±7.2 ab zj1 4.3±1.3 ab 17.9±4.2 c 19.2±4.4 bc 11.7±3.5 ab 18.9±4.5 ab zj2 8.7±1.3 b 19.6±1.8 c 23.3±2.2 bc 13.9±2.4 ab 9.4±0.6 a lp= lolium perenne kokomo, cd1= cynodon dactylon black jack, cd2= c. dactylon casinò royale, cd3= c. dactylon la paloma, pv1= paspalum vaginatum marina, pv2= p. vaginatum sea spray, zj1= zoysia japonica compadre, zj2= z. japonica zenith. values in a column with the same letter are not significantly different (p<0.05) according to tukey test. fig. 2 average color of the species under investigation during the trial period (assessment with 1-9 scale). bars = standard error. l.p. = lolium perenne, c.d. = cynodon dactylon, p.v. = paspalum vaginatum, z.j. = zoysia japonica. 56 adv. hort. sci., 2015 29(1): 53-58 habitus that makes it less competitive with opportunistic weed species in comparison to warm-season grasses. in addition, lolium, after being stressed by the high summer temperatures, was also the only species not treated with glyphosate, taking into account its vegetative activity during the winter. for zoysia, the infestation was easily attributable to a slow establishment that left plenty of space for the development of invasive species at the beginning of the trial. nevertheless, for all the species, despite the winter control with glyphosate, a manual control against weeds in the second year was necessary. after these operations, the presence of weeds was shown to be reduced, thanks to the ability of these highly-competitive species to suppress weed growth. multiple regression analysis this model permits the calculation of turf quality value (considered the dependent variable) through the other observed parameters. the model applied (a stepwise model) involves a mechanism of removal of the independent variables provided that they do not cause a decline in the power of the model. the elimination of the variables from the model is performed automatically by comparison with a threshold value of significance, which in this case was set to be equal to 0.05. from the analysis the following formula was obtained: global quality= -1.447+0.067*cover-0, 050*weeds+0.443*color (r2 = 0.884) the parameters were ordered starting from the one most related to overall quality to the one with the lowest influence on the formula. no variable was removed from the model, demonstrating the importance of all investigated parameters in describing the aesthetic value of a turf. the presence of weeds, as expected, produced a negative value, as it was inversely related to the aesthetic appearance of the turf. the very high determination coefficient indicated the good correlation between the independent variables and the dependant variable (turf quality). this type of analysis could be used in future investigations to predict the overall performance of a turf. winter dormancy table 2 shows the dates at the beginning and the end of the growing season, and the length of vegetation and dormancy periods for the species during the trial years. at the end of the first year of experimentation, dormancy took place in november for all the species, with differences of about two weeks between the earliest (zoysia) and latest (paspalum) entrance into dormancy. the period of dormancy during winter 2011-2012 was particularly long for paspalum, also because the growing season in the spring 2012 started at the end of april. cynodon and zoysia showed early vegetation activity at the end of march. the growing season for 2012 was longer for z. japonica (236 days), due to its early vegetative growth and the entrance into dormancy in mid-november, which was exceeded only by paspalum vaginatum. nearly the same duration was found for c. dactylon (233 days), with greater precocity in vegetative growth, but with an earlier loss of green color than with other species. the shortest growing season, as expected, was found in p. vaginatum (208 days), despite its late entrance into dormancy. the period of dormancy during winter 2012-2013 showed a substantial analogy to the previous year for zoysia (137 days in 2011/2012 and 142 in 2012/2013) and paspalum (151 days compared to 159) but a considerable lengthening for cynodon (124 days compare to 160 in the second year). this behavior could be connected to the fact that mean and maximum spring temperatures in 2013 were lower compared with the previous year. this trend definitely affected the vegetative growth of the earliest species (cynodon), highlighting the important role temperatures play in the vegetative activity of warm-season grasses. 4. discussion and conclusions in this study, cynodon dactylon emerged as the species with the best overall behavior and the best potential adaptation to the environment. it displayed good quality standards, rapid establishment, good overall appearance, high ground cover and a competitive behavior against weed table 2 dates of the beginning and the end of the growing season and length of vegetation and dormancy periods for the species during the years of trial species beginning end vegetation (days) dormancy (days) cynodon dactylon 16/11/2011 124 paspalum vaginatum 26/11/2011 151 zoysia japonica 08/11/2011 137 cynodon dactylon 20/03/2012 08/11/2012 233 paspalum vaginatum 26/04/2012 20/11/2012 208 zoysia japonica 25/03/2012 16/11/2012 236 cynodon dactylon 17/04/2013 160 paspalum vaginatum 28/04/2013 159 zoysia japonica 07/04/2013 142 57 seppoloni et al., performance of warm-season turfgrasses in an area of central italy infestation. the constancy of studied parameters along experimental trial are consistent with volterrani et al. (1997). the worst performance concerned color of this species that probably resulted remarkably affected by presence of thatch, confirming the observations of holm et al. (1991). paspalum vaginatum presented an overall satisfactory appearance. however, the species was highly susceptible to cold winters, in agreement with de luca et al. (2008), and tesi (2012) reported cold damages on paspalum with temperature around -7°c. anyway this species performed in a very good manner concerning other variables, as it displayed a dark green-blue color, as well as a good color retention during autumn, as reported previously by volterrani et al. (1996) and geren et al. (2009). given that the stability of the turf was compromised, this behavior establishes a limit to the use of this species in areas that border the mediterranean climate or with severe risks of low temperatures during winter. zoysia japonica exhibited a typical growing pattern, confirming that observed by patton and reicher (2007), and it showed very poor performance in the first year, slow development and not competitive attitude against weeds. similar results regarding this species were found by geren et al. (2009) in a comparative study among warm-season grasses in turkey. on the other hand, starting from the beginning of the second year, a gradual increase in quality, color and coverage was observed, and the species achieved remarkable results at the end of second growing season, in agreement with sladek et al., (2011). through multiple regression analysis it was possible to evaluate which of the tested parameters influenced turf quality the most. the results showed that the ground cover and the presence of weeds are strongly related with the overall aesthetic quality. the winter season revealed important differences in relation to the length of the period of dormancy among species. p. vaginatum had the longest period of dormancy (about 155 days as the average of the two years of experimentation). a delay in the vegetative activity in paspalum, in comparison to other warm-season grasses, was also reported by other authors in different climatic areas (duncan, 1996; miele et al., 2000; volterrani et al., 2001), as well as in both coastal and internal areas of tuscany (volterrani et al., 2000; pardini et al., 2002) and in the province of rome (croce et al., 2001). c. dactylon showed a rapid recovery from winter dormancy in warm springs, while a longer length of dormancy was observed during the cold spring 2013. z. japonica remained dormant for approximately 140 days during both years of the experiment. in general, in the interior zones of this study, the duration of dormancy was found to be longer than those verified by other experiments along the coastal areas of central italy (croce et al., 2004). for this reason, in this environment, it is necessary to plan and analyze costs and benefits associated with the use of warm-season grasses. where it is not possible to accept the absence of green color during winter, it is evident that the use of warm-season species can be extended to these areas only by adopting appropriate overseeding programs, but these interventions seem possible only in high standard turf such as those related to sport pitches. acknowledgements the authors acknowledge prof. marco volterrani for his valuable and constructive suggestions during the planning and development of this research work and green grass s.r.l. for its technical support for this project. research was funded by the italian ministry of agricultural and forest policies, “bando oiga 2009”. references argenti g., ferrari l., 2009 plant cover evolution and naturalisation of revegetated ski runs in an apennine ski resort (italy). iforest, biogeosciences and forestry, 2: 178182. beard j.b., 1973 turfgrass: science and culture. prenticehall, inc., englewood cliffs, nj, usa. bernardini v., 2007 i manti erbosi sportivi ad alto livello: l’esperienza dell’uso delle macroterme allo stadio olimpico di roma. tappeti erbosi, aspetti tecnici, ambientali e paesaggistici. i quaderni del c.i.r.a.a., felici editore, pp. 65-70. bigelow c.a., walker k.s., 2008 annual nitrogen fertility regime affects appearance and soil loss for three coolseason lawn species. proceedings of the 1st european turfgrass society conference, pisa, italy, pp. 47-48. bullitta s., caredda s., piluzza g., seddaiu g., dettori d., 2005 valutazione varietale di specie da tappeto erboso (iii) (uniformità, colore, aspetto estetico globale) campo prova ottava (sassari) 1999-2003. atti del convegno “inerbimenti e tappeti erbosi per l’agricoltura, l’ambiente e la società”. vol. 1. comunicazioni poster. istituto sperimentale per le colture foraggere, lodi, italy, p. c7. busey p., 2003 cultural management of weeds in turfgrass: a review. crop science, 4: 1899-1911. croce p., de luca a., mocioni m., volterrani m., beard j.b., 2001 warm-season turfgrass species and cultivar characterizations for a mediterranean climate. international turfgrass society research journal, 9: 3-7. croce p., de luca a., mocioni m., volterrani m., beard j.b., 2004 adaptability of warm season turfgrass species and cultivars in a mediterranean climate. acta horticulturae, 661: 365-368. de luca a., volterrani m., gaetani m., grossi n., croce p., mocioni m., lulli f., magni s., 2008 warmseason turgrass adaptation in northern italy. proceedings of the 1st european turfgrass society conference, pisa, italy, 2008, pp. 75-76. duncan r.r., 1996 the environmentally sound turfgrass of the future. seashore paspalum can withstand the test. a usga-sponsored research project, pp. 17-19. 58 adv. hort. sci., 2015 29(1): 53-58 easton z.m., petrovic a.m., 2005 impact of organic and mineral fertilizers on run-off from turf. better crops, 89: 16-17. gaetani m., lulli f., andreucci a., masini a., vittori g., volterrani m. 2013 sprouting and plant regeneration capability in saline conditions of seashore paspalum, manilagrass, and hybrid bermudagrass stolons. propagation of ornamental plants, 13: 57-64. geren h., avcioglu r., curaoglu m., 2009 performances of some warm-season turfgrasses under mediterranean conditions. african journal of biotechnology, 8: 4469-4474. holm l.g., plunknett d.l., pancho j.v., herberger j.p., 1991 the world’s worst weeds: distribution and biology. krieger publishing company, malabar, florida, usa. hull r.j., 1990 the psychological value of turf and ornamental plants. agronomy abstracts, 175. kir b., avcioglu r., demiroglu g., simic a., 2010 performance of some cool season turfgrass species in mediterranean environment: i. lolium perenne l., festuca arundinacea schreb., poa pratensis l., and agrostis tenuis sibth. turkish journal of field crops, 12: 174-179. linse s.j., mergen d.e., smith j.l., trlica m.j., 2001 upland erosion under a simulated most damaging storm. journal of range management, 54: 356-361. magni s., gaetani m., grossi n., caturegli l., la bella s., leto c., virga g., tuttolomondo t., lulli f., volterrani m., 2014 bermudagrass adaptation in the mediterranean climate: phenotypic traits of 44 accessions. adv. hort. sci., 28(1): 29-34. marchione v., 2008 performance of several cynodon dactylon and zoysia japonica cultivars in southern italy. proceedings of the 1st european turfgrass society conference, pisa, italy, 2008, pp. 125-126. miele s., volterrani m., grossi n., 2000 warm-season turfgrasses: results of a five-year study in tuscany. agricoltura mediterranea, 130: 196-202. panella a., croce p., de luca a., falcinelli m., modestini f.s., veronesi f., 2000 tappeti erbosi. calderini edagricole, bologna, italy. pardini a., tallarico r., parrini d., 2002 comportamento di cultivar di specie graminacee micro e macroterme da tappeto erboso in una località del mugello. rivista di agronomia, 36: 257-263. patton a., reicher z., 2007 zoysiagrass establishment rates. golf course management, march, pp. 98-101. piano e., 2005 inerbimenti e tappeti erbosi nella realtà italiana: motivazioni e finalità per lo sviluppo della ricerca. atti del convegno “inerbimenti e tappeti erbosi per l’agricoltura, l’ambiente e la società”. volume 1. comunicazioni poster. istituto sperimentale per le colture foraggere, lodi, italy, pp. 5-20. reyneri a., bruno g., 2008 effects of clipping management on the quality of a low maintenance tall fescue turf. proceedings of the 1st european turfgrass society conference, pisa, italy, pp. 159-160. roberts e.c., 1990 the significance of turf benefits. an overview. agronomy abstract, 174. schiavon m., barnes b., shaw d., henry j., baird j., 2013 strategies for converting tall fescue to warm-season turf in a mediterranean climate. horttechnology, 23: 442-448. sevostianova e., leinauer b., 2014 subsurface-applied tailored water: combining nutrient benefits with efficient turfgrass irrigation. crop science, 54: 1926-1938. sladek b.s., henry g.m., auld d.l., 2011 effect of genotype, planting date, and spacing on zoysiagrass establishment from vegetative plugs. hortscience, 46: 1194-1197. tesi r., 2012 importanza dei tappeti erbosi e ruolo delle poaceae macroterme. atti del convegno accademia dei georgofili, firenze, italy. turgeon a.j., 2002 turfgrass management. prentice hall inc., upper saddle river, nj, usa. volterrani m., gaetani m., miele s., 2000 la trasemina autunnale di specie microterme su tappeti erbosi realizzati con paspalum vaginatum swartz e cynodon dactylon x transvaalensis burtt. davy. rivista di agronomia, 34: 28-34. volterrani m., grossi n., pardini g., miele s., gaetani m., magni s., 1997 warm season turfgrass adaptation in italy. international turfgrass society research journal, 8: 1344 -1354. volterrani m., magni s., 2007 il tappeto erboso ieri, oggi e domani. tappeti erbosi, aspetti tecnici, ambientali e paesaggistici. i quaderni del c.i.r.a.a., felici editore, pp. 29-36. volterrani m., miele s., magni s., gaetani m., pardini g., 2001 bermudagrass and seashore paspalum winter overseeded with seven cool-season turfgrasses. international turfgrass society research journal, 9: 957-961. volterrani m., pardini g., grossi n., gaetani m., miele s., pietrini e., 1996 valutazione dell’adattabilità di specie graminacee macroterme da tappeti erbosi alle condizioni ambientali dell’italia centrale. italus hortus, 3: 1016. youngner v.b., marsh a.w., strohman r.a., gibeault v.a., spaulding s., 1981 water use and turf quality of warm-season and cool-season turfgrasses. proceedings of the fourth international turf research conference, 4: 251-257. impaginato 159 1. introduction banana (musa spp.) is a climacteric fruit; therefore, ripening process is induced by ethylene production via acc (1-aminocyclopropane 1-carboxylic acid) biosynthesis. the rate of respiration is followed by reaching a threshold level of ethylene within the cells of fruit then rises rapidly to a peak and subsequently falls as ripening progress. during fruit softening, starch is turned to sugars, the peel color changes to yellow and fruit flavor develop by losing its astringency (pathak et al., 2003). polyamines as natural compounds suppress ethylene synthesis by inhibition of ethylene biosynthesis enzymes activities (lee et al., 1997). they are present ubiquitously in plant organs. the main polyamines are putrescine (1, 4-diaminobutane), spermidine (n3-aminopropyl-1, 4-diaminobutane), and spermine [bis (n-3-aminopropyl)-1, 4-diaminobutane] which are essential in plant growth, differentiation and stress responses (valero and serrano, 2010). they are known to improve the storage life of fruits by inhibiting ethylene production and delaying the ripening process, respectively. polyamines and ethylene have opposite impacts on fruit ripening and senescence. thus, a balance between them is crucial to enhance and retard the fruit ripening process. in general, polyamines level declines throughout fruit senescence along with accelerating ethylene synthesis (valero et al., 2002). much researches have indicated the positive effects of pre and postharvest polyamines application on retarding fruit softening in mango (malik et al., 2003) and pear (franco-mora et al., 2005), reducing weight loss in apricot (martinez-romero et al., 2002), inhibition of ethylene production in peach (zokaee khosroshahi and esna-ashari, 2008), delaying ripening process in nectarine (torrigiani et al., 2004) and peach (bregoli et al., 2002), and maintaining ta at higher levels, diminishing the increase in tss, and declining color change in plum (khan et al., 2008). thus, the present study was carried out to evaluate the application of putrescine for extending quality and storage life of musa acuminata l. adv. hort. sci., 2016 30(3): 159-164 doi: 10.13128/ahs-20278 pre-storage putrescine treatment maintains quality and prolongs postharvest life of musa acuminata l. m.s. hosseini 1, s.m. zahedi 2, z. fakhar 3 (*) 1 department of horticultural science, hormozgan university, bandar abbas, iran. 2 department of horticultural science, faculty of agriculture, university of maragheh, 551181-8311 maragheh, iran. 3 department of horticultural science, college of agriculture and natural resources, university of tehran, 31587 karaj, iran. key words: firmness, polyphenol oxidase, postharvest, skin color, weight loss. abstract: the study was carried out to determine the effect of putrescine on quality and postharvest life of musa acuminata l. during storage. the fruits were dipped at different concentrations of putrescine (0.5, 1 and 2 mm for 30 min) and distilled water as ‘control’. changes in fruit quality attributes such as weight loss, firmness, skin color (l*, hue angle), total soluble solids (tss), titratable acidity (ta), ph, ascorbic acid, polyphenol oxidase (ppo) and polygalacturonase (pg) enzymatic activity were calculated at harvest and after 5, 10, 15 and 20 days of storage at 0±1°c, 80-85% relative humidity. weight loss, fruit softening, skin color changes, tss, ph, the activity of ppo and pg increased during fruit ripening but the rate of changes was significantly slowed in putrescine treated fruits. moreover, putrescine application maintained higher levels of ta, ascorbic acid and reduced the loss of sensory acceptability and decay incidence compared to control. in conclusion, the postharvest dip treatment of putrescine could be an effective means for extending the storage life of musa acuminata l. (*) corresponding author: za.fakhar@gmail.com received for publication 21 july 2016 accepted for publication 20 september 2016 copyright: © 2016 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2016 30(3): 159-164 160 2. materials and methods mature green bananas (musa acuminata l.) were harvested from a commercial orchard in minab, iran, and then transported to the laboratory for experiments. fruits uniform in size and color, without any noticeable defects, were selected and dipped in putrescine solution at different concentrations (0.5, 1 and 2 mm for 30 min) and distilled water as ‘control’. then all of treated and untreated fruits were stored at 13°c and 80-85% relative humidity. then, some physico-chemical attributes were measured at harvest and after 5, 10, 15 and 20 days of cold storage. quality parameters evaluation fruit weight was recorded just after harvest and a f t e r t h e d i f f e r e n t s a m p l i n g d a t e s a n d t h e n expressed as percentage of weight loss relative to the initial weight (soto-zamora et al., 2005). fruit firmness was measured using a fg-5020 penetrometer (lutron electronic enterprise co.) of 5 mm in diameter at 2 equatorial points and was expressed as newton (n). color was determined at opposite sides of each f r u i t f r o m e a c h r e p l i c a t e w i t h a m i n o l t a chromameter cr400; the following parameters were considered: l* (0= black; 100= white), a* (green to red) and b* (blue to yellow) then expressed as l* and hue angle (h°)= arctan (b* a*-1) (ozdemir, 2016). total soluble solids (tss) content was assessed by a digital refractometer (atago n1, japan) at 20˚c and expressed as a percent. titratable acidity (ta) was estimated by titrating 5 ml of diluted juice against 0.1 n naoh using phenolphthalein as an indicator and was expressed as percent malic acid (%). the ph of fruit juice was measured using a mtt65 (japan) ph meter calibrated by ph 4 and 7 buffer solutions. ascorbic acid assessment ascorbic acid content was estimated using the methods of marisa and wall (2006). polyphenol oxidase (ppo) and polygalactronase (pg) activities measurement ppo and pg were assessed using the procedure of marquez cardozo et al. (2015) and zhu et al. (2015) respectively. decay incidence and sensory acceptability determination fruit deteriorations were measured on individual fruit by visual observations. from each fruit 5 slices were obtained and fruits decay was recorded using the following formula: a/b ×100 in which a is the number of decayed fruit slices and b the initial number of all fruit slices. the fruits were rated by a panel of 10 judges on the basis of color, texture, taste and flavor and overall acceptability (as 1-2 unusable, 3-4 unsalable, 5-6 salable, 7-8 good, 9-10 very good). statistical analysis to estimate storability of fruit, a factorial design completely randomized was carried out in three replications. all data were analyzed using sas software package 9.4 for windows and mean comparisons were conducted using duncan’s multiple range tests. 3. results and discussion weight loss and firmness weight loss percentage increased in all the treatments along the storage. however, all putrescine concentrations demonstrated significantly lower weight loss than control. fruits treated with 2 mm putrescine exhibited the lowest weight loss amongst the putrescine concentrations during storage while highest weight loss was registered by control (fig. 1 a). the effect of putrescine on reducing weight loss fig. 1 the effect of putrescine at different concentrations (0.5, 1 and 2 mm) on weight loss (a) and firmness (b) of musa acuminata l. during storage. hosseini et al. prolonging storage life of banana by using putrescine 161 may be ascribed to conjugation of polyamines to the cell membrane phospholipids that result in cell membrane integrity (mirdehghan and rahimi, 2016). similar results have been reported in apricot (enas et al., 2010). as shown in figure 1b irrespective of treatments, fruit firmness decreased significantly over storage but putrescine treated fruits were observed firmer, and especially 2 mm putrescine treatments was more effective than others in keeping the firmness. it is suggested that polyamines maintain fruit firmness by their cross-linkage to the pectin substances carboxyl groups in the cell wall and lead to rigidification o f c e l l w a l l ; c o n s e q u e n t l y c e l l w a l l d e g r a d i n g enzymes activities of pectin methyl esterase (pme), pectin esterase (pe) and polygalactouronase (pg) are decreased (valero et al., 2002). the results are in line with peach (bregoli et al., 2002). color changes skin color alteration from green to yellow is a predominant index used for evaluating the stage of ripening in banana (gomes et al., 2013). as the storage time progressed, fruit color changed as a result of chlorophyll degradation along with carotenoid synthesis. however, putrescine treated fruits showed higher l* and hue angle than control (fig. 2 a and b). delayed color changes can be associated to the effect of putrescine as anti-senescence by reducing ethylene production and subsequently delaying fruit ripening as well as senescence (drake and chen, 2000). similar results have been observed in apricot (martinez-romero et al., 2002). total soluble solids (tss), titratable acidity (ta) and ph tss content and ta increased along the storage period while ph demonstrated reverse trend in all treated and untreated fruits (fig. 3 a, b and c). lower fig. 2 the effect of putrescine at different concentrations (0.5, 1 and 2 mm) on l* (a) and hue angle (b) of musa acuminata l. during storage. fig. 3 the effect of putrescine at different concentrations (0.5, 1 and 2 mm) on tss (a), ta (b) and ph (c) of musa acuminata l. during storage. adv. hort. sci., 2016 30(3): 159-164 162 values of tss, ta and higher value of ph content were observed in putrescine treated fruits compared to control (fig. 3). that is ascribed to the role of putrescine on delaying fruit ripening process by reducing ethylene production and respiration rate in fruit (valero et al., 2002). the results are in agreement with those observed in mango (malik and singh, 2006). ascorbic acid the content of ascorbic acid was significantly influenced by putrescine. the value of ascorbic acid was higher in treated fruits than control throughout the storage (fig. 4). it is possible that putrescine inhibits ascorbic acid oxidation by decreasing ascorbate oxidase activity and consequently maintaining ascorbic acid (ishaq et al., 2009). this result is in line with the finding of davarynejad et al. (2013). enzymatic activity of polyphenol oxidase (ppo) irrespective of treatments, the activity of ppo increased during ripening process and it was significantly higher in control than treated fruits (figure 5 a). this trend may be attributed to the role of putrescine on reducing polyphenol oxidase activity (koushesh saba et al., 2012). previously, it has been observed in kiwifruit (jhalegari et al., 2012). polygalacturonase (pg) activity pg is known as an important enzyme on fruit softening, whereas, a reduction in pg activity results in a delay in fruit softening and consequently an increase in the storage life (jhalegari et al., 2012). in this study, the activity of pg increased during the storage. as shown in figure 5 b, untreated fruits demonstrated the highest values of pg activity (1.74 mmol kg-1 s-1 on the 20th day of storage). fruits treated with 2 mm exhibited the lowest pg activity, followed by putrescine at 1 and 0.5 mm respectively. this trend is associated to declining fruit firmness and increasing fruit softening by the loss of membrane integrity (sitrit and bennett, 1998). decay incidence and sensory acceptability the highest rate of fruit decay percent was observed in control while all three concentrations of putrescine reduced the decay development significantly during storage; in particular, the fruits dipped in 2 mm putrescine showed the lowest decay incidence in comparison to others (fig. 6 a). while time passed, sensor acceptability declined. however, fruit treated by putrescine exhibited higher scores of sensor acceptability compared to control at the end storage (fig. 6 b). fig. 4 the effect of putrescine at different concentrations (0.5, 1 and 2 mm) on ascorbic acid of musa acuminata l. during storage. fig. 5 the effect of putrescine at different concentrations (0.5, 1 and 2 mm) ppo (a) and pg (b) activities of musa acuminata l. during storage. hosseini et al. prolonging storage life of banana by using putrescine 163 4. conclusions the effect of putrescine treatment at different concentrations (0.5, 1 and 2 mm for 30 min) was investigated to improve and extend storage life of b a n a n a ( m u s a a c u m i n a t a l . ) . a p p l i c a t i o n o f putrescine maintained fruit quality attributes such as firmness, color, tss, ta, ph and sensory acceptability. in addition, the reduction of weight loss, ppo, pg, and decay incidence were observed in putrescin t r e a t e d f r u i t s c o m p a r e d t o c o n t r o l . t h u s , t h e postharvest dip treatment of putrescine may be an effective tool for prolonging the storage life of musa acuminata l. references bregoli a.m., scaramagli s., costa g., sabatini e., ziosi v., biondi s., torrigiani p., 2002 peach (prunus persica) fruit ripening: aminoethoxyvinylglycine (avg) and exogenous polyamines affect ethylene emission and flesh firmness. physiol. plant., 114(3): 472481. davarynejad g.h., zarei m., ardakani e., nasrabadi m.e., 2013 influence of putrescine application on 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ciriaci t., rasori a., biondi s., cost g., 2004 preharvest polyamine and aminoethoxy vinyl glycine (avg) applications modulate fruit ripening in stark red gold nectarines (prunus persica) postharvest biol. technol., 33(3): 293-308. valero d., martinez-romero d., serrano m., 2002 the role of polyamines in the improvement of the shelf life of fruit food sci. technol., 13(6-7): 228-234. valero d., serrano m., 2010 postharvest biology and technology for preserving fruit quality. crc press, taylor & francis group, boca raton, fl, usa, pp. 288. zhu x., shen l., fu d., si z., wu b., chen w., li x., 2015 effects of the combination treatment of 1-mcp and ethylene on the ripening of harvested banana fruit. postharvest biol. technol., 107: 23-32. zokaee khosroshahi m.r., esna-ashari m., 2008 effect of exogenous putrescine treatment on the quality and storage life of peach (prunus persica) fruit. postharvest biol. technol., 1(3): 278-287. 75 1. introduction grape phylloxera (daktulosphaira vitifoliae), aphidlike gall-forming parasite, is an economically important homopteran pest of grape vines vitis vinifera l. world wide. in syria, there are more than 70,000 ha planted with grapevine and the annual production is about 540,000 t. grape phylloxera annually causes millions of dollars in losses in grape production. grape phylloxera causes direct damage to grapevine by forming damaging root galls. the fleshy galls formed on immature roots, by swelling of the root cortex, are called “nodosities”, while on mature roots they are called “tuberosities”; these latter are considered more damaging to the vine. these galls are metabolically active organs suited to match the nutritional requirements of phylloxera and can support populations with high reproductive rates, making this pest capable of destroying the root system of v. vinifera vines (granett et al., 2001). in most cases the swelling stops rootlet growth, and the affected portion dies. root injuries reduce the vines’ ability to absorb nutrients and water, causing decline in vigor and productivity. weakened plants probably become more susceptible to secondary infections from fungal diseases and other insects, and to environmental stresses. since there is not an effective control method for grape phylloxera, sanitation and quarantine can be considered required procedures to prevent the spread of this insect pest. insecticides and hot water dips are used as quarantine treatments (granett et al., 2001). makee et al. (2010) proposed that gamma irradiation could be economically very useful in quarantine treatments against phylloxera. however, once phylloxera is in a vineyard, the use of resistant rootstocks is the most common and effective means of managing phylloxera. it should be mentioned that some rootstocks were more resistant than others to grape phylloxera (makee et al., 2003). moreover, for unknown reasons, the resistance of some rootstocks may break down and farmers must replant vineyards (granett et al., 1983; song and granett, 1990; de benedictis and granett, 1993). because replanting is costly in money, time, and labor, additional ways to control this pest should be considered. all plants have active defense mechanisms against pathogen attacks. some plant growth-promoting rhizobacteria (pgpr) are able to reduce disease through the stimulation of inducible plant defense mechanisms that render the host plant more resistant to further pathogen ingress. this induced systemic resistance (isr) (pieterse et al., 2002) can be the basis of integrated plant disease management strategies (ramamoorthy et al., 2001; zehnder et al., 2001; saravanakumar et al., 2007). many studies in plants on pgpr against pathogens have been performed. however, only a few of them determined the protective effect of pgpr against insects (zehnder et al., 1997 a, b; zehnder et al., 2001; kloepper et al., 2004; vijayasamundeeswari et al., 2009; valenzuela-soto et al., 2010). a non-pathogenic pseudomonas putida strain (btp1) was shown to enhance the level the influence of a non-pathogenic pseudomonas putida strain btp1 on reproduction and development of grape phylloxera a. adam(1), h. makee, i. idris department of biotechnology, atomic energy commission of syria. p.o. box 6091, damascus, syria. key words: developmental time, fecundity, grape phylloxera, oviposition period, pgpr, pseudomonas putida btp1. abstract: some non-pathogenic rhizobacteria called plant growth promoting rhizobacteria (pgpr) possess the capacity to induce defense mechanisms effective in plant against pathogens. the effect of pseudomonas putida btp1 on reproduction and development of phylloxera, which infested the roots of our local grape variety “balady”, was evaluated. our results showed that the life table of grape phylloxera was different between treated and control plants. the percentage of matured females, developmental time, fecundity and oviposition period were reduced when plants were treated with bacteria. the results showed that the phylloxera resistance was influenced by root soaking duration in p. putida btp1 suspension. the present study provides good information on the possibility of using pseudomonas putida btp1 to increase the resistance of grape to phylloxera. adv. hort. sci., 2012 26(2): 75-80 (1) corresponding author: ascientific@aec.org.sy received for publication 1 march 2012. accepted for publication 7 june 2012. 76 of resistance in cucumber, bean and tomato against the fungal pathogens pythium aphanidermatum and botrytis cinerea, respectively (ongena et al., 1999; adam et al., 2008). these studies revealed that the disease-protective effect was associated with stimulation of defense mechanisms in host plant (ongena et al., 2000, 2004; adam et al., 2008). the main objective of the present study was to evaluate the ability of strain p. putida btp1 to protect grape roots against grape phylloxera. thus, the effect of concentrations and root soaking duration in p. putida btp1 suspension on percentage of matured females, developmental time, fecundity and oviposition period of local phylloxera strain were determined. 2. materials and methods establishment of the phylloxera colony grape phylloxera was originally collected from fieldinfested roots of the local grape varieties in southern parts of syria. the phylloxera colony was established following similar procedures to those mentioned by makee et al. (2003). fresh and healthy pieces of roots (4-7 mm in diameter and 5-7 cm long) of local grape cultivar helwani (v. vinifera) were taken and washed with tap water. each piece was wrapped with moist cotton wool around one end, and then 10 to 15 phylloxera eggs were placed on each piece. the infested root pieces were then placed on a wet filter paper disk inside a plastic petri dish (12 cm diameter). each dish had three to four root pieces. for ventilation purposes the petri dish lid was modified with a 1-1.5 cm cloth-screened hole. the edges of the dishes were sealed with parafilm and they were kept in plastic boxes with tightly fitting lids and incubated at 25±1°c, 70±5% rh and 24 hr darkness. the root pieces were replaced when they desiccated, rotted or the phylloxera became crowded. microbial strains and inoculum preparation p. putida strain btp1, isolated from barley roots, was originally selected for its specific features regarding pyoverdine-mediated iron transport (jacques et al., 1995; ongena et al., 2002); it was maintained and prepared for use in the isr assays as previously described by ongena et al. (2002). for bioassays, two different concentrations (108 and 2x108 cfu/ml) of bacterial suspension were prepared. effect of bacteria-treated roots on phylloxera fresh root pieces were soaked for 3 hr in solutions with various p. putida concentrations: 0 (roots were soaked in distilled water as a control), 108, and 2x108 cfu/ml. all root pieces were then left to air-dry. for each concentration five root pieces were taken. each root piece was infested with 50 newly-laid phylloxera eggs (<24 hr old) and then all root pieces were kept at 25±1°c, 70±5% rh and 24 hr darkness. evaluation procedure a daily microscope inspection of all phylloxera stages on all root pieces was carried out. the number of eggs hatched, feeding nymphs and adults were detected to calculate the percentage of emerged mature females on each root piece at each concentration. also, the mean developmental time (egg to egg) was determined. fecundity (total number of eggs) of phylloxera was evaluated by randomly choosing five individuals of root-feeding phylloxera females on each root piece at each concentration. thus, at each tested concentration 20 females were examined. all eggs laid by each female were observed and counted till the female’s death. additionally, the oviposition period (the time from the first laid egg to the natural death of individual ovipositing females) was recorded for the females chosen for the fecundity measurement. effect of root soaking duration in bacterial suspension on phylloxera fresh root pieces were soaked in solutions with various p. putida concentrations: 0, 108, and 2x108 cfu/ml. at each tested concentration the root pieces were soaked for various periods: 0, 3, 5 and 15 hr. all root pieces were then left to air-dry. at each concentration and soaking duration, five root pieces were taken. each root piece, at each concentration and soaking duration, was infested with 50 newly-laid phylloxera eggs (<24 hr old) and then all root pieces were kept at 25±1°c, 70±5% rh and 24 hr darkness. the same evaluation procedure as described above was followed to determine the percentage of emerged mature females, mean developmental time, fecundity and the oviposition period of phylloxera. statistical analysis all statistical analyses were performed using statistic program version 6 (statsoft, inc. 2003) at 5% level (p = 0.05). data were subjected to analysis of variance (anova) for the determination of differences in means between tested plants at each dose. differences between means were tested for significance using tukey hsd test. 3. results effect of bacterial treated roots on phylloxera table 1 shows that when root pieces were treated with p. putida, the percentage of emerged matured females was significantly affected compared to the control (f=84.69; df=15, 64; p<0.05). however, the percentage emerged of matured females was not significantly increased by increasing p. putida concentrations. the result illustrates that the developmental time of phylloxera was significantly decreased by the application of p. putida (f= 15; df = 2, 42; p < 0.05). there was a significant reduction in developmental time as the concentration of p. putida was increased (table 1). there were significant differences in the mean number of laid eggs between all tested root pieces, regardless of concen77 tration (f = 19.9; df = 2, 42; p< 0.05). on untreated root pieces, the mean number of eggs was significantly higher than that on treated ones (table 1). when phylloxera females were reared on p. putida-treated root pieces, the mean number of eggs was markedly reduced by increasing p. putida concentration. table 1 shows that the oviposition period of phylloxera on untreated root pieces was significantly longer than that on treated ones, irrespective of the concentration of p. putida (f = 25.5; df = 2, 42; p < 0.05). there was a significant difference in the oviposition period between p. putida-treated root pieces. the oviposition period on 108 cfu/ml-treated root pieces was significantly longer than that on 2x108 cfu/ml-treated ones. effect of root soaking duration in bacterial suspension on phylloxera there was a significant effect of the soaking duration in p. putida suspension on the percentage emerged of matured females, regardless of the concentration (f= 620.87; df= 11, 24; p<0.05) (fig. 1). at each concentration, the percentage of emerged matured females on un-soaked root pieces was significantly higher than that on soaked ones, regardless of soaking duration. when the root pieces were soaked, the percentage of emerged matured females with 3 hr soaking duration significantly differed from that on 5 and 15 hr, whatever the p. putida concentration. at each p. putida concentration, there was no significant difference in the percentage of emerged matured females between 5 and 15 hr soaking duration (fig. 1). there was a significant effect of soaking in p. putida suspension on the mean developmental time, regardless of the soaking duration and concentration (f= 16; df= 11, 168; p< 0.05) (fig. 2). at each concentration, the mean developmental time on un-soaked root pieces was significantly higher than that on soaked ones, regardless of soaking duration. the result illustrates that at 2x108 cfu/ml the observed differences in mean development between soaking durations were not significant. there was a significant effect of the soaking duration in p. putida suspension on mean number of eggs, regardless of the concentration (f= 112; df = 11, 168; p<0.05) (fig. 3). at each concentration, mean number of eggs on soaked root pieces was significantly higher than that on un-soaked ones, regardless of soaking duration. at concentration 0 (root pieces soaked with distilled water), there was no significant difference in the mean number of eggs between 3 and 5 hr soaking duration. however, the mean number of eggs was significantly reduced with 15 hr soaking duration. at 108 and 2x108 cfu/ml concentrations, the mean number of eggs on 3 hr soaking duration was significantly higher than that on 5 and 15 hr (fig. 3). table 1 mean percentage of matured females, developmental time, fecundity and oviposition when phylloxera was reared on p.putida-treated grape root pieces oviposition period/d (mean±se) fecundity (mean±se) developmental time/d (mean±se) % matured female (mean±se) bacterial concentration cfu/ml 14.2±0.54 a 44.73±0.85 a 28.27±0.57 a 78.16±1.48 a 0 12.07±0.27 b 40.07±1.3 b 26.53±0.27 b 36.33±0.88 b 108 9.53±.53 c 34.2±1.3 c 24.53±0.54 c 32.03±1.49 b 2x108 means, in a column, followed by the same letter are not significantly different at the p < 0.05 (tukey hsd test). fig. 1 effect of root soaking duration in bacterial suspension of percentage emerged matured females of phylloxera. fig. 2 effect of root soaking duration in bacterial suspension on mean developmental time of phylloxera. fig. 3 effect of root soaking duration in bacterial suspension on mean number of eggs of phylloxera. 78 a significant effect of the soaking duration in p. putida suspension on mean oviposition period was found, regardless of the concentration (f= 46; df = 11, 168; p<0.05) (fig. 4). at each concentration, the mean oviposition period on un-soaked root pieces was significantly higher than that on soaked ones, regardless of soaking duration. at both 0 and 108 cfu/ml, the mean oviposition period with 3 hr soaking duration was significantly higher than that with 15 hr. at 0 concentration, there was no significant difference in the mean oviposition period between on 3 and 5 hr soaking duration, while at 108 cfu/ml there was. at 2x108 cfu/ml, there was no significant difference in the mean oviposition period between 3, 5 and 15 hr soaking duration. 4. discussion and conclusions several studies reported the use of rhizobacteria as a biological control of pests (racke and sikora, 1992; zehnder et al., 1997 a, b). to our knowledge, the application of rhizobacteria against phylloxera has not been investigated. our study indicates that when p. putida btp1-treated roots were infested by phylloxera eggs, the percentage of matured females was negatively influenced (table 1). the result illustrates that the percentage of matured females was significantly decreased when the concentration and the soaking duration were increased (fig. 1). the reduction of the number of matured females on the treated roots could be attributed to the inability of phylloxera to feed. thus, nymphs fed on p. putida btp1-treated roots showed antifeeding behaviour. when heliothis zeae (boddie) diet was contaminated with the bacterium pseudomonas maltophila, a 60% reduction in adult emergence and high pupal and adult malformations were observed (bong and sikorowski, 1991). moreover, when chestnut was treated with pseudomonas fluorescens, 20% chestnut weevil mortality was recorded (yaman et al., 1999). therefore, the antifeeding behaviour of phylloxera nymphs on the pgpr treatments could be related to the reduction in the feeding stimulant. consequently, the developmental time was altered leading to early emergence of matured females (table 1). a comparison between p. putida btp1-treated and untreated roots showed that the phylloxera development on treated roots was faster than that on untreated ones. similar results were reported when cucumber beetles and american bollworm were fed on pgpr-treated cucumber plants and cotton bolls, respectively (zehnder et al., 1997 a, b; vijayasamundeeswari, 2009). our current study indicates that the developmental time was significantly shorter on treated root pieces soaked for 15 hr (fig. 2). qingwen et al. (1988) detected a reduction of relative growth rate, consumption rate and digestibility of feed when heliothis armigera fed on p. gladioli-treated cotton plants. it is known that fecundity could be considered an essential factor in assessing the effect of p. putida btp1on phylloxera. phylloxera fecundity on p. putida btp1-treated root pieces was distinctly lower than that on untreated ones. when phylloxera females were reared on treated roots they were unable to produce a normal number of eggs compared to the control, especially at high concentration (table 1). nevertheless, when soaking duration in p. putida btp1 suspension was prolonged, a defective reproductive capacity was obtained (fig. 3). the average number of eggs was 65.3, 40, 32 and 21.6 eggs on 108 cfu/ml-treated roots pieces soaked for 0, 3, 5 and 15 hr, respectively. while on 2x108 cfu/ml-treated roots pieces soaked for 0, 3, 5 and 15 hr, the average number of eggs was 63.7, 34.2, 23.6 and 17.5 eggs, respectively. inadequate nutrition and inability to establish good feeding sites could directly affect the number of eggs laid. therefore, phylloxera resistance could be reflected in a strong relationship between poor feeding and the reduction of insect reproduction (granett et al., 1983). thus, phylloxera produced more eggs on untreated roots compared with treated ones. correspondingly, the oviposition period of phylloxera on treated roots was markedly shorter than that on untreated ones (table 1). when the concentration of p. putida btp1 was increased a great reduction in the oviposition period was obtained. such reduction was noticeably increased by prolonging the root soaking duration in p. putida btp1 (fig. 4). it is known that the resistance mechanisms of phylloxera could be related to several factors including: 1) reduction of phylloxera fitness (antibiosis); 2) decrease in plant attractiveness to phylloxera (antixenosis) (granett et al., 2001). zehnder et al. (2001) mentioned that the pgpr treatment led to alteration in the plant metabolic pathway which elicited the induction of plant defense compounds. qingwen et al. (1998) reported that polyphenol and terpenoid content were increased with cotton treated with p. gladioli. the synthesis and formation of such materials in p. putida btp1-treated roots would have a negative influence on phylloxera feeding and development. the results illustrate that the soaking of grape roots with p. putida btp1 suspension for 5 hr and 15 hr, regardless of concentration, led to a great reduction in matured females, developmental time, fecundity and oviposition fig. 4 effect of root soaking duration in bacterial suspension on mean oviposition period of phylloxera. 79 period. such reduction might be attributed to increased bacterial cell concentration by prolonging the soaking duration. therefore, roots pieces soaked with 2x108 cfu/ml for 15 hr showed more resistance to phylloxera than lower concentrations and a shorter soaking duration. these results provide essential information about the relationship between p. putida btp1 and phylloxera resistance of grape plants. 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yaman m., demirbag z., belduz a.o., 1999 investigations on the bacterial flora as a potential biocontol agent of chestnut weevil, curculio elephas (coleoptera: curculionidae) in turkey. afr. j. mycol biotechnol., 54: 625-630. zehnder g., kloepper j., tuzun s., yao c., wei g., chambliss o., shelby r., 1997b insect feeding on cucumber mediated by rhizobacteria-induced plant resistance. entomologia experimentalis et applicata, 83: 81-85. zehnder g., kloepper j., yao c., wei g., 1997 a induction of systemic resistance in cucumber against cucumber beetles (coleoptera: chrysomelidae) by plant growth-promoting rhizobacteria. journal of economic entomology, 90: 391-396. zehnder g.w., murphy j.f., sikora e.j., kloepper j.w., 2001 application of rhizobacteria for induced resistance. eur. j. plant pathol., 107: 39-50. impaginato special issue on plant and food metabolomic this special issue has been realized with the contribution of the regione toscana “praf 2012-2015 misura 1.2 e)” program, “profumi e sapori della toscana: dalla caratterizzazione del volatoma alla valorizzazione dei prodotti alimentari di pregio (volatosca)” project. foreword throughout the nineteenth century and for much of the twentieth century, the definition of secondary metabolites simply meant that these substances, while present in plants, did not participate in the metabolic processes essential to the life of the organism. they were defined as by-products of primary metabolism or as excretory products or final products of metabolism, always pointing out that their irregular presence on plants meant that they were not indispensable. over the last 50 years, knowledge in this area has been greatly expanded, especially following the development of new isolation, separation and structural identification technologies (spectroscopy, nuclear magnetic resonance and mass spectrometry). the ease of structural identification has expanded the number of secondary metabolites to reach the unthinkable number of 200000 for many of which, however, remain uncertainty about their biochemical and physiological role. in recent years, however, many researches have progressively clarified the role of these substances and some of them are known to have a fundamental function. thus it has been for shikimic acid, for many years considered a simple metabolite of illicium anisatum, then revealed as a key molecule for synthesis of aromatic amino acids and, in turn, precursors of phenylpropanoids (flavonoids, coumarin, tannins and lignin). certainly these are not secondary functions! primary metabolism is essential to plants for growth and development, and secondary metabolism helps plants to interact with the environment. many plant metabolites are also industrially important (e.g.: the physiologically active alkaloids used in modern medicine). these metabolites are produced by plants through complex metabolic pathways. among the researchers in this area it is inevitable to remember j.b. harborne, who has been devoting some 50 years to the study of flavonoids and, more generally, to chemical ecology and plant chemosystematics. harborne’s work has called for experimentation on all other classes of secondary metabolites, alkaloids, nonprotein amino acids, glucosinolates, lectins, terpenes, steroids, tannins, flavonoids, phenylpropanoids, lignins, coumarin, waxes, etc. plant secondary metabolites are critical to various biological processes. the plant’s specific organization has led it to produce a large amount of secondary metabolites and probably selective pressure has left those molecules capable of conferring specific benefits to the plant: defense role against animal, fungal and bacterial parasites, but also those molecules that allow to establish relationships between plants of the same species or between different species. numerous contributions to this special number of ahs are devoted to ecological biochemistry and that explains the “non-secondary” role of so many metabolites; it is also hoped that the molecular biological tools will spread to the labs in order to have accurate information on the genes involved in the various secondary metabolism. plant genomes are variously estimated to contain 20000-60000 genes, and perhaps 15-25% of these genes encode enzymes for secondary metabolism. still a lot of research work has to be done both physiologically and biochemically on the secondary metabolism of many species, but i believe it is essential that laboratories use data availability about the genes of arabidopsis, an extraordinary model species, involved in secondary metabolism. thus, our knowledge of secondary metabolites, which have already made great advances over the last decades, will make further progress in both physiological and biochemical and technological terms, confirming that their traditional placement as “secondary metabolism products” was only due to lack of knowledge. amedeo alpi impaginato 249 abbaszade k. effect of preand postharvest salicylic acid treatment on quality characteristics of tomato during cold storage, 183 abdely c. effect of h2o2 pretreatment on the response of two seashore paspalum (paspalum vaginatum sw.) cultivars (salam and seaspray) to cold stress, 103 adam a. induced resistance in potato plants by a nonpathogenic pseudomonas putida btp1 against potato tuber moth (phthorimaea operculella zeller), 47 adamipour n. morpho-physiological alteration in common bermudagrass [cynodon dactylon (l.) pers.] subjected to limited irrigation and light condition, 141 ahkbari a. introduction of determination of optimum harvest date in afghanistan. sweet cherry: a case study, 231 akbari n. salicylic acid at different plant growth stages affects secondary metabolites and phisico-chemical parameters of greenhouse tomato, 151 a h m a d a . s e l e c t e d p o m e g r a n a t e g e r m p l a s m f r o m afghanistan: morphological variability and relationship among collected accessions, 225 a h m a d j. the phytosanitary status of the national collection of fruits and nuts of afghanistan and the private mother stock nurseries: a virus survey, 239 a m a d a . s e l e c t e d p o m e g r a n a t e g e r m p l a s m f r o m afghanistan: morphological variability and relationship among collected accessions, 225 alizadeh m. the usefulness of apricot gum as an organic additive in grapevine tissue culture media, 111 al-safadi b. ultrastructural changes in potato (solanum tuberosum) under nacl mediated salinity stress in vitro, 95 arbaoui m. effect of h2o2 pretreatment on the response of two seashore paspalum (paspalum vaginatum sw.) cultivars (salam and seaspray) to cold stress, 103 arena m. characterization and evaluation of berberis microphylla g. forst pollen grains, 31 aslmoshtaghi e. analysis of the effects of glomus etunicatum fungi and pseudomonas fluorescence bacteria symbiosis on some morphological and physiological characteristics of mexican lime (citrus aurantifolia l.) under drought stress conditions, 39 azizi f. introduction of determination of optimum harvest date in afghanistan. sweet cherry: a case study, 231 bagheri f. some characteristics of tuberose as affected by pre-harvest application of calcium chloride and gibberellic acid, 69 bahadoran m. some characteristics of tuberose as affected by pre-harvest application of calcium chloride and gibberellic acid, 69 baninaiem e. effect of preand postharvest salicylic acid treatment on quality characteristics of tomato during cold storage, 183 b e n h a m e d k . e f f e c t o f h 2o 2 p r e t r e a t m e n t o n t h e response of two seashore paspalum (paspalum vaginatum sw.) cultivars (salam and seaspray) to cold stress, 103 berti m. phenotypic characterisation of almond accessions collected in afghanistan, 207 b e r t i m . s e l e c t e d p o m e g r a n a t e g e r m p l a s m f r o m afghanistan: morphological variability and relationship among collected accessions, 225 bettaieb t. effect of h2o2 pretreatment on the response of two seashore paspalum (paspalum vaginatum sw.) cultivars (salam and seaspray) to cold stress, 103 caboni e. comparative characterization of fruit quality, phenols and antioxidant activity of de-pigmented “ghiaccio” and white flesh peaches, 175 carbone k. comparative characterization of fruit quality, phenols and antioxidant activity of de-pigmented “ghiaccio” and white flesh peaches, 175 ceccarelli d. comparative characterization of fruit quality, phenols and antioxidant activity of de-pigmented “ghiaccio” and white flesh peaches, 175 chand r.r. bioactivity of selected essential oils from medicinal plants found in fiji against the spiralling whiteflies (aleurodius dispersus russell), 165 chiechi a. photo-oxidation of ethylene over mesoporous tio2/sio2 catalysts, 75 ciappellano s. vegetable production using a simplified hydroponics system inside city of dead (cairo), 23 comparini d. optical evaluation of the shading properties of climbing fern lygodium japonicum used as a thermal buffering green wall plant, 67 creti p. photo-oxidation of ethylene over mesoporous tio2/sio2 catalysts, 75 cullen g.j. implications of investigating pollination and c r o s s c o m p a t i b i l i t y i n t h e a l m o n d v a r i e t i e s o f afghanistan, 217 das k. influence of organics, inorganic and biofertilizers o n g r o w t h , f r u i t q u a l i t y , a n d s o i l c h a r a c t e r s o f himsagar mango grown in new alluvial zone of west bengal, india, 81 dutta p. influence of organics, inorganic and biofertilizers o n g r o w t h , f r u i t q u a l i t y , a n d s o i l c h a r a c t e r s o f himsagar mango grown in new alluvial zone of west bengal, india, 81 elbey n. effect of h2o2 pretreatment on the response of index volume 30, 2016 author index 250 two seashore paspalum (paspalum vaginatum sw.) cultivars (salam and seaspray) to cold stress, 103 enayat n. introduction of determination of optimum harvest date in afghanistan. sweet cherry: a case study, 231 esmaeili s. kentucky bluegrass (poa pratensis l.) silicontreated turfgrass tolerance to shortand long-term salinity condition, 87 fakhar z. pre-storage putrescine treatment maintains quality and prolongs postharvest life of musa acuminata l., 159 ferrante a. vegetable production using a simplified hydroponics system inside city of dead (cairo), 23 g h o u s m . s el ec t ed p o megra n a t e germp l a s m f ro m afghanistan: morphological variability and relationship among collected accessions, 225 giordani e. from traditional orchards to advanced fruitculture: establishing the bases of commercial horticulture in afghanistan, 197 giordani e. phenotypic characterisation of almond accessions collected in afghanistan, 207 gi o r d ani e. selected pomegranate germplasm from afghanistan: morphological variability and relationship among collected accessions, 225 giro a. vegetable production using a simplified hydroponics system inside city of dead (cairo), 23 gopalan r.d. bioactivity of selected essential oils from medicinal plants found in fiji against the spiralling whiteflies (aleurodius dispersus russell), 165 hara a. optical evaluation of the shading properties of climbing fern lygodium japonicum used as a thermal buffering green wall plant, 59 hara y. optical evaluation of the shading properties of climbing fern lygodium japonicum used as a thermal buffering green wall plant, 59 hosseini m.s. pre-storage putrescine treatment maintains quality and prolongs postharvest life of musa acuminata l., 159 houssian k. induced resistance in potato plants by a nonpathogenic pseudomonas putida btp1 against potato tuber moth (phthorimaea operculella zeller), 47 ibrahim m. arbuscular mycorrhizal isolate and phosphogypsum effects on growth and nutrients acquisition of cotton (gossypium hirsutum l.), 121 idris i. induced resistance in potato plants by a non-pathogenic pseudomonas putida btp1 against potato tuber moth (phthorimaea operculella zeller), 47 iwase j. optical evaluation of the shading properties of climbing fern lygodium japonicum used as a thermal buffering green wall plant, 59 javanmardi j. salicylic acid at different plant growth stages affects secondary metabolites and phisico-chemical parameters of greenhouse tomato, 151 jokhan a.d. bioactivity of selected essential oils from medicinal plants found in fiji against the spiralling, whiteflies (aleurodius dispersus russell), 165 kawano t. optical evaluation of the shading properties of climbing fern lygodium japonicum used as a thermal buffering green wall plant, 59 khalil n. induced resistance in potato plants by a nonpathogenic pseudomonas putida btp1 against potato tuber moth (phthorimaea operculella zeller), 47 khorsha s. the usefulness of apricot gum as an organic additive in grapevine tissue culture media, 111 khosh-khui m. morpho-physiological alteration in common bermudagrass [cynodon dactylon (l.) pers.] subjected to limited irrigation and light condition, 141 laera a.m. photo-oxidation of ethylene over mesoporous tio2/sio2 catalysts, 75 lanzoni c. the phytosanitary status of the national collection of fruits and nuts of afghanistan and the private mother stock nurseries: a virus survey, 239 licciulli a. photo-oxidation of ethylene over mesoporous tio2/sio2 catalysts, 75 luvisi a. insight on trans-plasma membrane behavior of virus-infected plant cells, 135 luvisi a. occurrence of viruses in calla and peruvian lily in tuscan nurseries and evidence of new viral records in italy, 53 mashayekhi k. the usefulness of apricot gum as an organic additive in grapevine tissue culture media, 111 masini g. from traditional orchards to advanced fruitculture: establishing the bases of commercial horticulture in afghanistan, 197 materazzi a. occurrence of viruses in calla and peruvian lily in tuscan nurseries and evidence of new viral records in italy, 53 mirzaaliandastjerdi a. effect of preand postharvest salicylic acid treatment on quality characteristics of tomato during cold storage, 183 mortazavi s.n. some characteristics of tuberose as affected by pre-harvest application of calcium chloride and gibberellic acid, 69 murata y. optical evaluation of the shading properties of climbing fern lygodium japonicum used as a thermal buffering green wall plant, 59 nakar m. ultrastructural changes in potato (solanum tuberosum) under nacl mediated salinity stress in vitro, 95 nisi r. photo-oxidation of ethylene over mesoporous tio2/sio2 catalysts, 75 pal s. photo-oxidation of ethylene over mesoporous tio2/sio2 catalysts, 75 panattoni a. occurrence of viruses in calla and peruvian lily in tuscan nurseries and evidence of new viral records in italy, 53 patel a. influence of organics, inorganic and biofertilizers o n g r o w t h , f r u i t q u a l i t y , a n d s o i l c h a r a c t e r s o f himsagar mango grown in new alluvial zone of west bengal, india, 81 radice s. characterization and evaluation of berberis microphylla g. forst pollen grains, 31 rastegar s. effect of preand postharvest salicylic acid treatment on quality characteristics of tomato during 251 cold storage, 183 r a t t i c . t h e p h y t o s a n i t a r y s t a t u s o f t h e n a t i o n a l collection of fruits and nuts of afghanistan and the private mother stock nurseries: a virus survey, 239 refahi a. analysis of the effects of glomus etunicatum fungi and pseudomonas fluorescence bacteria symbiosis on some morphological and physiological characteristics of mexican lime (citrus aurantifolia l.) under drought stress conditions, 39 rehman s. the phytosanitary status of the national collection of fruits and nuts of afghanistan and the private mother stock nurseries: a virus survey, 239 rezgui s. effect of h2o2 pretreatment on the response of two seashore paspalum (paspalum vaginatum sw.) cultivars (salam and seaspray) to cold stress, 103 rinaldelli e. insight on trans-plasma membrane behavior of virus-infected plant cells, 135 rizzo d. occurrence of viruses in calla and peruvian lily in tuscan nurseries and evidence of new viral records in italy, 53 rubies autonell c. the phytosanitary status of the national collection of fruits and nuts of afghanistan and the private mother stock nurseries: a virus survey, 239 s a e e d i a . s e l e c t e d p o m e g r a n a t e g e r m p l a s m f r o m afghanistan: morphological variability and relationship among collected accessions, 225 sakata r. optical evaluation of the shading properties of climbing fern lygodium japonicum used as a thermal buffering green wall plant, 59 saleh b. dna changes in cotton (gossypium hirsutum l.) under salt stress as revealed by rapd marker, 13 salehi h. kentucky bluegrass (poa pratensis l.) silicontreated turfgrass tolerance to shortand long-term salinity condition, 87 salehi h. morpho-physiological alteration in common bermudagrass [cynodon dactylon (l.) pers.] subjected to limited irrigation and light condition, 141 samadi g.r. implications of investigating pollination and c r o s s c o m p a t i b i l i t y i n t h e a l m o n d v a r i e t i e s o f afghanistan, 217 sartori a. comparative characterization of fruit quality, phenols and antioxidant activity of de-pigmented “ghiaccio” and white flesh peaches, 175 shahcheraghi s.t. micropropagation of three endemic and endangered fig (ficus carica l.) genotypes, 129 shahsavar a.r. analysis of the effects of glomus etunicatum fungi and pseudomonas fluorescence bacteria symbiosis on some morphological and physiological characteristics of mexican lime (citrus aurantifolia l.) under drought stress conditions, 39 shekafandeh a. micropropagation of three endemic and endangered fig (ficus carica l.) genotypes, 129 sivakumar r. impact of drought on flowering, yield and quality parameters in diverse genotypes of tomato (solanum lycopersicum l.), 3 soufi s. effect of h2o2 pretreatment on the response of two seashore paspalum (paspalum vaginatum sw.) cultivars (salam and seaspray) to cold stress, 103 srividhya s. impact of drought on flowering, yield and quality parameters in diverse genotypes of tomato (solanum lycopersicum l.), 3 st a n i k z a i s. selected pomegranate germplasm from afghanistan: morphological variability and relationship among collected accessions, 225 stefani l. occurrence of viruses in calla and peruvian lily in tuscan nurseries and evidence of new viral records in italy, 53 suzuki t. optical evaluation of the shading properties of climbing fern lygodium japonicum used as a thermal buffering green wall plant, 59 takaichi h. optical evaluation of the shading properties of climbing fern lygodium japonicum used as a thermal buffering green wall plant, 59 talento c. comparative characterization of fruit quality, phenols and antioxidant activity of de-pigmented “ghiaccio” and white flesh peaches, 175 terlizzi m. comparative characterization of fruit quality, phenols and antioxidant activity of de-pigmented “ghiaccio” and white flesh peaches, 175 valori f. introduction of determination of optimum harvest date in afghanistan. sweet cherry: a case study, 231 wali adel m. introduction of determination of optimum harvest date in afghanistan. sweet cherry: a case study, 231 yaqubi m.r. implications of investigating pollination and c r o s s c o m p a t i b i l i t y i n t h e a l m o n d v a r i e t i e s o f afghanistan, 217 yaqubi m.r. introduction of determination of optimum harvest date in afghanistan. sweet cherry: a case study, 231 yaqubi m.r. phenotypic characterisation of almond accessions collected in afghanistan, 207 yaqubi m.r. selected pomegranate germplasm from afghanistan: morphological variability and relationship among collected accessions, 225 z a d r a n b . s e l e c t e d p o m e g r a n a t e g e r m p l a s m f r o m afghanistan: morphological variability and relationship among collected accessions, 225 zahedi s.m. pre-storage putrescine treatment maintains quality and prolongs postharvest life of musa acuminata l., 159 zarei m. analysis of the effects of glomus etunicatum fungi and pseudomonas fluorescence bacteria symbiosis on some morphological and physiological characteristics of mexican lime (citrus aurantifolia l.) under drought stress conditions, 39 zarghon a.h. implications of investigating pollination and c r o s s c o m p a t i b i l i t y i n t h e a l m o n d v a r i e t i e s o f afghanistan, 217 35 1. introduction pepino (solanum muricatum ait.), a little-known herbaceous subshrub solanaceous plant, is native of the tropical and subtropical andes in south america. it is cultivated for its edible juicy, scented and sweet fruits (prohens et al., 1996) and has been introduced to different countries, becoming a specialty fruit (ahumada and cantwell, 1996). today, pepino is a species of increasing economic interest and it has considerable potential for future exploitation (prohens et al., 1996). the cultural techniques used in modern tomato growing have been adapted with slight modification to pepino management (dennis et al., 1985). symbiotic associations between arbuscular mycorrhizal fungi (amf) and plant roots in the natural environment provide a range of benefits to the host plant, however many conventional agricultural practices are detrimental to amf (gosling et al., 2006). organic farming systems may be less detrimental to amf, because they exclude the use of chemical fertilizers and most biocides and generally have diverse rotations. organic matter influences the nutrient profile, soil structure, water holding capacity and ph, all of which directly and or indirectly influence amf development (bagyaraj, 1991). addition of organic amendments to soil has been reported to enhance plant biomass, mycorrhizal infectivity, and proliferation of am fungal hyphae in soil (joner and jakobsen, 1995; dai et al., 2011). vermicompost as an organic fertilizer provides some essential nutrients for supporting plant growth compared with chemical fertilizers. to the best of our knowledge, there is no published report about the association of pepino with amf in literature. the objectives of the present study were to determine: 1) the association level of two amf species with pepino plant; 2) the possible beneficial effects of symbiosis on plant physiology and fruit quality; and 3) the benefits of vermicompost on amf association with pepino. 2. materials and methods pepino (cv. kanseola) mother plants were obtained from mashad ferdowsi university research station, iran. cuttings with an average 20 cm length and four buds were rooted in a 1:1 (v:v) peat and sand mixture for four weeks. general cultural practices were according to lopez et al. (2000). influence of arbuscular mycorrhizal fungi on physiology and fruit quality of pepino (solanum muricatum ait.) in vermicompost amended medium j. javanmardi* (1), m. zarei**, m. saei* * department of horticulture, college of agriculture, shiraz university, shiraz, iran. ** department of soil sciences, college of agriculture, shiraz university, shiraz, iran. key words: fruit quality characteristics, mycorrhizal symbiosis, organic fertilizer, pepino. abstract: the association level of pepino (solanum muricatum ait.) with two arbuscular mycorrhizal fungi (amf) species (glomus etunicatum and g. versiforme) was evaluated for the first time. the first part of experiment showed 30 and 50% root colonization for the two amf species, respectively, while the second part of study was a pot experiment under greenhouse conditions. the effects of vermicompost and root inoculation with g. etunicatum and g. versiforme on reproductive stage, yield and fruit quality of pepino were investigated. treatments included two levels of vermicompost (0 and 20% v/v) and inoculation with the two fungi species along with a non inoculated control. application of vermicompost increased the number of flowers, fruits and fruit weight, but decreased the number of days from plant setting to first flower and fruit set, fruit dry matter percent, fruit titratable acidity and vitamin c content. inoculation with g. versiforme increased fruit dry matter percent, fruit titratable acidity and fruit vitamin c content compared with the non inoculated control (nic) plants. plants inoculated with g. etunicatum showed greater fruit weight and juice ph compared to nic plants. amf inoculation in vermicompost amended pots led to 14 and 10 days earlier flowering for g. versiforme and g. etunicatum, respectively compared to those not amended with vermicompost. g. etunicatum in vermicompost supplemented medium hastened fruit set by 5.5 days compared to those without vermicompost application. fruit quality characteristics were affected differently for the two amf-inoculated plants in presence of vermicompost. adv. hort. sci., 2014 28(1): 35-42 (1) corresponding author: javanm@shirazu.ac.ir received for publication 12 april 2014 accepted for publication 30 may 2014 36 amf inocula amf spores were obtained from the department of soil science, faculty of agriculture, shiraz university, iran. glomus versiforme was isolated from a non-contaminated area of anguran mine, zanjan, iran (zarei et al., 2008) and g. etunicatum was provided from tabriz university, iran; these fungi are abundant in iranian soils (aliasgharzadeh et al., 2001; kariman et al., 2005). mycorrhizal inocula were prepared through the trap culture of maize (zea mays l.) on culture medium composed of autoclaved soil/quartz-sand (<1 mm) (1:4, v/v). simultaneously, some pots containing non autoclaved soil were kept without any spore inoculation to preserve the naturally-occurring microbial association and used for non-inoculated control (nic) treatments. after four and a half months, at the beginning of the maize reproductive period, shoots were removed and the contents of pots (mycorrhizal roots plus soil possessing fungal spores and mycelia) were maintained in polyethylene bags at 4°c. the potential of inoculants (spore numbers of 10-12 g-1 substrate and root colonization of 80-85%) for spore extraction, number, and evaluation of root colonization were measured based on the method described by zarei et al. (2008). examination of arbuscular mycorrhizal symbiosis the association level of pepino plant with two amf species along with a nic was evaluated in a completely randomized design with three replications. the presence of am propagules and the percentage of root colonization was determined after eight weeks of inoculation. the grid-line intersect method was used after cleaning washed roots in 10% koh and staining with 0.01% fuchsin acid in lactoglycerol according to the method described by kormanik and mcgraw (1982). the amf species colonizing roots of pepino plants were used for the main experiment. main experiment the experiment was carried out in a polyethylene greenhouse using 10 l pots. the experimental design was 2×3 factorial with four replications (four plants each) in a completely randomized arrangement. the first factor consisted of control (v 0 : no vermicompost) and v 1 : vermicompost application at 20% (v/v) to soil. the second factor was soil inoculation with g. etunicatum and g. versiforme. non-inoculated pots were considered as control (nic). four-week-old rooted cuttings were planted in pots which contained the described media for treatments. the physical and chemical properties of soil and vermicompost are presented in table 1. for the mycorrhizal treatments 50 g of each amf inoculum was used as a thin layer near the roots of cuttings. nic treatments consisted of adding 50 g of media from control maize trap culture pots (contained nonautoclaved soil with no spore inoculation) as described earlier (see section 2.1). pots containing pepino plants were arranged in rows, 1 m apart and 0.4 m between pots. plants were trained into three main branches. a total amount of 35 g·m-2 of organic soluble fertilizer biomin464-sp (jh biotech, inc. ventura, ca) was applied to fertigated plants during the experimental period. irrigation frequency from transplanting to harvest varied from 2 to 4 days with 0.4 1 l per irrigation based on plant requirements. plant reproductive phase measurements to evaluate the effects of colonization with amf and vermicompost application on the pepino reproductive stage, the following characteristics were assayed: number of days to first flower formation, number of flowers in the first and second truss, number of days from transplanting to first fruit formation, fruit number and fruit set percentage, and fruit fresh weight. fruit quality factors including vitamin c content at maturity [using the method described by association of official agricultural chemists (aoac, 1984)], titratable acidity (gutiérrez-miceli et al., 2007), fruit juice acidity (using ph meter), soluble solid content (using refractometer), and dry matter percent were assayed after harvest. statistical analyses the experiment was arranged in a completely randomized design. four replicates per treatment were used, each with four plants. data were analyzed using jmp statistical software, version 5.1 (sas institute inc., cary, nc, usa). if the interaction was significant, it was used to explain results; if it was not significant, means were separated with least significant differences (lsd) test at p≤0.05. 3. results and discussions the anova revealed significant main and interaction effects of vermicompost and amf for most measured characteristics (table 2). determination of plant association with amf for the first time in literature, our results report the association of pepino with two amf. the results indicate that g. etunicatum and g. versiforme can colonize pepino table 1 physical and chemical properties of soil and vermicompost used for the experiment sand (%) silt (%) clay (%) ec (ds/m) ph n (%) p k soil 34 46 20 1.63 7.82 0.031 5.4 mg/kg 135 mg/kg vermicompost 5 8.25 1.45 1.75% 1.2% 37 roots up to 30 and 50 percent, respectively. it has been stated that the difference between root colonization percentages of glomus strains might be due to the fact that amf have a wide host range, yet certain combinations of hosts and fungi are more efficient than others for either the fungus or the host (douds and millner, 1999; gutierrezmiceli et al., 2008). van der heijden et al. (1998) showed that plant species differed in their dependency on amf. some results suggest that amf has some degree of hostspecificity (eom et al., 2000). root colonization the main and interaction effects of vermicompost and amf on root colonization percent were significant (table 2). root colonization percentages in vermicompost amended soils were about 25% greater than the v 0 treatment regardless of amf (table 3). the greater percentage of mycorrhization in vermicompost amended soils has been attributed to the humic substances found in vermicompost, resulting in an increased metabolism of soil microorganisms, and the nutrient uptake (atiyeh et al., 2002). there was no significant difference between colonized root length of g. versiforme and g. etunicatum in vermicompost amended soils, but compared to nic plants, a greater root colonization was observed (2.46 and 2.80 times, respectively) (table 4). mycorrhizal plants colonized well with introduced amf species. a 15% mean root colonization in nic plants shows that the soil used contained native amf populations. more than 100% greater root colonization was observed in the presence of vermicompost when compared with v 0 treatment (table 5). this could be due to greater organic matter available for growth and development of amf hyphae; it has been reported that amf mycelia can mineralize and enhance utilization of organic materials (feng et al., 2003). days to first flower formation the main and interaction effects of vermicompost and amf on the number of days to first flower formation were significant (table 2). the number of days from planting to first flower formation was much lower in pots amended with vermicompost than in v 0 treatments (table 3). previous studies showing earlier flowering due to vermicompost application on german chamomile, begonia, and coleus (tomati et al., 1983; tomati et al., 1987; azizi et al., 2008) are in agreement with our results. the reason for this has been attributed to the development of efficient photosynthetic structure, higher dry matter production, early initiation, and greater development of the reproductive system (krishna et al., 2008). inoculation with g. etunicatum gave earlier flowering than g. versiforme-inoculated plants (table 4). in agreement with our results, chrysanthemum cuttings inoculated with amf had a significantly shorter flowering time compared with non-inoculated plants (sohn et al., 2003). it is reported that in amf-inoculated tomato plants, the time between emergence and completion of fruit set (the duration of purely vegetative growth) decreased, while the duration of the reproduction period increased (bryla and koide, 1998). this is consistent with the idea that plant resource status serves as a partial control of the switch table 2 analyses of variance for vermicompost application and arbuscular mycorrhizal fungi inoculation on some pepino plant characteristics source of variation df mean squares root colonization days to first flower flower number in truss days from flowering to fruit set fruit number fruit fresh weight fruit dry matter percent fruit juice ph fruit titratable acidity total soluble solids fruit vitamin c content vermicompost 1 67** 1056.25** 28.44** 42.25** 27.39** 2321.47** 14.06** 0.06 ns 0.15** 0.13 ns 264.23** amf 2 26** 44.62** 0.16 ns 45.42** 4.64 ns 513.87* 90.97** 0.08** 0.21** 2.98 ns 165.20** vermicompost × amf 2 12* 32.92 ** 0.75 ns 28.15** 4.99 ns 18.16 ns 28.72** 0.11** 0.12** 5.92* 147.53** error 30 0.004 5.08 0.87 1.58 3.46 130.21 0.58 0.02 0.01 1.00 1.56 total 35 ns, *, *= non-significant, significant at 0.05 and 0.01, respectively. table 3 the main effects of vermicompost application on some pepino plant characteristics treatment root colonization percent days to first flower flower number in truss days from flowering to fruit set fruit number fruit weight (g) fruit dry matter percent fruit titratable acidity (ml/100 ml) fruit vitamin c (mg/100 ml) control 18 b 74.11 a 7.36 b 16.14 a 1.81 b 29.69 b 11.10 a 0.78 a 19.83 a vermicompost added 45 a 63.28 b 9.14 a 13.97 b 3.56 a 45.75 a 9.85 b 0.65 b 14.41 b lsd value at p≤0.05 0.04 1.53 0.63 0.70 1.07 7.77 0.52 0.07 0.85 values in each column with the same letter are not significantly different using lsd test at p≤0.05. 38 from vegetative to reproductive growth (marschner, 1995). in mycorrhizal plants, greater root development leads to more phosphorus in vegetative and reproductive tissues, which eventually leads to early flowering (bryla and koide, 1998). this might explain the early flowering in this experiment due to inoculation with g. etunicatum. plants inoculated with g. versiforme and g. etunicatum in vermicompost amended pots had 15 and 10 days earlier flowering, respectively, compared to non vermicompost amended pots (table 5). the organic material provided by vermicompost can improve growth and development of amf inoculum (bending et al., 2004), which enhances nutrient uptake by the plant and hastens plant growth and development (mahmood and rizvi, 2010). flower number in truss a 24% increase in the number of flowers per truss was observed in vermicompost amended pots compared with v 0 treatments, however the effects of amf and coapplication of amf and vermicompost were not significant (tables 2, 3). previously, a 40% increase in flower number in strawberry was related to the increase in plant biological activity due to a vermicompost application rate of over 10 t/ha (arancon et al., 2004 b). a 20% (v/v) vermicompost application in the present study led to a very significant increase in flower number in trusses (table 2). some possible factors that improve flowering after vermicompost application have been attributed to the improvement in physical structure of growth medium, increased biological enzymatic activities, increased populations of beneficial microorganisms, or the presence of biologically active plant growth-influencing substances (plant growth regulators) in the vermicompost (arancon et al., 2008). our results on increased flower number due to vermicompost are in agreement with previous reports on eggplant and tomato (gajalakshmi and abbasi, 2002). days to first fruit set the main and interaction effects of vermicompost and amf on the number of days from flowering to first fruit set were significant (table 2). vermicompost amended pots showed fruit set occurring an average of 2.17 days earlier compared with v 0 treatments (table 3). non-inoculated control plants and plants inoculated with g. versiforme showed fruit set to be three days earlier than in plants inoculated with g. etunicatum (table 4). the two amf species showed different interactions with regard to the presence of vermicompost in the medium. those inoculated with g. etunicatum in the presence of vermicompost set fruit 5.58 days earlier compared to non vermicompost amended soil (table 5). it seems that the potential efficiency of g. etunicatum for earlier pepino fruit set is greater in the presence of vermicompost than with g. versiforme. nic plants and plants inoculated with g. versiforme with vermicompost added and non vermicompost added media showed no differences. fruit number our results show that vermicompost application was the primary contributing factor in increasing pepino fruit table 4 the main effects of arbuscular mycorrhizal fungi inoculation on some pepino plant characteristics treatment root colonization percent days to first flower days from flowering to fruit set fruit weight (g) fruit dry matter percent fruit juice ph fruit titratable acidity (ml/100 ml) fruit vitamin c (mg/100 ml) nic 15 b 68.08 ab 13.75 b 32.64 b 8.79 b 5.02 b 0.67 b 16.54 b glomus versiforme 37 a 70.85 a 14.12 b 35.41 b 13.65 a 4.99 b 0.87 a 21.08 a glomus etunicatum 42 a 67.15 b 17.29 a 45.10 a 8.98 b 5.15 a 0.62 b 13.73 c lsd value at p≤0.05 5.01 1.88 0.86 9.51 0.63 0.12 0.08 1.04 nic= non inoculated control. values in each column with the same letter are not significantly different using lsd test at p≤0.05. table 5 the interaction effects of vermicompost application and arbuscular mycorrhizal fungi inoculation on some pepino plant characteristics root colonization percent days to first flower days from flowering to fruit set fruit dry matter percent fruit juice ph fruit titratable acidity (ml/100 ml) total soluble solids (brix) vitamin c (mg/100 ml) no-vermicompost added non mycorrhizal control 5 c 72.17 b 13.58 b 8.06 d 5.09 ab 0.68 b 7.13 c 22.39 b glomus versiforme 22 b 78.12 a 14.75 b 15.96 a 4.88 c 1.04 a 9.38 a 24.42 a glomus etunicatum 27 b 72.04 b 20.08 a 9.28 c 5.06 ab 0.61 b 7.68 bc 12.66 e vermicompost added non mycorrhizal control 25 b 64.00 c 13.92 b 9.53 c 4.95 bc 0.65 b 8.15 bc 10.68 f glomus versiforme 53 a 63.58 c 13.50 b 11.35 b 5.10 ab 0.69 b 7.88 bc 17.74 c glomus etunicatum 58 a 62.25 c 14.50 b 8.68 cd 5.23 a 0.61 b 8.52 ab 14.80 d lsd value at p≤0.05 7.0 2.66 1.21 0.90 0.17 0.12 1.18 1.47 values in each column with the same letter are not significantly different using lsd test at p≤0.05 39 number (table 2). fruit number in vermicompost amended soils (an average of 3.56) was about 96% greater than v 0 treatments (1.81 fruits) (table 3). previously, increased yields of strawberry (arancon et al., 2004 b) and pepper (arancon et al., 2005) in vermicompost amended soils in field conditions were attributed to increased fruit number due to the availability of plant growth regulators and humic acids, which produced by the greatly increased microbial populations resulting from earthworm activity (arancon et al., 2004 a; b). according to our results, the simultaneous increased flower number and fruit set percentage considerably increased total yield. fruit fresh weight the main effects of vermicompost and amf on fruit fresh weight showed significant differences, but the interaction of vermicompost and amf was not significant (table 2). comparing fruit weight in vermicompost amended soils with v 0 treatments showed a 54% increase (table 3). this result is in agreement with previous studies on the application of vermicompost for eggplant (moraditochaee et al., 2011), greenhouse pepper (arancon et al., 2004 a), and tomato (arancon et al., 2003). it has been stated that the great microbial activity and populations in vermicompost are probably responsible for a considerable buildup of microbial populations and activity in soils. these improve the soil structure and have an indirect influence on root environment, nutrient absorption, plant growth (arancon et al., 2005), and yield (goswami et al., 2001). no significant differences were found between plants inoculated with g. versiforme and nic treatments. plants inoculated with g. etunicatum produced fruits with an average weight of 45.1 g, which was about 32% greater than nic and g. versiforme-treated plants (table 4). it has been reported that individual tomato fruit weight significantly increased when the plants were colonized with amf (bryla and koide, 1998). such evidence, which is in agreement with our results, also showed different increased levels of fruit fresh weight for other inoculated solanaceous plants, i.e. tomato plants inoculated with g. mosseae (abdel latef and chaoxing, 2010), chili pepper plants inoculated with g. intraradices (castillo et al., 2009), chileancho pepper inoculated with g. fasciculatum (mena-violante et al., 2006), and non-solanaceous cucumber (trimble and knowles, 1995). increased yields have been attributed to the increased yield components due to the positive effects of mycorrhiza including facilitated water and nutrient uptake through extension of root surfaces and increased photosynthesis (ortas et al., 1996; raman and mahadevan, 1996; tarkalson et al., 1998). fruit dry matter percent the main and interaction effects of vermicompost and amf on fruit dry matter percent showed significant differences (table 2). the v 0 treatments produced 12% greater fruit dry matter than pots amended with vermicompost, regardless of amf inoculation (table 3). results of fruit fresh weight and fruit dry matter percentage showed that vermicompost improved water uptake and partitioning in fruits, which had greater fresh weight (due to vermicompost) and lower dry matter percentages (table 3). inoculating pepino plants with g. versiforme increased the fruit dry matter percent to over 50% compared to those inoculated with g. etunicatum and plants not inoculated with amf (table 4). the differences could be related to the developmental pattern of amf species. an increased fruit dry matter percentage in amf plants has been attributed to improved water and nutrient uptake/translocation, higher photosynthesis (vamerali et al., 2003), and also to the pattern of dry matter distribution in inoculated plants, which pointed to a role of amf in carbon partitioning (mena-violante et al., 2006). co-application of vermicompost and amf showed different patterns for fruit dry matter percentage. g. versiforme in v 0 treatment produced about 40% greater fruit dry matter than vermicompost amended media, but no differences were observed between vermicompost and non vermicompost amended soils inoculated with g. etunicatum (table 5). fruit juice ph with regard to fruit juice ph, the main effects of amf and its interaction with vermicompost were highly significant (table 2). the ph of pepino plants inoculated with g. etunicatum was 0.13 higher than in nic plants. the difference between g. versiforme inoculated plants and nic plants was not significant for fruit juice ph (table 4). previously, inoculation of cucumber plants with g. intraradices gave no changes in fruit ph (rouphael et al., 2010). plants inoculated with g. etunicatum exhibited a higher fruit juice ph than g. versiforme inoculated plants in v 0 treatment, but the difference was not significant when vermicompost was used (table 5). fruit titratable acidity the main and interaction effects of vermicompost and inoculation with amf showed significant differences with nic plants for fruit juice titratable acidity (table 2). the plants grown in vermicompost amended soils (0.65 ml/100 ml fruit juice) had a 20% decrease in titratable acidity compared with plants in non-amended soils (0.78 ml/100 ml fruit juice) (table 3). in other studies, the effect of vermicompost on tomato fruit titratable acidity did not show significant differences (gutiérrez-miceli et al., 2007). the effect of inoculation of plants with g. versiforme showed a 40% increase in fruit titratable acidity compared with g. etunicatum inoculated plants and the control (table 4). our result is in agreement with a previous paper that reports a significant increase in fruit titratable acidity of amf inoculated tomato plants (regvar et al., 2003). a different reaction to vermicompost application was observed for inoculated plants with two different amfs (table 5). fruits of plants inoculated with g. versiforme in the v 0 treatment had a 0.5ml/100ml greater titratable acidity than fruits from g. etunicatum-inoculated plants. 40 it seems that the effect of amf on pepino fruit juice titratable acidity is amf-species dependent. total soluble solids the main effects of vermicompost and amf on total soluble solids were not significant but the interaction was significant (table 2). fruits of g. versiforme-inoculated plants in the v 0 treatment had 1.5 brix greater soluble solid content than those grown in vermicompost amended media. the differences between amf-inoculated and nic plants in vermicompost amended treatments were not significant (table 5). greater fruit total soluble solids in amf inoculated tomato plants, compared to non-inoculated plants, has been previously reported (subramanian et al., 2006). different microorganisms have been reported as involved in breaking down (mineralize) and releasing mineral nutrients of organic materials, to then be taken up by plant roots (linderman and davis, 2004). it seems that g. etunicatum has the ability to improve vermicompost utilization by pepino plant roots, which leads to more efficient photosynthetic activity and therefore greater total soluble solids in fruits. fruit vitamin c the main and interaction effects of vermicompost and inoculation with amf showed significant differences for fruit vitamin c content (table 2). fruits produced in vermicompost amended soils had 37% less vitamin c than fruits in the v 0 treatments (table 3). this could be related to a 1.5 times greater fresh fruit weight with constant vitamin c content in a unit volume. it seems that the level of vitamin c in pepino fruits is not affected by vermicompost application. different reports are available on the effect of vermicompost application on fruit ascorbic acid content: some show increased fruit vitamin c in tomato (sable et al., 2007), while others report no significant effect (roberts et al., 2007). the fruit vitamin c content was 53 and 27% greater in fruits from plants inoculated with g. versiforme as compared to fruits from plants inoculated with g. etunicatum and non mycorrhizal treatments, respectively (table 3). higher quantities of ascorbic acid in amf-inoculated tomato plants compared to non-inoculated plants has been previously reported (subramanian et al., 2006). the amf used in this experiment showed different reactions to vermicompost application. fruits from the v 0 treatment inoculated with g. versiforme and those inoculated with g. etunicatum in vermicompost amended soils had greater vitamin c than other treatments (table 5). it seems that the effect of amf on pepino fruit vitamin c is species dependent. those plants treated with g. etunicatum had higher vitamin c content when vermicompost was applied, while the same trend was not observed when vermicompost was applied to g. versiforme-inoculated plants. 4. conclusions despite the influence of amf on crop yield as documented in many reports on solanaceous plants, little is known about 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davenport et al., 2003; implay, 2003). moreover, salt stress causes nuclear deformation and subsequent nuclear degradation (katsuhara and kawaski, 1996). structural changes of nuclei caused by salt stress have been previously reported as well (werker et al., 1983). at present, there are several methods (physiological, biochemical, and molecular) available for detecting different kinds of dna damage but with some limitations. recently, molecular markers have been successfully applied to detect dna damage induced by different abiotic stresses, particularly salinity. among others, rapd technique has been well documented as a sensitive means of detecting dna damage and shows potential as a reliable and reproducible assay for the detection of dna fragmentation and chromosomal mutations (citterio et al., 2002). the rapd marker has been extensively applied for salinity tolerance screening in plant breeding programs, such as in date palm (phoenix dactylifera l.) (kurup et al., 2009), aquatic plants hydrilla verticillata and ceratophyllum demersum (gupta and sarin, 2009), in euplotes vannus (protozoa, ciliophora) (zhou et al., 2011), and in acacia senegal (daffalla et al., 2011); in cotton (dojan et al., 2012) and in fish full-sib nile tilapia (oreochromis niloticus), blue tilapia (oreochromis aureus) and their diallel interspecific hybridization (el-zaeem, 2012); and recently, also in soybean (glycine max l.) (khan et al., 2013). rapd bands can be scored for genomic template stability (gts) evaluation to detect various types of dna damage and mutations (rearrangement, point mutations, small insertions or deletions of dna and polyploidy changes) which suggests that rapd bands may potentially form the basis of novel biomarker adv. hort. sci., 2016 30(1): 13-21 doi: 10.13128/ahs-18697 dna changes in cotton (gossypium hirsutum l.) under salt stress as revealed by rapd marker b. saleh department of molecular biology and biotechnology, aecs, p.o. box 6091, damascus, syria. key words: cotton, genomic template stability, rapd, salt stress. abstract: random amplified polymorphic dna (rapd) analysis was applied to evaluate dna changes among four upland cotton (gossypium hirsutum l.) varieties [niab 78 (n78), deir-ezzor 22 (de22), deltapine 50 (dp50) and aleppo 118 (a118)] grown under non-saline conditions (control) and salt stress (200 mm nacl) for seven weeks. changes in rapd profiles were measured as genomic template stability (gts%). the highest estimated gts% value was recorded for the two sensitive varieties, dp50 (79.1%) followed by a118 (58.2%); whereas, the lowest value was recorded for the two other tolerant varieties de22 (36.7%) followed by n78 (26.4%). based upon the data presented, rapd marker could be used as potential tool for early identification of cotton tolerance to salt stress. (*) corresponding author: ascientific@aec.org.sy received for publication 28 may 2015 accepted for publication 20 december 2015 copyright: © 2016 author(s). this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. adv. hort. sci., 2016 30(1): 13-21 14 assays for the detection of dna damage and mutations in the cells of bacteria, plants, and animals (savva, 1998; atienzar et al., 1999; tanee et al., 2012). it is well documented that genomic template stability ratios (gts) were calculated. gts implies qualitative measure reflecting changes in rapd profiles. changes in rapd and profiles were expressed as reductions in gts in relation to profiles obtained from control samples (gupta and sarin, 2009; aly, 2012; dojan et al., 2012; tanee et al., 2012). therefore, this investigation aimed to detect dna changes induced by nacl application by monitoring the rapd profiles of control and stressed plants in four upland cotton (gossypium hirsutum l.) varieties grown in syria. 2. materials and methods plant materials and growth conditions two local varieties were selected on the basis of their wide-ranging tolerance towards salinity: deirezzor 22 (de22) as salt-tolerant and aleppo 118 (a118) as salt-sensitive variety (saleh, 2011). these two varieties were compared with two introduced cotton varieties, niab 78 (n78) (known as salt-tolerant) and deltapine 50 (dp50) (known as salt-sensitive) under 0 and 200 mm nacl for seven weeks. seeds of upland cotton (g. hirsutum l.) were provided by the general commission for scientific agricultural research of syria (gcsar). seeds were soaked in distilled water for 24 h and then planted in pots filled with a 1:2 (v/v) mixture of perlite:peatmoss. germination was carried out in a greenhouse at 18°c, 12 h photoperiod, and relative humidity of 80%. seedlings were allowed to grow in a greenhouse under controlled conditions (temperature 25°c, 12 h photoperiod, and relative humidity 80%). seedlings were irrigated with tap water for one week before the initiation of nacl treatments. salt stress application was carried out by adding nacl (200 mm) to the water. plants were irrigated twice a week with water with or without salt. all solutions were changed twice a week. the same environmental conditions were maintained during the experiment. the experiment (five replicates/treatment) was carried out in the greenhouse for seven weeks. genomic dna extraction plant genomic dna was extracted from young leaves (bulk of five plants/variety) including the control and stressed plants (200 mm nacl) using ctab (cetyltrimethylammonium bromide) protocols described by doyle and doyle (1987) with minor modifications. leaf tissue (150 mg) was ground in liquid nitrogen and the powder was transferred to a 2 ml eppendorf tube, mixed with 900 μl of extraction buffer (100 mm tris-hcl, ph 8.0, 1.4 m nacl, 20 mm edta, 0.0018 ml β-mercaptoethanol, 2% ctab), and incubated at 65°c for 20 min. one volume of a chloroform:isoamyl alcohol mix (24:1, v/v) was added and centrifuged at 12,000 g for 10 min at 4°c. the aqueous phase was transferred to a fresh tube, and the dna was precipitated with an equal volume of cold isopropanol and kept at -20°c for 10 min. it was then centrifuged at 12,000 g for 10 min at 4°c, the supernatant was discarded, and dna was spooled out and washed with 1 m ammonium acetate and 100% ethanol. the cleaned dna pellet was air dried and dissolved in 100 μl of 0.1x te buffer (1 mm tris-hcl, 0.1 mm edta, ph 8.0). finally 5 μl of rnase (10 mg ml-1) were added and incubation for 30 min at 37°c was applied. dna concentration was quantified by dna spectrophotometer at 260/280 nm and adjusted to final concentration of 10 ng μl-1. dna was stored at -80°c until needed. rapd marker twenty-three rapd primers from operon technologies inc. (usa) and three primers from the university of british columbia were tested to detect dna changes in stressed plants, and their respective controls, for four cotton varieties. rapd marker was performed as described by williams et al. (1990) with a minor modification. pcr amplification reaction was carried out in 25 μl reaction volume containing 1xpcr buffer, 2 mm mgcl2, 0.25 mm dntps, 25 pmol primer, 1.5 u of taq dna polymerase and 30 ng template dna. pcr amplification was performed in a t-gradient thermal cycler (bio-rad; t gradient) programmed to fulfill 42 cycles after an initial denaturation cycle for 4 min at 94°c. each cycle consisted of a denaturation step (1 min at 94°c), an annealing step (2 min at 35°c), and an extension step (for 2 min at 72°c). a final extension cycle was performed for 7 min at 72°c. the pcr products were separated on a 1.5% ethidium bromidestained agarose gel (bio-rad) in 0.5xtbe buffer. electrophoresis was performed for 3 h at 85v and visualized with a uv transilluminator. band sizes were determined by comparison with a 1 kb dna ladder mix, ready for use (fermentas). saleh dna changes in cotton under salt stress 15 rapd data analysis and genomic template stability (gts) estimations dna changes induced in treated plants compared to their respective controls were screened by rapd assay. the polymorphism was calculated in relation to the appearance of new bands and disappearance of bands in treated plants, compared to control band patterns. genomic template stability (gts%) was calculated as follows: gst% = (1a/n) x 100 where (a) is the average number of changes in the dna profile and (n) the number of total bands in the control. polymorphism observed in rapd profiles included disappearance of a normal band and appearance of a new band in comparison to the cotrol rapd profiles (atienzar et al., 2002). 3. results a set of 26 random10-mer primers was used to detect the dna changes among four cotton varieties under salt stress application compared to their respective controls. rapd fragment sizes ranged from 200 to 3000 bp. the generated band characteristics for the four varieties (including control and stressed plants) are summarized in table 1. the total number of characteristic bands (common observed bands in control and stressed plants for the four examined varieties) was 29. the amplification products produced from 26 rapd primers are listed in table 2 in terms of loss or appearance of new bands (number and size) under salt stress compared to their respective controls for each variety separately. the rapd analysis carried out on the four cotton varieties produced a number of distinct fragments which varied according to each tested primer. twelve of the 26 rapd primers (opa02, opb05, opc08, opd08, opd20, opj07, opk13, opk17, opr12, opy10, ubc132 and ubc159) produced polymorphic bands under saline conditions for the four tested varieties (table 2). figure 1 shows the amplification products using opa02, opb17 and opy10 rapd primers with template dna from the four varieties under control and saline conditions (200 mm nacl). changes in dna pattern induced by nacl treatment in the four tested cotton varieties were detected based on estimated genomic template stability (gts%) (table 3). in this respect, it was found that the highest gts% was recorded in salt-sensitive cotton, whereas the lowest was found among salt-tolerant varieties (table 3). 4. discussion and conclusions detection of dna changes in cotton via salt stress was assessed using rapd marker system. as shown in table 1, characteristic bands ranged between 0 (opb17, opd20, ope15, opj01, opj07, opk12, opk13, opk17 and ubc132) and 2 (opc08 and opt18), whereas the highest number (three) was yielded by opa04, ope07, opg11, opq01 and opq18 rapd primers (table 1). our findings reveal that nine out of the 26 tested rapd primers generattable 1 characteristic bands identified for the four tested cotton varieties using 26 rapd primers primer name sequence (5' 3') characteristic bands (number and size) opa02 tgccgagctg (1) 950 opa04 aatcgggctg (3) 250, 550 & 1600 opb05 tgcgcccttc (1) 1500 opb17 agggaacgag 0 opc08 tggaccggtg (2) 500 & 800 opc13 aagcctcgtc (1) 450 opd08 gtgtgcccca (1) 1200 opd20 ggtctacacc 0 ope07 agatgcagcc (3) 550, 650 & 800 ope15 acgcacaacc 0 opg11 tgcccgtcgt (3) 700, 1200 & 2100 opj01 cccggcataa 0 opj07 cctctcgaca 0 opk12 tggccctcac 0 opk13 ggttgtaccc 0 opk17 cccagctgtg 0 opq01 gggacgatgg (3) 450, 600 & 1000 opq18 aggctgggtg (3) 650, 750 & 1200 opr09 tgagcacgag (1) 250 opr12 acaggtgcgt (1) 300 opt18 gatgccagac (2) 1200 & 1350 opw17 gtcctgggtt (1) 2000 opy10 caaacgtggg (1) 650 ubc132 agggatctcc 0 ubc159 gagcccgtag (1) 1600 ubc702 gggagaaggg (1) 400 total 29 adv. hort. sci., 2016 30(1): 13-21 16 table 2 markers identified by 26 rapd primers for the four tested cotton varieties under salt stress compared to their respective controls. dna changes induced by nacl treatment using rapd marker as described by loss or appearance of new bands (number and size) under salt stress compared to their respective controls for each variety separately primer name n78 de22 dp50 a118 total polymorphic bandsc t c t c t c t opa02 9 8 9 9 ₋ (4) 400, 650, 1800 & 2500 (3) 650, 800 & 2500 (2) 1800 & 2500 (2) 1800 & 2500 16 ₊ (2) 600 & 1500 (3) 500, 600 & 900 nd nd opa04 3 3 3 3 ₋ nd nd nd nd 5 ₊ (3) 400, 500 & 1000 (2) 400 & 500 nd nd opb05 5 5 4 5 ₋ (4) 450, 650, 1000 & 2000 (3) 450, 1000 & 2000 (1) 900 (1) 2000 14 ₊ (3) 500, 700 & 1100 (2) 500 & 1100 nd nd opb17 7 9 8 8 ₋ (5) 600, 700, 1100, 1200 & 1800 (4) 350, 800, 1500 & 1800 nd (5) 400, 600, 800, 1000 & 1800 20 ₊ (2) 400 & 650 (2) 700 & 1100 nd (2) 450 & 750 opc08 4 5 5 4 ₋ (2) 1850 & 1900 (1) 1200 (1) 1850 nd 10 ₊ (2) 300 & 900 (2) 1350 & 1900 nd (2) 1850 & 1900 opc13 6 6 5 5 ₋ (1) 1000 (2) 500 & 1000 nd nd 12 ₊ (5) 300, 800, 900, 1100 & 1350 (4) 300, 1100, 1350 & 1500 nd nd opd08 4 3 4 3 ₋ (2) 450 & 700 (2) 450 & 750 (1) 900 (2) 450 & 800 15 ₊ (2) 650 & 1350 (4) 500, 600, 800 & 1350 (1) 800 (1) 600 opd20 6 4 2 2 ₋ (4) 650, 850, 1100 & 1850 (3) 700, 900 & 1350 (1) 800 (1) 300 13 ₊ (1) 1350 (1) 800 (1) 300 (1) 200 ope07 8 8 8 8 ₋ (5) 1000, 1300, 1800, 2300 & 3000 (1) 1800 nd nd 7 ₊ (1) 1100 nd nd nd ope15 5 4 6 6 ₋ (1) 200 (2) 800 & 1800 nd nd 8 ₊ (4) 800,1300, 1500 & 1800 (1)1500 nd nd opg11 3 3 3 3 ₋ nd nd nd nd 2 ₊ (1) 600 (1) 600 nd nd opj01 7 6 7 7 ₋ (5) 400, 600, 850, 1200 & 2000 (3) 400, 600 & 850 nd (2) 600 & 2000 20 ₊ (4) 350, 500, 950 & 1800 (4) 350, 500, 950 & 1800 nd (2) 500 & 800 opj07 3 4 2 3 ₋ (3) 500, 800 & 1600 (4) 300, 500, 700 & 1600 (2) 550 & 1350 (1) 1100 21 ₊ (3) 700, 900 & 1200 (3) 550, 1000 & 1200 (2) 450 & 1600 (3) 450, 550 & 650 to be continuedt(-) loss bands, (+) gains bands, (nd) no differences. saleh dna changes in cotton under salt stress 17 primer name n78 de22 dp50 a118 total polymorphic bands c t c t c t c t opk12 3 3 2 2 ₋ (3) 700, 900 & 1350 (3) 800, 900 & 1350 nd nd 11 ₊ (3) 850, 950 & 1100 (2) 500 & 1000 nd nd opk13 4 4 3 3 ₋ (2) 650 & 1100 (2) 350 & 1100 (1) 200 (1) 490 20 ₊ (4) 450, 750, 1200 & 1350 (4) 500, 750, 1200 & 1350 (3) 300, 500 & 1100 (3) 700, 1200 & 1350 opk17 8 4 4 6 ₋ (4) 400, 1500, 1850 & 2500 (1) 1850 (2) 700 & 1000 (4) 300, 400, 1000 & 1850 19 ₊ (3) 300, 350 & 800 (2) 350 & 1200 (2) 500 & 1500 (1) 1500 opq01 9 4 4 4 ₋ (5) 300, 800, 1100, 1200 & 1800 (1) 1100 nd nd 10 ₊ (3) 200, 700 & 900 (1) 300 nd nd opq18 7 3 6 6 ₋ (4) 650, 900, 1350 & 1500 nd nd nd 9 ₊ (2) 550 & 1600 (2) 1500 & 1600 nd (1) 1600 opr09 6 3 4 4 ₋ (4) 400, 550, 1100 & 3000 (1) 600 nd (1) 600 9 ₊ (2) 600 & 900 (1) 1000 nd nd opr12 11 10 7 7 ₋ (6) 200, 450, 600, 1100, 2100 & 2250 (6) 250, 500, 800, 900, 1350 & 2250 (1) 900 (3) 200, 1200 & 1350 28 ₊ (4) 250, 500, 900 & 1800 (4) 700, 1000, 1100 & 1800 (2) 1350 & 2250 (2) 450 & 1000 opt18 5 8 10 10 ₋ (1) 650 (2) 1800 & 2000 nd nd 9 ₊ (4) 400, 500, 1000 & 2250 (1) 950 nd (1) 2000 opw17 5 4 3 3 ₋ (2) 600 & 1200 (3) 300, 900 & 1500 nd nd 17 ₊ (5) 450, 500, 800, 1350 & 1500 (4) 300, 450, 500 & 800 nd (3) 250, 450 & 800 opy10 4 5 4 4 ₋ (1) 3000 (2) 1500 & 3000 (1) 400 (3) 400, 900 & 2500 12 ₊ (3) 400, 850 & 2500 (2) 400 & 2500 nd nd ubc132 5 5 4 5 ₋ (4) 1000, 1350, 1500 & 3000 (3) 550, 600 & 3000 (1) 1200 (3) 400, 1000 & 1200 17 ₊ (3) 600, 1200 & 1800 (3) 1000, 1350 & 1500 nd nd ubc159 3 6 3 3 ₋ (1) 500 (2) 1350 & 2000 (2) 500 & 700 (2) 650 & 2000 19 ₊ (5) 550, 700, 950, 1100 & 1350 (3) 300, 700 & 900 (2) 450 & 600 (2) 500 & 700 ubc702 3 3 3 3 ₋ (1) 800 (2) 750 & 800 nd nd 7 ₊ (2) 600 & 2500 (1) 2500 nd (1) 2500 table 2 (continued) adv. hort. sci., 2016 30(1): 13-21 18 ed no characteristic bands for the for tested cotton varieties (table 1). it is worth noting that primer opr12 identified more polymorphisms (28) than any other primer tested (ranging between two for primer opg11 and 21 for primer opj07) (table 2). whereas, the banding patterns produced by primers opa04, opc13, ope07, ope15, opg11, opk12 and opq01 were not polymorphic for varieties dp50 and a118 (table 2). another investigation demonstrated varietal variation in salt tolerance among these cotton varieties based on various examined physiological indices (saleh, 2011). according to the study, the de22 variety could relatively be classified as salt tolerant variety to other tested varieties. dojan et al. (2012) reported the potential of rapd markers for the detection of dna damage induced by nacl in cotton. likewise, the rapd marker has the potential to be applied in environmental pollution detection, e.g. gupta and sarin (2009) applied the same marker to detect pollution by cadmium (cd) in two aquatic plants. zhou et al. (2011) also used rapd bands to indicate dna damage in euplotes vannus (protozoa, ciliophora) induced by nitrofurazone in marine ciliates. previously, aly (2012) used the same marker for genotoxic effect detection of cd stress on egyptian clover and sudan grass plants. changes in rapd profiles were also measured as genomic template stability (gts) and the data suggest noticeable genomic template instability (table 3). reduction in gts values was observed under salt stress, compared to their respective controls for the four tested varieties (table 3). similarly, genetic instability induced by nacl treatment of cotton was reflected by changes in rapd profiles: disappearance fig. 1 rapd banding profiles generated by opa02, opb17 and opy10 primers in the four tested cotton varieties showing dna changes induced by nacl application for seven weeks, c: control, t: treated plants. m: 1 kb dna ladder mix, ready for use. primer name n78 de22 dp50 a118 control 200 mm nacl 200 mm nacl 200 mm nacl 200 mm nacl opa02 100 33.3 25 77.8 77.8 opa04 100 0 33.3 100 100 opb05 100 40 0 75 80 opb17 100 0 33.3 100 12.5 opc08 100 0 40 80 50 opc13 100 0 0 100 100 opd08 100 0 100 50 0 opd20 100 16.7 0 0 0 ope07 100 25 87.5 100 100 ope15 100 0 25 100 100 opg11 100 66.7 66.7 100 100 opj01 100 28.6 16.7 100 42.9 opj07 100 100 75 100 33.3 opk12 100 100 66.7 100 100 opk13 100 50 50 33.3 33.3 opk17 100 12.5 25 0 16.7 opq01 100 11.1 50 100 100 opq18 100 14.3 33.3 100 83.3 opr09 100 0 33.3 100 75 opr12 100 9.1 0 57.2 28.6 opt18 100 0 62.5 100 90 opw17 100 40 75 100 0 opy10 100 0 20 75 50 ubc132 100 40 20 75 40 ubc159 100 100 16.7 33.3 33.3 ubc702 100 0 0 100 66.7 mean 100 26.4 36.7 79.1 58.2 table 3 genomic template stability (gts%) estimated by 26 rapd primers for the four tested cotton varieties under salt stress compared to their respective controls saleh dna changes in cotton under salt stresss 19 of bands and appearance of new bands occurred in the profiles in comparison to those of the controls (fig. 1, table 3). our data supports the suggestion by dojan et al. (2012) that detected dna changes, induced by nacl, using rapd marker could be explained as previously reported by atienzar et al. (1999). it has been demonstrated that dna damage levels could be reflected in gts (atienzar et al., 1999). the later investigation suggested that the loss of bands may be attributed to genomic rearrangements or to point mutations causing alterations in oligonucleotide priming sites, while appearance of new bands could be related to the presence of oligonucleotide priming sites which become accessible to oligonucleotide primers after structural changes (dna mutation, deletions or homologous recombination). table 3 reveals that gts% values decreased with salt application for the four tested varieties. our data show that the highest estimated gts value was recorded for the two sensitive varieties, dp5 (79.1%) followed by a118 (58.2%); whereas, the lowest was recorded for the two tolerant varieties, n78 (26.4%) followed by de22 (36.7%) (table 3). gupta and sarin (2009) reported that the genomic template stability test was significantly affected by heavy metal stress, while aly (2012) reported that gts values decreased obviously with an increase in cadmium (cd) concentration in egyptian clover and sudan grass seedlings. on the other hand, tanee et al. (2012) used gts to identify the vanda species (orchidaceae) of thailand. our results are in accordance with dojan et al. (2012) who reported that there is positive correlation between gts and other parameters (stem and leaf growth and stem length) under nacl stress in cotton. in this respect, the estimated gts values in the current investigation were positively correlated with various physiological indices (biomass and leaf k+/na+ ratio) tested under nacl application in cotton (saleh, 2011). moreover, a positive relationship was also determined between gts values and recent findings (saleh, 2013) based on physiological indices (relative water content, osmotic potential and salt tolerance index) among the same tested varieties (dojan et al., 2012). overall, the lowest estimated gts values combined with the highest polymorphism level recorded for the tolerant varieties (n78 and de22) (where, % polymorphic level was 68.5, 60.9, 21.3 and 36.4% for n78, de22, dp50 and a118, respectively exposed to 200 mm nacl for 7 weeks, using the same marker) could explain their salinity tolerance compared to the other tested varieties. however, the lowest estimated gts value recorded for n78 and de22 varieties could be attributed to genetic variation, inducing new protein in relation to salinity tolerance. it has been successfully demonstrated that environmental constraint induced variation in dna methylation pattern as a developed epigenetic mechanism after exposure to abiotic stress (zhong and wang, 2007; peng and zhang, 2009). our findings could be supported by the data provided in zhong and wang (2007), where genotyping variation in wheat (triticum aestivum l.) cultivar salinity tolerance was reported. in this respect, the study mentioned that the salt-sensitive wheat variety had a lower methylation rate compared to salt-tolerant ones. recently, saleh (2013) reported that n78 and de22 varieties showed a better protection mechanism against salinity damage than the other tested varieties, demonstrating variation in salt tolerance among cotton varieties based on physiological indices. likewise, in the same investigation comparing the protein profiles between control plants and those salts treated using sds-page showed protein changes under salt treatment compared to their respective control. in this respect, the expression of ~19, ~21 and ~26 kda for n78 and ~21 kda protein for de22, was highly increased by salt treatment, indicating that it could play a role in salt stress response. on the other hand, newly synthesized protein of ~30 kda was recorded for both de22 and n78 varieties under saline treatment which was not observed in their respective controls. the other two tested varieties (dp50 and a118) showed decreases in the same protein bands (~19, 26 and 34 kda) under saline conditions, with respect to their respective controls, reflecting their sensitivity to salt stress. salinity promotes the synthesis of salt stress-specific proteins; many of these proteins were suggested to protect the cell against the adverse effect of salt stress. accumulation of these proteins is a common response to salt stress (kong-ngern et al., 2005; metwali et al., 2011). it is worth noting that identified bands in de22 and n78 (salt-tolerant varieties), which were not amplified in salt-sensitive varieties (dp50 and a118), could be related to gene(s) involved in salinity tolerance. these findings are in accordance with kurup et al. (2009). adv. hort. sci., 2016 30(1): 13-21 20 dna variation could be exploited in plant breeding programs to improve salinity tolerance in germplasm. overall, the lowest estimated gts values for nb78 and de22 varieties (known as salt-tolerant) reflect their highest polymorphic values. based on the current results, the rapd marker was useful to establish specific dna markers associated with nacl stress. therefore, the rapd marker could be used as useful tool in plant breeding programs for early identification of cotton tolerance to salt stress. acknowledgements the author would like to thank prof. othman i, director general of aecs and prof. mirali n, head of molecular biology and biotechnology department for their support, and also to the plant biotechnology group for technical assistance. references aly a.a., 2012 application of dna (rapd) and ultrastructure to detect the effect of cadmium stress in egyptian clover and sudan grass plantlets. j. stress physiol. & biochem., 8: 241-257. 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cotton. journal of stress physiology & biochemistry, 9: 113-123. savva d., 1998 use of dna fingerprinting to detect genotoxic effects. ecotoxicol. environ. safe., 41: 103-106. tanee t., chadmuk p., sudmoon r., arunrat c., noikotr k., 2012 genetic analysis for identification, genomic template stability in hybrids and barcodes of the vanda species (orchidaceae) of thailand. african j. biotech., 11: 11772-11781. usda, 2011 syria cotton and products annual cotton report. gain report, www.fas.usda.gov. werker e., lerner h.r., weimberg r., poljakoof-mayber a., 1983 structural changes occurring in nuclei of barley root cells in response to a combined effect of salinity and ageing. am. j. bot., 70: 222-225. williams j.g.k., kubelik a.r., livak k.j., rafalski a.j., tingey s.v., 1990 dna polymorphisms amplified by arbitrary primers are useful as genetic markers. nucleic acids res., 18: 6531-6535. zhong l., wang j.b., 2007 the role of dna hypermethylation in salt resistance of triticum aestivum l. journal of wuhan botanical research, 25: 102-104. zhou l., li j., lin x., al-rasheid k.a.s., 2011 use of rapd to detect dna damage induced by nitrofurazone in marine ciliate euplotes vannus (protozoa, ciliophora). aquat. toxicol., 103: 225-232. impaginato 39 1. introduction biological and non-biological stresses, which are mostly due to adverse weather conditions, are main factors in yield reduction (wu et al., 2006). there is much evidence that mycorrhizal fungi cause variations in plant-water relations and improve drought tolerance. improvement in plant-water relations is affected by direct and indirect mechanisms (davies et al., 1993). in general, plants that have mycorrhizal symbiosis grow and perform better as they absorb more nutrients and water from the soil. these plants are also more tolerant towards environmental stresses including biotic and abiotic stresses (porcel and ruiz-lozano, 2004). most varieties of citrus, like orange, trifoliate orange, cleopatra mandarins, swingle citrumelo, and citrange, are very dependent, because of their hairy roots, on glomus species (davies et al., 1993). plant adaptations to arid climate conditions, morphological and physiological changes, and concentration of novel metabolites along with structural variations, increase their efficiencies in stress conditions (wu et al., 2006). when plants are under drought stress, osmotic adjustments occur to reduce potential water loss. this phenomenon leads to good water flow maintenance from the soil to plant roots (porcel and ruizlozano, 2004). g. versiforme fungus increased leaf water potentials of trifoliate orange and mandarin seedlings under both drought stress and enoughwater-supply conditions (wu et al., 2006, 2008). moreover, when trifoliate orange seedlings were under drought stress, the leaf relative water content (rwc) significantly increased compared to plants with no fungus (wu et al., 2006). in mandarin seedlings, plant height, leaf area and number of leaves per plant, decreased under drought stress conditions, while all those factors were improved using g. versiforme fungi (wu and zou, 2009). in citrus plants, g. versiforme fungi increased growth and adv. hort. sci., 2016 30(1): 39-45 doi: 10.13128/ahs-18700 analysis of the effects of glomus etunicatum fungi and pseudomonas fluorescence bacteria symbiosis on some morphological and physiological characteristics of mexican lime (citrus aurantifolia l.) under drought stress conditions a.r. shahsavar 1 (*), a. refahi 1, m. zarei 2, e. aslmoshtaghi 1 1 department of horticultural science, college of agriculture, shiraz university, shiraz, iran. 2 department of soil science, college of agriculture, shiraz university, shiraz, iran. key words: chlorophyll content, drought deficit, leaf water potential, mexican lime. abstract: to analyze the effects of glomus etunicatum fungi and pseudomonas fluorescence bacteria on some morphological and physiological characteristics of mexican lime plant under drought stress conditions, a factorial experiment was conducted. this experiment was based on a completely randomized design with three replicates; each replicate was composed of two pots. the factors used consisted of g. etunicatum fungi and control, pseudomonas fluorescence bacteria and control, and drought stress at three levels (-0.35, -0.47, and -0.6 bars). the analyzed characteristics were leaf chlorophyll content, leaf temperature, rate of net photosynthesis, transpiration, leaf relative water content (rwc), and percentage of root colonization. data analysis revealed that both fungi and bacteria increased leaf chlorophyll content, net photosynthesis rate, transpiration, and leaf rwc. moreover, the presence of fungi reduced leaf temperature while inoculation of bacteria had no effects on that the parameter. in addition, with the increase of irrigation periods, leaf temperature and transpiration were also increased. results showed that root colonization percentage dropped with increased irrigation and the highest root colonization percentage was observed in simultaneous inoculations of fungi and bacteria with a two-day irrigation period. (*) corresponding author: shahsava@shirazu.ac.ir received for publication 31 october 2015 accepted for publication 4 february 2016 copyright: © 2016 author(s). this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. adv. hort. sci., 2016 30(1): 39-45 40 biomass while they reduced their colonization percentage. these increases were attributed to the improvement of fungi water absorptions and increases in the length and volume of plant fungal roots (faber et al., 1991; bryla and duniway, 1997; wu et al., 2011). in these plants, root colonization increases with a decrease in drought stress (augé, 2001). under drought stress conditions, g. versiforme fungi increased fresh and dry weight of plant roots and shoots and increased the root colonization percentage (wu and xia, 2006). g. intraradices fungus, under drought stress conditions, increased root growth and respiration rate of rough lemon (leyv and syvestern, 2006). many studies showed that g. etunicatum fungi could affect plant-water relations of host plants including citrus, under both drought stress and enough-water-supply conditions (wu et al., 2006). therefore, they cause higher water use efficiency and this water use efficiency in mycorrhizal plants becomes even more tangible in drought stress conditions (davies et al., 1993). glomus etunicatum fungus increased phosphorus, potassium, zinc, and copper in pistachio trees planted under sufficient water supply conditions and also increased nitrogen and calcium in pistachio trees planted under drought stress conditions. however, this fungus did not change the magnesium concentration (abbaspour et al., 2011). it is reported that pseudomonas bacteria enhances growth and yield of some plants (rodriguez and fraga, 1999). construction of active metabolites such as vitamins, amino acids, and indole acetic bacteria may have a direct effect on the growth and metabolite contents of piriformospora indica and mycorrhizal fungi. as a helpful microorganism, it seems that bacteria supports fungal performances (vivas et al., 2003). plant inoculations with different types of pseudomonas bacteria in drought stress situations increased plant proline contents, thus the plants’ water levels were maintained and their protein contents and membranes remained safe from drought stress damage (yoshiba et al., 1997). inoculation with pseudomonas species, led to moderation of drought stress effects, improvement of plant growth and increase of proline, soluble sugars and amino acids production, explaining their effectiveness in absorbing water and nutrients from the soil (wu et al., 2008). these types of bacteria also help the plant maintain its rwc and lwl (leaf water loss) levels under drought stress conditions. studies have shown that mycorrhizal plants absorb more co2 in the presence of light. hence, their photosynthesis rates are also higher. the increase of co2 absorption in mycorrhizal plants is related to a decrease of liquid-phase resistance of mesophyll cells to co2 transmission (wu and zou, 2009). miller (2000) reported that in mycorrhizal plants, due to the increase of photosynthesis materials and rate, water use efficiency increased per water use unit. mycorrhiza can increase plant weight, leaf area, and plant pigments, and these increases may be attributed to the improvement of fungi water and phosphorus absorptions (bethlenfalvay et al., 1988; davies et al., 1993). glomus etunicatum and pseudomonas bacteria have positive effects on plant growth and employing them, instead of fertilizers, is considered a positive approach to reduce fertilizer use (davies et al., 1993). despite the lack of comprehensive scientific investigations on the horticultural characteristics of mexican lime (citrus aurantifolia swingle cv. mexican lime) as a rootstock, its seed availability for propagation and some its characteristics, such as good crop load and vigorous habit of grafted cultivars as scion, have made it a favorite in fars province, iran. considering the positive effects of fungi and bacteria in symbiosis with some plant roots, the aim of this study was to investigate the effects of g. etunicatum fungi and pseudomonas fluorescence bacteria and their interactions on some morphological and physiological characteristics of mexican lime plant under drought stress conditions. 2. materials and methods preparation and inoculation of plant materials mexican lime seedlings, six months of age and disease-free, were provided in khafr city of fars province, iran. they were transferred to the greenhouse. planting soil mixture in ratio 1:1:1 (sand:soil: leaf compost) was sterilized and 2.7 kg were placed in plastic pots. the arbuscular mycorrhizal fungi isolate used in this study was g. etunicatum supplied from the soil lab of the faculty of shiraz university. the lyophilized fungal inoculum of pseudomonas fluorescence was supplied from tehran university school of soil and water and was prepared as follows. to prepare a solution containing growth-stimulating bacteria, a nutrient broth (nb) medium was applied. first, 0.8 g of nb was dissolved in 100 mg of distilled water and then media were sterilized by autoclaving at 121°c and pressure of 1.1 atm for 25 shahsavar et al. effects of glomus etunicatum and pseudomonas fluorescence on mexican lime under drought stresss 41 min. a lyophilised pre-culture vial was first suspended in 0.3 ml of nutritive medium. one drop (1 ml) of that suspension was added to 5 ml of nutritive medium and incubated on an orbital shaker at 28°c for 24 h. this final preparation of medium was used as the inoculum. after the incubation period, roots were placed in a solution containing bacteria for 30 min. moreover, to ensure its effectiveness, 10 cc of the solution containing bacteria were added to each pot. for drought stress treatments, pots containing 2.7 kg soil without a seedling were selected and their moisture contents were equilibrated with the previously measured field capacity. the wet soils of the pots were weighed daily for 15 days, always at the time. daily water reductions and moisture curves were graphed. using those diagrams, irrigation periods were identified for every 2, 4 and, 6 days. for g. etunicatum fungus inoculation, 70 g of inoculum containing spores, hyphae, and root fragments were introduced 5 cm beneath the soil surface in the pots, and mixed thoroughly. equal to the amount of added inoculum, hyphae, and mycelium to the fungal treatment pots, inoculum without hyphae and mycelium was added to control pots. for bacteria inoculation, seedlings were placed in a solution containing pseudomonas fluorescence bacteria for 30 min and were then planted into pots. for fungi and bacteria treatments, bacteria-inoculated seedlings were planted in pots in which fungus was previously added. one seedling was planted per pot and, two months later, water treatments were applied. after six months, the implants were removed. the study was conducted using a factorial experiment, based on a completely randomized design with three replications in two replicate pots. factors used in the experiment were: 1) g. etunicatum fungus in two levels of g. etunicatum and control; 2) growth stimulating bacteria in two levels of pseudomonas fluorescence and control; 3) drought stress at three levels. the kormanik and mcgraw method (kormanik and mcgraw, 1982) was used to measure colonization percentage. in this method, 2 g of roots previously stored in faa (formaldehyde acetic acid ethanol) were washed with water three or four times and were placed in falcon tubes containing 10% koh solution for 24 h at room temperature. the color of the solution was almost yellow or light yellow. the solution was then poured out and the roots were again washed with water three or four times. the samples were placed in 2% hydrochloric acid for at least 15 min for staining. the acid was poured out and a colored solution was poured over the acidic roots. acid fuchsine stain was used in this study; the ratio of the fuchsine acid colored solution was 14 ml lactic acid, 1 ml glycerin and, 1 ml water. the roots and the solution were kept at room temperature for 24 h. the coloring solution was then removed. besides, due to elimination of extra colors, the coloring solution was poured on the roots. after 6-12 h, fungal organs such as arbuscules, hyphae, and vesicles were observed under a light microscope and colonization was calculated as a percentage. after application of water stress treatments, leaf chlorophyll content was measured with a spad-502 chlorophyllmeter using three fully-expanded leaves to find an average for chlorophyll content. leaf temperature factors, net photosynthesis and transpiration rates were measured by portable photosynthesis meter (lci, adc, england). relative water content was determined by using ten 7 mm-diameter leaf discs. leaf discs for each treatment were weighed (fw). they were hydrated until saturation (constant weight) for 48 h at 5°c in darkness (tw). the leaf discs were then dried in an oven at 105°c for 24 h (dw). relative water content was calculated according to the following expression (filella et al., 1998): rwc% = (fw-dw)/(tw–dw) × 100 statistical analysis the data were analyzed for significance (p<0.050) by anova (analysis of variance) with mean separation by duncan’s multiple range test. 3. results and discussion leaf relative water content (rwc) analysis of the effects of interaction between inoculation of g. etunicatum fungi and pseudomonas fluorescence bacteria on mexican lime leaf rwc, at different irrigation periods, identified that the maximum leaf rwc was observed in simultaneous inoculation of fungi and bacteria with the two-day irrigation period (74.7%). the general results indicate that the leaf rwc decreased with the increase in irrigation period, while inoculation with fungi or bacteria significantly increased rwc in all irrigation periods (table 1). osmotic adjustment is one of the most important factors in plant drought tolerance and it is closely related to rwc (haley et al., 1993). when plants are under a drought stress condition, osmotic adjustadv. hort. sci., 2016 30(1): 39-45 42 ment occurs to reduce water potential and maintain a good flow of water from the soil to the plant roots. plants with mycorrhizal fungi have more osmotic adjustment potentials than plants without fungi (porcel and ruiz-lozano, 2004). manette et al. (1988) reported that plants which are under drought stress conditions have specific morphological and physiological characteristics that enable them to store more water. clarke and craig (1982) stated that plants under drought stress conditions loose their water content more slowly. they also indicated that there are significant relationships between water content of the loss of leaves, plant drought tolerance, and leaves ability to retain water content (clarke and craig, 1982). therefore, mycorrhizal plants have higher osmotic adjustment and are more capable of retaining their water content. chlorophyll content chlorophyll content decreased with the increase of irrigation periods. in addition, inoculations of g. etunicatum fungi and pseudomonas fluorescence bacteria increased leaf chlorophyll content. analysis of the effects of interaction between inoculation of g. etunicatum fungi and pseudomonas fluorescence bacteria leaf chlorophyll content identified that the maximum leaf chlorophyll content was observed when both fungi and bacteria were inoculated and there was a two-day irrigation period (634.7). the lowest chlorophyll content was observed in the treatment without fungi and bacteria inoculations with six-day irrigation periods (table 2). analysis of the effects of g. etunicatum fungi and pseudomonas fluorescence bacteria inoculations on chlorophyll content of the mexican lime leaves in the current study revealed that the chlorophyll content decreased with an increase of drought stress periods. however, inoculations of fungi and bacteria largely reduced the deleterious effects of drought. this can be explained by the fact that in drought stress conditions, the chlorophyllase enzyme becomes activated while its activation results in the loss of chlorophyll content (shaharoona et al., 2008). under drought, oxygen free radicals, which are damaging to various cellular organelles, are formed. one of the most sensitive organelles to drought stress and free radicals is chloroplast (kaya et al., 2003). g. etunicatum fungi and pseudomonas fluorescence bacteria, by increasing antioxidant content and antioxidant enzyme activities, cause a loss of detrimental free radicals and consequently preserve plant chlorophyll content (molinari et al., 2007). they also increase the absorption of elements such as magnesium, iron, and nitrogen that lead to the plant’s production of more chlorophyll (molinari et al., 2007). leaf temperature our results indicate that the increase of irrigation periods led to an increase of leaf temperature. the presence of g. etunicatum fungi decreased leaf temperature while pseudomonas fluorescence bacteria inoculation had no effect on it. analysis of the effects of interaction between inoculation of g. etunicatum fungi and pseudomonas fluorescence bacteria on leaf temperature revealed that the minimum leaf temperature was with simultaneous inoculation of fungi without bacteria and a two-day irrigation period (31.47°c). likewise, the maximum temperature was observed in the treatment without fungi and bacteria inoculations and a six-day irrigation periods (table 3). rate of net photosynthesis analysis of the net photosynthesis rate of mexican lime revealed that it declined with the increase of irrigation periods: the maximum and minimum rates were observed with twoand six-day irriirrigation periods (day) ge + ge pf + pf pf + pf 2 634.7 a 574.3 b 565.4 b 529.6 c 4 578.2 b 512.9 cd 511.7 cd 441.7 f 6 503.8 d 484.6 e 479.3 e 320.5 g table 2 effects of g. etunicatum fungus and pseudomonas florescence bacteria inoculations on mexican lime leaf chlorophyll content with different irrigation periods (spad value) in each column, means followed by different letters differ significantly at p≤0.05 according to duncan's multiple range test. ge + = g. etunicatum presence; ge = g. etunicatum absence. pf + = pseudomonas florescence presence; pf = pseudomonas florescence absence. irrigation periods (day) ge + ge pf + pf pf + pf 2 74.7 a 73.5 ab 72.9 ab 72.6 ab 4 71.9 b 70.3 b 69.5 bc 67.6 c 6 70.2 b 69.5 bc 68.4 bc 66.3 c table 1 effects of g. etunicatum fungus and pseudomonas florescence bacteria inoculations on mexican lime leaf rwc with different irrigation periods (%) in each column, means followed by different letters differ significantly at p≤0.05 according to duncan's multiple range test. ge + = g. etunicatum presence; ge = g. etunicatum absence. pf + = pseudomonas florescence presence; pf = pseudomonas florescence absence. shahsavar et al. effects of glomus etunicatum and pseudomonas fluorescence on mexican lime under drought stress 43 gation periods, respectively. the results also indicated that the presence of g. etunicatum fungi and pseudomonas fluorescence bacteria increased the plants’ rate of net photosynthesis. analysis of the effects of interaction between inoculation of g. etunicatum fungi and pseudomonas fluorescence bacteria identified that the maximum rate was observed in simultaneous inoculation of both fungi and bacteria and with a two-day irrigation period (12.3 micromole/m2/s)(table 4). rate of transpiration analysis of the effects of interaction between inoculation of g. etunicatum fungi and pseudomonas fluorescence bacteria on mexican lime transpiration rate in plants grown with different irrigation periods identified that the highest rate was observed in simultaneous inoculation of both fungi and bacteria and a two-day irrigation period (10.25 micromole/m2/s). likewise, the minimum transpiration rate was observed in the treatment without fungi and bacteria inoculations and a six-day irrigation period. the overall results showed that the leaf transpiration rate increased with the increase of irrigation period (table 5). wu and xia (2006) specified that under drought stress conditions, g. versiforme fungi increase leaf water potential, photosynthesis rate, respiration rate, rwc, and stomatal conductance of mandarin seedlings; however, leaf temperature is decreased compared to plants without fungi. effects of irrigation period on leaf temperature, photosynthesis rate, and transpiration showed that with the increase of irrigation period, they all declined (figueiredo, 2008). this can be explained by the fact that under drought condition, more stomata are closed; with a loss of evaporation, the leaf surface loses less heat and the leaf temperature increases (dietz and foyer, 1986.). moreover, because of stomata closure, less water is lost and the transpiration rate decreases. it should be noted that stomata closure causes less carbon dioxide to enter into the leaf, resulting in a lower rate of photosynthesis (zhang et al., 2010). the presence of g . e t u n i c a t u m fungi and inoculation with pseudomonas fluorescence bacteria leads to better water absorption and higher drought stress tolerance, thus increasing the plant’s rate of photosynthesis. many studies have reported the effects of g. etunicatum fungi on increasing photosynthesis rate (johnson et al., 1986), increasing root hydraulic conductivity for water uptake (graham and syvertsen, 1984), and increasing transpiration rate (leyv and syvestern, 2006). root colonization percentage results of the present study showed that root colonization occurred in the presence of g. etunicatum fungi and p s e u d o m o n a s f l u o r e s c e n c e bacteria. moreover, an increase of irrigation period led to a decrease of root colonization percentage. analysis of the effects of interaction between inoculation of g. irrigation periods (day) ge + ge pf + pf pf + pf 2 32.59 de 31.47 e 33.16 d 33.05 d 4 34.25 d 33.87 d 34.92 c 36.50 b 6 35.94 b 36.35 b 36.28 b 38.41 a table 3 effects of g. etunicatum fungus and pseudomonas florescence bacteria inoculations on mexican lime leaf temperature with different irrigation periods (°c) in each column, means followed by different letters differ significantly at p≤0.05 according to duncan's multiple range test. ge + = g. etunicatum presence; ge = g. etunicatum absence. pf + = pseudomonas florescence presence; pf = pseudomonas florescence absence. table 4 effects of g. etunicatum fungus and pseudomonas florescence bacteria inoculations on mexican lime photosynthesis rate with different irrigation periods (micromole/m2/s) in each column, means followed by different letters differ significantly at p≤0.05 according to duncan's multiple range test. ge + = g. etunicatum presence; ge = g. etunicatum absence. pf + = pseudomonas florescence presence; pf = pseudomonas florescence absence. irrigation periods (day) ge + ge pf + pf pf + pf 2 12.3 a 11.6 ab 11.4 b 10.50 c 4 10.2 c 10.2 c 10.1 c 9.06 d 6 9.51 d 8.52 de 8.37 e 6.48 f table 5 effects of g. etunicatum fungus and pseudomonas florescence bacteria inoculations on mexican lime transpiration rate with different irrigation periods (micromole/m2/s) in each column, means followed by different letters differ significantly at p≤0.05 according to duncan's multiple range test. ge + = g. etunicatum presence; ge = g. etunicatum absence. pf + = pseudomonas florescence presence; pf = pseudomonas florescence absence. irrigation periods (day) ge + ge pf + pf pf + pf 2 10.25 a 9.96 b 10.07 ab 9.83 b 4 9.68 c 9.58 c 9.16 d 8.74 e 6 9.17 d 9.72 b 8.91 e 8.65 e adv. hort. sci., 2016 30(1): 39-45 44 etunicatum fungi and pseudomonas fluorescence bacteria on mexican lime percentage of root colonization revealed that the maximum percentage was observed in simultaneous inoculation of both fungi and bacteria with a two-day irrigation period (49.66%) (table 6). as previously mentioned, root colonization occurred only in the presence of g. etunicatum fungi and its percentage dropped with an increase in irrigation period. until now, no specific reason has been proposed for the reduction of colonization in drought stress conditions. probably water is one important element in fungi growth. the formation of secondary metabolites that prevent fungi growth in the plant roots is also a possible explanation. wu et al. (2006) reported that, in the case of citrus roots, the highest colonization percentage of mycorrhizal fungi occurs when the roots are not under drought stress conditions, which is consistent with the present study results. regarding other types of citrus, they found similar results in their subsequent studies (wu et al., 2006, 2008). in order to utilize root colonization of fungi and bacteria capacities in sustainable agriculture, there must be appropriate establishment of both fungi and bacteria on the plant roots. accordingly, observation of mexican lime root colonization percentage in the current investigation was a very important and valuable factor. in addition, specification of the appropriate colonization percentage for effective interaction between fungi and plant is an important issue. 4. conclusions the results of the current study and other research projects in this field have shown the practical and scientific advantages of g. etunicatum fungi and pseudomonas fluorescence bacteria applications in arid or semi-arid areas. the synergistic effect, which was observed between g. etunicatum fungi and pseudomonas fluorescence bacteria, could increase most of the plant characteristics such as leaf chlorophyll content, net photosynthesis and transpiration rates, leaf rwc and root colonization percentage which provide the material energy and information for plant growth, development and reproduction. pseudomonas fluorescence bacteria could reduce the negative effects of drought stress less than g. etunicatum fungi. using their hyphae and extra/intra root mycelia, g. etunicatum fungi expand root evacuation area for better uptakes of water and nutrients. arbuscular mycorrhizal fungi can be integrated in soil management to achieve low-cost sustainable agricultural systems, offering a sustainable and environmentally safe treatment to improve drought tolerance. consequently, using these fungi as well as pseudomonas fluorescence bacteria can be very effective in achieving the goals of sustainable agriculture. references abbaspour h., saeidsarand s., afshar h., 2011 i m p r o v i n g d r o u g h t t o l e r a n c e o f pistacia vera l . seedlings by arbuscular mycorrhiza under greenhouse conditions. j. medicinal plants research, 5: 70657072. augé r.m., 2001 water relations, drought and vesiculararbuscular mycorrhizal symbiosis. micorrhiza, 11: 3-42. bethlenfalvay g.j., brown m.s., ames r.n., thomas r., 1988 effects of drought on host and endophyte development in mycorrhizal soybeans in relation to water use and phosphate uptake. physiol. plant., 72: 565-571. bryla d.r., duniway j.m., 1997 effects of mycorrhizal infection on drought tolerance and recovery in safflower and wheat. plant and soil., 197(1): 95-103. clarke j.m., craig t.n., 1982 excised-leaf water retention capability as an indicator of drought resistance of triticum genotypes. can. j. plant sci., 62: 571-578. davies f.t., potter j.r., linderman r.g., 1993 drought resistance of mycorrhizal pepper plants independent of leaf p concentration response in gas exchange and water relations. physiologia plantarum., 87: 45-53. dietz k.j., foyer c., 1986 the relationship between phosphate status and photosynthesis in leaves. planta, 167(3): 376-381. faber b.a., zasoske r.j., munns d.n., shackel k., 1991 table 6 effects of g. etunicatum fungus and pseudomonas florescence bacteria inoculations on mexican lime root colonization percentage with different irrigation periods (%) irrigation periods (day) ge + ge pf + pf pf + pf 2 49.66 a 42.36 ab 0 d 0 d 4 38.73 b 36.87 bc 0 d 0 d 6 34.24 c 35.12 c 0 d 0 d in each column, means followed by different letters differ significantly at p≤0.05 according to duncan's multiple range test. ge + = g. etunicatum presence; ge = g. etunicatum absence. pf + = pseudomonas florescence presence; pf = pseudomonas florescence absence. shahsavar et al. effects of glomus etunicatum and pseudomonas fluorescence on mexican lime under drought stresss 45 a method for measuring hyphal nutrition and water uptake in mycorrhizal plants. can. j. bot., 69: 87-94. figueiredo v.b., 2008 alleviation of drought stress in the common bean (phaseolus vulgaris l.) by co-inoculation with paenibacillus polymyxa and rhizobium tropici. appl. soil ecol., 40: 182-188. filella i., llusia j., pin j.o., pen j.u., 1998 leaf gas exchange and fluorescence of phillyrea latifolia, pistacia lentiscus and quercus ilex saplings in severe drought and high temperature conditions. environ. exp. bot., 39: 213-220. graham h., syvertsen j.p., 1984 influence of vesiculararbuscular mycorrhiza on the hydraulic conductivity of roots of two citrus rootstocks. new phytologist., 97: 277-284. haley s.d., quick j.s., morgan j.a., 1993 excised-leaf water status evaluation and associations in field-grown winter wheat. can. j. plant sci., 73:55-63. johnson c.r., duke e.r., koch k.e., 1986 accumulation of phosphorus, dry matter and betaine during nacl stress of split-root citrus seedlings colonized with vesicular-arbuscular mycorrhizal fungi on zero, one or two halves. new phytologist., 104: 583-590. kaya c., higgs d., kirnak h., tas i., 2003 mycorrhizal colonization improves fruit yield and water use efficiency in watermelon (citrullus lanatus thunb.) grown under well-watered and water-stressed conditions. plant soil., 253: 287-292. kormanik p.p., mcgraw a.c., 1982 quantification of vesicular-arbuscular mycorrhizae in plant root, pp. 3745. in: schenk n.c. (ed.) methods and principles of mycorrhizal research. the american phytopathological society, st. paul, mn, usa, pp. 244. leyv y., syvestern j.p., 2006 effect of drought stress and vesicular arbuscular mycorrhiza on citrus transpiration and hydraulic conductivity of roots. j. plant physiol., 85: 25-31. manette a.s., richard c.j., carver b.f., mornhinweg d.w., 1988 water relations in winter wheat as drought resistance indicators. crop sci., 28: 526-531. miller m.h., 2000 arbuscular mycorrhizae and the phosphorus nutrition of maize: a review of guelph studies. can. j. plant sci., 80: 47-52. molinari h.b., marur c.j., daros e., marilia campos k.f., carvalho j.f., filho j.c., pereira l.f., vieira l.g., 2007 evaluation of the stress inducible production of proline in transgenic sugarcane (saccharum spp.): osmotic adjustment, chlorophyll fluorescence and oxidative stress. physiol. plant., 130: 218-229. porcel r., ruiz-lozano j.m., 2004 arbuscular mycorrhizal influence on leaf water potential, solute accumulation, and oxidative stress in soybean plants subjected to drought stress. j. exp. bot., 55: 1743-1750. rodriguez h., fraga r., 1999 phosphate solubilizing bacteria and their role in plant growth promotion. biotech. adv., 17: 319-339. shaharoona b., naveed m., arshad m., zahir z.a., 2008 fertilizer-dependent efficiency of pseudomonads for improving growth, yield, and nutrient use efficiency of wheat (triticum aestivum l.). appl. microbiol. biotechnol., 79: 147-155. vivas a., juan b., ruiz-lozano m., 2003 influence of a bacillus sp. on physiological activities of two arbuscular mycorrhizal fungi and on plant responses to peginduced drought stress. mycorrhiza, 13: 249-256. wu q.s., xia r.x., 2006 arbuscular mycorrhizal fungi influence growth, osmotic adjustment and photosynthesis of citrus under well-watered and water stress conditions. j. plant physiol., 163: 417-425. wu q.s., xia r.x., zou y.n., 2006 reactive oxygen metabolism in nonmycorrhizal citrus (poncirus trifoliata) seedlings subjected to water stress. j. plant physiol., 163: 1101-1110. wu q.s., xia r.x., zou y.n., 2008 improved soil structure and citrus growth after inoculation with three arbuscular mycorrhizal fungi under drought stress. euro. j. soil biol., 44(1): 122-128. wu q.s., zou y.n., 2009 the effect of dual application of arbuscular mycorrhizal fungi and polyamin upon growth and nutrient uptake of trifolia orange seedling. not. bot. agrobot. cluj., 37(2): 95-98. wu q.s., zou y.n., he x., luo p., 2011 arbuscular mycorrhizal fungi can alter some root characters and physiological status in trifoliate orange (poncirus trifoliata l. raf.) seedlings. plant growth regul., 65: 273-278 yoshiba y., kiyosue t., nakashima k., yamaguchi-shinozaki k., shinozaki k., 1997 regulation of levels of proline as an osmolyte in plants under water stress. plant cell physiol., 38: 1095-1102. zhang y., zhong c.l., chen y., chen z., jiang q.b., wu c., pinyopusarek k., 2010 improving drought tolerance of causarina equisetifilia seedlings by arbuscular mycorrhizal under glasshouse conditions. new for., 40(3): 261-271. impaginato 121 1. introduction acquisition of mineral nutrients is important to plant growth and productivity. the ability of plants to acquire nutrients may be associated with root colonization with arbuscular mycorrhizal fungi (amf) (clark and zeto, 1996). amf are obligatory biotrophic symbionts occurring in nearly all natural and agricultural soils and commonly colonize roots of many plant species (smith and read, 1997). acquisition of mineral nutrients by plants with amf depends on factors such as soil ph, soil nutrient deficiencies, amf isolate, and plant species (sylvia and williams, 1992). previous studies showed a positive response of cotton to amf (liu et al., 1994; defeng et al., 1998; ibrahim, 2010). in subsistence agriculture systems, it is important to use indigenous amf that are ecotypically adapted to the site (davies et al., 2005). native amf can grow and function better in soils from which they are isolated, e.g. agricultural systems (calvente et al., 2004). phosphogypsum (pg) is the main by-product of the industrial production of phosphoric acid by treatment of rock phosphate with sulfuric acid. calcium sulfate is the dominant component in pg. pg contains the radioactive materials 226ra and 210po, phosphorus, silicon, fe, cu, and f– (al-masri et al., 2004). studies suggest that pg can be used in the improvement of soil structure, plant growth and agricultural production (alcordo and rechcigl, 1993), enhancing seedling emergence (vyshpolsky et al., 2010), and increasing available s and p (al-oudat et al., 1998). the use of pg as a fertilizer in agriculture has been practiced in many parts of the world (enamorado et al., 2009) without constituting environmental hazards to soil and crop tissue (al-oudat et al., 2011). application of pg (a poorly soluble source of p) to soil may become available to plants by solubilization f r o m a m f ( a l k a r a k i a n d a l o m o u s h , 2 0 0 2 ) . solubilization of pg might insure a continuous supply of p without inhibiting root amf colonization (cui et al., 2014). in addition, enhanced acquisition of nutriadv. hort. sci., 2016 30(3): 121-128 doi: 10.13128/ahs-20247 arbuscular mycorrhizal isolate and phosphogypsum effects on growth and nutrients acquisition of cotton (gossypium hirsutum l.) m. ibrahim department of agriculture, atomic energy commission of syria, p.o. box 6091, damascus, syria. key words: arbuscular mycorrhizal fungi, gossypium hirsutum, indigenous, phosphogypsum. abstract: cotton was grown in pots with added phosphogypsum (pg) to evaluate the effect of indigenous arbuscular mycorrhizal fungi (amf) and phosphogypsum on cotton growth and acquisition of phosphorus (p), potassium (k), calcium (ca), manganese (mn), iron (fe), copper (cu), and zinc (zn). amf isolate was a mixture of glomus intraradices, glomus viscosum, and glomus mosseae previously isolated from a cotton field. shoot dry biomass was enhanced significantly by both indigenous amf and pg. shoot dry biomass and seed cotton yields were enhanced by the amf and pg combination and even more when pg in compost was added to mycorrhizal plants. p content in amf with pg and in amf with pg/compost treated plants was, respectively, 209.3 and 278.7%, significantly higher than control. acquisition of k, ca, and micronutrients was significantly enhanced by the combination of amf and pg. the treatment of amf with pg/compost induced the highest contents in mn, fe, cu and zn which were found to be, respectively, 287, 201, 192.8, and 171% higher compared to control. results indicate that cotton growth responded to indigenous amf in soils amended with pg. combination of amf with pg added in compost can ensure satisfactory benefits for cotton growth in low input, sustainable cropping systems. (*) corresponding author: ascientific@aec.org.sy received for publication 21 april 2016 accepted for publication 1 july 2016 copyright: © 2016 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2016 30(3): 121-128 122 ents by amf plants in combination with pg has been reported (al-karaki and al-omoush, 2002; bai et al., 2011). compost increases soil organic carbon (el mrabet et al., 2014) and could increase the release of micronutrients in the soil, making them more available to plants (habashy et al., 2008; jan et al., 2014). on the other hand, addition of pg during manure composting decreased the amount of ammonia lost by volatilization (prochnow et al., 1995). therefore, addition of compost and the introduction of mycorrhizal technology may become an effective way of applying pg to soils with p deficiency. the objective of this research was to determine the effects of indigenous amf, in combination with syrian pg alone or integrated in compost, on growth and mineral nutrients acquisition of a syrian cotton variety. 2. materials and methods the experiment was conducted in pots during the summer season (may-september 2013) at deral h a j a r res ea rc h s t a t i o n , l o c a t ed s o u t h ea s t o f damascus, syria (33°21’ n, 36°28’ e) at 617 m above sea level. the area is located within an arid region in which the total annual precipitation is 120 mm. sandy clay loam soil was air dried, sieved to pass a 3 mm screen, and pasteurized at 5 kgy of gamma ray (gr) with 60co source using a gamma irradiator (robo, russa). phosphogypsum (pg) was previously collected from the area near the phosphoric acid factory in homs (180 km n of damascus). a total of 20 composite samples of pg were obtained from the levels corresponding to pile ages of approximately 1, 3-6 and 7-12 years, from the top, middle and bottom layers, respectively (each sample weighed 1 kg). pg samples were ground, homogenized, and sieving through a 0.5 mm sieve. before planting, soil was mixed with pg alone (at a rate of 30 g kg-1 dry soil) and pg integrated in compost (pg/compost mixture was prepared to be applied at a rate of 30 g of compost plus 30 g of pg per kg dry soil). compost was pasteurized at 120°c for 20 min in the autoclave. apart from pg, no chemical fertilizers were added during the experiment. mycorrhizal inoculum was a mixture of glomus intraradices (schenck & smith), glomus viscosum (nicolson), and glomus mosseae (nicol. & gerd.) gerd. & trappe. amf inoculum was previously isolated (ibrahim, 2010) from a cotton field at der-alhajar research station and multiplied in pot cultures using onion (allium cepa) as a host. the inoculum consisted of fragments of onion root and spores mixed with soil. half of the pots were inoculated with amf inoculum (100 g per pot) at the time of sowing. nonmycorrhizal pots were prepared by mixing the same amount of sterilized amf inoculums. the treatments were: t1, no amf inoculation without pg; t2, amf inoculation without pg; t3, no amf inoculation with pg; t4, amf inoculation with pg; t5, no amf inoculation with pg/compost; and t6, amf inoculation with pg/compost. seeds of cotton (gossypium hirsutum cv. aleppo 33/1) were sterilized in 20% naclo for 1 min and subsequently rinsed with sterilized water. they were then sown five per pot and placed to grow under natural conditions. ten days after emergence, seedlings were thinned to one per pot; the roots of discarded plants were left in the soil to avoid removing the amf inoculum. plants were watered with tap water as needed. growth parameters, such as plant height and fresh weight, were measured at the physiological maturity stage. the total shoot dry weights were measured after oven drying to constant weight at 70°c. boll weight and number of mature bolls per plant at the first handpicking were recorded. the seed cotton yield and percent lint of each plant was determined at one handpicking for all treatments. the vegetative portion of the plants was ground to a fine powder (0.5 mm). nitrogen was determined using the kjeldahl method and phosphorus was determined colorimetrically using a spectrophotometer (thermo spectronic, uk), while determination of ca, k, fe, zn, cu, mn was performed by x-ray fluorescence (xrf). root samples were rinsed free of soil, cut into 1 cm fragments, thoroughly mixed, cleared with koh and stained with acid fuchsin in lactoglycerol. percent root colonization and percent root length colonized by amf were determined micros c o p i c a l l y u s i n g a g r i d l i n e i n t e r c e p t m e t h o d (giovannetti and mosse, 1980). the experimental design was randomized complete blocks with four replications. data were subjected to analysis of variance by the sas program (sas institut inc, 2004) and means were compared using the least significant difference (lsd) test at a probability level of p≤0.05. ibrahim am isolate and phosphogypsum effects on cotton 123 3. results addition of pg to the soil significantly decreased ph (table 1). amf root colonization was between 22.8 and 30.8% regardless of pg addition; the percent was higher for plants grown without added pg t h a n a d d e d p g a l o n e . a d d i n g p g i n c o m p o s t increased the percent of amf root colonization of cotton plants (table 1). the percentage of mycorrhizal root length was 66.3, 54.5 and 73.3% in the amf plants, amf plus pg, and amf with pg/compost treated plants, respectively (table 1). no amf root colonization was noted for plants grown without amf. significant differences between mycorrhizal and nonmycorrhizal plants were noted for shoot dry biomass regardless of pg addition (table 1). shoot dry biomass was significantly enhanced by indigenous amf and when the mixture of pg/compost was added to soil. application of pg to soil significantly increased shoot dry biomass for both mycorrhizal and nonmycorrhizal plants. shoot fresh weight significantly increased in amf inoculated and pg treated plants in comparison to the control (table 1). plant height at harvest was between 38.3 and 73.8 cm, and it was significantly enhanced by amf inoculation and pg addition compared to control. the maximum plant height was observed for mycorrhizal cotton plants grown with pg/compost mixture. the growth response of cotton plants to amf, pg, and amf plus pg treatments increased by 59.6, 49.4, and 76.3% over control, respectively (table 2). in addition, the growth response of cotton to the combination of amf with pg/compost was higher by 118.9% over control (table 2). yield components of cotton under different treatments are shown in table 2. the number of bolls per plant was significantly increased by both amf and pg in comparison with control. the plants showed the highest number of bolls when pg/compost mixture was added to amf plants (t6). the increase in boll number led to an increase in seed cotton yield, which was improved by both amf and pg. seed cotton yield varied between 50.83 g plant-1 (4574 kg ha-1 on the basis of a density of nine plants m-2) and 18.38 g plant-1 (1654 kg ha-1). the highest seed yield of cotton was observed in amf plants with added pg/compost. boll weight was generally higher in amf plants and pg treated plants than control and it was significantly higher with the amf plus pg/compost treatment compared to other treatments. percent lint varied between 47.3 and 34.3%, and amf inoculation increased it significantly. in addition, lint percentage was significantly increased by pg and this increase was clearly noted when pg was contained in compost. n and p concentrations in the vegetative portions of plants were significantly affected by amf inoculation (table 3, fig. 1). the concentrations of n and p table 1 experimental soil ph, mycorrhizal root colonization, and some growth parameters of cotton plants grown with different treatments of arbuscular mycorrhizal fungi (amf) and added phosphogypsum (pg) treatment amf colonization (%) mycorrhizal root length (%) soil ph plant height (cm) fresh biomass (g plant-1) dry biomass (g plant-1) control 0 0 8.1 a 38.3 e 141.72 c 44.40 e amf 24.60 b 66.30 b 8.1 a 55.0 d 256.30 ab 70.38 cd pg 0 0 7.5 b 58.0 cd 240.86 b 65.79 d amf+pg 22.80 bc 54.50 bc 7.5 b 62.0 bc 295.87 a 78.05 b pg/compost 0 0 7.2 c 64.3 b 255.43 ab 72.97 bc amf+pg/compost 30.80 a 73.30 a 7.1 c 73.8 a 316.52 a 96.64 a mean values within columns followed by different letters are significantly different at p<0.05. table 2 growth response and some yield components of cotton plants grown with different treatments of arbuscular mycorrhizal fungi (amf) and added phosphogypsum (pg) treatment growth response (%) boll number (per plant) boll weight (g) seed cotton yield (g plant-1) lint (%) control 0 5.3 c 4.9 c 18.4 d 34.3 f amf 59.6 bc 7.8 b 5.1 b 32.9 c 37.9 e pg 49.4 c 7.3 b 5.2 b 29.8 c 42.3 d amf+pg 76.3 b 8.3 b 5.3 b 36.9 b 42.9 c pg/compost 64.7 bc 8.0 b 5.5 b 36.5 b 46.4 b amf+pg/compost 118.9 a 9.5 a 6.1 a 50.8 a 47.3 a mean values within columns followed by different letters are significantly different at p<0.05. values are mean (n= 4). growth response (%)= (dwamf dwcontrol) x100/dwcontrol. lint (%)= (lint weight/seed cotton weight) x 100. seed cotton= seed + lint. table 3 concentrations of k, ca, and micronutrients in vegetative portion of cotton plants grown with different treatments of arbuscular mycorrhizal fungi (amf) and added phosphogypsum (pg) treatment k (mg g-1 dm) ca (mg g-1 dm) mn (µg g-1 dm) fe (µg g-1 dm) cu (µg g-1 dm) zn (µg g-1 dm) control 22.28 c 30.42 e 85.05 c 593.8 d 3.63 d 15.23 d amf 28.57 b 33.44 d 127.75 b 676.3 c 4.62 c 18.78 b pg 23.19 c 34.65 d 104.75 bc 688.3 c 3.78 d 16.28 cd amf+pg 29.44 b 40.28 b 154.75 a 797.5 b 4.69 c 20.58 a pg/compost 29.61 b 36.72 c 116.25 b 766.0 b 5.12 bc 16.68 c amf+pg/compost 32.67 a 43.61 a 172.75 a 930.8 a 5.59 a 21.88 a mean values within columns followed by different letters are significantly different at p<0.05. adv. hort. sci., 2016 30(3): 121-128 124 were generally higher for mycorrhizal than for nonmycorrhizal plants. pg significantly increased n concentration only when combined with compost for nonmycorrhizal (t5) and mycorrhizal plants (t6), while it significantly increased p concentration in nonmycorrhizal and mycorrhizal plants. the role of the combination of amf and pg in improving plant p content was noted (table 4). data indicate that maximum plant p contents of 107.8 and 131.9 mg plant-1 were found in amf plus pg and amf with pg/compost treatments, respectively, which was significantly higher (p≤0.05) by 209.3 and 278.7%, respectively, over control (table 5). the concentrations of k and ca were significantly higher in the vegetative portion of mycorrhizal compared to nonmycorrhizal plants regardless of pg (table 3). pg significantly increased ca concentration in nonmycorrhizal and mycorrhizal plants; added alone it had no significant effect on k concentration in either group. the concentrations of both elements increased significantly when pg/compost mixture was added to mycorrhizal plants. higher concentrations of mn, fe, cu, and zn were noted for mycorrhizal than for nonmycorrhizal plants (table 3) and pg significantly increased fe concentration. mn and zn concentrations were increased by pg only in amf plants while pg had no significant effect fig. 1 concentrations of n and p in vegetative portion of cotton plants grown at different treatments (t1= no amf without pg; t2= amf without pg; t3= no amf with pg; t4= amf with pg; t5= no amf with pg/compost; and t6= amf with pg/compost). table 4 contents of nutrients in cotton plants grown with different treatments of arbuscular mycorrhizal fungi (amf) and added phosphogypsum (pg) treatment nutrient content (mg plant-1) n p k ca mn fe cu zn control 708.0 e 35.2 e 465.3 e 634.0 e 1.77 d 12.5 d 0.08 d 0.32 e amf 1165.7 c 86.5 c 884.8 c 1036.8 c 3.97 c 21.0 c 0.14 c 0.58 c pg 890.4 d 45.2 d 595.0 d 887.5 d 2.69 d 17.6 c 0.10 d 0.42 d amf+pg 1331.8 b 107.8 b 1015.0 b 1388.5 b 5.35 b 27.5 b 0.16 c 0.71 b pg/compost 1402.7 b 82.3 c 1013.7 b 1262.0 b 4.02 c 26.5 b 0.18 bc 0.57 c amf+pg/compost 1627.8 a 131.9 a 1276.5 a 1705.1 a 6.76 a 36.4 a 0.22 a 0.85 a mean values within columns followed by different letters are significantly different at p<0.05. table 5 percentage change in nutrient contents (nc) due to pg amendment and amf inoculation of cotton plants treatment nutrient content change (%) n p k ca mn fe cu zn amf 65.4 c 148.5 c 91.8 b 65.2 c 128.6 c 73.3 bc 90.6 b 85.0 c pg 25.9 d 30.0 d 28.2 c 40.6 c 56.2 d 44.4 cd 29.3 c 31.8 d amf+pg 89.1 bc 209.3 b 119.1 b 120.9 b 207.3 b 126.3 b 115.6 b 123.9 b pg/compost 97.7 b 135.6 c 118.5 b 100.0 b 129.2 c 115.3 b 87.4 b 80.4 c amf+ pg/compost 130.9 a 278.7 a 177.5 a 171.2 a 287.1 a 201.1 a 192.8 a 171.0 a data in the same column followed by the same letter are not significantly different (p<0.05). nutrient content (nc) change=(ncamf ncnonamf)x100/ncnonamf. ibrahim am isolate and phosphogypsum effects on cotton 125 on zn and mn concentration in nonmycorrhizal plants. also, pg had no significant effect on cu concentration in either amf or non-amf plants. the highest concentrations of mn, fe, cu, and zn were observed at amf plus pg/compost treated plants compared to other treatments. plant contents of k, ca, mn, fe, cu, and zn were significantly higher for mycorrhizal than for nonmycorrhizal plants (table 4). the data revealed that maximum plant uptake of k, ca, mn, fe, cu, and zn was found in the treatment of indigenous amf with pg/compost, which was significantly (p≤0.05) higher by 177.5, 171.2, 287.1, 201.1, 192.8, and 171%, respectively, over control (table 5). 4. discussion and conclusions the soil p concentration in this study was low (3 mg kg-1), and this nutrient normally has to be added to this soil to provide sufficient p for plant growth. addition of poorly soluble forms of p, such as phosphogypsum, to soil had no negative effect on amf root colonization, as was reported by cui et al. (2014). this may be because amf root colonization often depends on given amounts of soluble p in the soil at the time of root colonization (stribley et al., 1980). in particular, greater root infection was found with amf inoculation plus pg/compost treatment and it was possibly due to the improvement of the rooting zone environment which stimulated better root proliferation (nagahashi et al., 1996; van der heijden and kuyper, 2001). soil ph affects the availability of nutrients and how the nutrients react with each other. application of pg to soil lowered soil ph, a result which is in agreement with literature reports of previous studies (al-karaki and al-omoush, 2002; lee et al., 2009). the lower soil ph caused by pg might be attributed to the release of phosphoric acid and sulfuric acid contained in pg. the increase in cotton plant biomass by pg corroborates reports by zhang et al. (2014) who showed that amendment of pg significantly increased shoot biomass in tobacco, regardless of amf inoculation. according to quintero et al. (2014), increased dry matter of tomato by pg can be ascribed, at least in part, to an increase in water use efficiency. greater fresh biomass and plant height of inoculated cotton compared to control was noted in this study, which is in accordance with other earlier studies on cotton (afek et al., 1991; defeng et al., 1998). enhanced cotton growth with the combination of amf and pg agrees with previous studies conducted with different plant species such as wheat (al-karaki and al-omoush, 2002), maize (bai et al., 2011), tomato (cui et al., 2014), and shallot (gu et al., 2012). improved growth of amf and pg treated plants may have been due to improved soil p availability. the trend noted for n concentration and biomass of amf plus pg/compost treated plants might be due to the compost releasing its nitrogen gradually to the soil/crop to produce a greater number of leaves. strong mycorrhizal effects on cotton were also observed when looking at the nutrients uptake. higher k and ca in mycorrhizal than in nonmycorrhizal cotton is supported by liu et al. (2002) who reported that amf enhanced acquisition of the nutrients that move mainly by mass flow. pg/compost had a positive effect on ca and k concentration in m y c o r r h i z a l c o t t o n w h i c h c o u l d b e d u e t o a n improvement in soil organic matter and exchangeable ca and k by compost (adeleye et al., 2010). el mrabet et al. (2014) also showed that bio-compost improved soil k-extractable. increasing p uptake of plants due to amf inoculation has been widely reported (deguchi et al. 2007; sharif et al., 2009; ibrahim, 2010). in our study, increased p concentration and uptake by pg addition to mycorrhizal cotton is supported by zhang et al. (2015) who reported that pg amendment significantly increased the concentration and absorption of p in mycorrhizal and nonmycorrhizal tobacco plants. also, gu et al. (2012) found that p concentration in shallot was increased by increasing pg, and the combination of pg and amf colonization can improve p uptake by shallot to different degrees. our results show that indigenous amf increased the concentrations of cu, zn, fe, and mn in cotton; similar results were obtained in cotton inoculated with different species of amf (liu et al., 1994; ibrahim, 2010). our result regarding enhanced acquisition of p and micronutrients in amf cotton grown with pg is supported by al-karaki and alomoush (2002) in their work on mycorrhizal wheat grown with pg. the extension of amf hyphae, beyond the root zone, provides p and other nutrients to plants during growth stages. the ability of the hyphae to extend the root system should be especially beneficial in the case of cotton because its roots have a low density per unit soil volume (mcmichael, 1990). in this case, adv. hort. sci., 2016 30(3): 121-128 126 amf likely contributed p (and other mineral nutrients) from soil and pg particles with which roots would not make contact. high absorption of zn, cu, and fe may be due to greater p uptake by amf plants (clark and zeto, 1996; davies et al., 2005). on the other hand, the positive response of cotton to amf inoculation for nutrient concentration could be due to the effectiveness of the amf isolate in improving soil properties and nutrient availability. previous reports showed that improvement of plant growth and nutrients acquisition by the combination of pg and amf depends on compatibility between plant species, the rate of pg added, and the amf isolate. bai et al. (2011) reported that shoot growth of pg treated maize strain (40 g kg-1) was sign i f i c a n t l y e n h a n c e d w h e n i n o c u l a t e d w i t h diversispora spurcum, but was significantly inhibited when inoculated with glomus aggregatum. gu et al. (2012) reported that the treatment of pg40 addition with glomus mosseae inoculation had a significant effect in improving shallot biomass and p, s uptake. according to zhang et al. (2014), the combination of pg40 and g. aggregatum inoculation had the most desirable effects on tobacco growth. u n d e r t h e c o n d i t i o n s i n t h i s s t u d y , a d d e d pg/compost enhanced p and nutrient concentrations of mycorrhizal plants. previous reports have shown that pg application induced changes in soil chemical properties (decreased soil ph and enhanced ece, available p, so4, exchangeable k, ca and mg) (aloudat et al., 1998; lee et al., 2009). in addition, organic fertilizers and amf inoculation could improve soil physico-chemical properties (warnock et al., 2007). amf colonization enhances soil aggregation by exuding the glycoprotein, glomalin, from extraradical hyphae (wright and upadhyaya, 1998). the improved soil structure enhances air and water percolation, improves root system access to soil water and nutrients, and improves crop production (celik et al., 2004). therefore, the increase in nutrients noted in amf and pg/compost treatments could be due to improved soil structure and to increased release of nutrients in the soil, which become more available to the plant (habashy et al., 2008; jan et al., 2014). enhanced acquisition of nutrients and plant growth by amf and pg was reflected by increased yield and yield components of cotton. previous studies showed that pg increased grain yield of barley, wheat, and cotton (al-oudat et al., 2011). cui et al. (2014) reported that tomato yield of amf or amf plus pg seedlings were significantly higher than those of the non-mycorrhizal seedlings. al-karaki and alomoush (2002) reported that grain yield of wheat was enhanced by pg, and even more so when roots were colonized with amf. cotton inoculation with indigenous arbuscular mycorrhizal fungi (amf) and soil amendment with phosphogypsum (pg) enhanced nutrients acquisition from soil and improved growth and yield of cotton. however, mycorrhizal plants grown with pg and compost mixture had greater growth and yield than plants grown with pg alone. therefore, the combination of amf with pg added in compost can ensure satisfactory benefits for cotton growth and yield in low input, sustainable cropping systems. acknowledgements the author would like to thank the atomic energy commission of syria for encouragement and technical support. references adeleye e.o., ayeni l.s., ojeniyi s.o., 2010 effect of poultry manure on soil physic-chemical 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read d.j., 1997 mycorrhizal symbiosis. academic press, london, uk, pp. 606. stribley d.p., tinker p.b., snellgrove r.c., 1980 effect of vesicular-arbuscular mycorrhizal fungi on the relations of plant growth, internal phosphorus concentrations and soil phosphate analyses. j. soil sci., 31: 655-672. sylvia d.m., williams s.e., 1992 vesicular-arbuscular mycorrhiza and environmental stress, pp. 101-124. in: bethlenfalvay g.j., and r.j. linderman (eds.) mycorrhizae in sustainable agriculture. amer. soc. agron., madison, wi, usa, pp. 124. van der heijden e.w., kuyper t.w., 2001 does origin of mycorrhizal fungus or mycorrhizal plant influence adv. hort. sci., 2016 30(3): 121-128 128 effectiveness of the mycorrhizal symbiosis? plant soil, 230: 161-174. vyshpolsky f., mukhamedjanov k., bekbaev u., ibatullin s., yuldashev t., noble a.d., mirzabaev a., aw-hassan a., qadir m., 2010 optimizing the rate and timing of phosphogypsum application to magnesium-affected soils for crop yield and water 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apple. the top producer of peaches is china, followed by the eu with italy, greece and spain being the biggest european producers (faostat, 2015). at present, the demand from the market to develop and introduce new varieties with different characteristics, which could make possible to expand cultivation areas and production calendars and improve technology with regards to production and post-harvest handling of these delicate fruits, is increasing. however, breeders have traditionally selected new cultivars mainly for external fruit traits (i.e. size and appearance), with organoleptic and nutritional characteristics being a secondary goal. in spite of this, fruit quality is fundamental for the acceptance of different cultivars by consumers, due to the high competition in the markets with the presence of numerous new varieties, other fruits and other foods (iglesias and echeverria, 2009). abbot (1999) indicates that food quality is a concept, which includes sensory, mechanical and functional properties as well as chemical composition and nutritional values. the latter is a key point as fruit has long been promoted for its health benefits in preventing various cancer and age-related diseases (bazzano et al., 2002; liu, 2003; casacchia and sofo, 2013). this is due to the presence of high added value bioactive compounds, named phytochemicals (iriti and faoro, 2006). these compounds have strong antioxidant properties that enable them to scavenge free radicals, donate hydrogen, chelate metals, break radical chain reactions, and quench singlet oxygen in vitro and in vivo (dai and mumper, 2010). all these properties enable them to act in the prevention of oxidative stress-related diseases (pandey and rizvi, 2009). among phytochemicals, the most abundant class present in fruit is that of polyphenols (manach et al., 2004). phenolic rings have the capacity to scavenge free radicals, first of all hydroxyl ones, by virtue adv. hort. sci., 2016 30(3): 175-181 doi: 10.13128/ahs-20280 comparative characterization of fruit quality, phenols and antioxidant activity of de-pigmented “ghiaccio” and white flesh peaches d. ceccarelli, c. talento, a. sartori, m. terlizzi, e. caboni, k. carbone (*) consiglio per la ricerca in agricoltura e analisi dell’economia agraria, crea, centro di ricerca per la frutticoltura, via di fioranello, 52, 00134 roma, italy. key words: antioxidant capacity, depigmented peaches, phenolic compounds, phytochemicals, prunus persica. abstract: quality traits and nutraceutical potential of new de-pigmented peaches (“ghiaccio”) were investigated and compared with those of four white flesh peaches. total soluble solids (tss) were measured by digital refractometer, and titratable acidity (ta) by volumetric titration. total phenolic (tpc) and anthocyanin content (tac) were analysed spectrophotometrically and the antioxidant capacity (ac) evaluated by dpph• assay. a strong influence of genotype on quality traits and phytochemical profile of “ghiaccio” peaches was observed. “ghiaccio” series showed, on average, a higher tpc content than that of white flesh peaches, both in flesh and peels (+129% and +14%, respectively). the peels of all peaches analysed were significantly richer in tpc than the flesh. tac was not detectable in de-pigmented genotypes; on the contrary, in the white flesh peaches, it was higher in the peels than in the flesh. ac correlated well with tpc. data confirm, for all peaches analysed, the influence of genotype and fruit tissue on the nutraceutical properties. among genotypes, the best candidates for “ghiaccio” peaches with enriched nutraceutical properties are the advanced selections gø and gx. observed differences in the nutraceutical potential among “ghiaccio” series may open new opportunities for breeding de-pigmented peach varieties with a higher nutritional value. (*) corresponding author: katya.carbone@crea.gov.it received for publication 14 july 2016 accepted for publication 16 november 2016 copyright: © 2016 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2016 30(3): 175-181 176 of the aromatic hydroxylation at the ortho-position (xia et al., 2010). these compounds are distributed in every part of the fruit in different ratios, focusing more in the peels (rich in anthocyanins, hydroxycinnamic acids and flavans) and in the seeds (rich in proanthocyanidins and flavans) (lachman et al., 2009). peaches, even though having a lower antiradical capacity than other fruits, are ones of the most important commodities consumed worldwide, both as fresh and processed product (i.e. fruit juice, jam or canned) (cantin et al., 2009). as a consequence, breeding programs aimed to increase peach nutraceutical value is desirable. “ghiaccio” peach series is a new type of peach variety, resulting from a breeding program c o n d u c t e d i n t h e p a s t y e a r s a t t h e f r u i t t r e e research centre of rome with the aim to obtain varieties with enhanced postharvest fruit characteristics and an improved resistance to disease and pests (nicotra et al., 2001). their progenitor is a stony hard-type peach cultivar, the korean “yumyeong” (kim et al., 1978), from which “ghiaccio” selections have been obtained by self-pollination (nicotra et al., 2001). peaches of “ghiaccio” series have different ripening times but similar pomological traits (nicotra et al., 2001). to the best of our knowledge, there is no phytochemical and nutritional characterisation of “ghiaccio” peaches in literature, making this study quite relevant, providing breeders and consumers with experimental data on this emerging varieties. hence, the aim of the present study was to characterise the quality and nutraceutical properties of these new genotypes by measuring their total polyphenolic and total anthocyanin content, and their relative antioxidant capacity. a comparison with four commercial white flesh peach cultivars was also p e r f o r m e d . t h e u l t i m a t e g o a l w a s t o s e l e c t “ g h i a c c i o ” p e a c h g e n o t y p e s w i t h e n h a n c e d nutraceutical traits, to provide breeders with new varieties having more healthful properties, making them competitive with other fruits known for their healthy properties. 2. materials and methods chemicals all used reagents were of analytical spectrophotometric grade (carlo erba, rome, italy). cyanidin chloride was purchased from extrasyntese (genay cedex, france). folin-ciocalteu reagent, malic acid, chlorogenic acid, 2,2-diphenyl-1-picrylhydrazyl radical (dpph•), and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox) were purchased from sigma-aldrich (milan, italy). plant materials peach fruits from five early-to-late ripening “ghiaccio” genotypes (table 1) and four white flesh peach cultivars were collected in the experimental orchards of the fruit tree research centre of rome (crea-fru, italy), at fully ripening phase. all plants (six years old) were grafted on the same rootstock (gf677), spaced at 4.5x2.5 m and standard pruning, drip irrigation and cultural practices were performed. for each genotype, 15 undamaged and disease-free fruits were collected in order to have three replications of five fruits each for the analysis. traits cultivar/selection ghiaccio ø (g ø) ghiaccio 1® (g1) ghiaccio x (gx) ghiaccio 2® (g2) ghiaccio 3® (g3) size (g) large (168) very large (178) very large (217) very large (205) very large (200) shape oblate oblate oblate oblate oblate shape of pistil end weakly depressed weakly depressed weakly depressed weakly depressed weakly depressed symmetry symmetric symmetric asymmetric symmetric symmetric prominence of suture weak weak weak weak weak ground colour cream white cream white cream white cream white cream white over colour present absent absent present absent hue of over colour pink pink density of pubescence medium sparse medium very sparse very sparse firmness of flesh very firm very firm very firm very firm very firm ground colour of flesh cream white cream white cream white cream white cream white sweetness high high high high high acidity low low low low low ripening time (y) -20 -8 -5 11 25 table 1 pomological and phenological traits of “ghiaccio” series (z) (z) data were detected as community of plant varity office (cpvo) descriptors. (y) the reference cv. rome star ripens in center italy between 25th-30th of july. ceccarelli et al. comparative characterization of de-pigmented “ghiaccio” and white flesh peaches 177 fruit quality attributes after harvesting, fruits were washed, stoned and homogenized, and the homogenate samples were analysed for total soluble solid (tss) content using a digital refractometer (refracto 30 px, mettler toledo, milan, italy); data are given as °brix. the method for analysis of titratable acidity (ta) was based on titration of the acids present in the fruit juice with sodium hydroxide (0.1 n). data are given as g malic acid l-1, since this is the dominant organic acid in peach (david et al., 1956). the ph value was measured using a digital ph-meter (785 dmp, methrom, milan, italy). every analysis was replicated three times. extraction of bioactive compounds fruits from the different genotypes were carefully separated in two different tissues: peel and flesh. the peel fraction was removed from the whole fruit with a sharp knife and immediately frozen with liquid nitrogen, placed in a plastic freezer bag, and stored at -80°c until evaluation. the flesh fraction consisted of a peeled wedge, which was chopped into small pieces, frozen in liquid nitrogen, and also stored at 80°c. samples (5 g) of the different fractions were extracted with a 25 ml hydro alcoholic solution (methanol:water= 70:30, v/v) acidified with hcl (0.005 n) and homogenized with an ultra-turrax blender (ultra turrax t25, ika, milan, italy) at 9000 rpm. then, the homogenates were allowed to stand for 2 hours at 37°c under magnetic stirring to rich a complete solvent extraction. extracts were centrifuged at 8400 rpm for 15 min at 5°c and then the obtained supernatants were analysed as follows. extractions were repeated on three independent samples of the initial homogenate to give triplicate readings. determination of total polyphenol content (tpc) tpc of both flesh and peel samples was determ i n e d u s i n g t h e f o l i n c i o c a l t e u ( f c ) m e t h o d (waterhouse, 2002). tpc was calculated from a calibration curve, using chlorogenic acid as a standard. results were expressed as milligrams of chlorogenic acid equivalents (cae) per 100 g fresh weight (fw). every analysis was replicated three times. determination of total anthocyanin content (tac) tac of both flesh and peel fractions was estimated according to the method of mondello et al. (2000). tac was calculated from a calibration curve, using cyanidin chloride (cc) as a standard. results were expressed on a fresh weight basis as milligrams of cc equivalents (cce) x 100 g fw. every analysis was replicated three times. measurement of antioxidant capacity (ac) ac was assessed by measuring the effect of the bioactive extracts on the content of 2,2-diphenyl-1, picrylhydrazyl radical (dpph•) according to brandwilliams et al. (1995). all the measurements were made in triplicate. ac was expressed as micrograms of trolox equivalents (te) x mg fw. statistical analysis data analysis was performed with spss 17.0 software (spss, inc., chicago, illinois). all measurements were performed at least in triplicate and data were reported, where not specified differently, as means ± standard error of the mean (se). an exploratory data analysis was made to check the data normal distribution (shapiro-wilkinson test) and the equality of variances (levene’s test). when these conditions were met (tss, ta, ac), data were subjected to one-way analysis of variance (anova) and comparisons between means were determined according to tukey’s hsd test. significant differences were accepted at p<0.05 and represented by different letters. when anova assumptions were violated (tpc), even after mathematical transformation of data, a n o n p a r a m e t r i c d a t a a n a l y s i s w a s c a r r i e d o u t (kruskal-wallis non parametric test) and significant mean differences were established using the mannwhitney test for independent and non-parametric procedures and a bonferroni’s correction to set the critical value for significance for each test. box-plots were used to display the range, median and distribution density of phytochemicals and ac in the peel and flesh of genotypes analysed. spearman’s correlation coefficient (ρ) was used to determine the correlation among variables in the non-parametric analysis (p<0.01). 3. results and discussion fruit quality attributes table 1 shows the main pomological and phenological traits of “ghiaccio” series analysed. ghiaccio means ice in italian to remind consumers that the colour of the fruit is white or pale cream. they are very different from the common peach type grown in europe and the u.s. these peaches are characterized by totally white cream skin and flesh, very firm flesh, high sugar content (up to 17°brix), 2025 days longevity on the tree, a great productivity and a noteworthy shelf life. moreover, this variety adv. hort. sci., 2016 30(3): 175-181 178 shows a great resistance against diseases, rottenness and pathogenic agents making it particularly suitable for organic practice (nicotra et al., 2001). the “ g h i a c c i o ” s e r i e s i n c l u d e s “ g h i a c c i o 1 ” ( g 1 ) , “ghiaccio 2” (g2) and “ghiaccio 3” (g3) genotypes, w h i c h h a v e b e e n a l r e a d y l i c e n s e d , w h i l e t w o a d v a n c e d s e l e c t i o n s , “ g h i a c c i o ø ” ( g ø ) a n d “ghiaccio x” (gx) are still under evaluation. all genotypes analysed were harvested between early july and late august and, generally, significant differences in quality traits were detected (table 2). in “ghiaccio” series, the highest tss level (15.2 °brix) was observed in g1 samples, while the lowest one in gø ones (9.1°brix). all samples analysed showed a tss content greater than 8 °brix, which represents the minimum tss content established by t h e e u t o m a r k e t p e a c h e s a n d n e c t a r i n e s [ c o m m i s s i o n r e g u l a t i o n ( e c ) n o . 1 8 6 1 / 2 0 0 4 , 10/28/2004]. moreover, “ghiaccio” genotypes showed tss similar to the white flesh peaches analysed. ta values ranged from 3.18 (g3) to 4.23 (g1) g malic acid l-1, with significant differences among selections (table 2). on average, these values are lower than that of the progenitor and “ghiaccio” series can be collocated in sub acidic peach varieties (crisosto et al., 2001). these results are relevant since the acceptance of new cultivars by consumers, which is the ultimate goal of breeders, is linked to the fruit quality. in fact, acid levels, expressed in terms of ph and ta, and sugar concentrations, reported as tss, affect flavour perception of the fruit influencing peach sensory profile and consumer acceptance of peach fruits (crisosto et al., 2001; crisosto and crisosto, 2005). in the present study, we also reported the tss/ta ratio as the relationship between these parameters has an important role in fruit consumer acceptance (diaz-mula et al., 2009). among “ghiaccio” peaches, tss/ta values ranged from 3.62 for g3 to 3.16 for g2, being the latter significant different from the other genotypes of the series. in the fresh market, consumers desire large shaped and flavourful fruit with a high sugar content and low to moderate acidity and these new genotypes appear to respond adequately to such requests. in this regard, “ghiaccio” series showed, on average, a ta content lower than that of the white flesh cultivars analysed, which is reflected in a higher value of the tss/ta ratio. bioactive compounds distribution within the peach genotypes and relative antioxidant capacity fruit antioxidant potential varies in relation to the phytochemical moieties present, and variations can occur among genotypes within a single species (van der sluis et al., 2001; cantin et al., 2009). moreover, it is well known that the content of phytochemicals can vary within different tissues (carbone et al., 2011). in figure 1, box-plots showed the distribution of polyphenols, and the antiradical activity between t h e p e e l a n d t h e f l e s h o f “ g h i a c c i o ” p e a c h e s analysed, independently from the genotype. tpc was significantly higher in the peach peel extracts than in the flesh ones (+103%). these data are consistent with those reported in the literature about the influence of the type of fruit tissue on the accumulation of nutraceutical substances (carbone et al., 2011; table 2 fruit quality attributes of genotype analysed (mean±se) tss= total soluble solids. ta= titratable acidity. nd = not determined. significant differences were accepted at p<0.05 and represented by different letters on the column, within the cultivars or within the groups (“ghiaccio” series and white flesh cultivars). genotype harvest date tss (°brix) ta (g l-1 malic acid) tss/ta ratio ghiaccio 1 july, 22nd 15.20±0.03 e 4.23±0.06 b 3.58±0.04 d ghiaccio 2 august, 11th 13.00±0.03 d 4.13±0.01 b 3.16±0.02 c ghiaccio 3 august, 25th 11.5±0.1 b 3.18±0.02 a 3.62±0.05 d ghiaccio ø july, 7th 9.10±0.07 a nd nd ghiaccio x july, 21st 12.10±0.07 c 3.43±0.04 a 3.54±0.06 d crizia june, 28th 9.6±0.4 a 7.96±0.03 c 1.21±0.05 a maria anna august, 4th 13.60±0.03 d 12.0±0.2 e 1.13±0.02 a redhaven bianca july, 21st 12.2±0.1 c 8.74±0.01 d 1.39±0.01 b silver late september, 8th 13.20±0.06 d 12.61±0.02 f 1.05±0.00 a "ghiaccio" series 12.2±0.5 a 3.7±0.1a 3.47±0.06 b white flesh cvs. 12.1±0.5 a 10.3±0.6 b 1.19±0.04 a ceccarelli et al. comparative characterization of de-pigmented “ghiaccio” and white flesh peaches 179 tomás-barberán et al., 2001). tables 3 and 4 show the flesh and peel phytochemical content and ac of different cultivars analysed. among “ghiaccio” genotypes, gø showed the highest tpc both in the peel and flesh (260 and 134 mg cae 100 g-1 fw, respectively), while g2 the lowest one (70 and 28 mg cae 100 g-1 fw, respectively) (tables 3 and 4). these findings highlight that tpc of “ghiaccio” series is related to the genotype, in agreement with those reported in literature, not only for other peaches but also for other fruit species (tomás-barberán et al., 2001; ceccarelli et al., 2016). interestingly, the average peel polyphenol content of “ghiaccio” series (175 mg cae 100 g-1 fw) was not significantly different from that of the white flesh cultivars analysed (table 3), while the average tpc of the flesh was significantly higher (table 4). obtained results emphasize the healthy properties of the flesh of these new de-pigmented peaches. as regards total anthocyanins, their content was not detectable in “ghiaccio” peaches both in the peel and the flesh. besides, white flesh peaches revealed the presence of these compounds in all tissue analysed. in agreement with the phytochemical data, genotype also influenced the ac of peaches analysed. among “ghiaccio” genotypes, the highest value of ac was found in gø (1.3 µg te mg-1 fw) and gx (1.5 µg table 3 phytochemicals and antioxidant capacity of the peel of genotypes analyzed (mean±se) genotype ac tpc tac ghiaccio 1 1.0±0.1 bc 232±6 e nd ghiaccio 2 0.53±0.08 a 70±2 a nd ghiaccio 3 0.7±0.2 ab 197±4 d nd ghiaccio ø 1.3 ±0.1 cd 260±4 f nd ghiaccio x 1.5±0.1 d 144±4 c nd crizia 1.34±0.03 cd 102±3 b 26.53±0.03 c maria anna 1.42±0.06 cd 260.9±0.7 f 34.55±0.06 d redhaven bianca 1.13±0.04 cd 100±3 b 19.70±0.05 a silver late 1.50±0.08 d 151±2 c 23.40±0.03 b “ghiaccio” series 1.00±0.08 a 175±14 a nd white flesh cvs. 1.35±0.04 b 154±14 a 26±2 ac= antioxidant capacity. tpc= total polyphenol content. tac= total anthocyanin content. nd = not determined. significant differences were accepted at p<0.05 and represented by different letters on the column, within the cultivars or within the groups (“ghiaccio” series and white flesh cultivars). table 4 phytochemicals and antioxidant capacity of the flesh of genotypes analyzed (mean±se) ac= antioxidant capacity. tpc= total polyphenol content. tac= total anthocyanin content. nd = not determined. significant differences were accepted at p<0.05 and represented by different letters on the column, within the cultivars or within the groups (“ghiaccio” series and white flesh cultivars). genotype ac tpc tac ghiaccio 1 0.286±0.007 cd 59±8 c nd ghiaccio 2 0.16±0.01 a 28±2 ab nd ghiaccio 3 0.325±0.007 d 126±2 d nd ghiaccio ø 0.308±0.007 d 134±4 d nd ghiaccio x 0.20±0.02 b 71±3 c nd crizia 0.256±0.005 c 23±2 a 7.54±0.01 c maria anna 0.157±0.003 a 38±2 b nd redhaven bianca 0.263±0.004 c 28±2 ab 1.67±0.01 a silver late 0.412±0.005 e 62±2 c 6.45±0.01 b “ghiaccio” series 0.25±0.01 a 86±8 b nd white flesh cvs. 0.27±0.02 a 38±3 a 4.1±0.9 fig. 1 box-plots for the phytochemicals and antioxidant capacity of different peach tissues. the line in the box indicates the median value of the data; the right and the left edges of the box respectively indicates the 75th and the 25th percentiles of the data set, the ends of the horizontal lines indicate the minimum and maximun data values; the point outside the box are outliers or suspected outliers. a) tpc= total polyphenol content (mg cae 100 g-1 fw). b) ac = antioxidant capacity (µg te mg-1 fw). outlier: value more than 1.5 and less than 3 box-lengths from end of box. adv. hort. sci., 2016 30(3): 175-181 180 te mg-1 fw) for what concern the peel and in gø (0.308 µg te mg-1 fw) and g3 (0.325 µg te mg-1 fw) for the flesh. on average, the antioxidant capacity of the peel was lower in “ghiaccio” series than in white flesh cultivars (table 3), probably due to the absence of anthocyanins in the peel of “ghiaccio” series. moreover, correlation analysis pointed out that the scavenging capacity against dpph• of peach extracts and tpc were significantly and positively correlated (ρ= 0.796; p<0.01). this result confirms previous reports on commercial white and yellow flesh cultivars, showing that phenolic compounds can be considered the main phytochemicals contributing to ac in peaches (vizzotto et al., 2007). 4. conclusions peach breeding has supplied a large number of improved cultivars each year to satisfy different market demands. nevertheless, there are some critical issues not yet fully addressed through breeding such as the increasing competition between peach and a broad range of other fruits, as well as the consumer eating habits, which are changed in the last years, with a growing emphasis on nutrition and health properties of food. to meet these challenges, it is necessary to explore new germplasm for the production of new cultivars with improved quality and nutritional characteristics as well as to provide fruits to the market for long periods of time. in this context, the “ghiaccio” series could be a right answer for breeders. the present study describes for the first time the quality traits, phytochemical composition and ac of five genotypes (three cultivars and two advanced selections) belonging to the “ghiaccio” peaches. data point out the key role played by the genotype also within the “ghiaccio” series, underling the importance of the varietal selection. among the genotypes analysed, the best candidates for a “ghiaccio” peach with enriched nutraceutical properties are the advanced selections gø and gx. in addition, data pointed out a higher nutraceutical potential of “ghiaccio” series than that of the commercial white flesh peaches analysed. finally, taking into account that the fruit characteristics (i.e. shape, colour and size) are very similar to each other, but with different ripening periods, “ghiaccio” genotypes, if grown together, would allow the producers to supply the markets with the same type of fruit for a long period of time (i.e. 75 days). references abbott j.a., 1999 quality measurement of fruits and vegetables. postharvest biol. technol., 15: 207-225. bazzano l.a., he j., ogden l.g., loria c.m., vupputuri s., myers l., whelton p.k., 2002 fruit and vegetable intake and risk of cardiovascular disease in us adults: the first national health and nutrition examination survey epidemiologic follow-up study. am. j. clin. nutr., 76: 93-99. brand-willams w., cuvelier m.e., berset c., 1995 use of a free radical method to evaluate antioxidant activity. lebensm. wiss.technol., 28: 25-30. cantin c.m., moreno m.a., gogorcena y., 2009 evaluation of the antioxidant capacity, phenolic compounds, and vitamin c content of different peach and nectarine (prunus persica (l.) batsch) breeding progenies. j. agric. food chem., 57: 4586-4592. carbone k., giannini b., picchi v., lo scalzo r., cecchini f., 2011 phenolic composition and free radical scavenging activity of different apple varieties in relation to the cultivar, tissue type and storage. food chem., 127(2): 493-500. casacchia t., sofo a., 2013 antioxidant compounds and nutraceutical benefits of mediterranean red fruit, pp. 107-136. in: carbone k. (ed.) cultivars: chemical properties, antioxidant activities and health benefits. nova science publisher, hauppagem, ny, usa, pp. 281. ceccarelli d., simeone a.m., nota p., piazza m.g., fideghelli c., caboni e., 2016 phenolic compounds (hydroxycinnamic acids, flavan-3-ols, flavonols) profile in fruit of italian peach varieties. plant biosystems, 150(6): 1370-1375. crisosto c.h., crisosto g.m., 2005 relationship between ripe soluble solids concentration (rssc) and consumer acceptance of high and low acid melting flesh peach and nectarine (prunus persica (l.) batsch) cultivars. postharvest biol. technol., 38: 239-246. crisosto c.h., day k.r., crisosto g.m., garner d., 2001 quality attributes of white flesh peaches and nectarines grown under california conditions. j. am. pomol. soc., 55: 45-51. dai j., mumper r.j., 2010 plant phenolics: extraction, analysis and their antioxidant and anticancer properties. molecules, 15: 7313-7352. david j.j., luh b.s., marsh g.l., 1956 organic acids in 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and antioxidant activity in grapes and wines. int. j. wine res., 1: 101-121. liu r.h., 2003 health benefits of fruit and vegetables are from additive and synergistic combinations of phytochemicals. am. j. clin. nutr., 78: 517s-520s. manach c., scalbert a., morand c., remesy c., jimenez l., 2004 polyphenols: food sources and bioavailability. am. j. clin. nutr., 79: 727-747. mondello l., cotroneo a., errante g., dugo g., dugo p., 2000 determination of anthocyanins in blood orange juices by hplc analysis. j. pharm. biomed. an., 23: 191-195. nicotra a., conte l., moser l., fantechi p., 2001 new types of high quality peaches: flat peaches (p. persica var. platicarpa) and “ghiaccio‟ peach series with long on tree fruit life. acta horticulturae, 592: 131-135. pandey k.b., rizvi s.i., 2009 current understanding of dietary polyphenols and their role in health and disease. curr. nutr. food sci., 5: 249-263. tomás-barberán f.a., gil m.i., cremin p., waterhouse a.l., hess-pierce b., kader a.a., 2001 hplcdad-esims analysis of phenolic compounds in nectarines, peaches and plums. j. agr. food chem., 49: 4748-4760. van der sluis a.a., dekker m., de jager a., wim jongen m.f., 2001 activity and concentration of polyphenolic antioxidants in: apple: effect of cultivar, harvest year, and storage conditions. j. agr. food chem., 49: 3606-3613. vizzotto m., cisneros-zevallos l., byrne d.h., 2007 large variation found in the phytochemical and antioxidant activity of peach and plum germplasm. j. am. soc. hortic. sci., 132: 344-340. waterhouse a.l., 2002 determination of total phenolics, pp. 1-8. in: wrolstad r.e., t.e. acree, e.a. decker, m.h. penner, d.s. reid, s.j. schwartz, c.f. shoemaker, d.m. smith, and p. sporns (eds.) current protocols in food analytical chemistry. john wiley & sons, inc., oxford, uk. xia e., deng g.f., guo y.j., li h.b., 2010 biological activities of polyphenols from grapes. int. j. mol. sci., 11(2): 622-646. impaginato 19 1. introduction milk thistle (silybum marianum l.) is one of the most important medicinal plants in the pharmaceutical industry worldwide, and is used in the production of flavonoids of the silymarin group (silybin, silidianin and silychristine) which are important in the modern pharmaceutical industry (ghavami and ramin, 2007). the origin of this plant has been reported to be the east mediterranean region (keville, 1991). water availability may influence physiological and biochemical properties and seed yield of this medicinal plant. water stress severely limits growth and yield of plants by reducing ground green cover (ghassemigolezani and ghasssemi, 2013), chlorophyll content of leaves, photochemical efficiency of photosystem ii (ghassemi-golezani and lotfi, 2012) and photosynthesis (munns et al., 2006). water stress during vegetative stages largely reduces plant height and biomass, while during reproductive stages it has the greatest negative impact on seed yield (ghassemigolezani et al., 2008). reports in oil crops indicated that water stress decreases oil and increases protein percentages of seeds. however, both oil and protein yields per unit area are decreased as a result of large reduction in seed yield per unit area due to water limitation (ghassemi-golezani and lotfi, 2013; ghassemi-golezani et al., 2015 b). some of the deleterious effects of environmental stresses on plant performance could be alleviated by foliar application of growth regulators such as salicylic acid (sa) (ghassemi-golezani et al., 2015 a). it has been reported that the exogenous application of sa induces plant tolerance to several abiotic stresses including drought tolerance in wheat (singh and usha, 2003), salinity tolerance in safflower (ghassemi-golezani and hosseinzadeh-mahootchi, 2015) and mung bean (ghassemi-golezani et al., 2015 a), heat tolerance in mustard (dat et al., 1998) and chilling tolerance in maize (janda et al., 1999). these studies suggest that sa may enhance the multiple types of stress tolerance in plants by interactive effects on several functional molecules. hayat et al. (2008) found that there was a significant increase in photosynthetic parameters, chlorophyll and proline contents, and antioxidant enzyme activities in sa treated tomato plants. loutfy et al. (2012) reported that sa induced drought tolerance and increased plant biomass, leaf relative water content, and the solute contents in four wheat cultivars. moreover, adv. hort. sci., 2017 31(1): 19-23 doi: 10.13128/ahs-20721 improving oil and flavonoid contents of milk thistle under water stress by salicylic acid k. ghassemi-golezani*, s. ghassemi, i. yaghoubian department of plant eco-physiology, faculty of agriculture, university of tabriz, tabriz, iran. key words: foliar application, plant biomass, seed yield, silybum marianum l., water deficit. abstract: adverse environmental conditions such as water deficit can limit production. however, some of these adverse effects may be overcome by application of plant growth regulators including salicylic acid (sa). thus, a field experiment was conducted in 2015 to evaluate the effects of sa (0 and 1 mm l-1) on yield components, seed yield and oil and flavonoid contents of milk thistle (silybum marianum l.) under different irrigation treatments (i1, i2, i3 and i4: irrigation after 70, 110, 150 and 190 mm evaporation from class a pan, respectively). the experiment was arranged as split-plot based on randomized complete block (rcb) design in three replicates. irrigation treatments and sa levels were located in the main and sub plots, respectively. the results indicated that plant biomass, seeds per plant, 1000 seed weight, seed yield per unit area and harvest index of milk thistle decreased as a consequence of water stress. oil percentage and yield were also reduced, but flavonoid content enhanced with increasing water deficit. all these traits were considerably augmented by foliar application of sa under non-stress and stressful conditions. therefore, it was conclude that sa can be used to improve field performance of milk thistle under different environmental conditions. (*) corresponding author: golezani@gmail.com received for publication 11 october 2016 accepted for publication 20 december 2016 copyright: © 2017 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2017 31(1): 19-23 20 foliar spray of sa decreased the inhibitory effects of drought on phillyrea angustifolia (munné-bosch and penuelas, 2003). abd el-lateef gharib (2006) stated that oil content of basil and marjoram significantly increased with application of sa. however, the responses of some medicinal plants to sa treatment in stressful conditions were not documented so far. thus, this research was undertaken to evaluate the effects of foliar application of sa on seed yield and oil and flavonoid contents of milk thistle under different irrigation intervals. 2. materials and methods seeds of milk thistle (silybum marianum l.) were obtained from pakan bazr, isfahan, iran. the experiment was conducted in 2015 at the research farm of the faculty of agriculture, university of tabriz, iran (latitude 38° 05′ n, longitude 46° 17′ e, altitude 1360 m above sea level). the climate is characterized by mean annual precipitation of 245.75 mm per year, mean annual maximum temperature of 16.6°c and mean annual minimum temperature of 4.2°c. the field experiment was arranged as split-plot based on randomized complete block design in three replications, with irrigation intervals (i1, i2, i3, i4: irrigation after 70, 110, 150 and 190 mm evaporation from class a pan, respectively) in main plots and two levels of salicylic acid (sa; 0 and 1 mm l-1) in sub-plots. seeds of milk thistle were treated with 3.3 g/kg benomyl and then were sown by hand on 28 may 2015 in 3 cm depth of a sandy loam soil. each plot consisted of 6 rows of 3 m length, spaced 25 cm apart. all plots were regularly irrigated up to seedling establishment, but thereafter irrigations were carried out according to treatments. weeds were frequently controlled by hand during crop growth and development. salicylic acid (sa; 0 and 1 mm) was sprayed at vegetative and flowering stages. plant biomass and seed yield at maturity, plants in 1 m2 (8 plants) of the middle part of each plot were harvested and seeds per plant, 1000 seed weight and seed yield per unit area were determined. then above ground biomass was ovendried at 80°c for 48 hours and weighed and subsequently harvest index was calculated. oil extraction oil was extracted from 3 g mature seeds of each plot in petroleum ether for 5 hours using a soxhlet system according to the aocs method (aocs, 1993). oil content was determined as a percentage for each sample and then oil yield per unit area was calculated as: oil yield = seed yield × oil percentage flavonoid extraction powdered air-dried mature seeds (1 g) were extracted in a soxhelt extractor with 100 ml ethanol for an hour and the extract filtered. three ml of the extract was placed in a 15 ml volumetric flask. then 0.3 ml nano2 (1:20) and after 5 minutes 3 ml alcl3 (1:10) and 6 minutes later 2 ml of 1 mol litre-1 naoh were added and the total was made up to 10 ml with distilled water. the solution was mixed well again and the absorbance was measured against a blank at 510 nm with a halo db-20 spectrophotometer (zhuang et al., 1992). the flavonoid content was calculated using the following linear equation: a = 0.01069c 0.001163 where a is the absorbance and c is the flavonoid content in µg/g. analysis of variance analysis of variance of the data appropriate to the experimental design and comparison of means at p≤0.05 were carried out, using genstat 12 and mstatc softwares. excel software was used to draw figures. 3. results analysis of variance (table 1) showed that plant biomass, seeds per plant, 1000 seeds weight, seed yield per unit area and harvest index were significantly affected by water limitation and sa, but the interaction of irrigation × sa was only significant for 1000 seed weight and harvest index. source of variation df mean square plant biomass seeds per plant 1000 seeds weight seed yield harvest index replication 2 1198 2162 0.1163 61.7 0.0129 irrigation (i) 3 38104 ** 56241 ** 4.6906 * 3121.7 ** 4.1715 ** error 6 2795 4424 0.2785 140 0.1257 sa 1 837851 ** 1718420 ** 15.0417 ** 71195.6 ** 48.4504 ** i × sa 3 2633 ns 7585 ns 0.9128 ** 262 ns 3.3993 ** error 8 3987 7553 0.1212 259.2 0.1058 cv (%) 8.8 9.2 1.5 9.2 1.4 table 1 analysis of variance of the data for plant biomass, yield components and seed yield of milk thistle affected by irrigation treatments and salicylic acid (sa) treatments ns, * and ** no significant and significant at p≤0.05 and p≤0.01, respectively. ghassemi-golezani et al. oil and flavonoid of milk thistle affected by watering and salicylic acid 21 plant biomass, seeds per plant and seed yield per unit area decreased with decreasing water availability, but all these traits were considerably enhanced by foliar application of sa. reduction in seeds per plant was only significant under severe water stress (i4), with no significant difference among i1, i2 and i3 treatments. differences in plant biomass and seed yield between i1 and i2 and also between i2 and i3 were not statistically significant. application of sa improved plant biomass, seeds per plant and seed yield by about 71%, 79% and 91%, respectively (table 2). one thousand seeds weight (fig. 1a) and harvest index (fig. 1b) of untreated plants with sa gradually decreased as water stress increased. but, these reductions were only significant under severe water deficit. in contrast, sa treated plants did not show significant reduction in seed weight and harvest index due to water limitation. oil percentage, oil yield and flavonoid content were significantly affected by irrigation and sa treatments, but the interaction of irrigation × sa was not significant for these traits (table 3). oil percentage and yield of milk thistle decreased, but flavonoid content increased as a result of water stress. seed oil percentage and yield and flavonoid content were significantly enhanced by foliar spray of sa. this superiority was more pronounced for oil yield per unit area (table 4). 4. discussion and conclusions reduction in plant biomass due to water stress (table 2) was associated with diminishing leaf area expansion and plant growth during vegetative stages (ghassemi-golezani et al., 2009) and also with early leaf senescence (hugh and richard, 2003). drought fig. 1 mean seed weight (a) and harvest index (b) of milk thistle affected by irrigation and sa treatments. i1, i2, i3, i4= irrigation after 70, 110, 150 and 190 mm evaporation, respectively. sa0, sa1= 0 and 1 mm salicylic acid, respectively. different letters in each column indicate significant difference at p≤0.05. treatments plant biomas (g/m2) seeds per plant seed yield (g/m2) irrigation i1 797.2 a 1034.8 a 197.0 a i2 746.2 ab 984.0 a 185.2 ab i3 705.0 b 939.7 a 172.5 b i4 609.0 c 808.8 b 143.9 c salicylic acid irrigation 528 b 674 b 120.2 b sa1 901 a 1209 a 229.1 a table 2 means of plant biomass, seeds per plant and seed yield of milk thistle for irrigation and salicylic acid (sa) treatments different letters in each column indicate significant difference at p≤ 0.05. source of variation df oil content oil yield flavonoid content replication 2 2.2604 23.12 1.0208 irrigation (i) 3 24.2749 ** 367.88 ** 20.0397 ** error 6 0.6199 5.49 15046 salicylic acid (sa) 1 5.9004 ** 3294.13 ** 26.8182 ** i × sa 3 0.0849 ns 0.59 ns 0.2903 ns error 8 0.1625 10.8 0.3488 cv (%) 2 9.2 1.2 table 3 analysis of variance of oil percentage and yield and flavonoid content of milk thistle affected by irrigation and sa treatments ns, * and ** no significant and significant at p≤0.05 and p≤0.01, respectively. adv. hort. sci., 2017 31(1): 19-23 22 stress decreases water potential of plant, leading to stomata closure and reduction in photosynthesis rate and leaf growth (ozturk, 1999), which ultimately decreases plant biomass. this reduction in plant biomass resulted in decreasing the number of seeds per plant (table 2), 1000 seeds weight (fig. 1a) and consequently seed yield (table 2) and harvest index (fig. 1b). the losses in plant biomass and seed yield due to water deficit have also been reported for sesame (kim et al., 2007), dill (ghassemi-golezani et al., 2008), maize (ghassemi-golezani and dalil, 2011) and safflower (ghassemi-golezani et al., 2016). application of sa largely improved seed yield of milk thistle by enhancing plant biomass, seeds per plant (table 2), 1000 seeds weight and harvest index (fig. 1). sa influences a wide variety of plant processes, including stomatal regulation, chlorophyll content and photosynthesis (yildirim et al., 2008). ghassemigolezani and lotfi (2015) found that exogenous application of sa enhances maximum quantum efficiency of psii (fv/fm) and performance index (pi) in mung bean plants. in another report, ghassemigolezani and hosseinzadeh-mahootchi (2015) stated that chlorophyll content index (cci), photosystem ii efficiency (fv/fm), relative water content (rwc), leaf area index (lai) and finally seed yield of safflower were augmented by foliar application of sa. the low oil percentage due to water deficit (table 4) may be resulted from the short seed filling duration (ghassemi-golezani and lotfi, 2013) and low seed weight (fig. 1a). adequate irrigation during plant growth and development can likely increase seed weight and oil storage. decreasing oil yield per unit area as a consequence of water limitation (table 4) strongly related with reduction in seed yield under stressful condition (table 2). it was similarly reported that water limitation significantly decreases seed and oil yields of sunflower (soleimanzadeh et al., 2010) a n d m a i z e ( g h a s s e m i g o l e z a n i e t a l . , 2 0 1 5 b ) . increasing seed yield (table 2) and oil percentage by application of sa resulted in considerably higher oil yield of milk thistle (table 4). similar pattern of oil yield improvement by foliar spray of sa was observed in ocimum basilicum and origanium hortensis plants (abd el-lateef gharib, 2006). with decreasing photosynthesis rate under water stress, carbons from the photosynthesis cycle shift to the shikimic acid pathway in order to produce higher flavonoid content (table 4). it was found that phenolics and flavonoids are able to regulate plant growth and improve the physiological efficiency and can enhance effective partitioning of accumulates from the sources to the sinks in plants (ghasemzadeh et al., 2010). stimulation of flavonoid accumulation by sa treatment (table 4) may protect plants from certain biotic and abiotic stresses (dučaiová et al., 2013). an increase in secondary metabolites content w a s a l s o d e t e c t e d i n c h a m o m i l e ( m a t r i c a r i a c h a m o m i l l a l . ) p l a n t s a s a r e s u l t o f s a s p r a y (dučaiová et al., 2013). this result suggests that flavonoid accumulation is a biochemical response to water stress, and sa can induce flavonoid synthesis, providing an effective protection of milk thistle plants from stress. water stress reduced plant biomass, seeds per plant, 1000 seeds weight, seed yield, harvest index, oil percentage and consequently oil yield of milk thistle. however, flavonoid content of seeds increased with decreasing water availability. all these traits were considerably enhanced by foliar spray of sa under different irrigation intervals. this suggests that exogenous application of sa could be an effective way for improving field production of milk thistle under different environmental conditions. acknowledgements we appreciate the financial support of this work by the university of 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turk. j. agric. for., 23: 531-540. singh b., usha k., 2003 salicylic acid induced physiological and biochemical changes in wheat seedlings under water stress. plant growth regul., 39: 137-141. soleimanzadeh h., habibi d., ardakani m.r., paknejad f., rejali f., 2010 response of sunflower (helianthus annuus l.) to drought stress under different potassium levels. world applied sciences journal, 8: 443-448. yildirim e., turan m., guvenc i., 2008 effect of foliar salicylic acid applications on growth, chlorophyll and mineral content of cucumber (cucumis sativus l.) grown under salt stress. j. plant nutrit., 31: 593-612. zhuang x.p., lu y.y., yang g.s., 1992 extraction and determination of flavonoid in ginkgo. chinese herbal medicine, 23: 122-124. impaginato 217 1. introduction it is known that most almond [prunus dulcis (mill.) d.a. webb, syn. prunus amygdalus batch] varieties in the world, and until relatively recently, all the major commercial varieties, are self incompatible. this means that each variety needs to be grown with another variety to ensure pollination. not only that, each variety has its own specific incompatibility genes, and has to be pollinated by another variety with a different set of incompatibility genes (micke, 1996). in 2007, the perennial horticulture development project (phdp), funded by the european commission (ec) for the rehabilitation and development of the horticulture industry in afghanistan, began the collection of varieties of fruits and nuts in afghanistan, with a view to characterisation of those varieties and promotion of the best varieties for the development of commercial horticulture. the programme envisaged the distribution of varieties through a system of certified tree production with information provided as to how to plant and grow superior orchards. since every almond orchard that would be planted in afghanistan would need to be planted with a combination of two or more inter compatible varieties, it seemed essential that work be done on identifying suitable combinations of varieties. the phdp programme also included the development of the production of the superior afghan almond types, which include a range of paper shell types that are very much appreciated in the indian market, and which command very high prices. these are exemplified by the sattarbai and qambari types, which are elongated crescent shaped nuts with a paper shell that in the most prized varieties opens along a lateral fissure to expose the kernel inside. since there was no way of predicting the composition of the afghan germplasm in respect of incompatibility genes, a start was made in 2008 on testing the various combinations of varieties. this paper reports on the performed activities and adv. hort. sci., 2016 30(4): 217-223 doi: 10.13128/ahs-20347 short note implications of investigating pollination and cross compatibility in the almond varieties of afghanistan g.j. cullen 1, g.r. samadi 2, a.h. zarghon 3, m.r. yaqubi 3 1 independent consultant afghanistan national horticulture development organisation (anhdo), kabul, afghanistan. 2 kabul university, kabul, afghanistan. 3 afghanistan national horticulture development organisation (anhdo), kabul , afghanistan. key words: breeding, incompatibility groups, national collection, prunus amygdalus, prunus dulcis. abstract: survey and collection of almond accessions for a national collection of fruit and nuts of afghanistan began in 2007. investigations into cross compatibility of almond accessions began in 2008 on in situ collected exemplar trees, and in 2010 on trees in the ex situ collections. the methods varied in relation to specific trials, nevertheless as an average 150 flower buds were isolated ahead of flowering on 7 one-year-old shoots per tree, and used as pollen donors or receptors. the initial trials on in situ accessions were performed on one single tree per variety, while six trees were used for the tests carried on in the ex situ collections. fruit set percentage from self-pollination, cross-pollination and open pollination was calculated. all native afghan varieties tested were shown to be self incompatible. various problems related to weather and other conditions worked against a comprehensive testing of all combinations of varieties, although many useful and surprising conclusions were reached. (*) corresponding author: samadigr@gmail.com received for publication 11 january 2016 accepted for publication 13 july 2016 copyright: © 2016 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2016 30(4): 217-223 218 the first results achieved related to selfand intercompatibility of almond varieties of afghanistan, together with the some notes on the gained experience and the implications of these findings in the almond industry of afghanistan. as illustrated in the following paragraph, different trials have been defined and set up in different locations taking into account farmer knowledge and tradition, local operative conditions and environmental aspects. 2. in situ tree pollination trials in the kunduz, samangan and balkh provinces during the 2007 season, the phdp had collected budwood from some 84 separate accessions from around afghanistan, and budded these onto almond seedling rootstocks at six different centres with a view to planting out duplicate collections in the northern afghanistan centres of mazar-e sharif and kunduz in spring 2009. it had already been ascertained by phdp, in particular through a comprehensive nursery survey, that the main centre of the important almond varieties was in the north of afghanistan, especially in the provinces of kunduz, samangan and balkh provinces. while it was obvious that testing almond pollination attributes would be much easier when the germplasm was collected into the variety collections, it was considered an urgent priority for phdp to find out about the pollination compatibilities of the main varieties. many stories were being reported from the field by the phdp staff and by associated projects, s u c h a s r o o t s o f p e a c e a l m o n d i n d u s t r y development project, concerning the lack of knowledge among many afghan farmers about pollination requirements of almonds. in general, the need for pollination was unknown, and some farmers were insecticide spraying bees that were in the almond blossom, on the assumption that bees were “eating the flowers”. while traditionally many orchards included a range of varieties, they did not necessarily overlap in flowering time, nor were they necessarily c o m p a t i b l e . o n e g r o w e r w a s s u p p o s e d l y s o impressed with the results of the nonpareil variety that he planted a whole large block to that single variety, and then wondered why results were disappointing. not only was solving the almond pollination problem considered an urgent priority for phdp because of the lack of knowledge of most small farmers, but a s a d o n o r f u n d e d p r o j e c t w o r k i n g w i t h i n t h e ministry of agriculture, phdp had to demonstrate meaningful results ahead of planting out the national collections of the different species of fruits and nuts. so in 2008, a simple trial at three sites was designed, using the original in situ trees from which the budwood for the germplasm collection was collected. materials and methods the first experiment to study the self and cross incompatibility of almond accessions in the northern part of afghanistan took place from february 2008 (prior to flowering) to august, 2008 (harvesting of fruit). three groups of six in situ trees were selected for their proximity in single orchards or in closely nearby orchards, in khulm (balkh province), in aybak ( s a m a n g a n p r o v i n c e ) a n d c h a r d a r a ( k u n d u z province). each tree was selected before flowering and lengths of branch with 100-200 flower buds were selected and marked. branches used to donate or receive pollen were bagged ahead of flowering using cotton muslin cloth to avoid ingress of bees or other pollinating insects. all the trial was done using single in situ trees, so on each tree there were bags for ten sites (branch) to donate pollen of uncontaminated flowers, bags for ten sites to receive pollen from five other trees (including two replicates for each cross), bags for two sites for self pollination (two replicates), and two sites marked for natural pollination (open pollination). so there were 7 treatments on each tree. pollination, either self or cross pollination, was done on three successive days, to cover the period of flowering, by bringing flowers from the reserved branches to the receptor branches. protective bagging was removed only to allow the hand pollination, until a few days after flower fall, when the protection was removed. t h e s t ru ct u re o f t h e t ri a l wa s b a s ed o n t h e hypothesis that all the varieties were self incompatible, so that any fruit set was due to the pollen transferred by the hand crossing. for each tree there was also two self pollination replicates, where the tree’s own pollen was used to pollinate flowers. if there had been any self pollination, this would have invalidated the results of the crossing between different trees. results and discussion it was noted that the general level of fruit set on the control sections of branch (open pollination) was generally quite low. hand pollination as practised in this trial would increase the setting of fruit up to ten fold. this indicated a lot of problems with the current practices, probably mostly to do with the absence of cullen et al. ramifications of investigations into pollination and cross compatibility of almond varieties in afghanistan 219 pollinating insects. the condition of the flower buds after what had been a very hard winter, the fertility status, the temperatures and humidity at flowering could all have had an effect. presentation of data to farmers and discussion with them indicates a lack of knowledge of the need for pollination which results in the deliberate killing of bees, as they think the bees eat the flowers, and the planting of large areas to single varieties, with resulting nil crop. t h e d a t a o b t a i n e d i s r e p o r t e d i n t a b l e 1 . comparing the fruit set percentage obtained by selfing and intercrossed pairs or the average of all crosscombinations. it was shown that all six almond successions were self incompatible. on the other hand, the basic criteria for a successful crosscombination was fruit set with hand pollination equal to or higher than fruit set with natural background pollination (open pollination). the criteria for a non-successful combination was fruit set equal or lower than that with self pollination. the low fruit set with natural pollination brought the boundary line between successful/unsuccessful cross to be too near, so there were a lot of inconclusive results. hence, the information from this trial is generally disappointing. fruit set was low in the orchards, but occasionally there were some interesting results. the carmel flowered much later than other varieties, including nonpareil. the variety carmel 167 was too late flowering to be of use with the local varieties and nonpareil 171. nonpareil 171 was pollinated by abdul wahidi 1003, but the reverse cross had nil pollination. 3. pollination trials in the ex situ collections the almond pollination trials in phdp were done in 2008 with in situ trees at three locations in kunduz, samangan, and khulm. for the 2010 and 2011 trials, the same principles were followed, with cross pollination in all combinations of varieties in sets of six varieties. in the national collections, which for almonds are situated in the centres in balkh and kunduz, there are six identical trees of each almond accession, and this layout was exploited to greatly simplify the trials. out of the six trees, one tree was used to provide pollen to the other five varieties, and for self pollination, and each of the other received pollen from one of the other five trees in that trial set. in 2010, the numbers of flowers available for the trials were in many cases too few to permit valid results to be drawn, and most of the varieties in balkh and kunduz suffered damage from a late frost. in 2011, many varieties set reasonable amounts of fruit for drawing conclusions, but other varieties female (receptor) male (pollinizer)/fruit set % location: khulm, balkh province qambari 143 bellabai 144 sattarbai sufi 145 zang kaftar 148 sattarbai bakhmali 149 sattarbai no.4 154 qambari 143 0.2 12 10 4 13 2 bellabai 144 30 1 17 8:05 0 4 sattarbai sufi 145 13 10 0 8 11 2 zang kaftar 148 9 3 0 2 3 2 sattarbai bakhmali 149 14 0 3 16 0 0 sattarbai no.4 154 2 10 3 7 2 0.4 cross-pollination average 13.6 7 6.6 8.75 5.8 2 location: kunduz marawaja kaghazi 166 carmel 167 sattarbai 168 qaharbai 170 nonpareil 171 abdul wahidi 1003 marawaja kaghazi 166 0 0 0 1 0 1 carmel 167 0 1 11 3 0 0 sattarbai 168 2 0 0 0 2 0 qaharbai 170 1 0 1 0 0 6 nonpareil 171 2 2 7 4 1 8 abdul wahidi 1003 1 0 0 15 0 0.3 cross-pollination average 1.2 0.4 3.8 4.6 0.4 3 location: aybak, samangan province sattarbai 156 sattarbai guldar 157 sattarbai bakhmali 159 qaharbai 160 khairodini 161 shokorbai 162 sattarbai sais 156 3 42 42 35 57 26 sattarbai guldar 157 1 0 0 2 1 0 sattarbai bakhmali 159 5 3 6 3 24 9 qaharbai 160 22 15 27 0 20 6 khairodini 161 16 32 8 11 2 6 shokorbai 162 12 1 3 8 8 1 cross-pollination average 11.2 18.6 16 11.8 22 9.4 table 1 pollination data from mature in situ exemplar trees of the national collection of almond varieties, khulm, balkh province, 2008 summery results, based on final percentage. adv. hort. sci., 2016 30(4): 217-223 220 seemed to still remain in a juvenile phase. this did not allow for maximum values to be extracted from the trials. additional pollination trials were undertaken in the badam bagh kabul centre, in the plots of the almond variety demonstration, which included a limited number of accessions. it was considered important that information was collected about the condition of the trees for setting fruit. this is done by allowing a number of flowers to be open to natural pollination from bees and other insects. bees were introduced to the national collections to maximise natural pollination. the proposition was that if any variety was not showing good fruit set with this natural open pollination, then any cross pollination efforts by hand would also not show good fruit set. results from those varieties could clearly not then be used to indicate incompatibility in that cross. after the mostly rather inconclusive pollination trials in 2010 and 2011, by 2012 the trees were quite large, and only individual branches needed to be marked before flowers opened. materials and methods a straight length of branch with an estimated 200+ flowers were chosen for the trial. the beginning and end of the selected length of branch were marked and any flower buds above and below the marked length of branch could be stripped off to allow easy counting. for each variety, the enumerators were asked to pick two trees at either end of the plot, to get a good average of the open pollinated count. the layout of the pollination trial is shown for blocks of six trees, which is the standard layout in the national collections. by using all six trees available in the accession, it can be seen that a set of six varieties can easily be managed (fig. 1). the varieties for the pollination trials were every year chosen based on the relative importance of their nuts in the commercial market. a meeting was held with almond traders in october 2008, where the traders were asked to assign monetary values to a range of samples of almonds in shell collected from the exemplar trees of the national collection of almonds. this selection of the most important varieties was important, as the number of possible combinations of almond accessions to be tested ran into many thousands. results and discussion the 2012 trials repeated some combinations which did not give clear results in 2010 or 2011, and quite a lot of useful information was obtained. the larger number of combinations tested also allowed for progress in identifying varieties that did not combine with each other, and the start of working out some incompatibility groups. once a variety could be allocated to an incompatibility group, predictions can be made on what would be suitable combinations, without actually doing a field test. ahead of the 2010 trials it was suggested that two replicates should be made. however, for each combination there is the reverse cross, which makes a second replication. to obtain reasonable numbers for e s t i m a t i o n o f p o l l i n a t i o n c o m p a t i b i l i t y , i t w a s arranged that at least 100 flowers should be pollinated in each combination, but it was not considered necessary to pollinate more than 200 flowers for each combination. after 2010, the flowers were protected from bee pollination by nylon netting rather than cotton muslin. the nylon netting did not absorb moisture during rain, thus avoiding damage to flowers in wet and windy conditions. a substantial number of results were obtained from the 2012 trials. unfortunately in 2013, there were severe late frosts in the north of afghanistan, fig. 1 layouts of almond national collection blocks for cross pollination. each box in the diagram represents one tree in each variety block. each variety is coded by a letter, in order to show the systematic layout of the trial. the code sheets would represent a different accession in each year's trials. cullen et al. ramifications of investigations into pollination and cross compatibility of almond varieties in afghanistan 221 which meant that no results were obtained that year. late frosts also affected pollination trials in the north of afghanistan in 2014, and results were limited to the almonds in demonstration plots in kabul, which is a much later flowering site and has not lost a crop due to frost since planting out almond varieties in 2009. 4. successful combinations when tested for cross pollination compatibility 2008-2012 s o m e 9 3 d i f f e r e n t c o m b i n a t i o n s o f a f g h a n almond accessions (186 combinations when the reverse cross is taken into account) have been shown to be successful. this is calculated before the accession names have since been updated/rationalised in the national register of almond varieties published in 2014. no further information can be deduced from a list of compatible varieties. what is more useful for making further deductions about the possibilities of successful combinations is the classification of pollination groups (kester et al., 1994), which is derived from information about combinations of varieties which do not successfully cross pollinate, as below. from the information about incompatible combinations above, the following attempt was made to classify the different varieties in incompatibility groups, based on results up to 2012. only later was it decided to work on the assumption that sattarbai accessions 168, 771 and 1001 were all the same variety. it was also decided without the incompatibility data that sattarbai 142 should be reclassified as qambari, thus the incompatibility information would indicate that accessions 142 and 143 can be treated as different clones of the same variety. the results are reported in table 2 and 3, where the confirmed incompatible combinations and the individuated incompatibility groups are indicated. at this regard, it is worth noticing that group a can’t be said to be definitively separated from the others, due to non sufficient data at the end of the 2012 trials. conversely, groups b, c and d are considered different. 5. cross pollination for variety verification a range of similar sattarbai accessions, collected under accession number 168, 771 and 1001 from different growers had proven to be very similar when characterised across a range of leaf, flower and fruit characters per the upov standards (upov, 2011). accession numbers 168 and 1001 were also included in the set of nut samples to be assessed by the almond traders in october 2008, and were given almost identical market values ahead of all the other almond varieties. it seemed too much of a coincidence when such similar and high value varieties proved to be incompatible with each other. it is difficult to imagine a scenario whereby one of those three accessions is the parent of the other, because table 2 list of incompatible combinations of almond access i o n s b e l o n g i n g t o t h e n a t i o n a l c o l l e c t i o n o f aghanistan site year accessions khulm 2008 sattarbai bakhmali 149 x bellabai 144 khulm 2008 sattarbai bakhmali 149 x zang kaftar 148 khulm 2008 qambari 143 x sattarbai no. 4 -154 aybak 2008 shokurbai 162 x sattarbai guldar 157 kunduz 2011 qambari 143 x sattarbai 142 kunduz 2011 qambari 143 x qambari 2009 kunduz 2011 sattarbai 142 x qambari 143 kunduz 2011 sattarbai 142 x qambari 2009 kunduz 2011 qambari 2009 x sattarbai 142 kunduz 2011 sattarbai bakhmali 2008 x sattarbai no.4 -154 kunduz 2011 sattarbai 168 x sattarbai 771 mazar 2012 sattarbai 168 x sattarbai 1001 mazar 2012 sattarbai 771 x sattarbai sufi 145 mazar 2012 sattarbai 771 x sattarbai bakhmali 159 mazar 2012 sattarbai sais 777 x sattarbai guldar 157 kunduz 2012 khairodini 846 x abdulwahidi 153 a b c d e sattarbai bakhmali 149 qambari 143 shokorbai 162 sattarbai 168 khairodini 846 bellabai 144 sattarbai no.4 -154 sattarbai guldar 157 sattarbai 771 abdulwahidi 153 zang kaftar 148 sattarbai bakhmali 2008 sattarbai sais 777 sattarbai 1001 sattarbai 142 sattarbai sufi 145 qambari 2009 sattarbai bakhmali 159 table 3 incompatibility groups defined for some almond accessions of the national collection of almond of afghanistan this designation of incompatibility group does not refer to any other classification of almond incompatibility groups that might have been used by other authors. adv. hort. sci., 2016 30(4): 217-223 222 of the intrinsic nature of the incompatibility genes. the most logical explanation, because it is also the simplest, is that the three accession numbers 168, 771, and 1001 were just three different samples (clones) of the one variety that had spread across the north of afghanistan by vegetative propagation by different nursery growers. the three different clones had already been distributed to registered mother stock growers, and all three continue to be maintained under their accession numbers, but the final saplings are all marketed under the new name of “sattarbai mumtaz” (=”superior sattarbai”)(ministry of agriculture, irrigation and livestrock, 2014). following this rationalisation of the sattarbai accessions, it was decided to use this approach to try to identify other duplicates in the national collections. the accession cardinal seemed to be phenotypically very similar to the accession of carmel in the collection. since no other record of an almond variety called cardinal could be found, and because it was known that variety names gradually changed after introduction into afghanistan with unmanaged distribution of varieties, the hypothesis that cardinal was actually a misnamed carmel was tested by attempting cross pollination of the two accessions in the demonstration orchard in kabul. using the same protocol as with the other pollination testing, it was shown that cardinal and carmel would not cross with each other, with pollination in either direction. this was taken as confirmation that cardinal was the same as carmel and was therefore removed from the national collection. other pollination tests were done with the same o b j e c t i v e i n m i n d . a n a c c e s s i o n c o l l e c t e d a s nonpareil 6041 appeared to be very similar to carmel, but cross pollination with carmel set seed, indicating that 6041 was possibly an offspring of carmel, perhaps from a cross between nonpareil and carmel, which two varieties would have been introduced together into orchards in afghanistan in the 1990s. this accession, 6041, was also included in the sets of six varieties as was normally used in the cross pollination trials, and some of its offspring from crosses with superior afghan almond types continue to be grown and tested in the almond breeding programme. another accession, collected as sattarbai 6038, was considered to be very similar with the varieties ferragnes and ferraduel. again, cross pollination trials with all combinations of ferragnes, ferraduel and 6038 showed that accession 6038 was not either f e r r a g n e s o r f e r r a d u e l . a g a i n , f e r r a g n e s a n d ferraduel were also introduced into afghanistan in the 1990s as a combination to be planted together, so accession 6038 could be from a seedling resulting from a cross of those two varieties. 6. i n c o m p a t i b i l i t y i n a l m o n d v a r i e t i e s o f afghanistan: final remarks all the trials on pollination in afghanistan have been done within the context of a development project, the perennial horticulture development project (phdp) and its successor project phdpii. the projects have been successful in developing perennial horticulture from the very foundations of the industry, that is from deriving knowledge about how to use the native and imported germplasm to develop sustainable economic production systems. further development continues with continuing private and public sector programmes financed by the european union, within a programme framework that is now attracting other donors. the adaptive research programmes have been based on very simple and basic concepts that are implemented in the context of a country devastated by more than 35 years of almost continuous warfare and internal strife. almonds have been a traditional export for many years, and if a connection can be shown with the fergana valley to the north in tajikistan, then the afghans can claim an almond export industry to india and the persian gulf region for at least five hundred years (hiro, 2006). in terms of planting almond orchards, it is now possible to plant just two varieties in pairs that cross pollinate each other, in the same way there are the famous combinations of nonpareil and carmel in california, and ferragnes and ferraduel in france. a g o o d c o m b i n a t i o n f o r a f g h a n i s t a n c o u l d b e nonpareil planted with sattarbai mumtaz. what is clear however, is that there must be quite a few different incompatibility genotypes among the afghan almond germplasm, so the growers can be quite confident, that in the absence of any specific data on varietal combinations, the planting of four different varieties will almost certainly give good fruit set. it should also be noted that there is no reason to think that these four different varieties could not all be different sattarbai types. future work is provide information on the recommended combinations, to demonstrate the imporcullen et al. ramifications of investigations into pollination and cross compatibility of almond varieties in afghanistan 223 tance of pollination with bees, to ensure orchard growers understand the importance of planting mixtures of varieties, and taking remedial measures where they have single variety orchards. more combinations of varieties need to be tested for pollination compatibility, until recommendations can be made for all required commercial varieties. however, the testing of further combinations can probably wait until capacity in the plant biotechnology laboratory in kabul is increased to allow for direct examination of the incompatibility factors within the dna of each accession. the testing of almond pollination within the context of a development project has been simplified because of necessity, such that emasculation was not practised (kester et al., 1994). the theory of incompatibility has been extended to encompass pollination testing as an extra verification method in the identification of varieties. the results of the pollination trials, that are the seeds from the inter variety crosses, have been planted out and formed the beginnings of a selection process for almond crosses which is expected to result in the release of several improved lines to growers by 2017. the breeding programme has taken on a life of its own, and is now focused on achieving late flowering, productive afghan almond types. innovative methods of cross pollinating early and very late flowering almond lines have been developed, to ingress late flowering characters into the local germplasm. references hiro d., 2006 babur nama. journal of babur emperor. penguin classics, penguin books, london, england, pp. 384. k e s t e r c . e . , g r a d z i e l t . m . , m i c k e w . c . , 1 9 9 4 identifying pollen incompatibility groups in california almond cultivars. j. amer. soc. hort. sci., 119(1): 106109. ministry of agriculture, irrigation and livestock, 2014 national collection of varieties of fruits and nuts of afghanistan. almond. ministry of agriculture, irrigation and livestock, kabul, afghanistan, pp. 194. m i c k e w . c . , 1 9 9 6 a l m o n d p r o d u c t i o n m a n u a l . university of california anr, pp. 289. upov 2011 almond. guidelines for the conduct of tests for distinctness, uniformity and stability. ipov, geneva, switzerland. impaginato 231 1. introduction during the last decades, after over ten years of reconstruction projects, afghanistan horticulture has grown to the pre-war level. in 2014, the estimated horticulture crops extended over 340,000 ha that represents the 14% of the total irrigated land. the overall contribution of horticulture is valued at 1.4 billion (6.7%, to the total gdp) and the 34% of the agriculture gdp contribution with a crops employmen t gen era t ed es t i ma t ed i n 2 mi l l i o n p eo p l e involved to various degrees. principal crops are grapes ($330m), almonds ($120m), pomegranates ($100m), all of which are largely exported to the neighbour countries. by comparison, illicit crops (opium poppy, cannabis) in the same period are grown on 220,000 ha with an estimated value of about $1 billion. many important fruit and vegetables are origin from afghanistan (e.g. pistachio, walnut and pomegranate) and afghanistan has favourable climate condition suitable for high quality horticulture. the hypothetically development of the horticulture sector is huge as well as the potential international interest for their quality (pomegranates, apricots, almonds, raisins). despite the improvements, especially the road network, the horticulture sector claims a lack of massive interventions. as a consequence, the overall quality is still low due to a wellknown list of constraints: small land holdings, small commercial orchards, lack of standardized product, poor orchard management, lack of infrastructure and substandard storage, sorting, packaging, marketing, transport facilities. afghan quality control system in general, the quality of the agriculture products is the final result of a long list of interventions, controls and standards adopted by a nation, generically define as quality control system. the quality control system has the primary function of protecting its citizen by selecting active controls of the imported commodities at the borders and promote the safety of the exported goods. the lack adv. hort. sci., 2016 30(4): 231-237 doi: 10.13128/ahs-20351 introduction of determination of optimum harvest date in afghanistan. sweet cherry: a case study f. valori 1 (*), a. ahkbari 2, m.r. yaqubi 2 , n. enayat 2, f. azizi 3, m. wali adel 2 1 independent consultant, florence, italy. 2 afghanistan national horticulture development organization, kabul, afghanistan. 3 ministry of agriculture, irrigation and livestock, kabul, afghanistan. key words: harvest date, maturity index, sweet cherry. abstract: in the last decade, afghanistan has been under the political attention by several institution and organizations whose invested millions of dollars for the reconstruction of the country. part of such efforts were focused on the agricultural sector, as the principal source of income of afghanistan. mainly, perennial horticulture represents the one of the most challenging opportunity for the developing economies of afghanistan, and the high contribute of eu in such direction, shown significant results. however, the level of the export is still neglected and the reputation of afghan’ products has lost the ancient brittleness. the generic low hygienic conditions and poor quality of the products are the primary cause of such situation. in this scenario, phdp (perennial horticulture development project) first and anhdo (afghanistan national horticulture organization) then, tried to improve the quality of perennial horticulture crops. phdp has, for the first time created a certification scheme for the production of certified perennial plants, anhdo has the main goal of improving the quality of the fruit value chain of the principal crops in afghanistan. in this study the first protocol for the determination of the optimum harvest date (ohd) for cherry var. burlat is given, as a case study of the new course of horticulture in afghanistan. (*) corresponding author: federico.valori@gmail.com received for publication 10 march 2016 accepted for publication 5 september 2016 copyright: © 2016 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2016 30(4): 231-237 232 of an efficient quality control system, affects the horticulture sector in many ways. the general situation in afghanistan is critical in both the import/export directions: international requirements (rules and consumers education) on food are extremely rigid and difficult to overcome for afghan traders. from the import side, the situation is without control. the absence of an explicit laws with a specific phytosanitary standards regulation, allows to the neighbour countries to freely provide goods and services without any reliable control. the consequence is a risk for the food safety and human health aspects (e.g. ogm) and besides that also the religious point of view is involved. the respect of halal rules is in fact often not certified and the customs authority has technical difficulties to check the provided certificates. for the above mentioned reasons, the national quality control system of afghanistan is now under a deep revision: afghanistan has started the world trade organization (wto) membership path. this is a very important step forward for the quality control of the country, especially for the agricultura l c o m m o d i t i e s : j o i n t h e w t o w i l l a l l o w t o afghanistan to implement non-tariff barriers useful to protect his local market from the massive import from neighbours countries i.e china, pakistan and india on top (wto, 1995 a). beyond wto, the gap between the afghan food industries and the international competitors, is consistent. afghan industries may require large investment to make them efficient and competitive in a reasonable period of time. applying a protection policy is probably, the best way for afghanistan to fill the gap with its competitors. protection would act as incentive for such industries, the introduction of technical barriers agreements (tbt) may establish a more transparent trade with the neighbour countries and allow to afghanistan a better control of the imported commodities based on harmonised rules. application of tbt required the wto membership and the main obstacle to this is the lack of a food law that will clarify the different responsibilities among the ministries and will produce the conditions to the creation of the national quality control system. the responsibilities on the food safety are now divided between the ministry of public health (moph) and the ministry of agriculture irrigation and livestock (mail) but such division is not clear, with the consequence of an inefficient sanitary and phytosanitary system, with respect to the requested sps agreement (wto, 1995 b). at the best of our knowledge, the food law is still stuck in the parliament and afghanistan doesn’t adopts any procedures and a clear set of harmonized standards. such a law represents a necessary milestone for the future of agriculture sectors, but it is not sufficient to promote a real food quality control in afghanistan. the first goal of such law must be the protection of human health from risks created by uncontrolled trading, tools as quarantine measures, certification, borders checks, inspections network and laboratory tests are the pillars of the quality control. in the last ten years, the phdp has successfully developed a certification scheme for the horticulture crops and keep the horticulture to an upper level of quality in the whole afghanistan. moreover, phdp developed the national collection orchards (nc) where the local and international varieties were tested for their adaptive level in the specific afghanistan climate. within its activities, phdp has also set-up six pomology laboratories where, for the first time, local and introduced horticulture varieties were characterized under many aspects (e.g. upov, grafting, etc.). the phdp project ends up with a great numbers of varieties, however, not all these varieties were fully characterized and some basic information are still missing. specifically, the harvesting date and maturity index were not included as tested parameters because out of the scope of the project. horticulture private sector development project (hps) has, in one specific subjective (so3), as primary goal, the evaluation and the promotion of the fruit commercial varieties. anhdo is the implementing partner of hps and in close collaboration with mail (that has inherited the ownership of the pomology laboratories) has succeeded to the phdp cultural heritage and is developing the missing horticulture parameters: in the next two years at least 60 varieties of the major commercial horticulture crops will be fully characterized and studied for the pomology basic tests, including post-harvest tests, maturity indexes and harvesting date by hps specific sub objective (so2 adaptive research). anhdo/hps ambitions are high and even if the pomology laboratories are well equipped and the technical staff has been trained, the overall technical level is still critical due to the general environmental situations and only basic tests are possible. fruit quality in afghanistan: cherries as least development country, afghanistan has a total potential catchment not depending on the quality of the products and the domestic market has the peculiarity of accepting every type of fruits. the wide range of the existing social classes creates consumers valori et al. optimum harvest date in afghanistan. sweet cherry: a case study 233 with different tolerance and allow traders to sell all the product without any formal losses. the wellknown effect of the extended cherry shelf-life over the 7-10 days (loss of firmness, color and flavour, stem discoloration, desiccation and mould growth) will not affect the trade. however, such condition is changing with the increase of the consumer awareness. soon, farmers and traders will face, for the first time, the necessity of an improvement in the cherries quality. to be prepared, for the very first time in afghanistan, hps has set-up a formal protocol to start the characterization of the main commercial fruit varieties, with the final aim of helping farmers to harvest the crops in the optimum period for domestic consumption or international export. aim of the work the aim of the study is to provide a simple and pragmatic in-field tool for the anhdo/hps beneficiaries farmers, to identify the appropriate harvesting date and suitable maturity index for cherry cv. burlat. the proposed approach is a compromise between scientific research (that requires at least twenty sing l e o b s e r v a t i o n s ) a n d t h e r e a l c o n d i t i o n o f afghanistan that forced to reduce such observations to five. phdpii project provided initial data on the harvesting period of many varieties, based on visual observations. this study will confirm the hypothetical harvesting date and will provide (among different existing parameters) one or two easy and quick maturity indexes for the afghan farmers. determination of optimum harvest date of sweet cherry cv. burlat grown in kabul optimum harvest date (ohd) is defined as the date in which harvesting results in optimum fruit quality of the produce after long-term storage (baumann, 1998). typically, ohd is predicted using calculation models or through direct measurement of certain physiological fruit parameters and quality characteristics during a given period until harvest (çalhan et al., 2014). the first method required historical series of meteorological and phenological data that are not available in afghanistan. on the contrary, direct measurements of fruit characteristics, in specific intervals before the estimated harvest date, are more reliable for specific cultivars and applicable in afghanistan. this study was conducted to evaluate ohd through physical and chemical measurements of fruit quality as a maturity index for estimating proper harvest time of burlat cherries, in kabul. among the exiting maturity indexes, sugar content (ssc), titratable acidity (ta), ssc/ta ratio, skin color, and firmness have all been used as indices of cherry fruit maturity. however, skin color has long been accepted as the best indicator for the appropriate harvest maturity of sweet cherries (drake and elfving, 2002). 2. materials and methods cherry cv. burlat has leaves sawed, average green, slightly lengthy and pointed. burlat fruit has a dark red or purple skin, slightly flat and mean sized. the flesh is dark red, with sweet taste, juicy and firm. it is defined as early ripening, it is self-incompatible and it needs a pollinizer. fruit from the sweet cherry cv. burlat was harvested during 5 different dates (between 18.05.2015 and 27.05.2015) from the national collection in badam bagh, kabul, afghanistan. full bloom date of burlat was at 19.04.2015. the harvesting period was c o n c e n t r a t e d i n 1 0 d a y s d u e t o e x i s t i n g d a t a obtained from previous tests on the same station. in the national collection each varieties is replied 6 times in a row. fruits were collected early in the morning from branches with the same orientation (south-east) and all the fruit from the selected branches were immediately transferred to the pomology laboratory. in the laboratory, samples with defects (split, diseased or damaged fruit) were annotated and then rejected. the collected cherries were then divided into 5 maturity class according to their visual skin color: a) yellowish immature; b) light red nearly mature; c) red mature; d) dark red ripe; e) overripe. the number of cherries for each class was annotated. in this study only red and dark groups were considered. fruits parameters were collected by 24 fruits per class for a total of 48 samples. weight was measured by an electronic balance (kern emb-200) to an accuracy of 0.01 g; results were expressed as gram (g). fruit width and length was measured using manual calliper (verinier caliper 0-150 mm). results were expressed as millimeter (mm). total soluble solids concentration (tss) was determined with a temperature compensated digital refractometer (atago pal-1) previously calibrated with distilled water and results expressed as °brix. titratable acidity (ta), was estimated by juice titration with 0.1 n naoh to the titration end point of ph 8.1, monitored adv. hort. sci., 2016 30(4): 231-237 234 with a ph meter (hanna instruments hi3221) and expressed as malic acid content (mg/100 ml). flesh firmness was defined as the maximum load required to push the 6 mm diameter probe into the fruit firmness one side of each fruit, to a depth of 6mm with fruit texture analyser (fruit pressure tester ft 327). the results were expressed in newton (n). skin color was measured on 24 fruits per class using a minolta chromometer model cr 400 and average readings at six pre-determined points on the circumference of the fruits were recorded. the instrument was calibrated against a standard white color plate (y=93,9, x=0,313, y=0,321) (konika minolta, 2013), color space (xyz) (batu, 2004; žnidarčič and požrl et al., 2006). all the above mentioned parameters were measured between 18.05.2015 and 27.05.2015. the final dataset was statistically analysed by pearson correlation test, anova test and pca using systat 12 software and minitab 17. part of the graphs do not shown red group values at the latest observation. this happened because during the division in maturity classes immediately done after the harvest in the last part of the maturity process, the red group was without samples. such a situation is obviously due to the ripening of cherries and the lack of non-mature fruits. 3. results a s r e p o r t e d i n f i g u r e 1 , r e d g r o u p s w e i g h t increase at maximum rate to 24/05 and then starting decreasing. on the contrary, the dark group results do not shown significant variation of weight. this suggest that the maturity weight was reached before the start of the observations, and is confirmed by the red group trend that increases as confirmation of the maturity development. as reported in figure 2, height increase during the observation period. however, as expected, the height/diameter ratio is not changing (table 1). the difference between the first and the last observations reports an increase of 7.56%. this result is clearly explained by the shorter observation period where the contiguous observations are not significant. firmness (fig. 3), as expected, decrease during the observations period and drastically dropped between 24/05 and 26/05. considering the dark red and red groups separately (fig. 3), the decrease is 31.89% and 50.23% respectively. sugar content (fig. 4) is constantly increasing for both dark and red groups. the most significant increase is visible between 24/05 and 27/05, in which the sugar content passes from 20.27 °brix to 24.73 °brix for the dark group. in the last day of observation (27/05), none of the sampled cherries was classified as red group, as consequence of the ripening process. the ph values, in this study, do not provide a reliable maturity index, as the variation observed during the experimental period were significant but ranged within the instrument fig. 2 effects of different picked dates on 48 fruit height (mm) of sweet cherry cv. burlat. vertical bars represent standard error. means with different letter are significant different (tukeys’s hsd p<0.05). means with different letter are significant different (tukey’s hsd p<0.05). fig. 1 effects of different picked dates on 48 fruit weight (g) of sweet cherry cv. burlat fruits. vertical bars represent standard error. means with different letter are significant different (tukeys’s hsd p<0.05). table 1 effects of different picked date of ratio between height and diameters (h/d) on 48 fruits of sweet cherry cv. burlat date height-diameter ratio dark red group red group error d.r. error r. 5/18/15 0.852 a 0.893 a 0.017 0.018 5/20/15 0.859 a 0.879 a 0.018 0.018 5/24/15 0.833 a 0.839 a 0.018 0.019 5/26/15 0.834 a 0.861 a 0.018 0.018 5/27/15 0.984 b 0.018 valori et al. optimum harvest date in afghanistan. sweet cherry: a case study 235 error (fig. 5). the graph reported in figure 6, perfectly, explain and reflects the burlat maturity pattern of ta: the total acidity starts decreasing from the 26/05 in the dark group. such a result, compared with sugar content clearly states that cherry acidity is decreasing, while the sugar content is still growing (fig. 4). considering the path of the sugar content and the ta result, is possible to set the maturity days between 26/05 and 27/05. the same conclusion is obtained by looking the color xyz-cie results (fig. 7), in which the variation of color is stable in the same period. the identification of the most suitable parameter used as easy maturity index, passed through a first analysis of the data set using a pca analysis. however, the interpretation of a pca with more than 8 factors and at least three principal components, is not easy. to overcome such problem the pearson correlation matrix was used to reduce the number of parameters analysed with pca. the main correlations among x, y, z parameters are reported in table 2. the color components are very well correlated, and such strong correlations justify to use only one of the fig. 3 effects of different picked dates on 48 fruit firmness of sweet cherry cv. burlat compared with red and dark red group. vertical bars represent standard error. means with different letter are significant different (tukeys’s hsd p<0.05). fig. 4 effects of different picked dates on 48 fruit sugar content (brix°) of sweet cherry cv. burlat for both dark and red groups. vertical bars represent standard error. means with different letter are significant different (tukeys’s hsd p<0.05). fig. 5 effects of different picked dates on 48 fruit ph of sweet cherry cv. burlat. vertical bars represent standard error. means with different letter are significant different (tukeys’s hsd p<0.05). fig. 6 effects of different picked dates on 48 fruit sugar content and tritatable acidity (ta) of sweet cherry cv. burlat fruits for both dark and red groups. vertical bars represent standard error. means with different letter are significant different (tukeys’s hsd p<0.05). fig. 7 effects of different picked dates on 48 fruit xyz cie color of sweet cherry cv. burlat for both dark and red groups. vertical bars represent standard error. means with different letter are significant different (tukeys’s hsd p<0.05). table 2 pearson correlation for the xyz-cie parameters x y z x 1.000 y 0.993 1.000 z 0.834 0.883 1.000 adv. hort. sci., 2016 30(4): 231-237 236 three components in the pca (tabachnick and fidell, 2006). therefore, only x factor has been considered in the simplified pca test, because from the statistical analysis (fig. 7) such variable appears more sensible to the ripening process compared to y and z. table 3 reported all the other correlations. between diameter and weight the correlation is sufficient to justify the usage of the height and diameter ratio (h/d) to get an unique shape factor that will reduce the variability in the data set (tabachnick and fidell, 2006). sugar content and ph are well positively correlated (0.694), as expected. applying the above mentioned consideration, the simplified pca is reported in figure 8. the first three components explain 71.8% of the variability. table 4 reports loadings values for the first three components. ssc and ph have the highest loadings values and are positively correlated, thus able to explain almost all the variance of the dataset. also color has a high loading value (table 3) that suggests it as one of the possibly maturity index suitable for the afghan farmers. the parameter weight explains the remaining variance. weight, as very easy and cheap measure can be used as additional maturity index with sugar content, ph and color. 4. conclusions the cherry cv. burlat ohd is set for the year 2015 at 26th of may. such result must be verified in the next coming years and correlated with the meteorological situation. based on the whole results dataset, the suggested ‘burlat’ characteristic at the ohd are: height: 19.167±0.254 mm, weight: 6.705±0.126 mm, ph: 3.246±0.009, sugar content: 22.488±0.5°brix, ta: 15.433±0.450 g/ml, firmness 0.653±0.037 n, x: 6.391, y: 5.229, z: 5.368 (rgb=6b3540 html color). as explained, the aim of the study was providing a simple tool applicable in the field as maturity index to the afghan farmers. as reported, sugar content, ph, color and weight have very good chances to be used as maturity indexes. however, for the color determination, the required instrument is very expensive and not suitable for the afghan condition, in which the gross national income per capita is 640 usd (world bank, 2004). a similar conclusion is valid for the ph. sugar content is, thus, the suggested solution for farmers. it is relatively cheap and extremely easy to use. however, based on the laboratory results, hps may provide color gauge specific for each single varieties to the hps beneficiaries and confirm with ssc, weight, ph and color tests the validity of sugar content as maturity index. the same methodology applied for ‘burlat’, and explained in this study, was used with four other table 4 variance explained by components table 3 pearson correlation for all paramenters fig. 8 simplified pca using the following parameters: weight, ssc, ta, ph, h/d, firmness, x. weight height diameter grade firmness ssc x ph ta weight 1 height 0.274 1 diameter 0.599 0.485 1 grade 0.540 0.121 0.423 1 firmness -0.207 0.053 0.119 -0.098 1 ssc -0.134 -0.259 -0.339 0.035 -0.251 1 x -0.302 0.073 0.023 -0.382 0.417 -0.704 1 ph -0.199 -0.207 -0.315 -0.037 -0.260 0.694 -0.481 1 ta 0.129 0.045 0.153 0.262 -0.193 0.152 -0.306 -0.126 1 variable pc1 pc2 pc3 weight 0.027 -0.609 0.166 h/d -0.133 0.367 -0.724 firmness 0.317 0:35 -0.001 ssc -0.568 0.069 0.003 ph -0.532 0.733 0.166 ta 0.072 -0.477 -0.645 x 0.520 0.291 0.070 valori et al. optimum harvest date in afghanistan. sweet cherry: a case study 237 cherry cultivars: balck star, cherry pie, santina and stella compact, following the estimated ohd: ‘black star’: 6 june, ‘cherry pie’: 30 may-6 june, ‘santina’: 4 june, ‘stella compact’: 6-11 june. acknowledgements this study was funded by phdpii (europe aid/1293 2 0 / c / s e r / a f / 2 ) a n d h p s ( e u r o p e a i d / 1 3 3 872/l/af). in memory of dr. monica berti. references batu a., 2004 determination of acceptable firmness and color values of tomatoes. j. food engineering, 61(3): 471-475. baumann h., 1998 decrease of starch to predict the ‘optimum harvest date’ for apple storage. acta horticiculturae, 466: 41-46. çalhan ö., ebru o.c., güneyli a.,·eren i., demirtaş i., 2014 optimum harvest date of sweet cherry cv. lapins grown in isparta. türk tarım ve doğa bilimleri, 7(7): 1905-1910. drake s.r., elfving d.c., 2002 indicators of maturity a n d s t o r a g e q u a l i t y o f l a p i n s ’ s w e e t c h e r r y . horttechnology, 12(4): 687-690. konika minolta, 2013 precise color communication. color control from perception to instrumentation. konika minolta camera co. ltd. japan, pp. 62. tabachnick b.g., fidell l.s., 2006 using multivariate statistics. 5th ed. allyn & bacon, boston, ma, usa, pp. 1008. world bank, 2014 data indicators. country afghanistan available at http://data.worldbank.org/country/ afghanistan. wto, 1995 a agreement on agriculture (aoa). is an international treaty of the world trade organization. it was negotiated during the uruguay round of the general agreement on tariffs and trade, and entered into force with the establishment of the wto on january 1. world trade organization. wto, 1995 b agreement on the application of sanitary and phytosanitary measures (sps agreement). is an international treaty of the world trade organization. it was negotiated during the uruguay round of the general agreement on tariffs and trade, and entered into force with the establishment of the wto at the beginning of january. world trade organization. žnidarčič d., požrl t., 2006 comparative study of quality changes in tomato cv. ‘malike’ lycopersicon esculentum mill.) whilst stored at different temperatures. acta agriculturae slovenica, 87: 2. impaginato 3 1. introduction strawberry (fragaria x annanassa duch.) is one of the most important berries produced in the world. in argentina, 33000 tons are produced, mainly cultivar camarosa (gómez riera et al., 2013). it is highly accepted by consumers firstly because of its physical aspect and organoleptic and nutritional properties (shin et al., 2008; garriga et al., 2015). nevertheless, its postharvest quality rapidly declines due to the soft texture, high metabolic activity and susceptibility to bacterial and fungal rots. usually, there is quality decay during transport and commercialization (dotto et al., 2011). cell wall degradation is one of the principal causes of strawberry´s postharvest quality decay. as pectine synthesis occurs along the fruit maturation, they are less firmly attached to the cell wall. additionally, middle lamella´s debilitation and solubilization during fruit ripening diminishes cell cohesion (lara et al., 2004). calcium plays a preferential role on permeability and cell integrity and has a direct influence on fruit firmness and storage time (fernández et al., 2006). calcium functions as an intracellular cement because it forms calcium-pectine complexes that give firmness to vegetable tissues. calcium´s presence also favours pectic material insolubilization and inhibits i t s d e g r a d a t i o n b y p o l y g a l a c t u r o n a s e e n z y m e (alonso, 1995). an immersion in calcium at harvest could increase calcium´s content in strawberry, increasing fruit firmness (galetto et al., 2010) and thereby storage period. however, the key factor for quality maintenance is temperature optimum for strawberry is 0°c (mitcham et al., 2015). could an immersion in calcium allow an increase in storage temperature, maintaining fruit quality? other factors, such as growing system and subs t r a t e , c a n i n f l u e n c e s t r a w b e r r y ´ s q u a l i t y . greenhouse production improves organoleptic quality of fruits and vegetables because they are not exposed directly to air conditions as in open field (gruda, 2009). soilless production, moreover, diminishes incidence of diseases and pests (urresterazu, 2004). adv. hort. sci., 2017 31(1): 3-10 doi: 10.13128/ahs-20719 a calcium lactate treatment at harvest, growing system and refrigerated modified atmosphere can affect strawberry’s ´camarosa´ postharvest quality? m. harris (*), m.c. llorens, d. frezza universidad de buenos aires, facultad de agronomía, departamento de producción vegetal, cátedra de horticultura, avenida san martin 4453 (1417) ciudad autónoma de buenos aires, argentina. key words: nutritional quality, organoleptic quality, perlite, postharvest behaviour, soil, storage temperature. abstract: the aim of this work was to evaluate the effect of a calcium lactate treatment on postharvest behaviour, organoleptic and nutritional quality of strawberries (fragaria x ananassa duch., cv. camarosa) grown in different growing systems and stored in refrigerated modified atmosphere. strawberry grown in perlite and soil in greenhouse and soil in open field was harvested and dipped in a calcium lactate 1% solution. fruits were packed in modified atmosphere at 1°c and 8°c. at 1, 3 and 7 days of storage postharvest behaviour, organoleptic and nutritional quality was evaluated. calcium had a positive effect on fruit firmness and no differences were observed between storage temperatures in calcium treated fruits. organoleptic quality (except visual quality) was better in fruits grown in open field soil, regardless calcium treatment and storage temperature. nutritional quality was better in untreated fruits and stored at 1°c. (*) corresponding author: mharris@agro.uba.ar received for publication 6 june 2016 accepted for publication 12 december 2016 copyright: © 2017 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2017 31(1): 3-10 4 the aim of this work was to evaluate the effect of a calcium lactate treatment on postharvest behaviour, organoleptic and nutritional quality of strawberries grown in three growing systems and stored at two temperatures in modified atmosphere during 7 days of storage. 2. materials and methods plant material and growth conditions the experiment was conducted in the horticulture experimental field, school of agriculture, university of buenos aires (latitude 45° s, longitude 58° 31´ w, altitude 26 m asl). strawberry seedlings (commercial variety ‘camarosa’) were planted in three growing systems: soil in open field, soil and perlite in greenhouse. plant density was 7 plants m-2. soil treatments were covered with a black plastic mulching. all treatments were fertirrigated: nutrient solutions were formulated according to strawberry´s requirements (table 1). hourly intervals of temperature (°c), relative humidity (%) and radiation (w·m-2) were measured in open field and greenhouse with datalogger hobo. harvest mature strawberry fruits (at least 75% red colour) were harvested and immediately submerged in cold water to decrease fruit temperature. half of the fruits of each growing system were treated with a calcium lactate 1% solution during one minute. although calcium chloride is most commonly used, calcium lactate was used as a firming agent (codex alimentarius world health organization, 2015) because chloride can give a bitter taste to fruits (oms-oliu et al., 2010). sixty-five grams (g) of fruit were packed in a modified atmosphere (medium density polyethylene semi rigid container). modified atmosphere increases postharvest life in fruits and vegetables by reducing respiratory rate (sandhya, 2010). containers were stored at storage chambers at 1°c and 8°c. postharvest at 1, 3 and 7 days of storage, quality characteristics of fruits were evaluated as follows: postharvest behaviour oxygen and carbon dioxide concentration in the container was measured with dansensor gas analyzer and expressed as percentage. fresh weight loss. fruits were weighted and weight loss was expressed as percentage relative to the initial value. organoleptic quality visual quality. the overall visual and sanitary quality was determined by scoring each strawberry using a 1-10 hedonic scale, being 10 excellent and 6 the commercialization limit. colour was measured with minolta chromameter cr300. l, a, b, c, h parameters were determined in four spots in equatorial zone of three fruits per treatment. firmness was measured in equatorial zone with ludwig penetrometer fitted with a 3 mm diameter round probe. total soluble solids were determined with atago refractometer and expressed as °brix. nutritional quality ascorbic and dehidroascorbic acid was determined by liquid chromatography and expressed as mg of ascorbic acid 100 g-1 of fresh weight. antioxidant capacity. antioxidant capacity was estimated by determining the free-radical scavenging capacity evaluated with the stable radical dpph (adapted from brand williams et al., 1995 and leong and shui, 2002). two g of edible portion of the fruit was homogenized using a blender and inserted into a 50 ml centrifuge tube. twenty ml of 50% aqueous ethanol was added (1:10 w/v) and mixed in a vortex mixer for 15-30 seconds. the extract was centrifuged at 2000 g for 5 min al 4°c. the supernatant was filtered before using. a 25 mg solution of dpph (1,1 diphenyl-2-picrylhydrazyl) was prepared in methanol. for calibration curve, aliquots of ascorbic acid (0, 25, 50, 75, and 100 μl) solved in aqueous ethanol 50% (0.1 ml ml-1) were placed in tubes with in 3 ml dpph. absorbance at 517 was measured at 1, 10, 30, 60, 90 and 120 minutes. an aliquot of 50 ml of an antioxidant/fruit extract solution was added to 3 ml of the dpph solution. the decrease in absorbance at 517 macroelements microelements element concentration (mg l-1) element concentration (mg l-1) nitrogen 64 iron 2.8 phosphorus 31 sodium 1.2 potassium 200 manganese 0.5 sulfur 64 boron 0.5 magnesium 48 copper 0.02 -zinc 0.05 molybdenum 0.01 table 1 nutrient solution formulation harris et al. calcium lactate treatments effects on postharvest of strawberry cultivar camarosa 5 nm was measured at 0, 1, 5 and then every 10 minutes until the reaction reached a plateau. the decreased absorbance of dpph remaining at the steady-state was calculated and expressed as mg of a s c o r b i c a c i d ( a a ) e q u i v a l e n t s p e r 1 0 0 g o f homogenate (aeac). the aeac was calculated using the following equation: aeac=δ a x f x v x 100 x 1/w where δ a is the change of absorbance after addition of fruit extract, f is the inverse of the calibration curve slope, v is the volume of filtrate (ml) and w is the weight of homogenate used for extraction (g). statistical analysis a completely randomized factorial design with 3 replicates per treatment was used. the results were analyzed by multivariate analysis of variance repeated in time with a 5% significance level. tukey test to compare means was used (kuehl, 2001). infostat software was used (di rienzo et al., 2015). 3. results and discussion oxygen and carbon dioxide concentration in the container temperature, as expected, generated the most significant differences: strawberries stored at 1°c respired less than those stored at 8°c (fig. 1). nevertheless, in all cases, at equal temperatures, strawberries treated with calcium decreased the respiration. on the other hand, an interaction between growing systems and calcium treatment was observed (p<0,0001) both in oxygen and carbon dioxide levels: strawberries grown in soil systems (both open field and greenhouse) and without calcium treatment showed larger differences between storage temperatures: those stored at 8°c showed a high oxygen decrease and carbon dioxide increase, especially at the 7th day of storage. treated fruits, instead, showed similar behaviour during the seven days of storage. thus, calcium treatment could be considered as a regulator because differences between temperat u r e s i n t r e a t e d s t r a w b e r r i e s w e r e s m a l l e r . waghmare and annapure (2013) observed that a combination of modified atmosphere, calcium chloride and nitric acid treatment in chopped papayas stored at 5°c had a significant decrease of oxygen and increase of carbon dioxide compared to fruits only stored in modified atmosphere. equilibrium modified atmosphere was not established, especially those stored at low temperatures. this could be because medium density polyethylene was used and this material has low permeability to gases: 2600 cm 3/m 2.d.atm for oxygen and 7600 cm3/m2.d.atm for carbon dioxide (sandhya, 2010). additionally, as temperature increases, material permeability increases as well. thus, containers stored at 8°c had a higher permeability (oliveira et al., 2015). fig. 1 oxygen and carbon dioxide concentration (%) in containers at 1, 3 and 7 days of postharvest of strawberries grown in different growing systems, calcium lactate treatment and two storage temperatures. adv. hort. sci., 2017 31(1): 3-10 6 fresh weight loss maximum allowable weight loss in strawberry is 6% (laurin et al., 2003). all treatments, except one case, were below those values (fig. 2). the smaller w e i g h t l o s s w a s o b s e r v e d i n o p e n f i e l d f r u i t s (p<0.0001), independently storage temperature and calcium treatment. preharvest temperatures can affect postharvest shelf life. for example, fruits grown at high temperatures can exhibit water soaking (benkeblia et al., 2011). in this work, differences between greenhouse and open field temperatures reached 7°c. fruits harvested in greenhouse soil and soilless systems had surely higher temperature, what determined higher weight loss in those systems. visual quality fruits grown in perlite had better quality: 7.7% more than open field soil and 20% compared to greenhouse soil fruits (fig. 3). at the 7th day of postharvest, all the strawberries stored at 1°c prefig. 2 fresh weight loss (%) at 1, 3 and 7 days of postharvest of strawberries grown in different growing systems, calcium lactate treatment and two storage temperatures. maximum allowable weight loss (6%) is indicated. fig. 3 visual quality at 1, 3 and 7 days of postharvest of strawberries grown in different growing systems, calcium lactate treatment and two storage temperatures. each graph represents a combination of growing system and storage temperature. commercialization limit (6) is indicated. harris et al. calcium lactate treatments effects on postharvest of strawberry cultivar camarosa 7 sented visual and sanitary quality above commercialization level. fruits stored at 8°c, instead, were below that level and in most cases they had botrytis cinerea symptoms. temperature management is a key factor to minimize postharvest deterioration in strawberry. at high storage temperatures, fruits have higher respiration rates, and consequently, shorter postharvest shelf life (shin et al., 2008). colour significant differences were not observed for all colour parameters. shin et al. (2008) did not find colour changes during 7 days of storage of l c and h° values. firmness an interaction was observed between growing system and calcium treatment (p=0,0203): treated strawberries grown in soil systems, both open field and greenhouse had 20% more firmness than others. in perlite, difference between treated and untreated fruit was not significant (fig. 4). firmness increased in fruits stored at 1°c along the storage time. shin et al. (2007) also observed an increase in firmness along 4 days of storage at high (10.5°c ) and low (0.5°c) temperatures. the same authors investigated firmness during 12 days of storage and observed a positive tendency in fruits stored at 3°c and a decrease in those stored at 10°c (shin et al., 2008). firmness increase in low storage temperatures is due to physical changes in cell wall: cold produces an increase in pectin viscosity, which impacts positively in fruit firmness (lara et al., 2004). total soluble solids total soluble solid content (table 2) in all cases was above the minimum content recommended (7°brix) for the postharvest quality maintenance (mitcham et al., 2015). a significant interaction was found between temperature and growing system (p<0,0001): fruits stored at 8°c and grown in open table 2 total soluble solids (°brix) at 1, 3 and 7 days of postharvest of strawberries grown in different growing systems, calcium lactate treatment and two storage temperatures growing system storage 1°c storage 8°c 1 day 3 days 7 days 1 day 3 days 7 days ca with ca without ca with ca without ca with ca without ca with ca without ca with ca without ca with ca without soil open field 8 a bc 10 a d 7.63 ab ab 7.83 ab bc 8 a bc 6.33 a a 8.50 a bc 7.17 a a 9.17 ab cd 12 a e 8 a ab 9 ab cd soil greenhouse 8 a bc 7.33 b a 7.75 ab ab 8.67 bc cd 8.17 ab bc 9.33 ab de 8.33 a cd 8.17 bc bc 8.33 b cd 7.33 bc ab 6 b a 8.83 b e perlite greenhouse 8 a a 9.5 a bc 8 a a 9.17 cd ab 8 a a 8.83 bc ab 9 ab cd 6.5 ab a 8 bc ab 8.33 cd bc 10.5 c de 7.67 bc ab different capital letters indicate differences between row and different small letters indicate differences between columns (p<0.05). fig. 4 firmness (kpa) at 1, 3 and 7 days of postharvest of strawberries grown in different growing systems, calcium lactate treatment and two storage temperatures. adv. hort. sci., 2017 31(1): 3-10 8 field had higher content of soluble solids. it was only in the open field system that a difference between storage temperatures was found: those stored at 8°c had a 12.5% higher content of total soluble solids. calcium lactate immersion affected negatively the total soluble solid content. similar results were found by other authors with different calcium sources. singh et al. (2007) observed that weekly foliar applications of calcium chloride since flowering decreased total soluble solid content in 10% at harvest. dunn and able (2006), as well, found that a calcium deficiency during growth stage increased significantly soluble solids content in fruits. ascorbic acid content in almost all cases, ascorbic acid content (fig. 5) decreased during storage period. phillips et al. (2016) also observed a decrease in strawberries stored at 1.5°c during 7 days. only the fruits stored at freezers (-10 to -20°c) and ultra freezers (-55°c) maintained ascorbic acid contents similar to those observed at harvest time. low temperature storage is a key factor to maintain ascorbic acid content during postharvest (lee and kader, 2000). an interaction was observed between calcium treatment and storage temperature (p=0.0026). untreated fruits stored at 1°c had 26% more ascorbic acid compared to the others. many authors recognize that a calcium treatment at harvest enhances ascorbic acid content in fruit mainly due to an increase in fruit firmness (aghdam et al., 2013). nevertheless, other authors did not find differences between calcium treatment and the control (shaffie et al., 2010). regardless the calcium treatment, temperature was the key factor to maintain ascorbic acid content in strawberries. fruits grown in perlite had a 49% higher content compared to those grown at soil (both open field and greenhouse). these results are in agreement with de data reported by treftz and omaye (2015), who observed that soilless grown strawberries had a 74% higher content of ascorbic acid compared to soil grown fruits. for other fruits, the results were contradictory: isabelle et al. (2010) observed higher ascorbic acid content in pepper and özcelik and akilli (1999) in tomato. however, gruda (2005) didn´t find differences in strawberry. antioxidant capacity several interactions were observed for antioxidant capacity. untreated and greenhouse soil grown fruits presented 13% higher antioxidant capacity ( p = 0 , 0 0 0 3 ) ( t a b l e 3 ) . w a n g a n d z h e n g ( 2 0 0 1 ) observed that strawberries grown under higher temperature (day and night) had higher antioxidant capacity, as it was observed in this work, where differences of temperatures between greenhouse and fig. 5 ascorbic acid content (mg 100 g-1) at 1, 3 and 7 days of postharvest of strawberries grown in different growing systems, calcium lactate treatment and two storage temperatures. harris et al. calcium lactate treatments effects on postharvest of strawberry cultivar camarosa 9 open field reached 7°c. as well, untreated fruits stored at 1°c had 10% h i g h e r a n t i o x i d a n t c a p a c i t y ( p = 0 . 0 2 9 5 ) . m a n y authors, as described in 3.7, explain that a calcium treatment enhances ascorbic acid content (and consequently antioxidant capacity) due to an increase in cell wall firmness. nevertheless, other authors express that a disruption in cell wall composition increases antioxidant capacity, but this increase is different depending the product (reyes et al., 2006). with respect to storage temperature, shin et al. (2008) found significant differences since the 12th day. different capital letters indicate differences between columns and different small letters indicate differences between rows (p< 0.05). yield in the growing systems although yield was not an objective of this work, we observed that the perlite system had 35% and 17% higher yield than soil in greenhouse and soil in open field systems, respectively. 4. conclusions strawberry is highly accepted by consumers but its postharvest quality rapidly declines. a calcium lactate treatment can increase principally strawberry’s firmness. this, in addition to storage temperature and growing system can increase postharvest shelf life of fruits. in this research, the calcium lactate treatment had a positive effect on fruit firmness, especially in those grown in open field and greenhouse soil. furthermore, treated fruits had less respiration during storage time and there was no significant difference between storage temperatures in fruits treated with calcium lactate. equilibrium modified atmosphere was not established. the other variables were not affected or had a negative response to calcium lactate treatment. weight loss and organoleptic quality (except visual quality) were better in fruits grown in open field soil, regardless calcium treatment and storage temperature. on the other hand, nutritional quality was better in untreated fruits and stored at 1°c. in conclusion, even though temperature is substantial to maintain fruit quality at postharvest, a calcium lactate treatment could be useful to improve strawberry (cv. camarosa) quality during transport and commercialization and decrease incidence of postharvest diseases. acknowledgements the present work was funded by grants from buenos aires university references aghdam m.s., dokhanieh a.y., hassanpour h., rezapour fard j., 2013 enhancement of antioxidant capacity of cornelian cherry (cornus mas) fruit by postharvest calcium treatment. sci. hortic., 161: 160164. alonso j., rodriguez t., canet w., 1995 effect of calcium pretreatments on the texture of frozen cherries. role of pectinesterase in the changes in the pectic materials. j. agric. food chem., 43: 1011-1016. benkeblia n., tennant d.p.f., jawandha s.k., gill p.s., 2011 preharvest and harvest factors influencing the postharvest quality of tropical and subtropical fruits, pp. 112-142. in: yahia e. 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worldwide production of about 44 million tons each year f r o m 2 0 1 2 t o 2 0 1 6 , 2 8 m i l l i o n t o n s i n t h e e u ( c o m m i t t e e f o r t h e c o m m o n o r g a n i z a t i o n o f agricultural markets, 2016). sunflowers prove to be a protein source of great interest for human nutrition; moreover, the residues originating from oil extraction are rich in phenolic antioxidants, which account for 1-4% of the total mass with chlorogenic acid being the predominant component (weisz et al., 2009). polyphenols in sunflower seeds were identified and quantified after hplc analysis (aramendia et al., 2000; pedrosa et al., 2000). the main phenolic compounds present in both the kernel and hull besides chlorogenic acid are caffeic acid and caffeoylquinic derivatives. phenolic compounds in sunflower seeds have been shown to exert a high antioxidative potential, which might be beneficial from a biofunctional point of view and may be used as effective antioxidants for sunflower oils (de leonardis et al., 2003, 2005; anjum et al., 2012). organic agriculture is most widely used and its benefits concern overall the environment and the health of food, which is not contaminated with pesticides and synthetic fertilizers. however organic fertilizer of biological origin may lead in the long term to t h e “ c o n v e n t i o n a l i z a t i o n o f o r g a n i c f a r m i n g ” (darnhofer et al., 2010). in order to go deeper in the organic management, the montepaldi long term experiment (molte) trial in central italy has been set up to compare three agro-ecosystems with different management: two organic (old organic since 1992 and young organic since 2001) and one conventional (migliorini, 2014). the aim of this research is to follow the fate of polyphenols in teguments, kernels and oil from sunflower seeds grown under the above-mentioned three different conditions (one conventional and two organic) in order to assess whether such condition may affect the polyphenols profile and which compounds may be regarded as innovative parameters in order to deeper investigate the relation among soil condition, agricultural management and seed characteristics. 2. materials and methods the montepaldi long term experiment (molte) h a s b e e n a c t i v e s i n c e 1 9 9 1 i n t h e f a r m o f t h e university of florence (location montepaldi, san adv. hort. sci., 2017 31(2): 85-88 doi: 10.13128/ahs-20608 seeds and oil polyphenol content of sunflower (helianthus annuus l.) grown with different agricultural management a. romani 1, p. pinelli 1, v. moschini 2, d. heimler 2 (*) 1 dipartimento di statistica, informatica, applicazioni “g. parenti”, università degli studi di firenze, viale morgagni, 40, 50134 firenze, italy. 2 dipartimento di scienze delle produzioni agroalimentari e dell’ambiente, università degli studi di firenze, piazzale delle cascine, 18, 50144 firenze, italy. key words: organic management, polyphenol content, sunflower. abstract: using a long term experiment (20 and 11 years of organic cultivation on the same soil), sunflower was cultivated under organic management and in a different part of the same farm under conventional management. kernels, teguments and oils were analyzed for their polyphenols content. five caffeoylquinic acids were identified. no qualitative differences were found in the three cases, while quantitative differences have been pointed out and discussed. (*) corresponding author: daniela.heimler@unifi.it received for publication 2 may 2017 accepted for publication 15 may 2017 copyright: © 2017 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2017 31(2): 85-88 86 c a s c i a n o , v a l d i p e s a , l o n g . 1 1 ° 0 9 ’ 0 8 ’ ’ e , l a t . 43°40’16’’ n) covering a slightly sloping surface of about 15 ha at 90 m a.s.l. the molte experiment has a system approach and includes the following three different micro agro-ecosystem managements: a) “old organic” (oldo) with an area of 5.2 ha, divided into 4 fields under organic since 1992; b) “youngorganic” (yngo) with an area of 5.2 ha, divided into 4 fields (integrated farming) from 1992 to 2000 and converted into organic management since 2001; c) “conventional” (conv) area of 2.6 ha divided into 2 conventional fields, where farming techniques used were those normally used in the territory of the study area for conventional management. on october 28, 2013, after harvesting the whole sunflower plant, seeds were put in an oven at 60°c for 48 hours and then preserved in paper bags. seeds were manually dehulled in order to obtain kernel and tegument. phenolic compounds were extracted for 30 min from kernels (about 2 g) and from teguments (about 1 g) with 70:30 ethanol/water (25 and 15 ml, respectively). the extracts were evaporated under vacuum at room temperature and finally dissolved in 10 ml ethanol/water (70:30). cold-pressed seeds oils ( 2 5 m l ) w e r e e x t r a c t e d w i t h 3 x 2 5 m l o f 7 0 : 3 0 ethanol/water, adjusted to ph 2.0 with formic acid; each step involved an extraction for 30 min at room temperature. the extracts were combined and defatted with 3x50 ml hexane. the defatted extracts were evaporated to dryness under vacuum at room temperature and finally dissolved in ethanol/water (70:30) to a final volume of 4 ml. solvents and reagents all the solvents (hplc grade) and formic acid (acs reagent) were purchased from aldrich chemical c o m p a n y i n c . ( m i l w a u k e e , w i s c o n s i n , u s a ) . chlorogenic acid was obtained from extrasynthese s.a. (lyon, nord-genay, france). the hplc-grade water was obtained via double-distillation and purification with a labconco water pro ps polishing station (labconco corporation, kansas city, usa). hplc analysis hplc/dad analysis. the hplc/dad analyses were performed with an hp 1100l liquid chromatograph equipped with hp dad (agilent technologies, palo alto, ca, usa). a kinetex c18 column 100×2.1 mm, 5 μm (phenomenex) operating at 30°c was used. the eluents were h2o adjusted to ph 3.2 by formic acid and acetonitrile. a four-step linear solvent gradient was performed starting from 100% water up to 100% acetonitrile, with a flow rate of 0.2 ml/min for a 30min period (table 1). hplc/esi-ms analysis. the hplc-ms analyses were performed using an hp 1100l liquid chromatograph equipped with a dad and 1100 ms detectors. the interface was an hp 1100 msd api-electrospray (agilent technologies). mass spectrometer operating conditions were the following: gas temperature 350°c at a flow rate of 10.0 l/min, nebulizer pressure 30 psi, quadrupole temperature 30°c and capillary voltage 3500 v. the mass spectrometer operated in positive and negative ionization mode at 80-120 ev, for both ionization modes. 3. results and discussion in figure 1, the chromatographic profile of organic sunflower kernel, registered at 330 nm, maximum wavelength of absorbance of caffeic acid and its derivatives, is reported. eight compounds have been identified: 3-o-caffeoylquinic acid, chlorogenic acid, 4-o-caffeoylquinic acid, three derivatives of caffeic acid, 3,5-o-dicaffeoylquinic acid, and 4,5-o-dicaftable 1 linear solvent gradient system used in hplc-dad and hplc-ms analysis time (min) a b 0.10 100.0 0.0 5.00 80.0 20.0 7.00 80.0 20.0 13.00 70.0 30.0 18.00 70.0 30.0 22.00 30.0 70.0 26.00 30.0 70.0 30.00 0.0 100.0 fig. 1 hplc chromatogram of sunflower kernel extract recorded at 330 nm. peaks= 1. 3-o-caffeoylquinic acid; 2. chlorogenic acid; 3. 4-o-caffeoylquinic acid; 4-6. caffeic acid derivatives; 7. 3,5o-dicaffeoylquinic acid; 8. ac. 4,5o-dicaffeoylquinic acid. solvent a= h2o adjusted to ph 3.2 by hcooh. solvent b= ch3cn. romani et al. seeds and oil polyphenol content of sunflower 87 feoylquinic acid. in the shells the same compounds with the exception of 4-o-caffeoylquinic acid and the two dicaffeoylquinic acids were identified. the same caffeoyl and dicaffeoylquinic acid derivatives were already found by aramendia et al. (2000), pedrosa et al. (2000), and weisz et al. (2009). the qualitative pattern does not change along with the agricultural management. as concerns quantitative data, table 2 reports the quantitative data of kernel and teguments. total polyphenols amount is the lowest, in the case of kernel, when the plant grows on the old organic field, while it is almost the same with the other two managements; in the case of teguments, on the contrary, the lowest amount is observed in the case of conventional management. it should however be noted that in the case of teguments, polyphenols content is very low changing from about 1 0 % t o 2 % w i t h r e s p e c t t o k e r n e l c o n t e n t . c h l o r o g en i c a c i d i s t h e m a i n c o m p o u n d a n d i t accounts for about 90% in tegument. chlorogenic acid in our samples, both kernel and tegument, is about ten times more abundant with respect to the quantitative data reported by aramedia et al. (2000) and pedrosa et al. (2000). the comparison of quantitative data is however difficult since different techniques and sample treatments are involved (weisz et al., 2009). polyphenols content along with agriculture management has recently reviewed and in most vegetables grown under organic conditions, a higher content of polyphenols has been found; on the other hand a higher soil nitrogen availability decreases polyphenols content (heimler et al., 2017). our data support previous findings that indicate how old managed organic soil was the most efficient in term of c and n storage (migliorini et al., 2014). furthermore, when the individual compounds are taken into account the relative percentages of compounds from new organic and conventional managed soils are similar, while in the case of the old managed organic soil, notwithstanding the lowest total polyphenol content, a higher dicaffeoylquinic acids content was found (17% with respect to 12%). chlorogenic acid and d i c a f f eo yl q u i n i c a c i d s d eri ve f ro m t h e p h en yl propanoid pathway. generally, this pathway is induced by biotic and abiotic stress such as wounding, uv irradiation, or pathogen attack (moglia et al., 2008). no information is available on the regulation of dicaffeoylquinic acids in any plant species, even if the same regulation of chlorogenic acid synthesis could be foreseen (moglia et al., 2008). in table 3, the data of sunflower oil are reported. oil has been obtained by means of a mechanical oldo= old organic; youngo= young organic; co= conventional (see experimental section). nd= not determined. standard deviation within brackets. table 2 polyphenols (mg/g, fresh weight) in sunflower kernel and tegument compound agricultural management kernel tegument oldo youngo co oldo youngo co 3-o-caffeoylquinic acid 2.51(0.37) 4.57(0.82) 4.74(0.76) 0.08(0.01) 0.08(0.01) 0.05(0.01) chlorogenic acid 6.25(1.12) 11.39(1.94) 11.82(1.42) 0.95(0.18) 0.96(0.18) 0.41(0.07) 4-o-caffeoylquinic acid 0.44(0.08) 0.8(0.11) 0.83(0.13) 0.03(0.005) traces traces caffeic acid derivatives 0.21(0.04) 0.38(0.04) 0.39(0.06) traces n.d. traces 3,5-o-dicaffeoylquinic acid 1.3(0.24) n.d. n.d. n.d. n.d. n.d. 4,5-o-dicaffeoylquinic acid 0.77(0.11) 2.36(0.34) 2.45(0.27) n.d. n.d. n.d. total polyphenols 11.48 19.5 20.23 1.06 1.04 0.46 oldo= old organic; youngo= young organic; co= conventional (see experimental section). nd= not determined. standard deviation within brackets. table 3 polyphenols (mg/l) in sunflower oil compound agricultural management oldo youngo co 3-o-caffeoylquinic acid 0.232(0.192) 0.35(0.09) 0.176(0.057) chlorogenic acid 1.96(0.393) 2.43(0.11) 1.936(0.12) p-coumaroylquinic acid 2.36(0.318) 0.112(0.022) 0.592(0.079) 4-o-caffeoylquinic acid 0.048(0.005) 0.104(0.011) 0.08(0.02) caffeic acid derivatives 2.28(0.644) 0.328(0.079) 3.216(0.415) not identified polar compounds 0.504(0.011) 1.2(0.045) 1.456(0.429) total polyphenols 7.384 4.368 7.456 adv. hort. sci., 2017 31(2): 85-88 88 press, which is the only technology that allows the maintenance of polyphenols high content (bendini et al., 2011). oil yields are different (22.2% for old organic soil, 29.8% for young organic soil and 24.7% for conventional management) and, the young organic soil, with the highest yield, exhibits the lowest total polyphenol content. sunflower oil-amount of 10 mg kg-1 has been reported (de leonardis et al., 2005). polyphenols content of the three seeds oils (table 3) are almost in accordance with such amount. young managed organic soil gave rise to an oil with the smallest polyphenols content notwithstanding the high polyphenols content of almond and teguments. acknowledgements the authors acknowledge the regione toscana ‘‘praf 2012-2015 misura 1.2 e)’’ program (project volatosca). references anjum f.m., nadeem m., khan m.i., hussain s., 2012 nutritional and therapeutic potential of sunflower seeds: a review. brit. food j., 114: 544-552. aramendia m.a., garcia i.m., lafont f., lizaso j., marinas j.m., urbano f.j., 2000 rapid determination of chlorogenic acid and related compounds in sunflower seeds by high performance liquid chromatography/atmospheric pressure chemical ionization mass spectrometry. rapid commun. mass spectrom., 14: 1019-1022. bendini a., barbieri s., valli e., buchecker k., canavani m., gallina toschi t., 2011 quality evaluation of cold pressed sunflower oils by sensory and chemical analysis. eur. j. lipid sci. technol., 113: 1375-1384. committee for the common organization of agricultural markets, 2016 oilseeds and protein crops, market situation. http://ec.europa.eu/agricult u r e / s i t e s / a g r i c u l t u r e / f i l e s / c e r e a l s / presentations/cereals-oilseeds/market-situationoilseeds_en.pdf darnhofer i., lindenthal t., bartel-kratochvil r., zollitsh w., 2010 conventionalization of organic farming practices: from structural criteria towards an assessment based on organic principles. a review. agron. sust. dev., 30: 67-81. de leonardis a., macciola v., di domenico n., 2005 a first pilot study to produce a food antioxidant from sunflower seed shells (helianthus annuus). eur. j. lipid sci. technol., 107: 220-227. de leonardis a., macciola v., di rocco a., 2003 oxidative stabilization of cold pressed sunflower oil using phenolic compound of the same seed. j. sci. food agric., 83: 523-528. heimler d., romani a., ieri f., 2017 plant polyphenol content, soil fertilization and agricultural management: a review. eur. food res. technol., 243(7): 1107-1115. miglioriini p., moschini v., tittarelli f., ciaccia c., benedettelli s., vazzana c., canaliu s., 2014 agronomic performance, carbon storage and nitrogen utilization of long-term organic and conventional stockless arable systems in mediterranean area. europ. j. agron., 52: 138-145. moglia a., lanteri s., comino c., acquadro a., de vos r., beekwilder j., 2008 stress-induced biosynthesis of dicaffeoylquinic acids in globe artichoke. j. agric. food chem., 56: 8641-8649. pedrosa m.m., muzquiz m., vallejo c.g., burbano c., c u a d r a r o c . , a y e t g . , r o b r e d o l . m . , 2 0 0 0 determination of caffeic and chlorogenic acids and their derivatives in different sunflower seeds. j. sci. food agric., 80: 459-464. w e i s z g . m . , k a m m e r e r d . r . , c a r l e r . , 2 0 0 9 – identification and quantification of phenolic compounds from sunflower (helianthus annuus l.) kernels and shells by hplc-dad/esi-ms. food chem. 115: 758-765. 151 1. introduction the genus ocimum (lamiaceae family), collectively called basil, comprises between 50 and 150 species of herbs and shrubs (darrah, 1980). basil is native to asia (india, pakistan, iran, thailand, and other countries) and can be observed growing wild in tropical and sub-tropical regions (makri and kintzios, 2008). the essential oil profiles of this group of plants are extremely variable, such that several aroma compounds can be found in chemotypes of basil such as citral, eugenol, linalool, methylchavicol, and methylcinnamate that are traded in the international essential oil market (simon et al., 1999). the diversity within basil species has been accentuated by centuries of cultivation and cross compatibility, which has lead to great variation in morphology and chemical profile (javanmardi et al., 2002). basil species have antioxidant, antimicrobial and antitumor activities that are due to the presence of phenolic acids and aromatic compounds (hussain et al., 2008). lemon basil (ocimum × citriodorum vis.), a hybrid of sweet basil (ocimum basilicum) and american basil (ocimum americanum), is a herb grown primarily in northeastern africa and southern asia (fisher and phillips, 2006). it is naturalized in asia and cultivated for its lemon-scented leaves due to the essential oils citral and neral as predominant compounds (grayer et al., 1996). lemon basil is characterized by its small stature, early flowering, and small, narrow leaves. application of organic sources of nutrients, with no or very little use of inorganic fertilizers, is rapidly gaining favor (anwar et al., 2005). compost tea is a highly concentrated microbial solution produced by extracting beneficial microbes from compost. compost tea is produced by mixing compost with water and incubating it for a defined period, either actively aerating (aerated compost tea, act) or not (non-aerated compost tea, nct) and with or without additives that are intended to increase microbial population densities during production (scheuerell and mahaffee, 2002; ingham, 2005). it is a source of foliar and soil nutrients, contains chelated micronutrients for easy plant absorption and the nutrients are in biologically available forms for both plant and microbial uptake (hendawy, 2008). many researchers have pointed out the efficacy of organic manures, compost and compost teas in increasing vegetative growth, biomass and essential oil yield of sweet marjoram (gharib et al., 2008), cumin (safwat and badran, 2002), fennel (azzaz et al., 2009) and sweet basil (khalid et al., 2006). improvements in yield and quality following application of these organic-based substances has been attributed to an enhancement of the beneficial microbial communities in soil, an improvement of mineral absorption conditions for plants, and a stimulation of defense compounds, growth regulators or phytohormones in effects of chicken manure and vermicompost teas on herb yield, secondary metabolites and antioxidant activity of lemon basil (ocimum × citriodorum vis.) j. javanmardi(1), e. ghorbani department of horticultural sciences, faculty of agriculture, shiraz university, shiraz, iran. key words: essential oil, flavonoids, organic agriculture, sustainable agriculture, total phenolics. abstract: effects of chicken manure tea (cmt) and vermicompost tea (vct) as soil drench on vegetative growth, herb yield, essential oil content, total phenolics, total flavonoids and antioxidant activity of lemon basil (ocimum × citriodorum vis.) was evaluated in a two-year field experiment. the greatest plant height, number of leaves and flowers, shoot fresh and dry weight and leaf chlorophyll content were obtained using cmt at either 1:5 or 1:10 dilutions with no significant differences. the highest number of lateral branches and flavonoid content were obtained when cmt at 1:5 dilution was applied. essential oil content was at its highest level (0.618%) when cmt or vct were used at 1:10 dilution, while the greatest total phenolic content and total antioxidant activity were obtained at 1:5 dilution of vct. the results emphasize the possibility of using organic-based compost teas for enhancing herbal yield and important secondary metabolites in aromatic medicinal plants. adv. hort. sci., 2012 26(3-4): 151-157 (1) corresponding author: javann@shirazu.ac.ir. received for publication 27 june 2012 accepted for publication 31 january 2013 152 plants (pant et al., 2009). various liquid manures or their extracts are known to serve primarily as a source of soluble plant nutrients, growth stimulants and disease suppressors (khalid et al., 2006). several studies have reported the effects of compost tea on suppression of certain plant diseases such as damping– off caused by phytium ultimum (scheuerell and mahaffee, 2004), gray mold (botrytis cinerea) (scheuerell and mahaffee, 2006), alternaria solani and phytophthora infestans (koné et al., 2010). however, relatively little work has been done to investigate the effect of vermicompost or manure teas on yield, nutritional and quality factors, secondary metabolites and antioxidant activity of vegetable herbs. in the present work the effects of chicken manure and vermicompost teas as soil drench on growth characteristics, chlorophyll content, essential oil yield, total phenolics, total flavonoids and antioxidant activity of lemon basil were evaluated. the significance of this study lies in the possibility of applying compost teas as soil amendments to improve yield components and secondary metabolites in organic agriculture. 2. materials and methods site description, plant material and experimental design the experiment was carried out in two subsequent years, 2010 and 2011, in a field 1810 m above sea level in a silty-loam soil. three soil samples from 0-30 cm depth were collected and sent to a certified local soil laboratory for analysis. the chemical properties of the soil are shown in table 1. the local maximum-minimum mean temperatures and relative humidity during the growing period were 28.7-12.6°c and 47.3%, respectively. seeds of lemon basil (ocimum × citriodorum vis.) were sown in double rows (20 cm apart) with 50 cm spacing. seedlings were thinned three weeks after sowing to 15 cm between plants within the rows. plants were harvested at ground level when they were at full bloom stage (90 days after sowing) for further analyses. irrigation (using a drip-tube system), hand weeding, and other management practices were performed when required throughout the growing period. the experiment was carried out in a complete randomized block design with three replicates per treatment, each of which consisted of 20 plants. five fertilization treatments were applied as soil drench of de-ionized water (control), chicken manure tea (cmt) at 1:5 and 1:10 water dilution (v/v) and vermicompost tea (vct) at 1:5 and 1:10 water dilution (v/v). tea preparation and application method vermicompost and chicken manure teas were prepared as described by javanmardi (2010). briefly, vermicompost and chicken manure were separately mixed with tap water at ratios of 1:5 and 1:10 (v/v) in loosely covered 14 l plastic containers. water was allowed to stand for 24 h for passive chlorine removal before mixing. the mixtures were aerated using an aquarium pump for 72 h brewing time in a shaded area. solutions were filtered through cheesecloth before application. chemical properties for teas are presented in table 2. treatments were started four weeks after sowing and applied five times as soil drench with 600 ml of solution per plant at weekly intervals. fresh solutions were prepared for each application interval. vegetative growth parameters at full bloom stage ten central plants from each replicate (to avoid marginal effect) were cut at ground level and plant height (cm), number of branches per plant, number of flowers, number of leaves, and fresh and dry weight of herb (g per plant) were recorded. chemical analysis chlorophyll content. chlorophyll content was determined as described by saini et al. (2001). randomly selected samples of fully expanded leaves (0.5 g) were used. samples were homogenized with 5 ml of acetone (80% v/v) using a pestle and mortar and filtered through filter paper (whatman no. 2). the process was conducted in the dark to avoid photo bleaching. absorbance was measured with a uv-visible spectrophotometer (camspec m108, spectronic instruments, leeds, uk) at 652 nm and total chlorophyll content calculated using: total chlorophyll (mg·g-1 fw) = [d 652 ×v]×v/w where: v is the total volume of acetone extract (ml) and w, the fresh sample weight (g). essential oil content. quantitative determination of the essential oil obtained from lemon basil subjected to the different treatments was achieved by placing the air-dried herbage in a 2 l flask with distilled water (1:15 w/v) and using a clevenger apparatus, as described by charles and table 1 chemical properties of soil organic matter (%) total n (%) available phosphorus as p (mg·kg-1) bray method available potassium as k (mg·kg-1) fe (mg·kg-1) cu (mg·kg-1) mn (mg·kg-1) zn (mg·kg-1) ph ec (ds·m-1) 1.21 0.05 13.50 540 4.88 1.19 0.39 0.23 7.73 1.50 153 simon (1990). the average essential oil content of aerial parts is reported as percent of plant dry matter. sample preparation for total phenolic and flavonoid content determination. samples were prepared using the method described previously by javanmardi et al. (2003). briefly, 250 mg of dried plant material from each replicate were ground and dissolved in 10 ml of 80% acetone. sample extracts were rotated for 1 h in the dark and centrifuged at 5400 g for 10 min. one ml of supernatant was dried under vacuum at 45°c and kept at -18°c for further use. each sample was dissolved in 1 ml acetone prior to analysis for total phenolic and flavonoid determination. total phenolic compound analysis. the amount of total phenolics in extracts was determined with the folin-ciocalteau reagent using the method of spanos and wrolstad (1990), as described by javanmardi et al. (2003). to 50 ml of each sample, 2.5 ml of 1/10 dilution of folin-ciocalteau reagent and 2 ml of na 2 co 3 (7.5%, w/v) were added and incubated at 45°c for 15 min. the absorbance of all samples was measured at 765 nm using a uv-visible spectrophotometer (camspec m108, spectronic instruments, leeds, uk). gallic acid was used as standard and results are expressed as mg of gallic acid equivalent per g of dry weight (mg gae/g dw). total flavonoid analysis. the method described by adom and liu (2002) was adopted for total flavonoid content analysis. to 0.5 ml of extract, 2.5 ml distilled water were added followed by 0.15 ml of 5% nano 2 solution. the mixture was left to stand for 6 min at room temperature before adding 0.3 ml 10% alcl 3 .6h 2 o solution. the mixture was left for an additional 5 min, then 1 ml of 1 m naoh added and made up to 5 ml with distilled water. the solution was vortexed and the uv absorbance at 510 nm was recorded against catechin as reference. the result is expressed as µg/g dw. total antioxidant activity assay. the antioxidant activity of samples was determined by free radical scavenging activity assay using 1,1-diphenyl-2-picryl-hydrazil (ddph) reagent according to brand-williams et al. (1995). the ground leaves (1 g) were extracted with 50% methanol, 50% water. to 0.75 ml of the extract sample, 1.5 ml of freshly prepared methanolic dpph solution (20 µg·ml-1) were added and stirred. the decolorizing process was recorded after 5 min of reaction at 517 nm and compared with a blank control. the total antioxidant activity is expressed in % calculated as (control absorbance sample absorbance /control absorbance) × 100. statistical analysis the experiment was carried out for two years in a randomized complete block design (rcbd) with three replicates, each of which consisted of 10 plants. data were analyzed using one-way analysis of variance (one-way anova) and means of two years for each trait were compared with least significant difference (lsd) at p≤0.05 by spss12 (spss inc., chicago, il) computer software for windows. the data presented in tables and figures are mean values ± standard errors of two years of data for three replicates. pearson correlation analysis using spss12 (spss inc., chicago, il) was performed to assess the relationship between total phenolics, total flavonoids and essential oils with total antioxidant activity. 3. results and discussion the combined analysis of data from the experiment did not show any significant differences for all traits (data not shown). growth parameters analyses of variance showed that the growth parameters of lemon basil including plant height, number of lateral branches, leaves and flowers, shoot fresh and dry table 2 chemical properties of vermicompost tea (vct) and chicken manure tea (cmt) vermicompost tea (1:5) vermicompost tea (1:10) chicken manure tea (1:5) chicken manure tea (1:10) organic matter (%) total n (%) 6.30 3.14 2.21 1.10 phosphorus as p (%) 1.01 0.50 1.31 0.65 potassium as k (%) 6.18 3.08 5.61 2.81 fe (mg·l-1) 9.20 4.51 9.00 4.48 cu (mg·l-1) 0.07 0.03 5.00 2.48 zn (mg·l-1) 25.0 12.0 3.20 1.60 mn (mg·l-1) 0.18 0.89 0.08 0.04 ph 8.22 8.22 8.29 8.29 ec (ds·m-1) 4.41 2.20 2.82 1.41 154 weight at full bloom stage were significantly affected by vct and cmt (table 3). plant height and number of leaves the greatest plant height and leaf number were observed at 1:5 cmt, which was not significantly different from 1:10 dilution. other treatments did not show significant differences for plant height and leaf number (table 3). the same result has been reported in plantago plants under 300 ml·l-1 compost tea application (hendawy, 2008). the increasing effects of organic manure and bio-fertilizers on plant height on fennel (azzaz et al., 2009) and peppermint (swaefy et al., 2007) have been previously reported. number of lateral branches the highest lateral branch number was observed at 1:5 cmt dilution, however it was not significantly different from 1:10 cmt. the differences among other treatments (1:5 and 1:10 vmt and control) with regard to plant lateral branches were not significant (table 3). a promoting effect of organic fertilizers on the number of branches was observed also by azzaz et al. (2009) in fennel plants, especially when organic fertilizers were used in combination with bio-fertilizers. number of flowers the highest flower numbers were observed in lemon basil plants treated with cmt (1:5 and 1:10 dilutions) and vct (1:5 dilution) with no significant differences. control plants and vct-treated plants at 1:10 dilution showed lower flower numbers (table 3). an increased number of flowers in plantago plants under 300 ml·l-1 compost tea application has previously been reported (hendawy, 2008). shoot fresh and dry weight the highest shoot fresh and dry weights were observed at 1:5 cmt, which was not significantly different from 1:10 dilution. other treatments did not show significant differences for these parameters (table 3). the promoting effect of chicken manure tea on herb yield may be attributed to the micronutrient content and to the action of living micro-organisms and microbial metabolites which stimulate plant growth (diver, 2002; carpenter, 2005). higher fresh and dry weights have also been attributed to the availability of macronutrients, especially nitrogen, and/or to the improvement of soil water-holding capacity (el-sherbeny et al., 2005). furthermore, it has been stated that organic manure activates many species of living organisms which release phytohormones and may stimulate plant growth and absorption of nutrients (naguib and aziz, 2003). the same increased vegetative growth characters (including shoot fresh and dry weights) of basil plants under organic farming has been previously reported (khalid et al., 2006). chemical parameters leaf chlorophyll content. the highest leaf chlorophyll content was observed at 1:5 cmt which was not different from 1:10 cmt dilution. other treatments did not show significant differences for this parameter (table 3). the promoting effect of highly n-containing chicken manure tea on chlo rophyll contents might be attributed to the fact that n is a constituent of chlorophyll molecule. moreover, nitrogen is the main constituent of all amino acids and lipids that act as structural compounds of the chloroplast (al-tarwneh, 2005). in sweet basil, chlorophyll content was significantly higher when organic manure compost was applied than in non-fertilized control plants (taie et al., 2010). essential oil content. the essential oil content of lemon basil was affected by dilution levels of organic compost teas. the 1:10 dilution of vct and cmt was higher in essential oil content compared to 1:5 dilutions (fig. 1). the highest essential oil content was found in the 1:10 vct treatment and it was about 3.12 times higher than in control plants. previously, the highest essential oil content in ocimum basilicum was obtained following soil application of vermicompost at 10 t·ha-1 level compared to application of 10 t·ha-1 farmyard manure and control treatments (anwar et al., 2005). in marjoram plants, aqueous extract of compost increased essential oil percentage and yield (gharib et al., 2008). in basil as a source of essential oils and aroma compounds (simon et al., 1990), the increase in essential oil yield has been attributed to increase in vegetative growth or changes in leaf oil gland population (gharib et al., 2008). our data are in agreement with a previous work that found a higher essential oil percentage in batable 3 effect of different dilutions of chicken manure tea (cmt) and vermicompost tea (vct) on plant height, number of leaves, lateral branches and flowers, shoot fresh and dry weight and leaf chlorophyll content of lemon basil plants treatment plant height (cm) no. of leaves no. of lateral branches no. of flowers shoot fresh weight (g) shoot dry weight (g) chlorophyll (mg·g-1 fw) control 27.30±0.89 130.03±12.15 6.80±0.84 6.57±0.90 8.10±1.01 1.45±0.18 0.71±0.016 cmt (1:5) 30.47±1.27 225.93±22.80 10.23±0.92 11.80±1.62 17.63±2.44 2.64±0.36 0.82±0.012 cmt (1:10) 29.23±1.09 197.30±22.50 8.07±0.85 10.27±1.48 14.27±2.41 2.51±0.48 0.80±0.013 vmt (1:5) 27.57±0.64 136.70±13.58 6.77±0.93 10.47±1.80 8.37±0.92 1.60±0.18 0.74±0.016 vmt (1:10) 27.30±1.01 131.13±12.88 5.20±0.72 6.84±0.91 7.87±1.07 1.53±0.17 0.74±0.015 lsd value (p=0.05) 2.65 47.11 2.34 3.85 4.58 0.79 0.039 data (per plant) are mean values of 3 replicates ± standard errors. 155 sil due to application of organic fertilizers as compared to control plants (taie et al., 2010). fig. 1 effect of different dilutions of chicken manure tea (cmt) and vermicompost tea (vct) as soil drench on essential oil percent of lemon basil. vertical bars show standard errors of the means (n=3). total phenolic content. application of vct at 1:5 dilution gave the highest total phenolic content. the differences between cmt and 1:10 dilution of vct were not significant (fig. 2). previously, sousa et al. (2005) and taie et al. (2010) reported that total phenolic contents achieved by organic culture were higher than those from conventional practice in tronchuda cabbage (brassica oleracea l. var. costata dc) and sweet basil. asami et al. (2003) and wang and lin (2002) also observed consistently higher levels of total phenolics in organically-grown crops compared with those produced by conventional agricultural practices. the amount of total phenolic content of lemon basil in this experiment is in the range of previously reported total phenolic content of different sweet basil accessions (javanmardi et al., 2003). plant phenolics constitute one of the major groups of compounds acting as primary antioxidants or free radical terminators (lukmanul-hakkim et al., 2008). total flavonoids. the highest total flavonoid content was observed in 1:5 cmt-treated lemon basil plants and it was over 1.4 times higher than that produced in control plants. the differences between the two dilutions of vct were not significant (fig. 3). in previous studies, the amounts of surface flavonoids in ocimum x citriodorum specimens were reported in a range of 0.2 to 5.7 mg g-1 (grayer et al., 2004). our finding is in agreement with a previous work that reported a significant increase in flavonoid content of ocimum basilicum due to compost or compost tea application in comparison with control plants (khalid et al., 2006). it has been stated that the antioxidant activity in basil is largely due to the presence of phenolic components, including flavonoids and phenylpropanoids (juliani and simon, 2002). fig. 3 effect of different dilutions of chicken manure tea (cmt) and vermicompost tea (vct) as soil drench on total flavonoids of lemon basil. vertical bars show standard errors of the means (n=3). total antioxidant activity. the antioxidant activity of lemon basil extracts is shown in figure 4. the total antioxidant activity (taa) ranged from 48.28% (control) to 58.42% (vct at 1:5 dilution). there were no statistically significant differences between cmt dilutions and 1:10 dilution of vct. pearson correlation analysis between secondary metabolites (total phenolics, total flavonoids and essential oils) fig. 4 effect of different dilutions of chicken manure tea (cmt) and vermicompost tea (vct) as soil drench on total antioxidant activity of lemon basil. vertical bars show standard errors of the means (n=3). fig. 2 effect of different dilutions of chicken manure tea (cmt) and vermicompost tea (vct) as soil drench on total phenolic content of lemon basil. vertical bars show standard errors of the means (n=3). 156 with total antioxidant activity showed a significant positive correlation coefficient of r2=0.91 between the amount of total phenolics and total antioxidant activity (table 4). this means that about 91% of total antioxidant activity in lemon basil plants was due to phenolic compounds. other secondary metabolites did not show significant contributions to the antioxidant activity. the same correlation was previously reported in sweet basil (ocimum basilicum l.) accessions (javanmardi et al., 2003). the antioxidant activity of phenolics is mainly due to their redox properties, which allow them to act as reducing agent, hydrogen donors, single oxygen quenchers and having possible metal chelating activity (rice-evans et al., 1995). table 4 pearson’s correlation coefficients between total phenolics, total antioxidant activity, total flavonoids and essential oil percent of treated lemon basil plants total phenolics total flavonoids essential oil content total antioxidant activity 0.919 0.071 ns 0.320 ns data (per plant) are mean values of 3 replicates ± standard errors. 4. conclusions the findings of this study indicate that fertilization with chicken manure and vermicompost organic teas improves herbal and essential oil yields as well as antioxidative agents such as total phenolics in lemon basil plants. the results point to the beneficial effects of compost tea as possible nutrition sources on growth characteristics and essential oil yields of basil. organic nutrition systems have environmental advantages such as a beneficial impact on soil properties and the production of safe plants. 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(lepidoptera: tortricidae) is considered the most important insect pest of apple in syria and insecticides are widely used to control it. such control methods are costly, nonselective, environmentally unsafe and effective for only a short period in the treated area. moreover, c. pomonella has already developed resistance to various insecticides (varela et al., 1993). therefore, a more reliable and environmentally safe control method is required. knipling (1970) and myers et al. (1998) reported that the sterile insect technique (sit) can be considered an important component of an area-wide approach to insect control programs. the possibility of applying sit as an alternative control method to suppress c. pomonella populations has been determined by many researchers (bloem et al., 1999, 2001). this approach relies on mass-rearing and release of both sexes of irradiated moths into wild pest populations. the success of this technique against codling moth depends largely on the release of sexually competitive insects capable of locating and carrying out mating with several feral individuals (knipling, 1981). it is widely agreed that insects with a long lifespan, good dispersal pattern, high incidence of mating, ability to transfer sperm successfully, and a noticeable fecundity would exhibit an acceptable level of mating competitiveness (carpentar et al., 1989). mating in lepidoptera involves the following sequence of events: copulation, spermatophore transfer, insemination and egg fertilization. bues et al. (1992) observed that most lepidopteran females tend to mate within 24 h of emergence. the effect of adult age on mating varies considerably between species as well as sex. previous studies have stated that younger females of pectinophora gossypiella saunders (lingren et al., 1988), eoreuma loftini dyar (spurgeon et al., 1995), lymantria dispar l. (proshold, 1996), and phthorimaea operculella zeller (makee and saour, 2001) had a higher occurrence of successful matings compared to older individuals. on the contrary, young females of ephestia kuehniella zeller showed a low mating incidence. however, the newly emerged males of e. kuehniella were more likely to transfer spermatophores than older males (calvert and corbet, 1973). the effect of adult age on reproductive capacity is well known in several lepidopteran species (ellis and steele, 1982). it has been shown that the reproduction of plutella xylostella (l.) females was significantly reduced by age (nemoto et al., 1992). insect weight plays an important role in mating and reproduction. large males of ephestia elutella hübner were more likely to mate than smaller ones (phelan and barker 1986). similarly, a significant relationship between body size and successful mating has also been reported in the dipteran drosophila melanogaster meigen (partridge and farquhor, 1983). studies have shown that in hemipteran podisus macufactors influencing mating incidence and reproduction in codling moth cydia pomonella l. (lepidoptera: tortricidae) h. makee*(1), i. idris, k. hussian atomic energy commision of syria (aecs), syrian arab republic, p.o. box 6091, damascus, syria. key words: cydia pomonella, fecundity, fertility, mating ability, mating frequency. abstract: the codling moth cydia pomonella l. is a primary pest of apple and various studies have been performed to assess the possibility of applying sterile insect technique as a control method against this pest. in support of this technique, the present work aims to examine the effects of adult age and weight on mating ability, number of matings, fecundity and fertility in c. pomonella. the relationship between number of matings, fecundity, and fertility of females was also studied. female and male weights were found to have an effect on the number of times individuals mate, but male weight only influenced mating success. unlike male weight, female weight affected fecundity and fertility. negative correlations were found between mating success, fecundity and fertility and adult age. multiply-mated females and those which did not accept a second mating showed higher fecundity and fertility than their counterparts that were not given the opportunity to remate. our results provide essential information necessary to increase the effectiveness of sterile insect technique as a control method against c. pomonella. adv. hort. sci., 2012 26(3-4): 180-186 (1) corresponding author: hmakee@aec.org.sy. received for publication 29 february 2012 accepted for publication 15 february 2013 181 liventris and p. connexivus the heavier females presented a better reproductive rate (evans, 1982; zanuncio et al., 1992). to maximize the mating competitiveness of released insects, the influence of adult age and weight on mating incidence and reproduction capacity of codling moth were examined. in addition, the effect of the number of matings on fecundity and fertility of this species was tested. 2. materials and methods a c. pomonella colony has been maintained in our laboratory for several years and was used for the present study. newly emerged adults were crossed in petri dishes (12 cm dim), five pairs in each. a wet cotton wool was placed in each petri dish as drinking source. after 3-4 days, the dishes which had eggs were collected and soaked with 2% sodium hypochlorite solution for 2 min for egg sterilization. then the dishes were washed with tap water and left to dry. eggs were checked daily for hatching. newly hatched larvae were placed on artificial media consisted of the following ingredients: agar-agar, maize, wheat germ, casein, yeast, wesson salts, benzoic acid, fumidil, ascorbic acid, vitamins and nipagine (anisimov, personal communication). all insect stages were kept under constant temperature at 25±1°c with 70±5% rh, and a photoperiod of 16:8 h (l:d). in all the experiments, pupae were sexed and individually placed in small plastic tubes until eclosion. for oviposition, newly emerged females and males were paired in petri dishes (12 cm dim) having a feeding source (a wet cotton wool). “mating incidence” in this study is used to indicate successful spermatophore transfer and/or spermatophore presence in the bursa copulatrix of the female. the number of matings is reflected by the number of spermatophores in the bursa copulatrix. the effect of adult age ten different groups of virgin females (n= 20 in each group) aged 1-10 d were employed. females of each group were individually paired with newly emerged males (<18 h). after 24 h, males were removed and females were kept for oviposition until death. all eggs were collected, counted and allowed to hatch. after death, the females were dissected and examined for the presence of spermatophores in the bursa copulatrix. ten different groups of virgin males (n=20 in each group) aged 1-10 d were examined to determine the effect of male age on successful spermatophore transfer. males of each group were individually paired with newly emerged virgin females (<18 h). after 24 h, males were removed and females were kept for oviposition until death. all eggs were collected, counted and allowed to hatch. after death, the females were dissected and examined for the presence of spermatophores. the effect of adult weight to determine the influence of male and female weight on incidence of mating, number of matings, fecundity and fertility pupae were weighed, sexed, and divided into three groups based on weight: light, medium and heavy. female pupal weights were, respectively, 34-37, 39-42 and 44-47 mg, while male pupal weights were, respectively, 25-31, 36-45 and 47-49 mg. emerged females and males in each group were individually paired with newly emerged adults of the opposite sex. in male and female groups, males were paired with females weighing 36.2±0.5 mg, while females were paired with males weighing 30.6±1.3 mg. in each group, the females and males were kept together until death. all eggs were collected, counted and left to hatch. the females were dissected and the number of spermatophores in the bursa copulatrix were counted. to determine the relationship between adult weight and fecundity and fertility, only mated females were considered. effect of sex ratio on mating ability two experiments were carried out. in the first experiment, 1-d-old males (n = 20) were individually confined with three newly emerged females (1 male: 3 females). males and females were kept together for 24 h, after which time the females were dissected and the number of spermatophores was determined. in the second experiment, 1-d-old females (n= 20) were singly confined with three newly emerged males (1 female: 3 males). after 24 h, females were removed, dissected and the number of spermatophores was determined. a ratio of 1 female: 1 male was used as a control group for the two experiments. effect of female multiple mating on fecundity and fertility two groups of virgin females were used. females of the first group (n= 20) were individually paired with newly emerged males. after 24 h, males were removed and females were kept for oviposition until death. in the second group, females (n= 45) were individually paired with 1-dold males. males were removed after 24 h and replaced with new 1-d-old males. the same procedure was followed for seven successive days. in both groups, all eggs were collected, counted and allowed to hatch. after death, the females were dissected to determine the presence and number of spermatophores. statistical analysis was carried out using the statistic program version 6 (statsoft, inc. 2003) at 5% level (p= 0.05). a simple linear regression analysis was done to study the relationship between adult age and incidence of mating and fertility. data were subjected to analysis of variance for determination of differences between means, which were tested for significance using tukey hsd test. the percentages were analyzed by applying normal approximation test (analysis of proportion). 3. results effect of adult age figure 1 illustrates the effect of male and female mating ability of c. pomonella with regard to age. a regres182 sion line was fitted to present the relationship between incidence of mating and adult age. the percentage of mating ability was significantly correlated with adult age (y= -7.2303x + 95.667, r2 = 0.77, p<0.05; y = -7.8788x + 90.133 r2 = 0.86, p<0.05 for females and males, respectively). a significant increase in mating ability was recorded when males and females became 2 d old. after that, a significant reduction in the mating ability was noticed 4 d and 5 d after male and female emergence (fig. 1). the mating ability of 1-d-old males and females was similar, and afterwards the mating ability of females was higher than that of males, regardless of adult age. fig. 1 effect of adult age on mating ability of codling moth. to determine the effect of adult age on fecundity and fertility only mated females were used in the analysis. figure 2 reveals that the number of eggs increased when the adults became 2-d-old; the number of eggs then significantly declined (f=13.19; d.f=9,190; p<0.05 for female and f=12.9; doff=9,190; p<0.05 for male). significant differences were noticed between males and females at each tested age, except when both sexes were 1-d-old (fig. 2). fig. 2 effect of adult age on mean fecundity of codling moth female. figure 3 illustrates that as females and males got older their fertility decreased. there was a strong relationship between adult age and fertility (y= -5.3455x + 87.8 r2 = 0.90 and y= -8.5939x + 91.867 r2= 0.93 for females and males, respectively). regardless of adult age, female fertility was significantly higher than that of males except at age 1 d. fig. 3 effect of adult age on adult fertility of codling moth. effect of body weight the percentage of mating ability of the females was not affected by their weight. no differences in mating ability were found among the three tested female weight groups (fig. 4). in contrast, the number of female matings was influenced by their weight. the mean number of matings of group 3 females (the heaviest) was significantly higher than that of groups 1 and 2 (f=8.6; d.f=2,74; p<0.05). however, the mean number of matings of group 2 females did not significantly differ from that of group 1 females (fig. 5). fig. 4 effect of body weight on mating ability of codling moth adults. fig. 5 effect of body weight on number of mating of codling moth adults. 183 a significant difference in the percentage of mating incidence was observed among male weight groups 1 and 3 (fig. 4). all males of group 3 (the heaviest) were able to transfer spermatophores, whereas only half of group 1 males (the lightest) was able to do so. the mating incidence of group 2 males did not significantly differ from that of group 3 (fig. 4). the mean number of matings of group 1 males was significantly lower than that of groups 2 and 3 (f=5.19; d.f=2,72; p<0.05). no differences in the number of matings were detected between males in groups 2 and 3 (fig. 5). results from the study show that female body weight significantly affects fecundity (f=6.24; d.f=2,74; p<0.05) and fertility (f=7.09; d.f=2,74; p<0.05) of the codling moth. the mean number of eggs laid and mean number of eggs hatch increased significantly when female weight increased (table 1). table 1 effect of female body weight on fecundity and fertility of c. pomonella female weight (mg) mean fecundity mean fertility 34-37 66.24±11.35 b 50.6±9.37 b 39-42 99.80±12.70 ab 68.9±11.74 b 44-47 134.11±16.03 a 112.5±14.14 a means followed by different letters (columns) are significantly different at p<0.05 (tukey hsd test). unlike female weight, male weight did not affect fecundity and fertility. there were no significant differences in fecundity and fertility between heavy and light males (table 2). table 2 effect of male body weight on fecundity and fertility of c. pomonella male weight (mg) mean fecundity fertility 25-31 63.16±12.85 a 52.36±10.8 a 36-45 85.04±9.88 a 57.80±8.6 a 47-49 95.32±12.2a 67.04±9.8 a means followed by different letters (columns) are significantly different at p<0.05 (tukey hsd test). effect of sex ratio on mating ability when c. pomonella females were confined with one or three males for 24 h, they mated only once. the incidence of female mating did not differ significantly when confined with one or three males (table 3). c. pomonella males mated only once when paired with a single virgin female for 24 h. when a male was confined with three females, it mated more than once (table 3). effect of female multiple mating on fecundity and fertility when c. pomonella females were exposed to newly emerged males for seven successive days (group 2 females), 18% of them mated once, more than half mated twice or three times and 18% mated four or five times (table 4). table 4 effects of repeated mating on the mean number of eggs and egg hatch percentage of c. pomonella females female group no. of matings females (%) mean no. of eggs/female±se mean fertility/female± se 1 1 60 93.6±19.9 b 93.6±19.9 b 2 0 7 134.7±25.6 b 0 1 18 237.1±32.1 a 237.1±32.1 a 2 35 225.8±20.5 a 225.8±20.5 a 3 22 260.9±27.9 a 260.9±27.9 a 4 11 238.6±8.5 a 238.6±8.5 a 5 7 270.0±25.8 a 270.0±25.8 a means and percentages followed by different letters (columns) are significantly different at p<0.05 (tukey hsd test). group 1= females were paired with the males only for 24 h (n = 20). group 2= females were paired with new virgin male every 24 h for 7 successive days (n = 45). the mean number of eggs and fertility of group 1 females, in which the females were not given an opportunity to remate, were significantly lower than those of group 2 females, in which the females were given a chance to remate (f=6.7; d.f=6,59; p<0.05 for fecundity, f=6.7; d.f=6,59; p<0.05, respectively). regardless of the number of matings of group 2 females, the mean number of eggs and fertility did not differ significantly. 4. discussion and conclusions the tendency and number of matings of c. pomonella may be affected by various factors. our results indicate that old males and females of this species were less likely to mate than young individuals. however, differences appeared when the patterns of mating ability of females and males were compared. the female mating ability was greater than that of males. however, the mating ability of females declined less rapidly than that of males with age (fig. 1). in lepidoptera, the ability to release adequate sex pheromone and/or to respond to the sex pheromone of the table 3 effect of number of females and males on number of mating of c. pomonella during a 24-h period sex sex ratio f:m % mated adult per no. of mating 0 1 2 female 1: 1 b35 ab a65 a 0 1: 3 b40 a a60 a 0 male 1: 1 b30 ab a70 a 0 3: 1 b20 b a60 a b20 percentages preceded by different capital letters (raws) and followed by different small letters (columns) are significantly different at p< 0.05 (normal approximation test). 184 opposite sex generally reduces with age (spurgeon et al., 1995; proshold, 1996; makee and saour, 2001). our result confirms that senescence might influence male moths to a greater degree than females. in the current study, male age greatly influenced female fecundity and fertility of c. pomonella (figs. 1 and 2). conflicting results have been reported on the impact of male age on female fecundity and fertility of c. pomonella. vickers (1997) reported that male age had no effect on female fecundity and fertility, whereas knight (2007) showed that female fecundity after mating with 1-d-old males was significantly lower than after mating with 3-d-old males. similarly, female fecundity and fertility of plutella xylostella l. decreased when the females mated with old males (nemoto et al., 1992; wang et al., 2011). female discrimination against older males has been demonstrated in several species (ritchie et al., 1995; jones and elgar, 2004). thus, c. pomonella females preferred mating with younger males since mating with older males diminishes female fecundity and fertility. such reduction in reproductively could be due to an age-correlated reduction in sperm quality (crow, 1997; hansen and price, 1999). the results from the present work clearly indicate both fecundity and fertility were affected by female age at mating, both of which decreased with an increase in age (figs. 1 and 2). similar effects have been observed previously in several species such as spodoptera exigua (hübner), (rogers and marti, 1996), lobesia botrana (dennis & schiffermüller) (torres-vila et al., 2002) and p. xylostella (wang et al., 2011). the reduction of egg production and viability when mating of c. pomenella was delayed after emergence could be related to utilization of the fat body, which is essential source of vitellogenins and lipids for oocyte maturation, for non reproduction metabolism by older females (barrer, 1976). unlike male weight, female weight did not play a role in mating ability in c. pomonella (fig. 4); both light and heavy females had similar mating tendencies. however, female weight did affect the number of matings (fig. 5). similar results were reported in p. operculella (makee and saour, 2001). male weight had important impact on mating ability and number of matings of c. pomonella (figs. 4 and 5). there may be two main reasons for the relationship between male weight, ability to produce and transfer spermatophore, and number of matings: (1) heavy males may be able to produce sufficient quantity of sex pheromone to attract females (thornhill and alcock, 1983; phelan and barker, 1986); (2) c. pomonella females tend to mate with heavier males, as confirmed by our data (i.e. they mated with 100 and 50% of heavy and light males, respectively) (fig. 4). our results show that reproductively of heavy females was greater than for light females (table 1). strong correlation between adult weight and fecundity has been noted in various insect species (evans, 1982). honek (1993) reported that genetic and environmental factors could influence insect weight. there are several environmental factors including food type and temperature (mohaghegh et al., 1999). generally, female weight partly reflects the size of fat-body. this organ is essential for oocyte maturation since it is a site of lipid and yolk protein synthesis (chapman, 1982). therefore, heavy females are able to produce more eggs since they have a larger fat-body. in contrast to female weight, male weight did not influence the fecundity and fertility of c. pomonella (tables 1 and 2). like most lepidopteran, when a c. pomonella male was paired with one female for 24 h, it was able to produce and transfer only one spermatophore (makee and saour, 2001). nevertheless, if several virgin females were available, c. pomonella males were able to transfer more than one spermatophore during one scotophase (table 3). a comparable result has been noted in males of grapholitha molesta busck, spodoptera frugiperda (j.e. smith) and p. operculella (george and howard, 1968; simmons and marti, 1992; makee and saour, 2001). nevertheless, c. pomonella females mated once in 24 h even when they were confined with three newly emerged males (table 3). thus, females needed a lapse of time to remate, regardless of the number of males available. a similar result was reported in p. operculella (makee and saour, 2001). after mating, females released special volatile materials that reduced their receptivity (tompkins and hall, 1981). when c. pomonella females were exposed to newly emerged males for seven successive days, only 18% of them mated once, 35% twice and 40% several times (table 4). c. pomonella females were capable of mating more than five times when they were paired with virgin males for seven successive days. conversely, p. operculella females were unable to mate more than three times when they were paired with virgin males for seven successive days (makee and saour, 2001). whatever the number of matings, the mean number of eggs and fertility of females were similar in both species. this may imply that once-mated females would not seek additional matings since they received sufficient effective sperm during their first mating. several studies reported that p. operculella, heliothis virescens f. and l. dispar females that did not receive an adequate quantity and quality of sperm during the first mating needed to remate (lingren et al., 1988; proshold, 1995; makee and saour, 2001). on the contrary, knight (2007) stated that c. pomonella females that had mated three times had a significantly higher fecundity than singly-mated moths. fecundity and fertility of females with an opportunity to remate were higher than those of once-mated females that were not allowed to remate (table 4). the relationship between the number of matings and the female’s reproductivity could be attributed to: (1) sperm replenishment which is required for egg fertilization; and (2) nutrients derived from spermatophores that are utilized by the female in egg production (greenfield, 1983). the present study provides useful information for situations where sterile insect technique could be considered against c. pomonella: (1) repeated releases of young sterile insects should be executed rather than one major release; (2) production of heavy insects is preferable in mass-rearing procedures; (3) the effectiveness of sterile in185 sect technique against c. pomonella would not restrain by the release of both sterile males and females, since during one scotophase c. pomonella males could remate whereas females were unable to do that. references barrer p.m., 1976 the influence of delayed mating on the reproduction of ephestia cautella walker (lepidoptera: phycitidae). j. stored prod. res., 12: 165-169. bloem s., bloem k.a., carpenter j.e., calkins c.o., 1999 inherited sterility in codling moth (lepidoptera: tortricidae): effect of sub-sterilising 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(lepidoptera: tortricidae). australian journal of entomology, 36: 179-182. wang x.-p., fang y.-l., zhang z.-n., 2011 effects of delayed mating on the fecundity, fertility and longevity of females of diamondback moth, plutella xylostella. insect science, 18: 305-310. zanuncio j.c., bragança m.a.l., díaz j.l.s., sartório r.c., 1992 avaliação dos parâmetros de fecundidade de fêmeas de podisus connexivus (hemiptera: pentatomidae) de diferentes pesos. rev. ceres., 39: 591-596. impaginato 141 1. introduction bermudagrass is a warm-season, c4, perennial grass. it has short, grey-green blades with rough edges, stems of 1 to 30 cm in length and a deep root system that can penetrate 2 m into the ground; however, most of the root mass is less than 60 cm deep (xu et al., 2011). among the many advantages of turfgrass areas are erosion and dust control, aquifer recharge and protection from pollutants, heat reduction in urban environments, reduction of noise and pollution, and providing human health and aesthetic benefits (stier et al., 2013). water scarcity is an increasing challenge to the turfgrass industry and may result in irrigation restrictions being imposed without regard for damage to turfgrass (beard and kenna, 2008). for turf managers, thriving in an industry where turf quality is of utmost importance is difficult when water is limiting. therefore, researches investigating turfgrass resistance to drought stress have become increasingly important (fry and huang, 2004). fu and huang (2001) investigated the effects of drought stress on two cool-season turfgrasses and found that moderate drought stress had not effects on morphological and physiological characteristics, however in intensive drought stress, antioxidant enzyme activities, chlorophyll content, relative water content and shoot dry weight were decreased. in addition to limited amounts of water, turfgrasses are impacted by low-light environments. shade is more problematic for warm-season turfgrasses to maintain quality given their higher light saturation point compared to cool-season turfgrasses (fry and huang, 2004). turfgrasses perform poorly in reduced light environments due to high traffic rate, daily mowing, and reduced photosynthesis. in shade, increased disease presence adversely affects cool-season turfgrass development, while morphological limitations, such adv. hort. sci., 2016 30(3): 141-149 doi: 10.13128/ahs-20250 morpho-physiological alteration in common bermudagrass [cynodon dactylon (l.) pers.] subjected to limited irrigation and light condition n. adamipour (*), h. salehi, m. khosh-khui department of horticulture science, college of agriculture, university of shiraz, shiraz, iran. key words: antioxidative enzymes, irrigation, photoperiod, turfgrass. abstract: bermudagrass (cynodon spp.) is the most popular warm-season turfgrass used in warm climatic regions of the world due to its recuperative ability, high traffic tolerance, heat tolerance, and relative drought and salt tolerance. however, shade is a microenvironment in which bermudagrass performs poorly. in order to evaluate the interaction of photoperiod and irrigation on [cynodon dactylon (l.) pers. california origin], a greenhouse experiment was conducted at the research greenhouse of the department of horticultural sciences, college of agriculture, shiraz university, shiraz, iran. the experiment was conducted with four field capacity regimes (25%, 50%, 75% and 100%) and three light durations (8, 12 and 16 h) in a completely randomized design factorial arrangements with four replications. results showed that decreasing field capacity and photoperiod decreased fresh and dry weights shoot and root, chlorophyll and starch contents and superoxide dismutase, catalase and ascorbate peroxidase activities. decreasing the field capacity and light duration increased proline content. reducing sugars and peroxidase enzyme in leaves increased with decreasing field capacity. shoot height and leaf area increased by shortening the photoperiod. in overall, results showed that, the increase in irrigation alleviates the destructive effects of reduced day lengths and vice versa. further studies are needed to clarify more the interaction between irrigation and light treatments at structural and ultrastructural levels, in common bermudagrass. (*) corresponding author: adamipournader@yahoo.com received for publication 9 may 2016 accepted for publication 27 july 2016 copyright: © 2016 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2016 30(3): 141-149 142 as reduced lateral stem growth, inhibits warm-season turfgrass development (beard, 1972). variations of shade responses among species and cultivars (jiang et al., 2004; trenholm and nagata, 2005; sladek et al., 2009) make it possible to select turfgrasses with superior shade tolerance. identifying morphological characteristics that are associated with superior shade performance based on genetic variation would add value to germplasm screening for shade tolerant species and cultivars. esmaili and salehi (2012) noted in bermudagrass that were treated with short photoperiod duration, verdure fresh and dry weight, shoot height, tiller density, leaf area and chlorophyll and relative water contents were decreased, however electrolyte leakage and proline content were increased. although bermudagrass, the most widely grown c4 turfgrass on an international basis (shearman, 2006), has been extensively studied, many challenges and questions still remain when light is a limiting growth factor. the main objective of the present study was to investigate the effects of both irrigation interval and light duration on growth and quality of common bermudagrass. 2. materials and methods plant material and experimental conditions this experiment was conducted at the research greenhouse of the department of horticultural sciences, college of agriculture, shiraz university, shiraz, iran (52◦32’e and 29◦36’n, 1810 m asl). seeds of common bermudagrass (cynodon dactylon [l.] pers. california origin) were weighed and cultured in plastic pots with 19 cm in diameter and 25 cm in height, without drainage (0.25 g pot−1) filled with 4 kg clay-loam soil with permanent wilting point (pwp) of 19% and field capacity (fc) 29%. watering was carried out daily prior to beginning of treatments. plants were kept in a greenhouse with 31/25°c (day/night) temperature and 35% relative humidity for one m o n t h b e f o r e t h e b e g i n n i n g o f t r e a t m e n t s . treatments were conducted at four irrigation levels (25%, 50%, 75% and 100% fc) and three photoperiod duration [8, 12 and 16 h as short day length (sdl), intermediate day length (idl) and long day length (ldl)]. watering was carried out daily before seed germination and after turf establishment. then, the t u r v e s w e r e w a t e r e d e q u a l l y w h e n r e q u i r e d . established turves were clipped from 3 cm above soil by a hand mower and were transferred to a covered frame which temperature, light (intensity and length) and relative humidity were controlled with digital sensors. the environmental condition of covered frame was 31°c, white and creamy fluorescent lamps one m above the pots with a constant light intensity of 3000 lux, and 35% relative humidity for applying simultaneous irrigation and photoperiod treatments. pots were weighed daily and set to different irrigation treatments (25, 50, 75 and 100% fc), during the whole of experiment. after three months, plants were harvested in order to measure morphological and biochemical traits. growth parameters growth parameters including, shoot height (cm), leaf area (cm2) and fresh and dry weights of shoot and root (g) were measured. dry weights were measured when the materials dried at 60°c for 48 h. chlorophyll content chlorophyll content was measured according to the method of saini et al. (2001) using the following formula: chlorophyll (mg/g f.w.) = [20.2(od 645 nm) + 8.02(od 663 nm) × v/ (f.w.×1000)] where: od is optical density, v is the final solution volume in ml and f.w. is tissue fresh weight in mg. proline content proline was determined according to the method described by bates et al. (1973). using spectrophotometer (biowave ii, england) at 520 nm wavelength, appropriate proline standards were included in calculation of its content in samples. total soluble sugars and starch analysis the total soluble sugars were measured using the method as previously described by dubois et al. (1956). the total soluble sugar content of samples was measured at 490 nm of absorbance and glucose solution was used at different concentrations for standard curve drawing. the starch content was quantified using the bradford method (mccready et al., 1950). the starch content was measured at absorbance of 630 nm and calculated using the standard curve of glucose and multiplying it by 0.92. antioxidant analysis fresh samples were homogenized in extraction buffer (0.1 m phosphate buffer ph 6.8) with mortar and pestle on ice. the homogenate was then centrifuged at 12,000 g for 15 min at 4°c and the supernatant was used as the crude extract for the superoxadamipour et al. morpho-physiological alteration in common bermudagrass subjected to limited irrigation and light condition 143 ide dismutase (sod), guaiacol peroxidase (pod), ascorbate peroxidase (apx) and catalase (cat). the sod, pod, apx and cat enzymes were estimated u s i n g t h e m e t h o d s p r e v i o u s l y d e s c r i b e d b y b e a u c h a m p a n d f r i d o v i c h ( 1 9 7 1 ) , c h a n c e a n d m a e h l y ( 1 9 9 5 ) , n a k a n o a n d a s a d a ( 1 9 8 1 ) a n d dhindsa et al. (1981), respectively. experimental design and data analysis this study was conducted in a completely randomized design with factorial arrangements and two factors: field capacity and photoperiod with four replicates. data were analyzed using statistical software (sas software) and mean comparisons were performed using lsd test at 5% level. 3. results and discussion results of analysis of variance (tables 1 and 2) showed that photoperiod (except for soluble sugar) and irrigation had significantly influenced the measured traits and also the interaction of photoperiod and irrigation had a significant effect on fresh and dry weights of shoot, proline content and the level of activity of superoxide dismutase. shoot height and leaf area shoot height and leaf area significantly declined by decreasing field capacity from 100% to 25% (table 3). shoot height and leaf area decreased (47.09% and 27.77%, respectively) at 25% fc compared to 100% fc (table 3). ryan (2011) reported that growth can be reduced through impairment of cell division and cell expansion which occurs at a lower water stress threshold rather than photosynthetic inhibition. fu and huang (2001) reported that shoot growth of both table 1 analysis of variance of photoperiod, field capacity and interaction between photoperiod and field capacity measured traits source of variability df shoot height (cm) leaf area (cm2) shoot fresh weight (g) shoot dry weight (g) root fresh weight (g) root dry weight (g) sugars of shoot (mg g-1 d.w.) photoperiod 2 38.45 ** 0.04** 212.24** 20.59** 36.59** 4.87** 5.77 ns field capacity 3 314.82** 0.33** 141.40** 141.40** 1337.54** 324.86** 79732.82** photoperiod * field capacity 6 0.0 ns 0.0 ns 1.25** 1.25** 0.0 ns 0.0 ns 0.0 ns error 33 0.17 0.002 0.24 0.24 0.00 0.08 7.01 cv 2.18 4.32 3.58 6.57 0.00 2.02 1.96 ** and ns significant at the 0.01 level and not significant respectively. table 2 analysis of variance of photoperiod, field capacity and interaction between photoperiod and field capacity measured traits ** and ns significant at the 0.01 level and not significant respectively. source of variability df superoxide dismutase (ug-1 fw) catalase (ug-1 fw) peroxidase (ug-1 fw) ascorbate peroxidase (ug-1 fw) chlorophyll (mg g-1 fw) proline (µmol g-1 fw) starch content (mg g-1 dw) photoperiod 2 4044.08** 97.06** 969.12** 28933.33** 0.59** 4.99** 3.33** field capacity 3 55360.44** 234.71** 5248.15** 257973.85** 1.74** 7238.96** 105235.35** photoperiod * field capacity 6 2.52 ns 0.0 ns 0.0 ns 0.0 ns 0.00 ns 2.27** 0.00 ns error 33 235,20 3.26 27.65 260.03 0.00 0.12 0.00 cv 9.91 5.65 6.67 1.79 0.0 2.68 0.00 variables photoperiod field capacity (%) mean 100% 75% 50% 25% shoot length ldl 22.25 d* 21.27 e 16.67 h 11.05 k 17.81 c (cm) idl 23.75 c 22.77 d 18.17 g 12.55 j 19.31 b sdl 25.35 a 24.37 b 19.77 f 14.15 i 20.91 a mean 23.78 a 22.80 b 18.20 c 12.58 d leaf area ldl 1.22 bc 1.22 bc 1.17 c 0.87 e 1.12 b (cm2) idl 1.23 bc 1.23 bc 1.18 c 0.88 e 1.13 b sdl 1.32 a 1.32 a 1.27 ab 0.97 d 1.22 a mean 1.26 a 1.25 a 1.20 b 0.91 c shoot fresh ldl 20.30 a 20.26 a 16.16 b 14.06 c 17.69 a weight (g) idl 16.32 b 16.26 b 12.81 d 9.13 e 13.63 b sdl 13.85 c 12.83 d 9.69 e 5.34 f 10.42 c mean 16.82 a 16.45 a 12.88 b 9.51 c shoot dry ldl 11.30 a 11.26 a 7.16 d 5.06 e 8.69 a weight (g) idl 10.32 b 10.26 b 6.81 d 3.13 f 7.63 b sdl 9.85 b 8.83 c 5.69 e 1.34 g 6.42 c mean 10.49 a 10.11 a 6.55 b 3.17 c root fresh ldl 39.78 a 38.69 b 29.54 g 16.99 j 31.25 a weight (g) idl 37.80 c 36.71 e 27.56 h 15.01 k 29.27 b sdl 36.81 d 35.72 f 26.57 i 14.02 l 28.28 c mean 38.13 a 37.04 b 27.89 c 15.34 d root dry ldl 19.78 a 18.69 b 13.46 d 8.54 f 15.12 a weight (g) idl 19.69 a 18.60 b 13.37 d 8.45 f 15.03 a sdl 18.82 b 17.73 c 12.50 e 7.43 g 14.12 b mean 19.43 a 18.34 b 13.11 c 8.14 d proline content ldl 5.57 i* 7.06 g 14.35 f 23.25 c 12.56 c (mol g−1 f.w.) idl 5.63 i 6.51 h 15.19 e 24.20 b 12.88 b sdl 5.66 i 6.89 gh 17.29 d 24.74 a 13.65 a mean 5.62 d 6.82 c 15.61 b 24.07 a *in each variable, data followed by the same letters (small letters for interactions and capital letters for means) are not significantly different using lsd at 5% level. ldl= long day length. idl= intermediate day length sdl= short day length. table 3 effect of field capacity and photoperiod and their interaction on shoot length, leaf area, shoot fresh and dry weight, root fresh and dry weight and chlorophyll content adv. hort. sci., 2016 30(3): 141-149 144 kentucky bluegrass and tall fescue generally were not affected by surface soil drying but under full drying, shoot growth declined for both species. the reduced leaf area is a modification to avoid evopo-transpiration loss and to increase water use efficiency in grasses which helps to tolerate water stress. low leaf surface area would reduce transpiration rate also by lowering stomatal activity (riaz et al., 2010). turf shoot height showed considerable difference in ldl treatments compared to sdl treatments. reducing photoperiod significantly increased the shoot height and leaf area (table 3). shoot height and leaf area increased significantly with shortening day length that it’s maximum and minimum decreased (14.82% and 8.19%, respectively) was observed at sld compared to ldl (table 3). similar results have reported on bermudagrass (tegg and lane, 2004) and zoysiagrass (qian and engelke, 1999). shoot fresh weight reducing field capacity from 100% fc to 25% fc significantly decreased the shoot fresh weight to 43.46% at 25% fc compared to 100% fc (table 3). riaz et al. (2010) demonstrated that, water deficit conditions had a significant inhibitory effect on shoot fresh and dry weights of three bermudagrass cultivars. the extended photoperiod (16 h) significantly increased fresh weight compared to shorter phot o p e r i o d s ( 1 2 h a n d 8 h ) . s h o o t f r e s h w e i g h t increased 41.09% under ldl compared to the sdl condition (table 3). sinclair et al. (2004) demonstrated that the extended photoperiod increased biomass accumulation of four grasses (‘pensacola’ bahiagrass, paspalum notatum flugge var. saurde parodi; ‘tifton 85’ bermudagrass, cynodon spp. l. pers.; ‘florakirk’ bermudagrass; and ‘florona’ stargrass, cynodon nlemfuensis vanderyst var. nlemfuensis) compared to short day condition. interaction between field capacity and photoperiod resulted in the highest and lowest fresh weight in 100% fc-ldl and 25% fc-sdl treatments (table 3). shoot dry weight different percentages of field capacity and photoperiod had significant effects on dry weight (table 3). reducing field capacity and photoperiod significantly decreased the dry weight. the shoot dry weight in 100% fc conditions decreased 69.78% compared to 25% fc condition (table 3). similar results have been reported on creeping bentgrass (agrostis stolonifera l.), rough bluegrass (poa trivialis l.), and perennial ryegrass (lolium perenne l.) ( p e s s a r a k l i a n d k o p e c , 2 0 0 8 ) , b e r m u d a g r a s s (cynodon dactylon l.) (riaz et al., 2010). the highest and lowest dry weight was observed in 100% fc-ldl and 25% fc-sdl treatments, respectively (table 3). burton et al. (1988) stated that day length was highly correlated with yield of ‘coastal’ bermudagrass, with yield reduction occurring in day lengths under 13 h. therefore, photoperiod influenced dry matter production of forage grasses. extended photoperiod throughout the cool-season in short-day length conditions substantially decreased forage yield (sinclair et al., 1997, 2001, 2003). root fresh weight r o o t f r e s h w e i g h t s i g n i f i c a n t l y d e c l i n e d b y decreasing field capacity from 100% to 25%. root fresh weight decreased (59.76%) at 25% fc compared to 100% fc (table 3). the impact of partially closing stomata limits co2 availability and reduces photosynthesis, which is vital to produce and translocate carbohydrates to roots to explore deeper moisture (huang, 2006). huang and gao (2000) found that severe leakage of organic solutes from roots in drying soil gives evidence that root death of tall fescue cultivars during drought stress may correlate with root desiccation. there was a significant difference between ldl, idl and sdl treatments and the highest and lowest root fresh weights were obtained in ldl and sdl treatments, respectively. root fresh weight decreased 9.50% at sdl compared to ldl (table 3). this is in agreement with wang et al. (2004) who reported that an increase in root growth is associated with extended light duration and is related to increase in internal cytokinin concentration and its increased activity in root tips. root dry weight as shown in table 3, reduction in field capacity decreased root dry weight of plants. the highest and lowest root dry weights were observed in 100% fc and 25% fc treatments, respectively and in 25% fc decreased 58.10% compared to 100% fc. pessarakli and kopec (2008) demonstrated that, water deficit conditions showed a significant decrease in root dry weight of three turfgrass species. the highest and lowest root dry weight was obtained in ldl and sdl treatments, respectively (table 3). root dry weight decreased 6.61% at sdl compared to ldl (table 3). beard (1972) reviewed the morphological responses of turfgrasses under shade based on the research conducted before 1995, and found alterations such as: reduced tillering and shoot density, longer internodes with a reduced stem diameter, increased leaf length, decreased leaf width, thinner leaves, more adamipour et al. morpho-physiological alteration in common bermudagrass subjected to limited irrigation and light condition 145 vertical leaf orientation, and fewer roots (mcbee and holt, 1966; almodares, 1980; dudeck and peacock, 1992). a shift in allocation of dry matter occurs in response to shade, resulting in more dry matter partitioning into shoots rather than roots (allard et al., 1991; dias-filho, 2000). in response to lower irradiance, accelerated leaf elongation and decrease in partitioning to root dry matter are adaptive strategies to enhance light capture (semchenko et al., 2012). proline content reducing field capacity and photoperiod significantly increased proline content in all plants. the highest amount of proline content was obtained in 25% fc and the lowest one was obtained in 100% fc treatment (table 3). this is in agreement with (etemadi et al., 2005) who demonstrated that the increase in drought increased proline content in bermudagrass (cynodon dactylon l.). during drought stress, plants respond to different stresses with changes they create in their physiological features. accumulation of soluble material in response to drought is a way to maintain turger. it seems that the accumulation of free proline in plants is the general reaction to the stress. however several other amino acids increase under drought and salinity stress. but the degree of changes is not comparable with proline accumulation (gzik, 1996). in a comparative study between perennial ryegrass and red fescue for the amount of resistance to the drought, it was seen that the amount of proline in red fescue was more than perennial ryegrass (bandurska and jozwiak, 2010). the highest and lowest proline content was obtained in sdl and ldl treatments, respectively (table 3). this is in agreement to the findings reported on the effects of decreased photoperiod on bermodagrass (esmaili and salehi, 2012). interaction between field capacity and photoperiod resulted in the highest and lowest proline content in 25% fc-sdl and 100% fcldl treatments (table 3). chlorophyll content field capacity and light durations had significant effects on leaf chlorophyll content. the highest and lowest chlorophyll content, were observed in 100% fc and 25% fc treatments, respectively (table 4). induction of drought has caused a reduction of electron carrier in photosynthesis and a reduction in chlorophyll content which has been reported by (zuily et al., 1990; moran et al., 1994). prolonged drought, heat, and the combined stresses could lead to loss of chlorophyll and lipid peroxidation, resulting in further turf quality decline (jiang and huang, 2001). water is required to facilitate photosynthesis in plants. low energy electrons are extracted from water and are energized through light energy captured by chlorophyll. these energized electrons enable the production of nadph and atp which are then used to reduce co2. co2 is taken up from the atmosphere through stomata. stomata are very sensitive to external environmental factors such as light, co2, water status, and temperature (hopkins and hüner, 2004). the loss of chlorophyll by the plant in an intense stress can be associated with photo oxidation and consequently oxidative stress (kato and shimizu, 1985). kaiser (1987) indicated that an irreversible decrease in plant photosynthetic capacity occurs as rwc declines below 30%, leading to cell death from membrane damage in chloroplasts. table 4 effect of field capacity and photoperiod and their interaction on proline, sugars and starch contents, a c t i v i t y o f s u p e r o x i d a s e d i s m u t a s e , c a t a l a s e , peroxidase, and ascorbate peroxidase enzymes variables photoperiod field capacity (%) mean 100% 75% 50% 25% chlorophyll ldl 1.81 a 1.78 b 1.48 e 0.99 j 1.52 a content idl 1.74 c 1.71 d 1.41 h 0.92 k 1.44 b (mg chl g−1f.w.) sdl 1.45 f 1.42 g 1.12 i 0.63 l 1.15 c mean 1.67 a 1.64 b 1.33 c 0.85 d sugars of shoot ldl 62.63 de 67.19 c 199.39 b 212.15 a 135.34 a (mg g−1d.w.) idl 61.81 e 66.37 cd 198.22 b 211.33 a 134.52 a sdl 61.45 e 66.02 cd 198.22 b 210.98 a 134.17 a mean 61.96 d 66.53 c 198.72 b 211.49 a starch content ldl 225.30 a 224.10 d 91.50 g 41.30 j 145.50 a (mg g−1 d.w.) idl 224.90 b 223.70 e 91.10 h 40.90 k 145.10 b sdl 224.30 c 223.20 f 90.60 i 40.40 l 144.60 c mean 224.80 a 223.70 b 91.10 c 40.90 d superoxide ldl 136.00 cd 148.50 c 266.00 a 116.00 def 166.62 a dismutase idl 128.50 cde 143.50 c 261.50 a 109.50 ef 160.75 a (ug-1 fw) sdl 106.00 fg 118.50 def 236.00 b 86.00 g 136.62 b mean 123.50 c 136.83 b 254.50 a 103.83 d catalase ldl 31.48 ef 34.45 cd 40.71 a 31.18 efg 34.45 a (ug-1 f.w.) idl 28.89 fgh 31.86 de 38.12 ab 28.59 ghi 31.86 b sdl 29.53 efg 29.53 efg 35.78 bc 26.25 i 29.53 c mean 28.98 c 31.95 b 38.20 a 28.67 c peroxidase ldl 68.40 fg 70.75 f 94.58 c 112.40 a 86.53 a (ug-1 f.w.) idl 60.80 hi 63.15 hi 86.99 d 104.81 b 78.94 b sdl 52.83 j 55.18 ji 79.02 e 96.84 c 70.97 c mean 60.68 c 63.03 c 86.86 b 104.68 a ascorbate ldl 869.64 de 879.64 d 1160.36 a 854.29 ef 940.98 a peroxidase idl 829.64 gh 839.64 fg 1120.36 b 814.29 hi 900.98 b (ug-1 f.w.) sdl 784.64 kj 794.64 ij 1075.36 c 769.29 k 855.98 c mean 827.97 b 837.97 b 1118.69 a 812.61 c *in each variable, data followed by the same letters (small letters for interactions and capital letters for means) are not significantly different using lsd at 5% level. ldl= long day length. idl= intermediate day length sdl= short day length. adv. hort. sci., 2016 30(3): 141-149 146 detrimental effects on chloroplast biochemistry or chlorophyll fluorescence occur when rwc drops below 60% in tall fescue (huang et al., 1998). surface drying had no effects on chlorophyll content in kentucky bluegrass (poa pratensis l.) and tall fescue (festuca arundinacea schreb.) while under full drying, chlorophyll content decreased in both grasses (fu and huang, 2001). our findings were in agreement are (fu and huang, 2001) who reported that amount of chlorophyll in bermudagrass under moderate stress is not reduced, but it will be reduced in the severe drought. chlorophyll content decreased with decreasing day length and the highest and lowest ones were observed in ldl and sdl treatments, respectively (table 4). shorting photoperiod caused decrease in chlorophyll content. in a research, the resistances to low light stress in both bermudagrass and paspalum have been examined and it was concluded that resistance to low light stress in the paspalum is more than bermudagrass (jiang et al., 2004). baldwin et al. (2008) reported that bermudagrass showed significant decrease in chlorophyll content in response to short day length condition. total soluble sugars and starch content regardless of photoperiod, decrease in field capacity significantly increased total soluble sugars in the shoot (table 4). starch content declined by decreasing field capacity from 100% to 25% (table 4). shoot starch content, were highest and lowest in 100% and 25% fc treatments, respectively (table 4). on the other hand, total soluble sugars during the drought can increase making these compounds nonphotosynthetic routes and growth stopping due to the destruction of in soluble sugars and their change to soluble sugars (hissao, 1973). although some researchers have suggested that the destruction of starch can also increase monosaccharaides (düring, 1992). the researchers stated that an increase of amylase in water stress causes starch degradation and the conversion of this large molecule into smaller units (movahhedi-dehnavi et al., 2004). different photoperiod had no significant effects on total soluble sugars (table 4). shoot starch content decreased by different light durations and the highest and lowest one was observed in ldl and sdl treatments, respectively (table 4). starch content decreased in response to shortening the photoperiod. some researchers have reported that prolonging photoperiod increases carbohydrates (hay and pederson, 1986; solhoug, 1991; wang et al., 1998). other researchers reported that the photoperiod had no effect on carbohydrates production (sicher et al., 1982; logendra and janes, 1992). antioxidant enzyme activities a p x , p o d , c a t a n d s o d e n z y m e s a c t i v i t i e s showed significant differences among field capacity and photoperiod treatments. the activities of apx were not significantly different between 100% fc and 75% fc treatments while were significantly increased in 50% fc treatment and minimum apx activity was observed at 25% fc treatment (table 4). bian and jiang (2009) investigated the accumulation of reactive species of oxygen and antioxidants activity and t h e p a t t e r n o f g e n e e x p r e s s i o n o f a n t i o x i d a n t enzymes in the kentuchy bluegrass in the drought c o n d i t i o n . t h e y o b s e r v e d t h a t d r o u g h t s t r e s s increased the activity of apx and cat and decreased sod and they stated that antioxidant enzymes and their gene expression might be different or occur in the immune system of kentuchy bluegrass roots and l e a v e s . p o d e n z y m e a c t i v i t i e s i n c r e a s e d w i t h decrease in field capacity levels. differences in leaf pod enzyme activities were not detected between 100% fc and 75% fc treatments. the maximum and minimum pod activity was obtained in 25% fc and 100% fc treatments, respectively (table 4). in a research on drought tolerance of three cultivars of creeping bentgrass, it was observed that long-term drought stress reduced the activity of antioxidants such as pod and increased lipid peroxidation and the ‘greenwich’ showed high resistance to drought (dacosta and huang, 2007). cat and sod enzymes activities significantly increased with decreasing field capacity from 100% to 50% then, declined in 25% fc treatment (table 4). shao et al. (2005) reported that in the of drought stress, the production amount of three enzymes, cat, sod and pod in resistant bermudagrass varieties have been significantly more than drought-sensitive ones. general declines in antioxidants, including cat were reported in the response of three species of creeping bentgrass to drought stress. moreover, they found that the species agrostis canina l. was the most resistant species to drought (dacosta and huang, 2007). liu et al. (2008) in a research, physiologically and morphologically investigated the five cultivars of kentuchy b l u e g r a s s u n d e r d r o u g h t a n d h e a t s t r e s s a n d observed that drought and heat stress simultaneously reduces sod enzyme in all cultivars and stated that an increase of sod enzyme activity cannot inhibit stress and would only delay free radicals accumulation. results of present study indicated that regardadamipour et al. morpho-physiological alteration in common bermudagrass subjected to limited irrigation and light condition 147 less of field capacity treatments, apx, pod, cat and sod enzymes activities significantly decreased in response to decreasing day length therefore, the m a x i m u m a n d m i n i m u m e n z y m e s a c t i v i t y w a s observed in ldl and sdl treatments (table 4). similar findings have been previously reported by (burritt and mackenzie, 2003) who stated that when the begonia plant is transferred from low light to bright light, cat activity increases. also, they stated that when the (picea abies l.) seedlings are transferred from low light to high light, the activity of cat enzyme decreases. xu et al. (2010) investigated the effect of nitric oxide and sodium nitroprusside in tall fescue under high light stress and concluded that using sodium nitroprusside reduces enzyme activity of sod, cat and apx, but using nitric oxide increases the activity of mentioned enzymes. jiang et al. (2005) demonstrated that, low light conditions showed a significant decrease in activity apx and cat of bermudagrss and paspalum. grace and logan (1996) reported that the cat enzyme activity varies depending on light intensity. the cat enzyme activity in schefflera [schefflera arboricola (hayata) merrill] and vinca (vinca major l.) plants did not change with a change in light intensity, but in mahonia (mahonia repens (lindley) don.), cat enzyme activity increased with an increase of light intensity. interaction between field capacity and photoperiod resulted in the highest and lowest sod enzyme activities in 50% fc-ldl and 25% fc-sdl treatments (table 4). 4. conclusions the results proved that the reduction in photoperiod led to a progressive increase in shoot height and leaf area, however, the increase in irrigation inhibited their progressive growths. additionally, the reduction in photoperiod caused a decrease in fresh and dry weight of root and shoot. however, the increase in irrigation led to alleviation of these negative effects during the day-time and thus increased the fresh and dry weight of root and shoot. therefore, it appears as though the increased irrigation might have contributed to the enlargement and flexibility of cells, which, in turn helped increasing the dry and fresh weight of root and shoot. the reduced photoperiod led to a reduction in chlorophyll and starch contents and enzymes activities, and the increased irrigation compensated this reduction to some extent. this phenomenon might be, at least in part, explained by the fact that irrigation reduced aba production, inhibited ros production and thus inhibited the closure of stomata. in overall, the increase in irrigation caused the destructive effects of reduced photoperiod to diminish, and vice versa. it seems that the interaction of photoperiod and irrigation treatments has superior effects on alleviating of the symptoms of stressed plants, than their separate. further studies are 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tropical fruit and considered king of all the fruits in india. the fruits are a rich source of iron and various anti-oxidants. in addition, mango is a rich source of vitamin a, e and selenium, which help to protect against heart disease and other such related aliments. nowadays, continuous use of chemical fertilizer without organic manure causes problems of soil health and fruit quality. fruit qualities are being deteriorated through the use of chemical fertilizers (huyskens-keil and schreiner, 2003). organic farming is currently gaining gradual momentum worldwide with growing awareness of health and environmental issues in agriculture and consumers demanding the production of organic fruit, thus offering an attractive source of rural income. organic farming in india has attracted many farmers throughout the country and different fruit crops like banana, papaya, pineapp l e a n d s a p o t a h a v e b e e n s u c c e s s f u l l y t e s t e d . according to neuhoff et al. (2011) working with oranges and ilic et al. (2013) tomato, organically grown fruits are rich in various minerals such as p, k, ca and mg. soil microbes were found to increase in organic systems compared to the conventional system of planting (dutta and kundu, 2011). however, the influence of organic, inorganic, and biofertilizer on growth characters and fruit quality of mango and soil properties are not well documented. hence, a study was initiated to evaluate their effect on fruit quality and soil properties. 2. materials and methods t h e s t u d y w a s c o n d u c t e d a t t h e u n i v e r s i t y research station, gayeshpur, bidhan chandra krishi viswavidyalaya, west bengal, india on 11 years old tree of mango cv. himsagar with 10×10 m spacing. the following eight treatments were imposed: v e r m i c o m p o s t a t 5 k g / p l a n t / y e a r , f y m a t 1 0 k g / p l a n t / y e a r , i n o r g a n i c f e r t i l i z e r ( n p k a t 1000:500:1000 g/plant/year), 50% vermicompost + adv. hort. sci., 2016 30(2): 81-85 doi: 10.13128/ahs-19133 influence of organics, inorganic and biofertilizers on growth, fruit quality, and soil characters of himsagar mango grown in new alluvial zone of west bengal, india p. dutta (*), k. das, a. patel department of fruits and orchard management, faculty of horticulture, bidhan chandra krishi viswavidyalaya, p.o. mohanpur, 741252 dt. nadia, west bengal, india. key words: growth characters, mangifera indica, quality, shelf life, soil microbial population. abstract: a field study with organic manures, inorganic manures and biofertilizers alone and in combination was carried out on 11-year-old mango (mangifera indica l.) cv. himsagar spaced 10 m apart in a randomized block design at the university research station, gayeshpur, bckv, west bengal, india. the objective was to determine their effect on growth, soil characters, and fruit quality of mango grown in a new alluvial zone of west bengal. respiration, physiological loss in weight (plw) and shelf life at ambient room temperatures, and soil characters were also assessed. results revealed that among the eight treatments, biofertiliser (azotobacter + psm) along with 50% inorganic fertilizer significantly increased the growth characters of mango trees. this treatment also increased the physico-chemical character of fruit while biofertilizers alone improved the fruit quality, viz. total soluble solids, total sugar and β-carotene. fruits treated with biofertilizers also had increased shelf life with lower plw and respiration rate. soil microbial population and other soil characters were improved by application of biofertilizer as well. it is concluded that biofertilizer application in mango gives better growth, fruit quality, and soil health. (*) corresponding author: pallab_bckv@rediffmail.com received for publication 9 august 2015 accepted for publication 11 april 2016 copyright: © 2016 author(s). this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. adv. hort. sci., 2016 30(2): 81-85 82 50% inorganic fertilizer, 50% fym+50% inorganic fertilizer, biofertilizer (azotobacter at 150 g/plant) + psm at 100 g/plant), biofertilizer + 50% inorganic fertilizer, and control (no treatment). treatments were applied separately in split doses, once at fruit set and another at harvest the experiment was laid out in a randomized block design with three replications. mature fruits were harvested and brought to the laboratory for physico-chemical analyses following all standard procedures as described by ranganna (2000). total soluble solids were determined using a hand refractometer (0-32°brix). total soluble sugar content was analysed using fehlings’ a and b solution, according to the method described by ranganna (2000) and expressed as percentage. in this method, for inversion of room temperature an aliquot of claried and diluted solution was transferred for a flask. 10 ml of hcl (1:1) was added and allowed to stand at room temperature for 24 hrs. the solution was then neutralized with concentrated naoh solution and made to volume. an aliquot was taken and the total soluble sugars were determined as invert sugars using fehling’s a and b solution. titratable acidity (% malic acid) was estimated by titrating fruit juice (5 ml) to ph 8 against 0.1 m naoh using phenolphthalene as indicator. total carotenoids were estimated by the method described by ranganna (2000). five grams of fresh sample were taken, a few crystals of anhydrous sodium sulphate were added, and then crushed in 10 ml acetone with the help of a mortar and pestle. the supernatant was decanted into a beaker. the process was repeated twice or thrice and the combined supernatant was transferred to a separating funnel out on standing. petroleum ether (10 to 15 ml) was added in the separating funnel and rinsed, the pigment was then transferred to the petroleum ether phase by diluting the acetone with water or water containing 5% sodium sulphate. the extraction of the acetone phase with a small volume of petroleum ether was repeated, if necessary, until no more colour was extracted. the lower layer was discarded and the upper layer was collected in a 100 ml volumetric flask. the petroleum ether extract was filtered through anhydrous na2so4 and the volume was made up to 100 ml with petroleum ether. the optical density was recorded at 452 nm using petroleum ether as blank containing 3 ml acetone per 100 ml and expressed as µg 100 g-1 pulp. as carotenoids are light sensitive, all steps were performed under subdued light. shelf life was determined at ambient room temperature (34±1°c). the co2 evolution of fruits was determined by titration of residual ba(oh)2 i n t h e s o l u t i o n w i t h s t a n d a r d i z e d n / 1 0 h c l a s described by mitra et al. (1971). growth characters such as plant height, plant spread, and trunk girth were measured after fruit harvest. soil properties were recorded as per the standard procedure given by black (1965). soil microbial population was counted using the method described by collin and lyne (1985). statistical analysis was carried out according to the standard procedures. 3. results and discussion growth parameters table 1 reveals that different nutrient treatments significantly increased plant height, canopy, spread, and trunk girth. biofertilizer + half inorganic fertilizer gave maximum (6.72 m) plant height, canopy spread (6.37×6.92 m), and trunk girth (79.32 cm), followed by vermicompost (2.5 kg/plant/year) + half chemical fertilizer (rdf-npk at 1000:500:1000 g/plant/year); treatments plant height (m) canopy spread trunk girth (cm) (m) e-w n-s 1. vermicompost (5 kg/plant) 5.99 5.49 5.12 69.72 2. fym (10 kg/plant) 6.10 5.97 6.14 72.11 3. inorganic fertilizer (npk1000:500:1000 g/plant/yr.) 6.00 5.97 6.11 73.72 4. 50% vermicompost + 50% inorganic fertilizer 6.24 6.12 6.31 70.47 5. 50% fym + 50% inorganic fertilizer 6.11 6.00 6.09 73.15 6. biofertilizer (azotobacter @ 150g/plant + psm @ 100 g/plant) 5.99 5.84 5.91 70.37 7. biofertilizer + 50% inorganic fertilizer 6.72 6.37 6.92 79.32 8. control 5.97 5.82 5.90 71.41 sem± 1.01 0.72 1.11 2.40 cd (p=0.05) 3.11 1.92 3.31 3.14 table 1 growth characters of mango cv. himsagar as influenced by inorganic and organic manures dutta et al. organics, inorganic and biofertilizer influence on growth, quality and soil characters of himsangar mango. 83 the lowest measurements for these three parameters were found in untreated control plants. korwar et al. (2006) in aonla and shukla et al. (2009) in guava obtained similar results. these findings may be due to a better nutritional environment: application of organic matter improves soil health by improving t h e p h y s i c o c h e m i c a l a n d b i o l o g i c a l a c t i v i t i e s (schnitizer, 1991) and biofertilizer was found to enhance the rate of mineralization and availability of the nutrients, further enhancing plant growth (sahoo and singh, 2005). physico-chemical composition of fruit d i f f e r e n t n u t r i e n t t r e a t m e n t s s i g n i f i c a n t l y improved the physico-chemical composition of fruits (table 2). biofertilizer + half of inorganic fertilizer produced maximum fruit weight (285.15 g), yield (57.20 kg/plant), fruit length/breath (9.14/8.19 cm), followed by biofertlizer alone. also with regard to fruits, control trees gave minimum results. unlike the physical characters, the bio-chemical composition of fruits was more effected by biofertilizer alone. biofertilizer (azotobacter @ 150 g/plant + psm @ 100 g/plant/year) gave maximum total soluble solids (19.80°brix, total sugar (16.00%) and β-carotene (6123 µg/100 g) with minimum (0.16%) acidity. fruits treated with inorganic fertilizers showed minimum total solids (18.20°brix) and β-carotene (4792 µg/100 g) with maximum (0.31%) acidity. results revealed that fruits grown under organic manure/biofertilizer had better fruit quality. the increase in physicochemical parameters in fruits due to bio-fertilizer might be because of their role in nitrogen fixation, production of phytohormone-like substances and increased uptake of nitrogen as reported by dutta and kundu (2012). furthermore, micro-organisms are an important component of soil environment (arshad and frankemberger, 1992). thus, utilization of biofertilizer could be a better preposition for improving biological attributes of soil, which in turn may increase quality and productivity potential of various crops as reported by allen et al. (2002). soil nutrient status and soil bacterial population d i f f e r e n t n u t r i e n t t r e a t m e n t s s i g n i f i c a n t l y increased the soil ph and soil organic carbon (table 3). biofertilizer alone gave maximum (6.70) soil ph while inorganic fertilizer-treated soil the minimum table 2 fruit quality of himsagar mango as influenced by inorganic and organic manures treatments fruit weight (g) yield (kg/plant) fruit length/breadth (cm) tss (°brix) total sugar (%) acidity (%) β carotene (µg/100g) 1. vermicompost (5 kg/plant) 250.00 50.95 8.40/7.29 18.70 16.12 0.14 5720 2. fym (10 kg/plant) 151.42 50.35 8.31/7.31 18.90 15.91 0.17 5824 3. inorganic fertilizer (npk1000:500:1000 g/plant/yr) 248.43 51.75 8.00/7.44 18.20 15.33 0.31 4792 4. 50% vermicompost + 50% inorganic fertilizer 265.14 53.92 9.11/8.12 19.00 15.11 0.25 5012 5. 50% fym + 50% inorganic fertilizer 270.00 54.98 9.00/8.15 19.10 15.23 0.26 5170 6. biofertilizer (azotobacter @ 150 g/plant + psm @ 100 g/plant) 270.40 54.12 8.99/85.16 19.80 16.00 0.16 6123 7. biofertilizer + 50% inorganic fertilizer 285.15 57.20 9.14/8.19 19.60 15.92 0.18 5814 8. control 240.40 48.00 8.10/7.60 18.00 14.94 0.19 4914 sem± 1.15 0.70 1.01/0.72 0.51 0.07 0.01 4.31 cd (p=0.05) 3.72 2.10 3.11/1.97 1.37 0.21 0.03 12.39 treatments ph ec (1:2.5) (dsm-1) bulk density (g/cc) oc (%) soil microbial population (bacteria) (cfug-1 soil) 1. vermicompost (5 kg/plant) 6.57 0.263 1.21 0.90 1.6 × 106 2. fym (10 kg/plant) 6.59 0.137 1.29 0.84 5.9× 106 3. inorganic fertilizer (npk1000:500:1000 g/plant/yr.) 6.00 0.171 1.49 0.61 4.3× 105 4. 50% vermicompost + 50% inorganic fertilizer 6.45 0.214 1.12 0.72 5.1× 106 5. 50% fym + 50% inorganic fertilizer 6.42 0.219 1.41 0.74 5.4× 106 6. biofertilizer (azotobacter @ 50 g/plant + psm @ 100 g/plant) 6.70 0.198 1.31 0.78 8.3× 106 7. biofertilizer + 50% inorganic fertilizer 6.42 0.197 1.27 0.74 6.0× 106 8. control 6.10 0.111 1.69 0.60 6.9× 105 sem± 0.13 0.70 0.03 0.02 4.72 cd (p=0.05) 0.39 2.10 0.09 0.07 14.43 table 3 soil characters as influenced by inorganic and organic manures adv. hort. sci., 2016 30(2): 81-85 84 (6.00). organic carbon content of soil also varied significantly. vermicompost or fym-treated soil gave maximum (0.90/0.84%) content of organic carbon, followed by biofertilizer-treated soil (0.78%). soil organic carbon was at the lowest level in untreated control. the increase in organic carbon of soil may be due to the addition of organic matter through organic manure or microbes and recycling of organic materials in the form of crop residue, which brings the soil ph nearer to neutral and increases the nutrient availability. our results are in close conformity with earlier findings (dutta and kundu, 2012). the effective conductivity (ec) of soil solution depends on the presence of soluble salts. the concentrations of these salts affect the growth and absorption of water. different treatments significantly influenced the ec of soil. application of vermicompost led to the highest ec (0.263 dsm-1) in this study, whereas the control plot gave the lowest. this could be due to the fact that vermicompost contains salts, mostly cl and so4, but not at toxic levels (masciandaro et al., 1998). bulk density varied due to different treatments, with the untreated control giving maximum bulk density (1.69 g/cc) of soil. the soil microbial population varied with the different treatments: the m a x i m u m ( 8 . 3 x 1 0 6 c f u g 1 s o i l ) w a s f o u n d w i t h biofertilizer (azotobacter at 50 g/plant + psm at 100 g/plant). soil applied with biofertilizer showed more s o i l b a c t e r i a , w h e r e a s l e a s t w a s f o r m e d b y v e r m i c o m p o s t ( t a b l e 3 ) . s i m i l a r r e s u l t s w e r e obtained by dutta et al. (2010) in litchi. plw, shelf life and respiration of fruit physiological loss in weight (plw) of fruit varied significantly among the different treatments (table 4). untreated fruit had the highest (18.95%) plw while the lowest was found with fruit grown in biofertilizer (11.31%). fruits grown in biofertilizer showed maximum (10 days) shelf life with minimum respiration rate (109.72 mg/hr/kg fruit) during storage. untreated control fruit recorded maximum respiration and plw with minimum shelf life (5 days). improvement of shelf life due to application of biofertilizer was previously reported in mango (dutta and kundu, 2012). 4. conclusions the present study reveals that application of organic and biofertilizer are more beneficial for quality mango production and increase soil health. therefore, this approach can be spread among growers to improve the quality in mango orchards. references allen m.f., jasper d.a., zak j.c., 2002 microorganism, pp. 257-278. in: perrow m.r., and a.j. davy (eds.) handbook of ecological restoration. volune i. principles of restoration. part 4. manupulation of the biota. cambridge university press, cambridge, uk, pp. 445. arshad m., frankemberger jr. w.t., 1992 microbial production of plant growth regulators, pp. 307-348. in: meeting f.b. jr. (ed.) soil microbial ecology. application in agricultural and environmental management. marcel dekker, inc. new york, usa. black c.a., 1965 method of soil analysis. amer. soc. of agron. inc. publisher, madison, wisconsin, usa, vol. 9, parts 1 & 2, pp. 1572. collin c.h., lyne p.m., 1985 microbiological methods. butterworth co., london, uk, pp. 450. dutta p., kundu s., 2011 effect of organic manure and b i o f e r t i l i z e r s o n o r g a n i c p r o d u c t i o n o f m a n g o . treatment plw (%) shelf life (days) respiration co 2 evolution (mg/hr/kg fruit) 1. vermicompost (5 kg/plant) 12.62 8 120.41 2. fym (10 kg/plant) 12.44 9 118.32 3. inorganic fertilizer (npk1000:500:1000 g/plant/yr.) 17.92 6 170.41 4. 50% vermicompost + 50 % inorganic fertilizer 14.31 7 150.37 5. 50 % fym + 50 % inorganic fertilizer 14.45 2 159.37 6. biofertilizer (azotobacter @ 150 g/plant + psm @ 100 g/plant) 11.31 10 109.72 7. biofertilizer + 50% inorganic fertilizer 13.00 8 122.14 8. control 18.95 5 163.14 sem± 1.10 0.61 4.11 cd (p=0.05) 3.42 1.82 12.12 table 4 plw, shelf life and respiration of fruit at the end of storage life as affected by inorganic and organic manures dutta et al. organics, inorganic and biofertilizer influence on growth, quality and soil characters of himsangar mango. 85 proceedings of the third scientific conference of isofar, 1: 475-478. dutta p., kundu s., 2012 effect of biofertilizers on nutrient status and fruit quality of himsagar mango grown in new alluvial zones of west bengal. j. crop weed sci., 8(1): 72-74. dutta p., kundu s., biswas s., 2010 integrated nutrient management in litchi cv. bombai in new alluvgial zone of west bengal. indian j. hort., 67: 181-184. huyskens-keil s., schreiner m., 2003 quality of fruit and vegetable. j. appl. bot., 77: 147-151. i l i c z . s . , k a p o u l a s n . , m i l e n k o v i c l . , 2 0 1 3 micronutrient composition and quality characteristics of tomato (lycopersicon esculentum) from conventional and organic production. indian j. agric. sci., 83: 651-655. korwar g.r., pratibha g., ravi v., palanikumar d., 2006 influence of organics and inorganics on growth, yield of aonla (emblica officinalis) and soil quality in semi-arid tropics. indian j. agric. sci., 76: 457-461. m a s c i a n d a r o g . , c e c c a n t i b . , g a r c i a c . , 1 9 9 8 changes in soil biochemical and cracting properties induced by living much system. canadian j. soil sci., 77: 579-589. mitra d., guha j., chaudhuri s.k., 1971 studies in botany. vol, ii, moulik library, kolkata, india. n e u h o f f d . , v l a t s c h k o v v . , r a i g o n d . , 2 0 1 1 comparison of the quality of conventionally and organically grown oranges in spain. proceedings of the third scientific conference of isofar, pp. 487-490. ranganna s., 2000 manual of analysis of fruits and vegetables products. tata mcgrow hill publishing co. ltd. 3rd ed. new delhi, india. sahoo s.k., singh d.b., 2005 effect of different level of biofertilizers on growth, yield and quality of strawberry (fragania ananassa duch) cv. sweet charley. the orissa j. hort., 33: 82-85. schnitizer m., 1991 soil organic matter in the next 75 years. soil sci., 151: 41-59. shukla a.k., sarolia d.k., bhavana k., kaushik r.a., mahawer l.n., bairwa h.l., 2009 evaluation of substrate dynamics for integrated nutrient management under high density planting of guava cv. sardar. indian j. hort., 66: 461-464. 95 1. introduction citrus is one of the most important fruit crops in japan and also worldwide. various accessions of citrus species are adapted to the southwest of japan, and although they are cultivated in this region, almost all of them are nonnative, that is, they were introduced from abroad, arose as chance seedlings, were selected from bud sports, and were bred by artificial pollination. only two species, citrus tachibana (makino) tanaka (tachibana) and citrus depressa hayata (shiikuwasha) were present in japan before recorded history. c. tachibana mainly grows indigenously on the pacific side of the southwest of japan’s main islands (kyushu, shikoku, and honshu). c. tachibana was recorded in “kojiki”, the oldest chronicle in japan dating from the early 8th century. its indigenous trees were also found on the ryukyu islands (islands including the okinawa islands, sakishima islands, and amami islands, which were ruled by japan from the 17th to19th centuries) and taiwan (tanaka, 1931; lin and chen, 2006; inafuku-teramoto et al., 2010). c. depressa is indigenous to both the ryukyu islands and taiwan (tanaka, 1936; lin and chen, 2006). compared to c. tachibana, c. depressa is considered to be adapted to a warmer climate; the former is usually used as an ornamental for gardens and its fruit is inedible. on the other hand, fruit of c. depressa is in much demand as an ingredient for food and drinks, to garnish dishes similar to a lemon or lime, to make juice and jam, and as an additive to soy sauce and distilled spirits. recently, this fruit has attracted attention because it contains high levels of polymethoxyflavonoids, one of the most important health-promoting components of citrus (inafuku-teramoto et al., 2010). we have investigated the phylogenetic relationships of citrus and its relatives through the analysis of genes encoded in chloroplast dna (cpdna) (tshering et al., 2010, 2013). in our recent study (tshering et al., 2013) in which various citrus accessions were used as materials, we found that c. tachibana and c. depressa possess a characteristic cpdna genome based on the sequences of the chloroplast matk genes, which encode a maturase involved in splicing type ii introns from rna transcripts (hilu and liang, 1997; hilu et al., 2003; olmstead and palmer, 1994). there are many accessions in both species, and intraspecies diversity is found within each species (hirai et al., 1990; yamamoto et al., 1998; kinjo, 2007; inafuku-teramoto et al., 2010; yamamoto et al., 2011). characterization of chloroplast matk sequences of citrus tachibana and citrus depressa, two indigenous species in japan y. nagano1, s. inafuku-teramoto2,3, m. hashimoto4, t. mimura4, r. matsumoto4, m. yamamoto5(*) 1 analytical research center for experimental sciences, saga university, honjo-machi, saga 840-8502, japan. 2 faculty of agriculture, university of the ryukyus, nishihara, okinawa 903-0213, japan. 3 botswana-jica jatropha project, dar, sebele, gaborone, botswana. 4 faculty of agriculture, saga university, honjo-machi, saga 840-8502, japan. 5 faculty of agriculture, kagoshima university, korimoto, kagoshima 890-0065, japan. key words: cpdna, genetic resources, ryukyu islands, shiikuwasha, tachibana. abstract: citrus tachibana, c. nippokoreana, and c. depressa are indigenous mandarin species in japan. we deduced their phylogenetic relationships from nucleotide sequences of the chloroplast matk gene. the results indicate that c. tachibana, c. nippokoreana, and c. depressa accessions can be classified into two types: type a, all sixteen c. tachibana and six c. depressa; type b, eleven c. depressa and one c. nippokoreana. both type a and type b accessions of c. depressa were found on the okinawa islands, whereas only type b accessions of c. depressa were found on the sakishima and amami islands. this cpdna divergence seemed to indicate a polyphyletic origin of c. depressa. the matk genes of type a were found only in c. tachibana and some c. depressa. from these results, both species probably possess a characteristic chloroplast genome among various citrus species. adv. hort. sci., 2014 28(2): 95-99 (*) corresponding author: yamasa@agri.kagoshima-u.ac.jp received for publication 31 march 2014 accepted for publication 1 july 2014 96 however, a limited number of accessions were used in our previous study (tshering et al., 2013). therefore, for the present work, we analyzed the matk gene sequences of a number of c. tachibana and c. depressa plants grown in various regions in japan to reveal their characteristic profiles of the cpdna genome. c. nippokoreana (korai tachibana), a c. tachibana relative indigenous to hagi city, yamaguchi prefecture, japan, and cheju island, korea (kimura and taninaka, 1995), was also investigated. 2. materials and methods plant materials sixteen c. tachibana, one c. nippokoreana, 17 c. depressa, and 13 control accessions were used in this study. the sources of the materials are shown in table 1 and figure 1. pcr amplification and dna sequencing genomic dna was extracted from leaves using the dneasy plant mini kit (qiagen, valencia, ca, usa). by using this genomic dna as a template, the matk gene was amplified by pcr using proofreading primestar gxl dna polymerase (takara bio, ohtsu, shiga, japan). the primers used for pcr amplification of the matk gene were matk1f (5′-accgtatcgcactatgtatc-3′) and matk1r (5′-gaactagtcggatggagtag-3′). the amplified dna fragments were purified using the nucleospin gel and pcr clean-up kit (macherey-nagel, düren, germany). the primers used for sequencing of the matk gene were matk1f, matk2f (5′-acggttctttctccacgagt-3′), matk3f (5′-ggtccgatttctctgattct-3′), matk1r, matk2r (5′-agaatcagagaaatcggacc-3′), and matk3r (5′-actcgtggagaaagaaccgt-3′). the purified dna fragments were sequenced in both directions in an applied biosystems 3130 genetic analyzer (applied biosystems) with a bigdye terminator cycle sequencing ready reaction kit v. 3.1 (applied biosystems) as described previously (platt et al., 2007). sequence data were submitted to ddbj/genbank/ebi and were assigned accession numbers ranging from ab839905 to ab839932. the sequences of the accessions from no. 29 to no. 34 were deposited in our previous study (tshering et al., 2013). phylogenetic analyses the neighbor-joining (nj) and maximum likelihood (ml) methods from the mega (version 5.2.1) program (tamura et al., 2011) were used to create phylogenetic trees. the reliability of each branch was tested by bootstrap analysis with 1,000 replications. 3. results and discussion we constructed multiple sequence alignments of 1,630-bp fragments containing the matk gene from different citrus accessions. each sequence contained a 1,530-bp protein-coding sequence and 100 bp of the 3′ utr. one exception is the matk gene of trifoliate orange (poncirus trifoliata), which has a 6 bp insertion at the 3′ utr. of these, 23 bases were variable and six bases were phylogenetically informative. we created phylogenetic trees using the nj and ml methods. the topologies of the different trees were identical (data not shown). therefore, we present here only the ml tree (fig. 2). c. tachibana, c. depressa, and c. nippokoreana accessions were classified into two types as follows: type a: all 16 c. tachibana and six c. depressa [shiikuwasha-okinawa#1 (no. 17), shiikuwasha-okinawa#3 (no. 19), shiikuwasha-okinawa#6 (no. 22), and shiikuwashaoku (no. 26), kabishi (no. 29), and fusubuta (no. 31)]. type b: eleven c. depressa [shiikuwasha-taketomi (nohara) (no. 11), shiikuwasha-taketomi (takana) (no. 12), shiikuwasha-iriomote (no. 13), shiikuwasha-iriomote (katoura) (no. 14), shiikuwasha-kohama (ufudake) (no. 15), shiikuwasha-kohama (omori) (no. 16), ishikunibu (no. 28), mikanguwa (no. 30), kaachi (no. 32), shiikunin (no. 33), and shiikurubu (no. 34)] and one c. nippokoreana. none of the control accessions belonged to type a, whereas all seven control mandarin accessions belonged to type b. the other control accessions were clearly distinguished from type a and type b. this finding is consistent with the results of our previous study (tshering et al., 2013). all 16 c. tachibana accessions carried an identical matk sequence. previous studies (hirai et al., 1990; yamamoto and tominaga, 2003) reported that c. tachibana was genetically differentiated from citrus species originating from all fig. 1 collection sites of citrus tachibana, c. nippokoreana, and c. depressa in the present study. 97 table 1 citrus tachibana, c. nipponkoreana, and c. depressa accessions used in the present study no. accession latin name origin note 1 tachibana-dazaifu (uchi) citrus tachibana (makino) tanaka fukuoka, kyushu planted tree 2 tachibana-dazaifu (soto) c. tachibana (makino) tanaka fukuoka, kyushu planted tree 3 tachibana-heian jingu c. tachibana (makino) tanaka kyoto, honshu planted tree 4 tachibana-iwashimizu hachimangu c. tachibana (makino) tanaka kyoto, honshu planted tree 5 tachibana-kitano tenmangu c. tachibana (makino) tanaka kyoto, honshu planted tree 6 tachibana-toshijima (mie) c. tachibana (makino) tanaka mie, honshu native tree 7 tachibana-matsuoyama (kochi) c. tachibana (makino) tanaka kochi, shikoku native tree 8 tachibana-nangoku (kochi) c. tachibana (makino) tanaka kochi, shikoku planted tree 9 korai tachibana c. nippokoreana tanaka kochi, shikoku planted tree 10 tachibana-ishigakijima c. tachibana (makino) tanaka ishigaki-jima, sakishima native tree 11 shiikuwasha-taketomi (nohara) c. depressa hayata taketomi-jima, sakishima native tree 12 shiikuwasha-taketomi (takana) c. depressa hayata taketomi-jima, sakishima native tree 13 shiikuwasha-iriomote c. depressa hayata iriomote-jima, sakishima native tree 14 shiikuwasha-iriomote (katoura) c. depressa hayata iriomote-jima, sakishima native tree 15 shiikuwasha-kohama (ufudake) c. depressa hayata kohama-jima, sakishima native tree 16 shiikuwasha-kohama (omori) c. depressa hayata kohama-jima, sakishima native tree 17 shiikuwasha-okinawa#1 c. depressa hayata okinawa-honto native tree 18 tanibuta-okinawa#2 c. tachibana (makino) tanaka okinawa-honto native tree 19 shiikuwasha-okinawa#3 c. depressa hayata okinawa-honto native tree 20 tanibuta-okinawa#4 c. tachibana (makino) tanaka okinawa-honto native tree 21 tanibuta-okinawa#5 c. tachibana (makino) tanaka okinawa-honto native tree 22 shiikuwasha-okinawa#6 c. depressa hayata okinawa-honto native tree 23 tanibuta-okinawa#7 c. tachibana (makino) tanaka okinawa-honto native tree 24 tanibuta-okinawa#8 c. tachibana (makino) tanaka okinawa-honto native tree 25 garagara c. tachibana (makino) tanaka okinawa-honto native tree 26 shiikuwasha-oku c. depressa hayata okinawa-honto native tree 27 tanibuta c. tachibana (makino) tanaka okinawa-honto native tree 28 ishikunibu c. depressa hayata okinawa-honto native tree 29 kabishi c. depressa hayata okinawa-honto native tree 30 mikanguwa c. depressa hayata okinawa-honto native tree 31 fusubuta c. depressa hayata okinawa-honto native tree 32 kaachi c. depressa hayata okinawa-honto native tree 33 shiikunin c. depressa hayata tokuno-shima, amami native tree 34 shiikuribu c. depressa hayata okinoerabu-jima, amami native tree control accessions satsuma mandarin ‘aoshima’ c. unshiu marcow. ponkan ‘yoshida ponkan’ c. reticulata blanco mediterranean mandarin c. deliciosa ten. dancy c. tangerina hort. ex tanaka kinokuni ‘hirakishu’ c. kinokuni hort. ex tanaka sunki c. sunki (hayata) hort. ex tanaka cleopatra c. reshni hort. ex tanaka yuzu ‘yamane’ c. junos siebold ex tanaka sweet orange ‘fukuhara’ c. sinensis (l.) osbeck lemon ‘eureka’ c. limon (l.) burm. f. pummelo ‘mato buntan’ c. maxima (burm.) merr. citron ‘maru busshukan’ c. medica l.   trifoliate orange ‘standard’ poncirus trifoliata (l.) raf.     98 other countries except japan. the present study also confirmed that the matk sequence of c. tachibana was not identical to those of studied accessions originating from all other countries except japan. however, we found that the matk sequence of c. tachibana was identical to those of some investigated c. depressa accessions that are indigenous to the ryukyu islands, japan. this suggests that c. tachibana has been isolated from the mandarins elsewhere, and evolved in japan in unique ways. we found no diversity within species. however, further study considering more accessions is needed since the materials used here did not cover the entire area where c. tachibana grows. the matk sequence of c. nippokoreana was not identical to that of c. tachibana, indicting genetic differentiation between the two species. because it is considered that c. nippokoreana is related to c. tachibana (the japanese name “korai tachibana” means “tachibana from korea”), this finding is interesting. c. depressa accessions were divided into two types according to matk sequences. one was the same type as c. tachibana and the other was the same type as several mandarins such as c. reticulata and c. sunki. this result completely agrees with the results of our previous study (tshering et al., 2013). differentiation of the cpdna genome in c. depressa was also reported by urasaki et al. (2005) and yamamoto et al. (2013), who analyzed the trnl-trnf and trnf-trnvr regions, respectively. these results strongly suggest a polyphyletic origin of c. depressa. this divergence of matk genes was found in c. depressa accessions grown on okinawa-honto (the main island of okinawa islands) but not in those grown on the sakishima and amami islands. c. depressa possessing c. tachibana-type cpdna (a type) was found only on okinawa-honto. similar results were reported by yamamoto et al. (2013) who studied c. depressa on okinawa-honto and the amami islands. however, urasaki et al. (2005) found that c. depressa accessions possessed c. tachibana-type cpdna (trnl-trnf sequence) on the sakishima islands. thus, further study using many c. depressa accessions grown on various islands is necessary to resolve the distribution of each type. there is a possibility that type a c. depressa is genetically closer to c. tachibana than type b. however, this hypothesis is not supported since the proportion of common bands from random amplified polymorphic dna (rapd) analysis between c. depressa of type a and c. tachibana was not so different from that of type b and c. tachibana (yamamoto et al., 1998). since the origin and/or relationship of c. depressa to c. tachibana cannot be elucidated only by cpdna analysis, cpdna analysis combined with nuclear genome analysis such as simple sequence repeat (ssr), sequencerelated amplified polymorphism markers (sraps) (barkley et al., 2006; uzun et al., 2009), and restriction site-associated dna sequences (rad-seq) (baird et al., 2008) is considered to be necessary. for this purpose, structural analysis (barkley et al., 2006) seems to be informative. the present work demonstrates the characteristic profiles of the chloroplast genome of citrus tachibana and citrus depressa, two indigenous species in japan, using a number of accessions grown in various regions based on the results of matk sequencing. furthermore, the divergence of the cpdna genome of c. depressa seems to indicate a polyphyletic origin of this species. these findings are a contribution to progress in the study of the genetic resources in citrus and related genera. acknowledgements we would like to express our deep appreciation to dr. tetsuo koyama, kochi prefectural botanical garden for help in obtaining tachibana samples. we also acknowledge the help of staff of the following prestigious shrines, dazaifu tenmangu, kitano tenmangu, heian jingu, and iwashimizu hachimangu, for supplying tachibana samples from their sacred trees. references baird n.a., etter p.d., atwood t.s., currey m.c., shiver a.l., lewis z.a., selker e.u., cresko fig. 2 maximum likelihood tree of the matk genes from citrus tachibana, c. nippokoreana, and c. depressa and their control accessions. numbers at the nodes indicate bootstrap values (% over 1000 replicates). the scale bar shows the number of substitutions per site. 99 w.a., johnson e.a., 2008 rapid snp discovery and genetic mapping using sequenced rad markers. plos one, 3: e3376. barkley n.a., roose m.l., krueger r.r., federici c.t., 2006 assessing genetic diversity and population structure in a citrus germplasm collection utilizing simple sequence repeat markers (ssrs). theor. appl. genet., 112: 1519-1531. hilu k.w., borsch t., müller k., soltis d.e., soltis p.s., savolainen v., chase w., powell m.p., alice l.a., evans r., sauquet h., neinhuis c., slotta t.a.b., rohwer j.g., campbell c.s., chatrou l.w., 2003 angiosperm phylogeny based on matk sequence information. am. j. bot., 90: 1758-1776. hilu k.w., liang h., 1997 the matk gene: sequence variation and application in plant systematics. am. j. bot., 84: 830-839. hirai m., mitsue s., kita k., kajiura i., 1990 a survey and isozyme analysis of wild mandarin, tachibana (citrus tachibana (mak.) tanaka. growing in japan. j. japan. soc. hort. sci., 59: 1-7. inafuku-teramoto s., yamamoto m., kinjo h., kitajima a., wada k., kawamitsu y., 2010 local citrus genetic resources and their polymethoxyflavones content in northern part of okinawa island. hort. res., 9: 263-271. kimura k., taninaka t., 1995 acid citrus (in japanese), vol. 3. acid citrus in japan. harada co., tokushima, japan. kinjo h., 2007 acid citrus “shiikuwasha” in ryukyu island. studia citorologica, 17: 137-148. lin s.y., chen i.z., 2006 native citrus in taiwan and current statues of research and utilization. seed & nursery, 8: 1-12. olmstead r.g., palmer j.d., 1994 chloroplast dna systematics: a review of methods and data analysis. am. j. bot., 81: 1205-1224. platt a.r., woodhall r.w., george a.l., 2007 improved dna sequencing quality and efficiency using an optimized fast cycle sequencing protocol. biotechniques, 43: 58-62. tamura k., peterson d., peterson n., stecher g., nei m., kumar s., 2011 mega5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. mol. biol. evol., 28: 2731-2739. tanaka t., 1931 the discovery of citrus tachibana in formosa, and its scientific and industrial significance. studia citrologia, 5: 1-20. tanaka t., 1936 citrus studies. yokendo, tokyo, japan. tshering p., anai t., nagano y., matsumoto r., yamamoto m., 2010 phylogenetic relationships of citrus and its relatives based on rbcl gene sequences. tree. gen. genomes, 6: 931-939. tshering p., yamamoto m., uehara m., ide m., matsumoto n., matsumoto r., nagano y., 2013 phylogenetic relationships of citrus and its relatives based on matk gene sequences. plos one, 8: e62574. urasaki n., yoshida k., uehara t., inoue h., onda s., kinjyo h., kawano s., 2005 single nucleotide polymorphism in shiikuwasha (citrus depressa hayata) chloroplast dna, trnl-trnf. jpn. j. trop. agr., 49: 246-251. uzun a., yesiloglu t., aka-kacar y., tuzcu o., gulsen o., 2009 genetic diversity and relationships within citrus and related genera based on sequence related amplified polymorphism markers (sraps). sci. hortic., 121: 306-312. yamamoto m., kouno r., nakagawa t., usui t., kubo t., tominaga s., 2011 isozyme and dna analyses of local citrus germplasm on amami islands, japan. j. japan. soc. hort. sci., 80: 268-273. yamamoto m., matsuo y., kuniga t., matsumoto r., yamada y., 1998 isozyme and rapd analyses of shiikuwashas (citrus depressa hayata). bull. natl. inst. fruit tree sci., 30/31: 39-51. yamamoto m., tominaga s., 2003 high chromosomal variability of mandarin (citrus spp.) revealed by cma banding. euphytica, 129: 267-274. yamamoto m., tsuchimochi y., ninomiya t., koga t., kitajima a., yamasaki a., inafukuteramoto s., yang x., yang x., zhong g., nasir n., kubo t., tominaga s., 2013 diversity of chloroplast dna in various mandarins (citrus spp.) and other citrus demonstrated by caps analysis. j. japan. soc. hort. sci., 82: 106-113. 29 1. introduction bermudagrass is still the dominant warm-season turfgrass in warm to temperate climatic regions of the world. it is well adapted to a wide range of soil types, and its drought tolerance, recuperative ability, salt tolerance, wear tolerance, aggressive stoloniferous and rhizomatous growth habit, and overall appearance make bermudagrass an ideal turfgrass in many environments (taliaferro, 2003; shearman, 2006). bermudagrass includes several taxa of the genus cynodon (l.) rich. but the two species that represent the genetic pool from which the present cultivars descend are cynodon dactylon (l.) pers. var. dactylon and cynodon transvaalensis (burtt-davy) also known as african bermudagrass (taliaferro, 2003). c. transvaalensis is morphologically distinct from c. dactylon due to narrow erect pale leaves producing a fine textured turf with a yellowish-green colour (de wet and harlan, 1970; taliaferro, 1992). c. dactylon can be found as far north as 53° n latitude and from sea level to 3000 m altitude (taliaferro, 2003). asexual reproduction has played a role in bermudagrass enhancement as well. remarkable breeding progress has been obtained from inter-specific hybridization and mutation breeding. the inter-specific hybridization of c. dactylon and c. transvaalensis has been extensively used to obtain sterile cultivars for which clonal propagation is necessary due to a lack of viable seeds. among hybrid genotypes, a number of cultivars have been selected for plant size and morphology in response to the lower cutting height adopted over the years on golf greens. the more recently released “ultradwarf” cultivars have become routinely adopted thanks to an improved density, a slower vertical leaf extension and an increased dominance of stoloniferous growth relative to rhizomes at low mowing heights (beard and sifers, 1996). parameters used to evaluate cynodon turf typically include turfgrass quality, colour, percent spring green-up, establishment rates, leaf texture, and density. as the turfgrass industry moves towards more sustainable management practices, the types of parameters potentially related to better stress tolerance are increasingly important (baldwin and liu, 2013). one of the most important parameters is cold tolerance and rapidity of recovery from winter dormancy in the spring (anderson et al., 2007; patton et al., 2008). lowtemperature tolerance depends on a combination of sevbermudagrass adaptation in the mediterranean climate: phenotypic traits of 44 accessions s. magni*, m. gaetani*(1), n. grossi*, l. caturegli*, s. la bella**, c. leto**, g. virga**, t. tuttolomondo**, f. lulli***, m. volterrani* * dipartimento di scienze, agrarie, alimentari e agro-ambientali, università di pisa, via del borghetto, 80, 56127 pisa, italy. ** dipartimento di scienze agrarie e forestali, università di palermo, viale delle scienze, 208, 90128 palermo, italy. *** turf europe r&d, pisa, italy. key words: colour, cynodon dactylon, green-up, node density, quality, shoot density. abstract: the use of bermudagrass in the mediterranean area is increasing for its outstanding tolerance to heat and drought, and its aggressive growth and high recuperative potential make it particularly suited to heavily worn areas and appreciated for sports turfs. however, the overall performance of a given genetic type can be affected by the adaptation to a specific environment. the objective of this research was to determine the variability of a number of phenotypic traits that can affect bermudagrass turf performance on a wide range of bermudagrass accessions grown in two locations in italy. in may 2010, 44 accessions of bermudagrasses, grouped in “wild”, “improved” “hybrid” and “dwarf” types were transplanted in the center of field plots in pisa and palermo. in 2011, when the turf was completely established, the following traits were determined: shoot density, horizontal stem density, node density, leaf width, colour, quality, spring green-up, and fall colour retention. dwarf and hybrid types yielded the best aesthetic characteristics. with respect to colour retention and spring green-up, great variability was recorded within the groups. dwarf types presented the earliest dormancy, while the hybrid types were in general the ones to green-up first in spring. adv. hort. sci., 2014 28(1): 29-34 1 corresponding author: monica.gaetani@unipi.it received for publication 14 april 2014 accepted for publication 19 may 2014 30 eral factors, including environmental conditions, cultural practices, and especially genetic factors (blum, 1988; anderson and taliaferro, 2002). bermudagrass survives the dormancy period using its reserves of nonstructural carbohydrates and nitrogen compounds accumulated during the previous growing season in storage organs such as stolons and rhizomes (macolino et al., 2010; volterrani et al., 2012; giolo et al., 2013; pompeiano et al., 2013). in the last two decades several southern european universities have developed research programs to study warm-season turfgrass species, including bermudagrass (volterrani et al., 2008; lulli et al., 2011; lulli et al., 2012; nikolopoulou et al., 2012; agati et al., 2013; gómez de barreda et al., 2013), and in particular their adaptability to the mediterranean environment (volterrani and magni, 2004). the aim of our research was to determine the variability of a number of phenotypic traits and aesthetical characteristics that can affect bermudagrass turf performance in a wide range of bermudagrass accessions grown in two locations in italy. this information can provide further insight into bermudagrass adaptability in the mediterranean climate. 2. materials and methods plant material with the objective of expanding morphological diversity of the plant material, 44 accessions of bermudagrass [cynodon (l.) rich.], representative of both wild populations and cultivars, were included in the present study. group one included 13 entries that were called “wild types”, naturally occurring populations of c. dactylon (l.) pers. collected from contrasting environments supposed to generate a selective pressure. collection sites were located in italy (certes-1= warm temperate, salt affected soil; certes-2= warm temperate, fertile soil; certes-3= warm temperate, polluted soil; certes-13= warm temperate, fertile soil), france (certes-4= cool humid, fertile soil), greece (certes-5, -6 and -7= warm temperate, salt affected soils), croatia (certes-8= warm temperate, salt affected soil), argentina (certes-9= warm temperate, salt affected pastureland), united arab emirates (certes-10 and -11= warm arid, desert sand), and maldives (certes-12= tropical humid, salt affected soil). group two included 13 entries that were called “improved types”. these were experimental or commercial vegetative and seeded improved c. dactylon cultivars. group 3 included 11 entries called “hybrid types”, commercial or experimental inter-specific hybrids (cynodon dactylon x transvaalensis burtt.-davy) of which those labelled tifwere kindly provided by dr. w. hanna (university of georgia, usa). group four included seven entries called “dwarf types”, commercial interspecific dwarf and ultradwarf hybrid cultivars and two cynodon transvaalensis burtt.-davy accessions, one a commercial cultivar (uganda) and the other (roma) a line of african bermudagrass that was collected in a turf nursery in rome (italy) where the species was first introduced presumably as a weed. african bermudagrasses were included in the “dwarf types” due to their similarity in leaf texture, density and growth habit with the well-known hybrid dwarf bermudagrasses. 2010 turf establishment on 15 april 2010 at the university of pisa, italy, all the accessions were propagated in the greenhouse (24±5°c) in peat-filled honeycomb seed trays (7 cm2 area and 25 cm3 volume each cell). vegetatively propagated genotypes were planted as single stolon and seeded cultivars were seeded as single seed. on 13 may 2010 plants in the greenhouse were fertilized (30 kg ha-1 n, 10 kg ha-1 p, and 10 kg ha-1 k) using a soluble fertilizer (grow more inc., gardena, ca, usa). on 31 may 2010, plants were mown to 5 cm and transplanted into field plots in two locations in italy: the research station of the university of pisa (43°40’n, 10°19’e, 6 m a.s.l.) and the research station of the university of palermo (38°06’n, 13°20’e, 50 m a.s.l.). experimental plots were 1.5 by 1.5 m with 0.5 m bare soil pathways arranged in a randomized complete-block design with four replications. one plant of bermudagrass was transplanted in the centre of each plot. soil type at pisa was silt-loam (28% sand, 55% silt and 17% clay) with a ph of 7.8 and 18 g kg-1 organic matter while at palermo soil type was sandy clay loam (54% sand, 23% silt and 23% clay) with a ph of 7.6 and 14 g kg-1 organic matter. irrigation was applied as needed to encourage establishment. plots received 50 kg ha-1 n, 10 kg ha-1 p, and 40 kg ha-1 k per month from june to september 2010. in order to minimize weed competition, from two years before establishment, the experimental areas were treated twice a year with glyphosate [n-(phosphonomethyl) glycine] at 2.88 kg ha-1 a.i. the day before planting, oxadiazon [5-tert-butyl-3-(2,4-dichloro-5-isopropoxyphenyl)-1,3,4oxadiazol-2(3h)-one] was applied at 3.36 kg ha-1 a.i. plots were not mowed during the year of establishment to avoid genotype x mowing interaction. weeds occurring during the trial period were manually removed, even if it is not a standard cultivation technique, as the accessions could be differently injured by chemical removal. in the trial, no pesticides were applied. encroachment of stolons into adjacent plots was avoided by using toothpicks that redirected the growing tip back toward the plot centre. 2011 in 2011, in the first and second weeks of march at palermo and pisa, respectively, scalping was carried out. from the end of april (the end of green-up) to october 2011, the turf was mowed weekly with a reel mower (john deere 20sr7) at a mowing height of 2.5 cm. the irrigation program was adjusted according to soil temperature and evapotranspiration rate, with supplement irrigations applied as needed to prevent visual wilt of the turf (croce et al., 2004). plots received 50 kg ha-1 n, 20 kg ha-1 p, and 40 kg ha-1 k per month from may to august 2011. 31 weeds were manually removed inside the plots during the trial period as the accessions could be differently injured by chemical treatments. in the trial, no pesticides were applied. to avoid the encroachment of stolons into adjacent plots, the corridors were treated with glyphosate at 2.88 kg ha-1 a.i. every other week. no turf cultivation, or verticutting or phytosanitary treatment was practiced on the plots. monthly mean maximum and minimum temperatures recorded at the two trial sites are reported in table 1. there were 13 days in pisa with air temperatures below 0°c from november 2010 to march 2011; zero days were recorded in palermo for the same period. assessments spring green-up (15 march-15 may 2011) and fall colour retention (15 november 2011-15 january 2012) were estimated and expressed as percentage of green ground cover. in the second week of october at both testing sites a 50 cm2 core sample per plot was collected and the following parameters determined: leaf width (20 fully expanded leaves per plot measured with a precision vernier caliper and data reported in millimeters), shoot density (direct counting with data reported as shoot no cm-2), horizontal stem density (stolons and rhizomes collected after soil washing measured with a ruler and data reported as cm cm-2) and node density (nodes of stolons and rhizomes collected from the core samples counted and reported as no cm-2) (roche and loch, 2005; volterrani et al., 2008; volterrani et al., 2010; pompeiano et al., 2012). at the pisa location two additional parameters were determined: at 30-day intervals throughout the growing season (may-october) colour, with a rating scale of 1=light green and 9=dark green, and quality with a rating scale of 9 = best and 1 = poorest (morris and shearman, 2007; patton et al., 2009 ) were estimated. data were subjected to analysis of variance using costat software (monterey, ca, usa). to test the effects of location, accession and their interaction, a factorial combination was used. significantly different means were separated using fisher’s least significant difference (lsd) at the t-probability level of 0.05. 3. results the interaction of treatments was not statistically significant for any of the parameters recorded in pisa or palermo. for all the parameters, the location and accession mean effects were statistically significant. location effect is reported as average across accessions and accession effect is reported as average across locations. location mean effect for the location effect, shoot density and node density were on average higher in pisa (3.1 shoots cm-2 and 2.4 nodes cm-2 respectively) compared to palermo (0.8 and 1.0) (table 2). also horizontal stem density was higher in pisa (4.2 cm cm-2) while leaf width recorded in pisa was on average less (1.4 mm) compared to palermo (2.4 mm). spring green-up evaluations as average across locations showed green cover percentages (april 14) higher in palermo (81%) with respect to pisa (60%), while fall colour retention (december 17) showed higher green cover percentages in palermo (77%). accession mean effect shoot density all wild type entries, with the exception of certes 12 (2.4 shoot cm-2) which produced a density comparable to hybrid and improved types, had a similar shoot dentable 1 monthly mean air temperatures (°c) during the trial period (2011) at pisa and palermo month air temperature (°c) pisa palermo mean maximum mean minimum mean maximum mean minimum january 10.6 3.5 14.7 9.3 february 11.8 3.3 13.9 9.3 march 13.9 5.2 16.2 10.4 april 19.2 9.0 19.6 13.1 may 23.8 11.8 21.4 15.9 june 26.2 17.0 25.2 20.2 july 27.6 18.3 28.6 22.9 august 30.2 18.5 29.0 23.0 september 26.7 16.0 26.9 21.0 october 21.8 10.5 22.6 16.5 november 17.5 6.6 20.2 14.5 december 13.3 4.7 16.9 12.6 table 2 bermudagrass [cynodon (l.) rich.] shoot density, horizontal stem density, node density, leaf width. location effect averaged across accessions shoot density (n° cm-2) horizontal stem density (cm cm-2) node density (n° cm-2) leaf width (mm) spring green-up (%) fall colour retention (%) pisa 3.1 4.2 2.4 1.4 60 33 palermo 0.8 1.9 1.0 2.4 81 77 means are significantly different at the 0.05 level of probability as determined by fisher’s protected lsd. 32 sity with values ranging from 0.5 to 1.0 shoots cm-2 (table 3). the most dense improved type was wintergreen (2.3 shoots cm-2), hybrid type values ranged from 1.7 shoots cm-2 (patriot) to 3.8 shoots cm-2 (tif 00-1), while the most dense dwarf type was miniverde with 5.1 shoots cm-2. horizontal stem density the highest value was recorded for miniverde with 5.5 cm cm-2 while certes 12 had a slightly lower value (5.1 cm cm-2) (table 3). the variability within the different groups was high for this parameter with values ranging from 1.5 to 5.1 cm cm-2 (respectively for certes 1 and certes 3 versus certes 12) for the wild types, from 2.1 to 4.9 cm cm-2 (respectively for scotts r6la and yukon versus bull’s eye) for the improved types, from 2.7 to 4.3 cm cm-2 (for tif 00-18 compared to santa ana and tif 00-1) for the hybrid types, and from 1.6 to 5.1 cm cm-2 (respectively for tifdwarf and miniverde) for the dwarf types. node density certes 3 and miniverde were the entries with the lowest and the highest node density with 0.6 and 5.5 nodes cm-2, respectively (table 3). the variability within the groups was high for this parameter, with the exception of the hybrid types that had values ranging from 1.6 to 2.5 nodes cm-2 (respectively for tif 00-18 and santa ana). leaf width coarser leaves were found in the wild types with values ranging from 2.1 mm (certes 10) to 3.0 mm (certes 5, 6, 7) with certes 12 (1.1 mm) the exception (table 3). improved cd types had a leaf width ranging from 1.6 mm (wintergreen and yukon) to 2.2 mm (scotts r6la and sovereign). for hybrid types values ranged from 1.3 mm (tif 00-10) to 1.9 mm (tif 00-27), while for dwarf types values ranged from 1.0 mm (roma) to 1.5 mm (uganda). spring green-up green cover percentage evaluated in mid-april showed accessions scoring values above 80% and not statistically different from each other in each group (table 4). in more detail, the accession with the best score was certes 5 (93%); the lowest score was found in sovereign (24%). fall colour retention green colour retention evaluated in mid-december showed a great variability within the groups (table 4). the highest value was recorded for tif 00-2, with the lowest for three dwarf types, miniverde, tifdwarf and tifeagle (11%). colour in pisa, the highest and lowest values, barazur (score 8.4) and riviera (score 6.0) respectively, were recorded within the improved type group. in the wild type group, the values ranged from 6.1 (certes 1) to 7.4 (certes 4 and 9) (table 4). with the exception of barazur, the highest values were recorded within the hybrid type group with values ranging from 7.1 (tifsport) to 8.2 (patriot), while table 3 bermudagrass [cynodon (l.) rich.] accessions. shoot density, horizontal stem density, node density and leaf width. accession effect averaged across locations accessions shoot density (n° cm-2) horizontal stem density (cm cm-2) node density (n° cm-2) leaf width (mm) wild types (cd) certes-1 0.8 1.5 0.7 2.8 certes-2 0.7 1.7 0.8 2.8 certes-3 0.9 1.5 0.6 2.8 certes-4 0.8 2.9 1.8 2.5 certes-5 0.9 1.9 0.9 3.0 certes-6 1.0 3.1 1.7 3.0 certes-7 0.6 2.1 0.9 3.0 certes-8 0.5 1.8 0.8 2.9 certes-9 0.9 2.3 0.9 2.8 certes-10 0.9 2.1 0.9 2.1 certes-11 1.0 2.5 1.1 2.7 certes-12 2.4 5.1 3.0 1.1 certes-13 0.7 1.9 1.1 2.8 improved types (cd) argentina 1.0 4.0 1.8 2.1 barazur 2.0 3.1 2.1 1.7 bull’s eye 1.9 4.9 2.4 1.9 celebration 1.3 2.5 1.3 1.9 grand prix 2.2 2.9 1.6 1.8 princess 77 2.1 3.6 1.9 1.8 riviera 1.1 2.3 0.9 1.9 scotts r6la 1.3 2.1 1.0 2.2 sovereign 0.9 3.3 1.4 2.2 sr 9554 1.3 2.2 0.7 2.0 veracruz 1.3 3.4 1.6 1.8 wintergreen 2.3 2.9 1.5 1.6 yukon 1.2 2.1 1.1 1.6 hybrid types (cdxt) patriot 1.7 3.9 2.0 1.8 santa ana 3.1 4.3 2.5 1.4 tifsport 2.3 3.4 1.7 1.8 tifway 2.6 3.8 2.0 1.4 tif 00-1 3.8 4.3 2.3 1.5 tif 00-2 3.2 3.3 2.1 1.5 tif 00-7 3.2 3.2 2.2 1.5 tif 00-10 3.5 3.7 2.0 1.3 tif 00-18 2.7 2.7 1.6 1.4 tif 00-24 2.0 3.7 1.9 1.4 tif 00-27 2.3 3.6 2.1 1.9 dwarf types (cdxt/ct) champion 2.2 4.0 2.0 1.2 miniverde 5.1 5.5 5.5 1.3 tifdwarf 2.6 1.6 1.0 1.3 tifeagle 3.1 4.4 4.2 1.1 tifgreen 2.7 1.9 1.5 1.2 roma 4.1 3.7 2.2 1.0 uganda 3.0 4.7 2.6 1.5 lsd 0.05 0.7 1.6 1.2 0.3 33 for the dwarf types the values ranged from 7.0 (tifgreen) to 7.9 (miniverde). quality in pisa, the highest quality was recorded for the dwarf type miniverde with a score of 8.4, however no significant differences were recorded within this group (table 4). wild types ranged in quality from 4.3 (certes 1) to 7.0 (certes 12), while improved types ranged from 5.8 (riviera) to 7.7 (barazur and bull’s eye); the variability of ratings within both these groups is worthy of note. the hybrid types scored from 6.8 (tif 00-7) to 8.1 (patriot and tifway). 4. discussion and conclusions the study carried out on a pool of genetically and morphologically different entries belonging to the genus cynodon has highlighted a wide variability of aesthetic and morphological traits. morphological characteristics such as shoot density, node density, and horizontal stem density highlighted the better quality of the majority of dwarf and hybrid type cultivars, with improved types showing performances similar to those of wild types; certes 12 was the exception. the genetic differences among groups are reflected more clearly with regard to leaf width, with values getting lower going from wild to dwarf types, with the exception of certes 12. recovery from winter dormancy in the spring, expressed as spring green up, showed a great variability within and among the groups. this parameter is associated with carbohydrate reserves accumulated in storage organs as observed by macolino et al. (2010). other studies (volterrani et al., 2012) focused on carbohydrates in stolons in the first year of establishment and the relationship with growth and establishment rate. cold tolerance, expressed as fall colour retention, highlighted the better performances of the hybrid types with the dwarf types being the first cultivars in which dormancy begins. the parameters representing turf aesthetic quality (colour and turf quality), although they indicate a great variability within groups, showed improving mean values from wild to dwarf types and confirmed what patton et al. (2009) observed concerning the differences between improved and hybrid types. acknowledgements the authors wish to acknowledge dr. wayne hanna, the university of georgia, for providing experimental clones of hybrid bermudagrass. this trial was carried out within the project “sistemi avanzati per la produzione vivaistica di tappeti erbosi di specie macroterme ad uso multifunzionale a basso consutable 4 bermudagrass [cynodon (l.) rich.] accessions. spring greenup (april 14 2011) and fall colour retention (percentage of green colour) (17 december 2011). accession effect averaged across locations. colour (visual estimation based on a 1-9 scale) and quality (visual estimation based on a 1-9 scale) refer only to the pisa location (mean values may-october 2011) accessions spring green-up (%) fall colour retention (%) colour (1-9) quality (1-9) wild types (cd) certes-1 55 16 6.1 4.3 certes-2 75 32 7.0 5.7 certes-3 66 38 6.3 5.8 certes-4 65 38 7.4 6.8 certes-5 93 76 6.4 6.0 certes-6 73 49 7.2 6.4 certes-7 74 37 7.1 6.2 certes-8 59 33 6.6 6.2 certes-9 85 63 7.4 6.7 certes-10 51 53 6.7 5.9 certes-11 72 68 6.2 5.6 certes-12 79 54 6.8 7.0 certes-13 60 41 6.7 5.9 improved types (cd) argentina 66 45 6.4 6.1 barazur 55 43 8.4 7.7 bull’s eye 76 59 7.9 7.7 celebration 49 59 7.5 6.4 grand prix 77 78 6.8 6.9 princess 77 72 73 6.9 6.9 riviera 57 50 6.0 5.8 scotts r6la 73 57 6.9 6.4 sovereign 24 56 6.4 6.3 sr 9554 67 56 6.8 6.5 veracruz 71 61 6.8 6.9 wintergreen 83 69 7.1 6.5 yukon 70 35 7.3 7.1 hybrid types (cdxt) patriot 75 33 8.2 8.1 santa ana 77 73 7.4 7.9 tifsport 79 68 7.1 7.2 tifway 87 83 7.9 8.1 tif 00-1 86 77 7.9 7.5 tif 00-2 89 86 7.2 7.6 tif 00-7 62 75 7.9 6.8 tif 00-10 82 81 8.1 7.8 tif 00-18 84 80 8.0 8.0 tif 00-24 74 79 7.7 7.0 tif 00-27 87 81 7.4 7.6 dwarf types (cdxt/ct) champion 86 76 7.7 8.0 miniverde 53 11 7.9 8.4 tifdwarf 63 11 7.8 7.9 tifeagle 52 11 7.4 8.1 tifgreen 72 31 7.0 7.9 roma 75 56 7.4 8.0 uganda 84 78 7.8 7.9 lsd 0.05 17 7 0.5 0.7 34 mo idrico ed 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breeding for stress environments. crc press, boca raton, florida, usa, pp. 114-132. croce p., de luca a., mocioni m., volterrani m., beard j.b., 2004 adaptability of warmseason turfgrass species and cultivars in a mediterranean climate. acta horticulturae, 61: 365-368. de wet j.m., harlan j.r., 1970 biosystematics of cynodon l.c. rich (gramineae). taxon, 19: 565-569. giolo s., macolino m., barolo e., rimi f., 2013 stolons reserves and spring green-up of seeded bermudagrass cultivars in a transition zone environment. hortscience, 48(6): 780-784. gómez de barreda d., reed t.v., yu j., mccullough p.e., 2013 spring establishment of four warm-season turfgrasses after fall indaziflam applications. weed techn., 27(3): 448-453. lulli f., guglielminetti l., grossi n., armeni r., stefanini s., volterrani m., 2011 physiological and morphological factors influencing leaf, rhizome and stolon tensile strength in c4 turfgrass species. functional plant biology, 38: 919-926. lulli f., volterrani m., grossi n., armeni r., stefanini s., guglielminetti l., 2012 physiological and morphological factors influencing wear resistance and recovery in c3 and c4 turfgrass species. functional plant biology, 39: 214-221. macolino s., serena m., leinauer b., ziliotto u., 2010 preliminary finding on the correlation between water-soluble carbohydrate content in stolons and first year green-up of seeded bermudagrass cultivars. horttechnology, 20: 758-763. morris k.n., shearman r.c., 2007 ntep turfgrass evaluation guidelines (last update: march 2014). national turfgrass evaluation program. nikolopoulou a.e., nektarios p.a., aivalakis g., volterrani m., chronopoulos i., 2012 effects of rootzone co 2 and o 2 levels on seed germination and stolon growth of cynodon dactylon. acta agriculturae scandinavica, 62(1): 53-61. patton a., richardson m., karcher d., trappe j., 2009 2007 ntep bermudagrass trial-year 3 results. arkansas turfgrass report 2009. ark. ag. exp. stn. res. ser., 579: 20-24. patton a.i., 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green section record, 30(4): 10-12. taliaferro c.m., 2003 bermudagrass [cynodon (l.) rich.], pp. 235-256. in: casler m.d., and duncan r.r. (eds.) turfgrass biology, genetics, and breeding. wiley & sons, hoboken, new jersey, usa, pp. 384. volterrani m., grossi n., gaetani m., pompeiano a., 2010 zoysiagrass cultivar establishment rate and turf quality in central italy. acta horticulturae, 881: 313-316. volterrani m., grossi n., lulli f., gaetani m., 2008 establishment of warm season turfgrass species by transplant of single potted plants. acta horticulturae, 783: 77-84. volterrani m., magni s., 2004 species and growing media for sports turfs in mediterranean area. acta horticulturae, 661: 359-364. volterrani m., pompeiano a., magni s., guglielminetti l., 2012 carbohydrate content, characterization and localization in bermudagrass stolons during establishment. acta agriculturae scandinavica, 62(1): 62-69. 20 1. introduction the genus festuca includes more than 360 species that differ widely in appearance. less than ten species are used as turf, all in cool climates. festuca arundinacea schreb., tall fescue, is a deep-rooted, cool-season perennial grass. it shows vigorous growth in spring and autumn, and its extensive root system helps it to withstand drought conditions. the species is adapted to a wide range of soil and climatic conditions, but performs best where winter is rather mild. its requirement for relatively high mowing times limits its use as lawns in parks, golf course roughs and other areas mowed at 40 mm or more (wiecko, 2006). zeolites are crystalline, hydrated alumino silicates of alkali and earth metals that possess infinite, threedimensional crystal structures. they are further characterized by an ability to lose and gain water reversibly and exchange some of their constituent elements without major changes in structure. nearly 50 natural species of zeoiltes have been recognized and more than 100 species without natural counterparts have been synthesized in the laboratory (breck, 1974; meier and olsen, 1987; mumpton, 1999).the great effectiveness of zeolites as natural sources of trace elements supplementing npk and their high adsorption ability have been reported (kolyagin and kucherenko, 2003). therefore, natural zeolites, due to their structure and properties (inert and non-toxic), can be used as slow-release carriers of fertilizers (ramesh et al., 2011). a 10% addition of clinoptilolite to sand used in the construction of golf-course greens substantially reduced no 3 -leaching and increased fertilizer-n uptake by creeping bent-grass without disturbing the drainage, compaction, or playability of greens (ferguson et al., 1986; nus and brauen, 1991; hung, 1992). the addition of nh 4 + exchanged clinoptilolite in greenhouse experiments resulted in 59 and 53% increase in root weight of radishes in medium with light clay soil (lewis et al., 1984). the water efficiency of surface irrigation systems in iran is low and nitrogenous fertilizers should be added to soil at a higher level. therefore leaching of nitrogen fertilizers occurs resulting in underground water pollution particularly in soils with light texture. clinoptilolite, a naturally occurring zeolite with high exchange capacity, may be used to absorb ammonium and retard excess leaching of nitrate. these facts dictate improving water and fertilization management to decrease the pollution of underground water resources (abdi et al., 2004). the object of the present study was to investigate the effects on tall fescue growth of applying different amounts of natural zeolite and nitrogen to growth medium. 2. materials and methods a greenhouse pot trial was conducted in 2009 at the faculty of agriculture, shiraz university, shiraz, iran, at badjgah, 1810 m above mean sea level, 29° 36´n and 52° growth of tall fescue (festuca arundinacea schreb.) seedlings sown in soil mixed with nitrogen and natural zeolite s. eshghi (1), m. bahadoran, h. salehi department of horticultural science, college of agriculture, shiraz university, shiraz, iran. key words: clipping, soil mixture, vegetative variables. abstract: to determine the interaction of nitrogen and natural zeolite in culture medium on the vegetative growth of tall fescue (festuca arundinacea schreb.) ‘starlet’ a greenhouse experiment was conducted. a complete randomized design with factorial arrangements including two factors (nitrogen and zeolite) was employed for each treatment with four replications. treatments of nitrogen were 0, 0.06 and 0.12 g kg-1 in the soil mixture and treatments of zeolite were 0, 10, 20 and 30 g kg-1 in the soil mixture. application of zeolite and nitrogen had different effects on seedling height, fresh and dry weights of clippings before first, second and third mowings, chlorophyll and nitrogen content of clippings, and dry weights of roots. adding zeolite at the rate of 30 g kg-1 and nitrogen at the rate of 0.12 g kg-1 to culture medium significantly increased the height of turf seedlings. it is concluded that zeolite could absorb and slowly release nitrogen to the culture medium. adv. hort. sci., 2014 28(1): 20-24 (1) corresponding author: eshghi@shirazu.ac.ir received for publication 4 january 2014 accepted for publication 21 march 2014 21 32´e on tall fescue (festuca arundinacea schreb.) ‘starlet’ to evaluate n uptake from soil mixture with different amounts of zeolite. the soil samples were collected for soil nutrition analysis before the seeds were planted (table 1). seeds of tall fescue cultivar of starlet were planted in 3 kg pots with soil mixture (1:1 v/v field soil and sand). the soil mixture of pots contained 0, 10, 20 and 30 g kg-1 zeolite and 0, 0.06 and 0.12 g kg-1 nitrogen (in the form of urea) in different treatments (4 levels of zeolite × 3 levels of nitrogen × 4 replication= 48 pots). plants were maintained in a greenhouse under natural light (>850 µ mol m-2 s-1) with diurnal temperature 26±3°c and nocturnal temperature 20±3°c, and rh of 56±4%. turf grasses were irrigated every four days in spring and every two days in summer. one month after seed germination the first mowing was carried out; second and third mowings occurred 2 and 3 months after germination of seeds, respectively. seedling height before first, second and third mowings, fresh and dry clipping weights after each mowing, chlorophyll and nitrogen content of clipping, and dry weights of roots were measured. seedling heights were measured. chlorophyll content was determined by spectrophotometric method (saini and buvalda, 1998). the total amount of nitrogen (n) was measured using the kjeldahl digestion method. clippings and roots were weighed for fresh weight and then oven dried at 70oc (karl kolb 112sl) for 48 h and weighed for dry weight. this research was carried out in a complete randomized design with factorial arrangements including two factors (nitrogen and zeolite) for each treatment with four replications from april to august 2009. data were analyzed by mstatc software and mean values were compared using the lsd test at 5% level. 3. results seedling height before first, second, and third mowings (cm) increasing the content of zeolite in the culture medium to 0.12 g kg-1nitrogen, seedling height increased before all mowing times. the effect of zeolite alone on seedling height had shown increase, this increase was significant at 30 g kg-1 zeolite in soil mixture before first and second times of mowing. increasing the nitrogen concentration in the soil mixture had no regular and significant effect on seedling height before the three mowing times when compared to untreated control (table 2). fresh and dry clipping weights after first, second, and third mowings (g) as indicated in figures 1, 2, and 3, an increase in zeolite and nitrogen together in the soil mixture let to greater fresh table 1 some physical, chemical and nutritional characteristics of the soil used in the study cec ph n % p (mg kg-1) k (mg kg-1) oc (%) s.p. clay (%) silt (%) sand (%) 1.3 7.53 0.26 27.5 400 2.72 26 12 34 54 table 2 effect of nitrogen and zeolite on seedling height before first, second and third mowings of festuca arundinaceae schreb ‘starlet’ seedling height before first mowing (cm) means of zeolite seedling height before second mowing (cm) means of zeolite seedling height before third mowing (cm) means of zeolitezeolite g·kg-1 soil mixture zeolite g·kg-1 soil mixture zeolite g·kg-1 soil mixture 0 0.06 0.12 0 0.06 0.12 0 0.06 0.12 nitrogen g kg-1 in soil mixture 0 20.50 ab* 18.25 abc 13.75 cd 17.50 b 17.50 bcd 18.00 bcd 13.50 d 16.33 b 15.25 a 13.50 abc 11.25 bc 13.33 ab 10 22.50 ab 13.25 d 22.75 ab 19.50 ab 19.00 abc 13.50 d 18.75 abcd 17.08 b 14.25 abc 11.00 c 13.75 abc 13.00 b 20 16.50 bcd 20.0 abcd 22.00 ab 19.50 ab 16.00 cd 17.75 bd 22.00 ab 18.58 b 13.50 abc 14.00 abc 13.50 abc 13.67 ab 30 21.50 ab 23.00 ab 24.25 a 22.92 a 20.25 abc 22.00 ab 24.00 a 22.08 a 15.00 ab 15.25 a 15.50 a 15.25 a mean of nitrogen 20.25 a 18.63 a 20.69 a 18.19 a 17.81 a 19.56 a 14.50 a 13.44 a 13.50 a * in each column and row means followed by the same letter(s) (small letters for means and capital letters for means of rows and columns) are not significantly different using lsd test at 5% level. fig. 1 effect of nitrogen and zeolite on clipping fresh weight after first mowing (g) of festuca arundinaceae schreb‘starlet’.. 22 and dry weights of the clippings from the first, second, and third mowings. the means of zeolite had shown increase with increasing in zeolite content of soil mixture and this increase was significant only at 30 g kg-1 zeolite in soil mixture at fresh weight of clipping after all times of mowings. also 30 g kg-1 zeolite in soil mixture had shown significant increase at dry weight of clipping after first and second times of mowings. the mean nitrogen content in the soil mixture had no significant effect on the fresh and dry weights of clippings for all mowing times (figs. 1, 2, 3, 4, 5 and 6). chlorophyll and nitrogen content of clippings with increasing nitrogen and zeolite in the soil mixture, the chlorophyll content of clippings decreased in most treatments but it was not significant when compared with control. maximum chlorophyll content of clippings was found at 10 g/kg zeolite with 0.12 g/kg nitrogen in the soil mixture. the nitrogen content of clippings increased when zeolite and nitrogen increased in most treatments but this increase was not significant compared to control. maximum nitrogen content of clippings was noted at 0 g/ kg of zeolite and 0.12 g/kg of nitrogen in the soil mixture (table 3). dry weight of roots results of this study indicate that by increasing the zeolite content alone in culture medium, the mean dry weight of roots (compared with control) increased but not significantly. an increase in the culture medium of nitrogen alone did not significantly effect the mean of root dry weight; with increasing nitrogen only, dry weight of roots decreased. however, adding nitrogen and zeolite together to the culture medium increased the dry weight of roots (fig. 7). fig. 2 effect of nitrogen and zeolite on clipping dry weight after first mowing (g) of festuca arundinaceae schreb‘starlet’. fig. 3 effect of nitrogen and zeolite on clipping fresh weight after second mowing (g) of festuca arundinaceae schreb‘starlet’. fig. 4 effect of nitrogen and zeolite on clipping dry weight after second mowing (g) of festuca arundinaceae schreb‘starlet’. fig. 5 effect of nitrogen and zeolite on clipping fresh weight after third mowing (g) of festuca arundinaceae schreb‘starlet’. fig. 6 effect of nitrogen and zeolite on clipping dry weight after third mowing (g) of festuca arundinaceae schreb‘starlet’ 23 4. discussion and conclusions application of zeolite and nitrogen to the culture medium increased the seedling height before all mowing times. zeolite absorbs available n (nh 4 +) and releases it gradually to the medium for use by plants. results of our study indicate that nitrogen alone did not increase seedling height but combined treatment with zeolite and nitrogen increased seedling height at most levels. the findings of the present study are in agreement with those of hung and petrovic (1995) who reported that the application of zeolite improved nitrogen efficiency in soil about 16 to 22%. furthermore, zeolite reduced the leaching of ammonium and nitrate 86 to 99% from the soil. zeolite, with a high cation exchange capacity (cec), causes easy storage and release of nitrogen. omar et al. (2011) reported that the application of peat soil water and zeolite with urea significantly increased dry matter, n, p, k uptake and use efficiency in maize plants compared with urea without additives. our results are in accordance with those of sepaskhah and barzegar (2010) who showed that nitrogen and zeolite application resulted in higher grain protein contents in rice and nitrogen recovery efficiency. also aghaalikhani et al., (2011) reported that amending soil with zeolite, reduced nitrogen leaching, increased canola yield and nitrogen useefficiency by avoiding nitrogen leaching and improving soil physical properties so it may be a beneficial approach to decrease chemical fertilizer application rates and develop sustainable agriculture. in the present study, where zeolite and nitrogen were applied to the soil mixture, the chlorophyll content of clippings decreased while the nitrogen content increased. perhaps increased nitrogen resulted in a decrease of some elements and the amount of chlorophyll. our results are in disagreement with abdi et al. (2004) and nazari et al. (2007) who reported that zeolite increases the chlorophyll content of strawberry and african marigold. since the application of zeolite and nitrogen, compared to nitrogen application alone, increased the fresh and dry weights of clippings, it can be concluded that natural zeolite with its high exchange capacity may absorb ammonium and release it slowly. these results are in agreement with abdi et al. (2004) who reported that zeolite application increased shoot dry weight in strawberry. therefore, zeolite balances the amount of nitrogen availability for plants and retards excess leaching of nitrate. in conclusion, natural zeolite as a slow-release compound may be recommended along with nitrogen for use in growth media to improve growth of tall fescue. acknowledgements authors wish to thank mr. pourkhaloie, mr.jozi, miss kakoie and mr. zadnour for their help with the experiment. references abdi g.h., khosh-khui m., eshghi s., 2004 effects of natural zeoilte on growth and flowering of strawberry (fragaria × ananassa duch.). intern. j. agric. res., 1: 384-389. aghaalikhani m., gholamhoseini m., dolatabadian a., khodaei-joghan a., sadat asilan k., 2011 zeolite influences on nitrate leaching, nitrogen-use efficiency, yield and yield components of canola in sandy soil. archives agron. soil sci. doi., 10.1080/03650340.2011.572876. fig. 7 effect of nitrogen and zeolite on clipping dry weight of roots (g) of festuca arundinaceae schreb‘starlet table 3 effect of nitrogen and zeolite on chlorophyll and nitrogen content of clippings of festuca arundinaceae schreb ‘starlet’ chlorophyll content of clipping means of zeolite nitrogen content of clipping means of zeolitezeolite g·kg -1 soil mixture zeolite g·kg-1 soil mixture 0 0.06 0.12 0 0.06 0.12 nitrogen g kg-1 in soil mixture 0 20.40 a* 17.47 a 19.60 a 19.16 a 01.12 ab 01.22 ab 03.36 a 01.90 a 10 13.85 a 14.78 a 20.86 a 16.50 a 01.49 ab 01.37 ab 01.45 ab 01.43 a 20 19.98 a 15.04 a 17.25 a 17.42 a 01.01 b 01.40 ab 00.925 b 01.11 a 30 14.89 a 16.55 a 13.22 a 14.89 a 01.12 ab 01.41 ab 01.79 ab 01.44 a means of nitrogen 17.28 a 15.96 a 17.73 a 01.18 a 01.35 a 01.88 a * in each column and row means followed by the same letter(s) (small letters for means and capital letters for means of rows and columns) are not significantly different using lsd test at 5% level. 24 breck d.w., 1974 zeolite molecular sieves: structure, chemistry and use. wiley j., and sons inc., london, uk, pp. 771. ferguson g.a., ppper i.a., kneebone w.r., 1986 growth of creeping bentgrass on a new medium for turfgrass growth: clinoptilolite zeolite-amended sand. agron. j., 78: 1095-1098. hung z.t., 1992 clinoptilolite zeolite as amendment of sand for golf green root zones. ph.d. thesis, cornell univ., ithaca, ny, usa. hung z.t., petrovic a.m., 1995 clinoptilolite zeolite effect on evaporation rate and shoot growth rate of bentgrass on sand base grass. j. turfgrass manag., 25: 35-39. kolyagin y.s., kucherenko s.p., 2003 yield and longterm effect fertilizers. sakharnaya-svekla., 3:17-18. lewis m.d., moore f.d., goldsberry k.l., 1984 clinoptilolite a fertilizer n exchanger. hortscience, 18: 235-239. meier w.m., olsen d.h., 1987 atlas of zeolite structure types. butterworths, london, uk, 152. mupton f.a., 1999 la roca majica: use of natural zeolites in agriculture and industry. proc. natl. acad. sci. usa, 96: 3463-3470. nazari f., khosh-khui m., eshghi s., salehi h., 2007the effect of natural zeolite on vegetative growth, flower and physiological characteristics of african marigold (tagets erecta l. “queen”. hort. environ. biotechnol., 48(8): 241-245. nus j.l., brauen s.e., 1991 clinoptilolitic zeolite as an amendement for establishment of creeping bentgrass on sandy media. hortscience, 26: 117-119. omar l., ahmed o.h., majid n.m.a., 2011 enhancing nutrient use efficiency of maize (zea mays l.) from mixing urea with zeolite and peat soil water. intern. j. physic. sci., 6(14): 3330-3335. ramesh k., biswas a.k., somasundaram j., rao a.s., 2011 nanoporous zeolites in farming: current status and issues ahead. curr. sci., 99(6): 760-764. saini g.s., buvalda j.b., 1998 kiwifruit, pp. 135-156. in: schaffer b., and p.c. anderson (eds.) handbook of environmental physiology of fruit crops: temperate crops. crc press. inc. boca raton, florida, usa, pp. 112. sepaskhah a.r., barzegar m., 2010 -yield, water and nitrogen-use response of rice to zeolite and nitrogen fertilization in a semi-arid environment. agric. water manage., 98: 38-44. wiecko g., 2006 fundamentals of tropical turf management. biddles ltd, king’s lynn, uk, pp. 205. impaginato 31 1. introduction apple (malus domestica borkh) is one of the most important horticultural crops considered as the third major fruit in the world (garming, 2014). the main proportion (½ million tons) of apple production in iran is used for fresh consumption (iranian ministry of agriculture, 2016), while some problems such as tissue softening during storage, tissue browning due to physical damages at post-harvest handling, high water loss and some physiological disorders such as bitter pit, water core, scald and internal browning are the most dominant post-harvest restriction factors ( e s n a a s h a r i a n d z o k a e e k h o s r o s h a h i , 2 0 1 1 ) . generally, quality apple must be mature, firm, crispy and juicy with a good flavor composition and free from mechanical damage, physiological disorders, and pathological diseases (baldwin, 2002). however, reducing consumer acceptance or nutritional value usually happens in the period after harvesting. apples contain several health-promoting compounds functioning as antioxidants, or modulators of enzyme activity. peel of red apples contains higher antioxidant than their flesh. meanwhile, drougoudi et al. (2008) discovered a positive correlation between phenolic content and antioxidant capacity in both flesh and peel of apple. color is also another important factor regarding fruit evaluation. there is a close correlation between color and overall quality of fruits (ritenour and khemira, 2007). salicylic acid (sa) is a natural compound that functions as plant growth regulator. sa carries a high potential of controlling post-harvest losses of horticultural crops. it has been discovered that the sa is associated with a delay in fruit ripening, (srivastava and dwivedi, 2000; zhang et al., 2003; mo et al., 2008), induction of disease resistance (shafiee et al., 2010), increasing antioxidant and phenolic compounds (peng and jiang, 2006; geransayeh et al., 2 0 1 5 ) , m a i n t e n a n c e o f p o s t h a r v e s t q u a l i t y (srivastava and dwivedi, 2000; zhang et al., 2003; wang et al., 2006; mo et al., 2008; harindra et al., adv. hort. sci., 2017 31(1): 31-38 doi: 10.13128/ahs-20723 effect of preand post-harvest salicylic acid treatments on quality and antioxidant properties of ‘red delicious’ apples during cold storage m. hadian-deljou, m. esna-ashari*, h. sarikhani department of horticultural sciences, bu-ali sina university, hamedan, iran. key words: anthocyanin, firmness, phenolic compound, soluble solid content, weight loss. abstract: salicylic acid is a natural phenolic compound known as a plant hormone having positive effect on storage life and quality of fruits. this study aimed to investigate the effects of preand post-harvest application of salicylic acid on antioxidant properties and quality of ‘red delicious’ apples during 193 days cold (0±0.5°c) storage. both preand postharvest salicylic acid treatments did not affect soluble solid content, titratable acidity and fruit firmness, with the exception of 1 mm at pre-harvest application for titratable acidity. fruit juice ph was reduced in all fruits at the end of storage, while it was not quite uniform during storage. although there was no significant difference between the concentrations of salicylic acid in terms of fruit weight loss, but the highest amount of weight loss was observed in post-harvest treatments. salicylic acid application increased total phenolics and antioxidant activity at the earlier stages of storage showing the highest capacity with 2 mm followed by 1 and 4 mm salicylic acid concentrations, while 1 mm concentration belonged to the highest antioxidant capacity at the end of storage. anthocyanin content showed a gradual increase during storage until day 60, then decreased right afterwards. the highest amounts of anthocyanin were obtained from the concentrations of 1 and 2 mm salicylic acid in pre-harvest treatments, while 4 mm treatment was not encouraging. overall, salicylic acid treatments could increase apple storage life and quality for a short period of time only. (*) corresponding author: m.esnaashari@basu.ac.ir received for publication 6 december 2016 accepted for publication 7 february 2017 copyright: © 2017 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2017 31(1): 31-38 32 2015) and increasing storage life of horticultural crops. hence, sa is suggested to be utilized in postharvest handling of fruits and vegetables (peng and jiang, 2006) which necessitates further investigation into know-how of its application. wen et al. (2008) found that treatment of grape berries with sa could induce an increase in phenylpropanoid and phenylalanine ammonia-lyase (pal). qin et al. (2003) reported a significant increase in polyphenoloxidase, pal and β-1, 3-glucanase activity in cherries fruit through sa treatment. exogenous application of sa on tomato green mature fruits has shown a delay in b i o s y n t h e s i s o f s o m e b i o c h e m i c a l s i n c l u d i n g carotenoids, lycopene, ascorbic acid, total phenolics, free amino acids and γ-amino butyric acid that led to increase in the keeping quality of fruits (kant et al., 2016). pre-treatment of sa combined with lower storage temperature could provide a useful means of maintaining beneficial antioxidant activity during s t o r a g e o f n a v e l o r a n g e ( h u a n g e t a l . , 2 0 0 8 ) . exogenous application of sa enabled grape leaves to maintain relatively higher activities of antioxidant enzymes under normal temperature, heat, or cold stress. the effect of sa on alleviating chilling injury of peaches during cold storage may be attributed to its ability to induce antioxidant systems (wang et al., 2006). the aim of this study was to maintain apple quality through the application of sa controlling physiological damages as well as investigating content of phenolic compounds, anthocyanin and antioxidant activity of fruits during cold storage. 2. materials and methods plant materials and salicylic acid treatments apples (malus domestica borkh cv. red delicious) w e r e p r o v i d e d f r o m a 2 0 y e a r s o l d o r c h a r d i n horticultural research center, bu-ali sina university, hamedan, iran. this study was conducted as a factorial experiment based on a complete randomized block design with three replications. first stage of experiment launched on september when apples’ green skin had just started to turn reddish. four concentrations of sa including 0 (control), 1, 2 and 4 mm were sprayed on previously-selected trees, branches and fruits. sa concentrations were made by dissolving powdered sa (merck, germany) in hot water. in the second stage of experiment, non-treated fruits were first harvested according to the maturity index (starch test) using cornell starch-iodine chart in late september. starch test is a standard method of determining apple maturity to estimate optimum harvest dates well before picking fruit (blanpide and silsby, 1992). the harvested fruits then dipped in the same concentrations of sa solution for approximately three minutes at room temperature (25±1ºc). previously-treated apples were also harvested at this time. in this experiment, 15 apple fruit were used in each replicate for a totally of 45 fruit for each treatment. two apples per each replicate were used for the measurments of all parameters (except for the weight loss) at any time of determination (totally 6 times). three remaining apples from each replicate were kept to weigh at any time of determination for the evaluation of weight loss. for the packaging of the samples, each group of five apples was packed in a cubic plastic container with two small pores (2.5 mm in diameter) in each side, and stored at 0±0.5°c with 90% relative humidity and kept up to 193 days. measurements of all parameters started at the beginning of the storage and then continued until the day of 90 with 30 days intervals. two other measurements were taken 157 and 193 days of storage. soluble solid content, titratable acidity and juice ph apple juice was first prepared using a domestic electric apple juice maker available in the local shops. soluble solid content (ssc) was determined by measuring refractive index of the juice with a handhold refractometer (n1, atago co., tokyo, japan) at room t e m p e r a t u r e ( 2 5 ± 1 ° c ) a n d t h e r e s u l t s w e r e expressed as °brix. titratable acidity (ta) was measured by titration with a calibrated titrator using a solution of the juice and water (2/10 ratio) with 0.1 n naoh to ph 8.2±0.1 and converted to malic acid percentage. fruit juice ph was determined using an aqualitic digital ph meter (model al10 ph) with a gel electrode. fruit firmness and weight loss firmness values of each individual apple were measured at three points of their equatorial region after which the peel was removed by using a manual p e n e t r o m e t e r ( f d k ; w a g n e r i n s t r u m e n t s , greenwich, ct, usa) with a 2 mm diameter flat probe (zhang et al., 2009). total phenolic content total phenolic content (tpc) of treated fruits were determined through a slightly modified version of folin-ciocalteu’s method as suggested by slinkard and singleton (1977). chlorogenic acid was used as standard phenolic compound. extraction was performed by homogenizing 0.5 g flesh tissue powder in 3 ml of 85% meoh and the extract was filtered with hadian-deljou et al. effect of salicylic acid on quality and antioxidant properties of ‘red delicious’ during cold storage 33 no.1 whatman filter paper. sample extract (300 µl) was mixed with 1500 µl of folin-ciocalteu’s reagent. after 5 min, 1200 µl of sodium carbonate solution (7.0%, w/v) was added, and the mixture vortexed and allowed to stand at room temperature (25±1°c) in the dark for 90 min. the absorbance was read at 765 nm in a uv/vis spectrophotometer (carry 100, varian analytical instruments, walnut creek, ca, usa), and the total phenolic concentration was calculated from a calibration curve, using chlorogenic acid as the standard. results were expressed as mg l-1 chlorogenic acid equivalents. total anthocyanin content for the assessment of anthocyanin contents of the apples, only their peels were used through applying a ph differential protocol (giusti and wrolstad, 2003). the absorbance was measured at 510 and 700 nm with a uv/vis spectrophotometer (carry 100, varian analytical instruments, walnut creek, ca, usa). anthocyanin content was then calculated using the following equation and expressed as mg cyanidin 3-galactoside equivalent per gram of fresh weight. mg cya-3-gal /g fw = [(a 510 a 700 ) ph1 (a 510a 700 ) ph4.5 ]×mw×f×1000/ε×d where: a= absorbance, mw= molecular weight of cyanidin 3-galactoside = 445.2 [g/mol], f= dilution factor = 10, d= cell pathlengths [cm], ε= molar absorbance of cyanidin 3-galactoside = 34300 [l/ mol×cm] and 1000= factor for mg. total antioxidant activity antioxidant activity was estimated using the stable 1,1-diphenyl-2-picryl hydrazyl (dpph) free radical (arnous et al., 2001) with slight modifications. the absorbance was read at t = 0 and t = 30 min with a spectrophotometer (carry 100, varian analytical instruments, walnut creek, ca, usa). for all the above assessments (except the anthocyanin measurement), the apple flesh was used. statistical analysis statistical analysis (analysis of variance) of the data was performed with sas software (version 9.1, 2002-2003, sas institute inc., cary, nc, usa). means were compared with duncan’s multiple range test. differences at p=0.05 were considered as significant. 3. results and discussion soluble solids content, titratable acidity and juice ph soluble solids content increased gradually during storage with no significant differences between the treatments (fig. 1). with pre-harvest application of 1 mm sa, ta slightly increased until the 60th day of storage, when reached its maximum value being significant with the other treatments, but decreased gradually afterwards (fig. 2). fruit juice ph was reduced in all fruits at the end of storage, while it was not quite uniform during storage (table 1). ssc may increase during fruit ripening due to the action of sucrose-phosphate synthase, a key enzyme in sucrose biosynthesis. this enzyme is activated by ethylene and the ripening process itself during storage. because organic acids are substrates of respiration, their levels decrease during ripening. utilization of these compounds over post-harvest period is the main reason of increasing sweetness in originally fig. 1 effect of preand post-harvest salicylic acid treatments on ssc of ‘red delicious’ apples stored at 0±0.5°c for 193 days. comparison of the means was conducted through the duncans’ multiple range test (p<0.05). pr= pre-harvest; po= post-harvest; t0 to t3= control, 1, 2 and 4 mm sa treatment respectively. fig. 2 effect of preand post-harvest salicylic acid treatments on ta of ‘red delicious’ apples stored at 0±0.5°c for 193 days. comparison of the means was conducted through the duncans’ multiple range test (p<0.05). pr= pre-harvest; po= post-harvest; t0 to t3= control, 1, 2 and 4 mm sa treatment respectively. adv. hort. sci., 2017 31(1): 31-38 34 high-sugar, high-acid apples or insipidity and blandness of fruit when sugar and acid concentrations are initially low (jackson, 2003). similar to our founding have been reported by sayyari et al. (2011) in which ssc increased during storage in both control and treated fruits. as previously reported, in climacteric fruits such as apples, starch turns into sucrose during p o st-h arvest sto rage. mo et al . ( 2008) stu d i es showed ssc in treated fruits to be lower than the control, suggesting that sa slowed starch degradat i o n . t h i s c o u l d b e a p o s s i b l e r e a s o n w h y s s c increased in this study. however, dehydration of fruits during storage could be mentioned as another reason. organic acids are consumed in the process of respiration during post-harvest storage. it could be concluded that, the apples’ respiration rate in this study was possibly higher at the first two months of storage, and for this reason, the ta values were increased over this period, then lowered afterwards. srivastava and dwivedi (2000) stated that sa treatment has made ta fixed in banana. similar results have been reported while investigating chestnut by peng and jiang (2006). as the contents of organic acids in fruit are mainly dependent to the activities of their synthetic and hydrolytic enzymes, sa treatment was probably regulated the activities of related enzymes (ding et al., 2007). effects of salicylic acid on fruit softening fruits showed signs of softening during storage (fig. 3). firmness of apples was initially decreased in b o t h c o n t r o l a n d s a t r e a t e d f r u i t s , a n d t h e n appeared rapidly. similar results was found by ding et al. (2007) and sayyari et al. (2011) who reported that fruit firmness decreased during storage. a close relationship between the change at endogenous sa level and the rate of fruit ripening and softening in ki wi f ru i t was o b served b y z h an g et al . ( 2003) . softening could be the result of turgor loss, starch degradation, and most importantly cell wall degradation associated with weakening intercellular cohesive forces (jackson, 2003). sa decreases production of ethylene and inhibits activation of cell wall and membrane degrading enzymes such as polygalacturonase, lipoxygenase, cellulose and pectinemethylesterase resulting in the reduction of softening (srivastava and dwivedi, 2000; zhang et al., 2003). adding sa to the nutrient solution could induce firmness and delay the softening process in strawberry and apple fruits (shafiee et al., 2010; kazemi et al., 2011). effects of sa on weight loss sa-treated fruits demonstrated controlled weight loss during initial stages of post-harvest. the lowest amount of weight loss was observed in 2 mm concentration of sapre-harvest-treated fruits. higher concentrations of sa could induce fruit weight loss, but did not control it (fig. 4). it was observed that the apples kept their quality until the 157 days of storage, so that they were still suitable to be transferred to the market, but they gradually started to show visible defects on the peel losing their quality afterwards. therefore, we recommend our method to be suitable for the storage of red delicious apples for the period of five months. weight loss is caused by both dehydration and consumption of soluble solids during respiration. sa table 1 effect of preand post-harvest salicylic acid treatments on ph of ‘red delicious’ apples stored at 0±0.5°c for 193 days the same letters in any column show no significant difference between the data. comparison of the means was conducted through the duncans’ multiple range test (p<0.05). pr= pre-harvest; po= post-harvest; t0 to t3= control, 1, 2 and 4 mm sa treatment respectively. treatment days of storage 0 30 60 90 157 193 pr 4.55 a 4.34 b 4.36 a 4.39 a 4.28 a 4.18 a po 4.56 a 4.54 a 4.34 a 4.43 a 4.31 a 4.10 b t0 4.52 a 4.39 c 4.35 a 4.41 a 4.27 a 4.13 a t1 4.54 a 4.42 bc 4.35 a 4.42 a 4.31 a 4.14 a t2 4.58 a 4.49 a 4.33 a 4.39 a 4.28 a 4.13 a t3 4.55 a 4.46 ab 4.37 a 4.42 a 4.33 a 4.15 a prt 0 4.53 a 4.30 d 4.34 a 4.36 a 4.26 a 4.15 abc prt 1 4.57 a 4.34 cd 4.36 a 4.41 a 4.30 a 4.25 a prt 2 4.52 a 4.36 cd 4.34 a 4.38 a 4.26 a 4.19 ab prt 3 4.59 a 4.37 c 4.40 a 4.40 a 4.32 a 4.15 abc pot 0 4.40 a 4.49 b 4.36 a 4.46 a 4.28 a 4.12 bc pot 1 4.53 a 4.50 b 4.34 a 4.43 a 4.32 a 4.03 c pot 2 4.46 a 4.62 a 4.32 a 4.40 a 4.31 a 4.08 bc pot 3 4.51 a 4.55 b 4.34 a 4.43 a 4.34 a 4.16 ab fig. 3 effect of preand post-harvest salicylic acid treatments on firmness of ‘red delicious’ apples stored at 0±0.5°c for 193 days. comparison of the means was conducted through the duncans’ multiple range test (p<0.05). pr= pre-harvest; po= post-harvest; t0 to t3= control, 1, 2 and 4 mm sa treatment respectively. hadian-deljou et al. effect of salicylic acid on quality and antioxidant properties of ‘red delicious’ during cold storage 35 could decrease water loss and respiration rate through controlling degradation of cell wall and reducing ethylene biosynthesis (srivastava and d w i v e d i , 2 0 0 0 ) , r e s p e c t i v e l y . j o n a g o l d a p p l e s immersed in sa have shown a significant decrease in weight loss during cold storage (kazemi et al., 2011). in pear fruits, ratio of weight loss has been decreased at 0.5 mm sa after 48 h of treatment (imran et al., 2007). strawberries dipped in sa solution demonstrated less weight loss as well (shafiee et al., 2010). it seems that high concentrations of sa could induce aggregation of intoxicated materials such as certain polyphenols in later stages of storage, which is probably why plant tissues degrade followed by increasing weight loss. further investigations are needed to clarify this. effects of salicylic acid on accumulation of phenolic compounds application of sa partially induced accumulation of phenolic compounds and these effects were significant only from the 60th day until the end of storage (fig. 5). since then, sa-treated fruits contained higher quantities of phenols than the control. these results are in agreement with the findings of harindra et al. (2015) in grapes and geransayeh et al. (2015) in strawberries. in contrast with the results of this work, kant et al. (2016) reported a delay in the biosynthesis of total phenolics in tomato green mature fruits dipped in sa solution resulting in an increase in fruit quality that is possibly related to the different physiological status of tomato green mature fruits during storage. apples contain many bioactive compounds including phenols. sa may act as modulator of phenylpropanoid metabolism leading probably to the accumulation of phenolics correlated with the induction of enzymes which are involved in general phenylpropanoid metabolism, e.g. pal (godoyhernandez and loyola-vargas, 1997). it has shown that pal activity could be induced by sa elicitation in citrus (lafuente et al., 2001) and grapes (chen et al., 2006), which would result in the accumulation of plant secondary metabolites. effects of salicylic acid on anthocyanin content a n t h o c y a n i n c o n t e n t o f a p p l e s r e v e a l e d a n increasing trend until the 60th day of storage while decreased right afterwards (table 2). initially, effects fig. 4 effect of preand post-harvest salicylic acid treatments on weight loss of ‘red delicious’ apples stored at 0±0.5°c for 193 days. comparison of the means was conducted through the duncans’ multiple range test (p<0.05). pr= pre-harvest; po= post-harvest; t0 to t3= control, 1, 2 and 4 mm sa treatment respectively. fig. 5 effect of preand post-harvest salicylic acid treatments on tpc of ‘red delicious’ apples stored at 0±0.5°c for 193 days. comparison of the means was conducted through the duncans’ multiple range test (p<0.05). pr= pre-harvest; po= post-harvest; t0 to t3= control, 1, 2 and 4 mm sa treatment respectively. table 2 effect of preand post-harvest salicylic acid treatments on anthocyanin content (mg/g fw) of ‘red delicious’ apples stored at 0±0.5°c for 193 days the same letters in any column show no significant difference between the data. comparison of the means was conducted through the duncans’ multiple range test (p<0.05). pr= pre-harvest; po= post-harvest; t0 to t3= control, 1, 2 and 4 mm sa treatment respectively. treatment days of storage 0 30 60 90 157 193 pr 5.57 a 7.39 a 12.41 a 9.15 a 4.91 a 2.54 a po 3.74 a 5.53 b 10.92 a 9.10 a 5.13 a 2.22 a t0 5.75 a 6.32 ab 12.08 a 7.84 b 4.59 a 2.83 a t 1 7.37 a 8.29 a 13.08 a 11.62 a 5.07 a 1.54 a t 2 4.33 a 6.46 ab 11.63 a 10.02 ab 5.44 a 2.22 a t 3 3.29 a 4.68 b 10.06 a 7.29 b 4.85 a 2.91 a prt 0 5.26 a 5.66 bc 12.50 a 7.65 b 4.04 a 2.88 ab prt 1 5.68 a 8.54 ab 15.39 a 12.98 a 5.24 a 1.35 b prt 2 7.56 a 10.13 a 12.02 a 9.43 ab 5.94 a 2.47 ab prt 3 4.46 a 5.38 bc 9.69 a 6.46 b 3.93 a 3.64 a pot 0 5.17 a 7.14 abc 11.53 a 8.00 b 5.14 a 2.79 ab pot 1 5.67 a 7.98 ab 10.78 a 10.27 ab 4.96 a 1.70 ab pot 2 2.75 a 3.52 c 11.23 a 10.74 ab 5.02 a 2.03 ab pot 3 2.10 a 3.97 c 10.43 a 7.96 b 5.54 a 2.36 ab adv. hort. sci., 2017 31(1): 31-38 36 of sa on anthocyanin content were significant. the highest contents of anthocyanin were obtained in the concentrations of 1 and 2 mm at pre-harvest treatments with no significant differences between one and with 1mm of post-harvest treatment. similar result has been reported by jamali et al. (2013) who sprayed strawberry plants by sa together with nickel sulfate and found higher amounts of anthocyanin in strawberry fruits. anthocyanins degrade by polyphenol oxidase during post-harvest, and this might be the main reason behind the reduction of anthocyanin compounds. the role of sa on anthocyanin production is unknown, one may hypothesize that sa could activate the key enzyme (chalcone synthase) in the a n t h o c y a n i n b i o s y n t h e t i c p a t h w a y ( g o d o y hernandez and loyola-vargas, 1997). obinata et al. (2003) reported that sa could markedly increase the production of procyanidin in grape. total anthocyanin has also been increased with storage period in both control and treated pomegranates (sayyari et al., 2011). our results agree with findings of obinata et al. (2003) and sayyari et al. (2011). effects of salicylic acid on antioxidant activity antioxidant activity increased in all treatments during storage (table 3). antioxidant activity was increased in sa-treated fruits, but this process was not uniform, i.e. changes antioxidant activity was not f o l l o w e d f r o m f i x e d p a t t e r n d u r i n g s t o r a g e . antioxidative potential of apple, however, is known to depend on the concentration and composition of phenolics. numerous researchers have indicated that sa and its functional analogs have inhibitory effects on cat and pod activities or serve as substrates for pod. knorzer et al. (1999) reported, when applied exogenously at suitable concentrations, sa was found to enhance the efficiency of antioxidant system in plants. imran et al. (2007) and sayyari et al. (2011) found that sa have the capacity of increasing antioxidant, the result that confirms the present study. kazemi et al. (2011) also observed the higher ascorbic acid (vitamin c) content as well as peroxidase and superoxide dismutase activities in apples treated with sa exogenously. sa is possibly effective through the activation of responsible genes for producing antioxidant compounds (wang et al., 2006) and increasing activity of the antioxidant enzymes such as superoxide dismutase, peroxidase, catalase and ascorbate peroxidase (mo et al., 2008). 4. conclusions this study showed the effectiveness of preand post-harvest treatment of sa on the quality of ‘red delicious’ apples. application of sa affected on juice ph and anthocyanin content until the middle of storage, maintaining phenolic compounds and increasing a n t i o x i d a n t a c t i v i t y . n o s i g n i f i c a n t d i f f e r e n c e between sa treatments was seen in terms of ssc, ta and fruit firmness. the lowest weight loss was o b s e r v e d o n p r e h a r v e s t s a t r e a t m e n t w h e n sprayed, and the highest concentration of phenolic compounds was observed in post-harvest treatment w h e n d i p p e d . a n t h o c y a n i n c o n t e n t w a s a l s o increased during storage up to 60 days, but by the day 90, it was gradually decreased until the end of storage when reached to the lower level. as a conclusion, treatment of ‘red delicious’ apples with sa could properly maintain the quality of fruits and increase their post-harvest life and antioxidant capacity for at least 2 months. references arnous a., makris d.p., kefalas p., 2001 effect of principal polyphenolic components in relation to antioxidant characteristics of aged red wines. j. agric. food chem., 49: 5736-5742. baldwin e., 2002 fruit flavour, volatile metabolism and consumer perceptions, pp. 89-106. in: kneen m. (ed.) fruit quality and its biological basis. crc press, boca table 3 effect of preand post-harvest salicylic acid treatments on antioxidant activity (% of dpph inhibition) of ‘red delicious’ apples stored at 0±0.5°c for 193 days the same letters in any column show no significant difference between the data. comparison of the means was conducted through the duncans’ multiple range test (p<0.05). pr= pre-harvest; po= post-harvest; t0 to t3= control, 1, 2 and 4 mm sa treatment respectively. treatment days of storage 0 30 60 90 157 193 pr 60.20 a 74.34 a 62.00 b 73.67 b 78.67 a 82.18 a po 47.40 a 67.35 b 84.25 a 81.53 a 76.79 a 82.49 a t 0 59.29 a 62.43 c 55.62 b 68.22 b 72.50 c 78.76 b t 1 50.14 a 69.88 b 79.16 a 78.26 a 83.93 a 83.67 ab t 2 61.03 a 81.16 a 81.52 a 81.93 a 79.84 ab 80.36 b t 3 58.24 a 70.27 b 79.68 a 81.42 a 74.55 bc 86.33 a prt 0 58.46 a 70.85 cd 50.37 d 70.33 cd 82.55 a 63.54 c prt 1 61.94 a 68.59 d 68.09 b 73.09 bcd 84.57 a 77.94 b prt 2 56.35 a 78.56 b 60.33 c 75.35 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activates phenylalanine ammonia-lyase in grape berry in response to high temperature stress. j. plant growth regu., 55: 1-10. zhang h., wang l., ma l., dong y., jiang s., xu b., zheng x., 2009 biocontrol of major post-harvest pathogens on apple using rhodotorula glutinis and its effects on post-harvest quality parameters. biol. control., 48: 79-83. zhang y., chen k., chen q., zhang s., ren y., 2003 effects of acetylsalicylic acid (asa) and ethylene treatments on ripening and softening of post-harvest kiwifruit. acta botan. sinica, 45(12): 1447-1452. zhang y., chen k., zhang s., ferguson i., 2003 the r o l e o f s a l i c y l i c a c i d i n p o s t h a r v e s t r i p e n i n g o f kiwifruit. post-harvest biol. technol., 28: 67-74. impaginato 252 afghan accessions almond, 207, 217 pomegranate, 225 afghanistan special issue almond, 207, 217 fruitculture, 197 germplasm, 197, 207, 217, 225, 231, 239 phytosanitary status, 239 pomegranate, 225 sweet cherry, 231 aleurodicus dispersus russell see spiralling whiteflies almond afghan accessions, 207, 217 afghanistan special issue, 207, 217 breeding, 217 cross compatibility, 217 genetic resources, 207 germplasm, 207 imcompatibility groups, 217 kernel, 207 phenotipic characterisation, 207 phytosanitary status, 239 pollination, 217 alstroemeria spp. see peruvian lily antioxidant activity peach, 175 tomato, 151 antioxidative bermudagrass, 141 apple phytosanitary status, 239 apricot phytosanitary status, 239 apricot gum carrot, 111 grapevine, 111 stevia, 111 arbuscular mycorrhizal fungi cotton, 121 ascorbate peroxidase tomato, 183 ascorbic acid tomato, 3 carrot apricot gum, 111 callus culture, 111 carbon source, 111 organic additive, 111 tissue culture, 111 chemical composition spiralling whiteflies, 165 chlorophyll content kentucky bluegrass, 87 mexican lime, 39 citrus aurantifolia l. see mexican lime climateric fruits banana, 75, 159 ethylene, 75 gaseous phase photocatalysis, 75 high ethylene production, 75 liquid-crystal template, 75 mixed oxides, 75 modified storage environment, 75 post-harvest, 75 uniform ripening, 75 cold stress seashore paspalum, 103 cold tolerance seashore paspalum, 103 control activities spiralling whiteflies, 165 cotton arbuscular mycorrhizal fungi, 121 dna changes, 13 genome template stability, 13 growth, 121 mineral nutrients, 121 phosphogypsum, 121 rapd markers, 13 salt stress, 13 cross compatibility almond, 217 cut flower tuberose, 69 cymbopogon citratus spiralling whiteflies, 165 cynodon dactylon (l.) pers. see subject index banana climateric fruits, 75, 159 ethylene production, 159 putrescine treatment, 159 quality, 159 ripening process, 159 shelf life, 159 barberry germination, 31 pollen grains pollen size, 31 pollen viability, 31 berberis microphylla g. forst see barberry bermudagrass antioxidative, 141 irrigation, 141 morpho-physiological alteration, 141 photoperiod, 141 turfgrass, 141 biocontrol potato, 47 biofertilizers mango, 81 breeding almond, 217 building thermal control japanese climbing fern, 59 ‘burlat’ sweet cherry, 231 calla lily new viral evidence, 53 rt-pcr tests, 53 tuscan nurseries, 53 viruses occurrence, 53 callus culture carrot, 111 grapevine, 111 stevia, 111 cananga odorata spiralling whiteflies, 165 carbon source carrot, 111 grapevine, 111 stevia, 111 253 bermudagrass daucus carota l. see carrot decay tomato, 183 de-pigmented cultivar peach, 175 dna changes cotton, 13 drought tomato, 3 drought deficit mexican lime, 39 endangered genotypes fig, 129 environmental measurements japanese climbing fern, 59 essential oils spiralling whiteflies, 165 ethylene climateric fruits, 75 tuberose, 69 ethylene production banana, 159 euodia hortensis spiralling whiteflies, 165 explant fig, 129 ficus carica l. see fig fig endangered genotypes, 129 explant, 129 micropropagation, 129 rooting, 129 shoot proliferation, 129 single node, 129 flavanone glycoside potato, 95 flavonoids tomato, 151 flower abscission tomato, 3 food security tomato, 23 growth cotton, 121 growth characters mango, 81 harvest date sweet cherry, 231 heavy metals tomato, 23 high ethylene production climateric fruits, 75 ‘himsagar’ mango, 81 histological analysis potato, 95 hydrogen peroxide treatment seashore paspalum, 103 hydroponic system tomato, 23 imcopatibility groups almond, 217 inorganic manure mango, 81 ion fluxes grapevine, 135 tobacco, 135 irrigation bermudagrass, 141 japanese climbing fern building thermal control, 59 environmental measurements, 59 green roof, 59 green wall, 59 roof top garder, 59 urban agriculture, 59 kentucky bluegrass chlorophyll content, 87 proline content, 87 salt stress, 87 silicon treatments, 87 turfgrass, 87 kernel almond, 207 leaf water potential mexican lime, 39 liquid-crystal template climateric fruits, 75 fruitculture afghanistan special issue, 197 germplasm, 197 plant nursery, 197 traditional orchard, 197 fumigant toxicity spiralling whiteflies, 165 gaseous phase photocatalysis climateric fruits, 75 gc-ms test spiralling whiteflies, 165 genetic resources almond, 207 genome template stability cotton, 13 germination barberry, 31 germplasm aghanistan special issue, 197, 207, 217, 225, 231, 239 almond, 207 fruitculture, 197 phytosanitary status, 239 pomegranate, 225 ‘ghiaccio’ peach, 175 gibberellic acid tuberose, 69 glomus etunicatum mexican lime, 39 gossypium hirsutum l. see cotton grapevine apricot gum, 111 callus culture, 111 carbon source, 111 ion fluxes, 135 organic additive, 111 phytosanitary status, 239 plant-virus interaction, 135 potassium, 135 tissue culture, 111 trans-plasma membrane, 135 green roof japanese climbing fern, 59 green wall japanese climbing fern, 59 greenhouse conditions tomato, 151 254 longevity tuberose, 69 lycopene tomato, 3 lygodium japonicum thunb. sw. see japanese climbing fern macro elements tomato, 23 malus domestica borkh. see apple mangifera indica l. see mango mango biofertilizers, 81 growth characters, 81 ‘himsagar’, 81 inorganic manure, 81 organic manure, 81 quality, 81 shelf life, 81 soil microbial population, 81 maturity index sweet cherry, 231 mexican lime chlorophyll content, 39 drought deficit, 39 glomus etunicatum, 39 leaf water potential, 39 pseudomonas fluorescence, 39 micro elements tomato, 23 micropropagation fig, 129 middle east tomato, 23 mineral nutrients cotton, 121 mixed oxides climateric fruits, 75 modified storage environment climateric fruits, 75 morphological variability pomegranate, 225 morpho-physiological alteration bermudagrass, 141 mother stock nurseries phytosanitary status, 239 phytochemicals peach, 175 phytosanitary status aghanistan special issue, 239 almond, 239 apple, 239 apricot, 239 germplasm, 239 grapevine, 239 mother stock nurseries, 239 peach, 239 pear, 239 plum, 239 sweet cherry, 239 virus disease, 239 plant nursery fruitculture, 197 plant resistance potato, 47 plant-virus interaction grapevine, 135 tobacco, 135 plum phytosanitary status, 239 poa pratensis l. see kentucky bluegrass polianthes tuberose l. see tuberose pollen grains barberry, 31 pollen size barberry, 31 pollen viability barberry, 31 pollination almond, 217 pomegranate afghan accessions, 225 aghanistan special issue, 225 germplasm, 225 morphological variability, 225 nutraceutical properties, 225 relationship, 225 post harvest climateric fruits, 75 tomato, 183 tuberose, 69 potassium grapevine, 135 tobacco, 135 murraya koenigii spiralling whiteflies, 165 musa acuminata l. see banana new viral evidence calla lily, 53 peruvian lily, 53 nicotiana tabacum l. see tobacco nutraceutical properties peach, 175 pomegranate, 225 ocimum tenuiflorum spiralling whiteflies, 165 organic additive carrot, 111 grapevine, 111 stevia, 111 organic manure mango, 81 paspalum vaginatum sw. see seashore paspalum peach antioxidant activity, 175 de-pigmented cultivar, 175 ‘ghiaccio’, 175 nutraceutical properties, 175 phenolic compounds, 175 phytochemicals, 175 phytosanitary status, 239 quality, 175 white flesh, 175 pear phytosanitary status, 239 peruvian lily new viral evidence, 53 rt-pcr tests, 53 tuscan nurseries, 53 viruses occurrence, 53 pesticidal activities spiralling whiteflies, 165 phenolic compounds peach, 175 phenotypic characterisation almond, 207 phosphogypsum cotton, 121 photoperiod bermudagrass, 141 255 potato biocontrol, 47 flavanone glycoside, 95 histological analysis, 95 plant resistance, 47 potato tuber moth, 47 pseudomonas putida btpi, 47 rhizobacteria, 47 salt stress, 95 trichomes, 95 ultrastructural changes, 95 potato tuber moth potato, 47 pre-harvest treatment tomato, 183 tuberose, 69 proline content kentucky bluegrass, 87 prunus amygdalus batch. see almond prunus armeniaca l. see apricot prunus avium l. see sweet cherry prunus domestica l. see plum prunus dulcis mill. see almond prunus persica l. see peach pseudomonas fluorescence mexican lime, 39 pseudomonas putida btpi potato, 47 punica granatum l. see pomegranate putrescine treatment banana, 159 pyrus l. see pear quality banana, 159 mango, 81 peach, 175 tomato, 183 quality control system sweet cherry, 231 rapd markers cotton, 13 relationship pomegranate, 225 repellent test spiralling whiteflies, 165 cymbopogon citratus, 165 essential oils, 165 euodia hortensis, 165 fumigant toxicity, 165 gc-ms test murraya koenigii, 165 ocimum tenuiflorum, 165 pesticidal activities, 165 repellent test, 165 sps tomato, 3 stevia rebaudiana bert. see stevia stevia apricot gum, 111 callus culture, 111 carbon source, 111 organic additive, 111 tissue culture, 111 sustainable agriculture tomato, 23 sweet cherry afghanistan special issue, 231 ‘burlat’, 231 harvest date, 231 maturity index, 231 phytosanitary status, 239 quality control system, 231 tissue culture carrot, 111 grapevine, 111 stevia, 111 tobacco ion fluxes, 135 plant-virus interaction, 135 potassium, 135 trans-plasma membrane, 135 tomato antioxidant activity, 151 ascorbate peroxidase, 183 ascorbic acid, 3 decay, 183 drought, 3 flavonoids, 151 flower abscission, 3 food security, 23 greenhouse conditions, 151 heavy metals, 23 hydroponic system, 23 lycopene, 3 macro elements, 23 micro elements, 23 middle east, 23 quality, 183 post harvest, 183 pre-harvest treatment, 183 salicylic acid, 151, 183 rhizobacteria potato, 47 ripening process banana, 159 roof top garden japanese climbing fern, 59 rooting fig, 129 rt-pcr tests calla lily, 53 peruvian lily, 53 ‘salam’ seashore paspalum, 103 salicylic acid tomato, 151, 183 salt stress cotton, 13 kentucky bluegrass, 87 potato, 95 seashore paspalum cold stress, 103 cold tolerance, 103 hydrogene peroxide treatments, 103 ‘salam’, 103 ‘seaspray’, 103 ‘seaspray’ seashore paspalum, 103 shelf life banana, 159 mango, 81 shoot proliferation fig, 129 silicon treatment kentucky bluegrass, 87 single node fig, 129 soil microbial population mango, 81 solanum lycopersicon l. see tomato solanum tuberosum l. see potato soluble protein tomato, 3 spiralling whiteflies cananga odorata, 165 chemical composition, 165 control activities, 165 256 soluble protein, 3 sps, 3 sustainable agriculture, 23 total phenolics, 151 weight loss, 183 yield, 3 yield components, 151 total phenolics tomato, 151 traditional orchard fruitculture, 197 trans-plasma membrane grapevine, 135 tobacco, 135 trichomes potato, 95 tuberose cut flower, 69 virus disease phytosanitary status, 239 viruses occurrence calla lily, 53 peruvian lily, 53 vitis vinifera l. see grapevine weight loss tomato, 183 white flesh peach, 175 yield tomato, 3 yield components tomato, 151 zantedeschia aethiopica l. spreng. see calla lily ethylene, 69 gibberellic acid, 69 longevity, 69 post harvest, 69 pre-harvest treatment, 69 turfgrass bermudagrass, 141 kentucky bluegrass, 87 tuscan nurseries calla lily, 53 peruvian lily, 53 ultrastructural changes potato, 95 uniform ripening climateric fruits, 75 urban agriculture japanese climbing fern, 59 impaginato 53 1. introduction salinity stress is dependent on environmental condition (kozlowski and pallardy, 1997), farming, water management and genotype (kozlowski and pallardy, 1997). olive (olea europea l.) is one of the most valuable and widespread fruit trees in the mediterranean area. its cultivation is continuously being extended to irrigated land. furthermore, in mediterranean area salinity is becoming a major problem due to high rates of evaporation (kozlowski and pallardy, 1997). olive is considered a moderately salt tolerant plant (ayers and westcot, 1976; aragues et al., 2005; weissbein et al., 2008). in comparison with other mediterranean-grown tree crops, olive is more tolerant than citrus but less tolerant than date palm (ayers and westcot, 1976). the tolerance of olive cultivars are different to salinity stress (therios a n d m i s o p o l i n o s , 1 9 8 8 ; p e r i c a e t a l . , 2 0 0 4 ; chartzoulakis, 2005). the relationship between saline water and olive cultivation has been intensively studied for many years and significant progress has been made in the understanding of this topic (ayers and westcot, 1976; wiesman et al., 2004). it is generally well established that saline conditions limit the vegetative and reproductive development of olives mainly as a result of interference with the osmotic balance in the root system zone and detrimental effects caused by specific toxic accumulation of chloride and sodium ions in the leaves (weissbein et al., 2008). salt stress reduces water availability in soil solution as a result of an increased osmotic potential, inducing the generation of reactive oxygen species (ros) (zhu, 2001; melloni et al., 2003), the reduction of hormonal signals generated by the roots (munns, 2002), altered carbohydrate metabolism (gao et al., 1998), reduced t h e a c t i v i t y o f c er t a i n en z y m es ( m u n n s , 1 9 9 3 ; chartzoulakis, 2005) and ultimately impaired photosynthesis (chartzoulakis, 2005). therefore, these physiological changes result reduced growth in either reduced cell division, expansion or promoting cell death (hasegawa et al., 2000). furthermore these criteria make plant reduce growth rate and yield, chlorophyll destruction which lead to leaf senescence. the plant response to salinity stress is depenadv. hort. sci., 2017 31(1): 53-59 doi: 10.13128/ahs-20726 physiological responses of olive cultivars to salinity stress m. rahemi 1 (*), s. karimi 2, s. sedaghat 1, a. ali rostami 1 1 department of horticultural science, college of agriculture, shiraz university, shiraz, iran. 2 department of horticultural science, college of abouraihan, university of tehran, tehran, iran. key words: electrolyte conductivity, osmoregulation, proline, soluble carbohydrates. abstract: the aim of this study was to evaluate the tolerance of seven promising olive cultivars for southern parts of iran (‘amigdalilolia’, ‘dakal’, ‘zard’, ‘dezful’, ‘tokhm-e-kabki’, ‘shiraz’, and ‘conservalia’) against salinity stress. biochemical and physiological responses of the cultivars irrigated with saline water application (control, 4, 8, and 12 ds m -1 ) were evaluated and the tolerant cultivars were identified. in contrast to the tolerant cultivars, the sensitive ones continue to grow with lower rate and died under salinity stress. in general, growth indices of olive cultivars were reduced with increasing salinity stress and the lowest growth indices were obtained under 12 ds m-1 treatment. results indicated that the accumulation of higher levels of soluble carbohydrates and proline in the leaves of the tolerant cultivars helps them to deal with salinity stress. the results showed that saline waters up to 4 ds m-1 for irrigation can be used for olive cultivars, however, based on the result of this study, it is not recommended to use water sources with higher electric conductivities to irrigate sensitive olive cultivars. we concluded that the tolerant cultivars stopped growth and used their energy to defend against the salinity stress. (*) corresponding author: rahemi@shirazu.ac.ir received for publication 9 february 2016 accepted for publication 17 february 2017 copyright: © 2017 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2017 31(1): 53-59 54 dent on environmental condition, farming, water management and plant genotype. the aim of this study was to screen for the tolerance of seven olive cultivars from the southern parts of iran, against salinity stress. tolerance was evaluated over several biochemical (proline content, stored carbohydrate, total chlorophyll and starch concentration), and physiological (cell membrane injury) responses of these cultivars under salinity stress. 2. materials and methods experiment was carried out in the department of horticultural sciences, shiraz university during the growing season in 2012, using one year old cuttings of seven olive cultivars: ‘dakal’, ‘zard’, ‘shiraz’, ‘ t o k h m e k a b k i ’ , ‘ d e z f u l ’ , ‘ a m i g d a l i l o l i a ’ , a n d ‘konservalia’ with four replicates for each cultivar. the cuttings were transplanted into 15 kg pots containing soil mixture (1:1:1) of soil (pure soil), sand and leaf mould. the physicochemical characteristics of the soil are shown in table 1. during the establishment phase in greenhouse, olive cultivars were pruned uniformly in order to produce a single stem. the salinity stress treatments were applied by sub irrigation with different salinity levels [control (1.1), 4,8,12 ds/m]. in order to prevent salinity shock, the concentration of salts was gradually increased to reach a given level. the day and night temperature of the greenhouse was 35°c and 25°c, respectively. the saline water was prepared by dissolving sodium chloride (control, 4, 8, 12 ds/m) in the water. the pots were irrigated with saline water for 90 days. they were irrigated with saline water to the field capacity (fc) level, which was equivalent 20% of the dry weight of the soil of pot. the total shoot length of olive cultivars was measured at the beginning and at the end of the experiment. additionally, the number of fully expanded leaves and branches of each cultivar were recorded. at the end of the experiment, the average length of new shoot was measured. using the data collected at the start and the end of salinity stress treatments, the rate of these changes was calculated. total chlorophyll measurement total chlorophyll content was determined by spectrophotometer (saini et al., 2001). briefly, chlorophyll a and b contents were obtained by extraction in 85% acetone solution and measuring their absorbances using camp spec m501 single beam uv/vis spectrophotometer at λ= 663 nm and λ = 645 nm. the concentration of chlorophylls and carotenoids were calculated according to the following formula: total chlorophylls (mg /g fw) = [(20.2×od 645 nm + 8.02×od 663 nm ) × v] /(fw × 1000) where od is optical density, v is the final solution volume in ml and fw is tissue fresh weight in mg. v is the final solution volume in ml and fw is tissue fresh weight in mg. proline measurement free proline was extracted from 0.5 g samples of fully expanded and young leaves with 3%, sulfuric acid and estimated by using ninhydrin reagent, according to the protocol described by bates et al. (1973). the absorbance of the fraction with toluene was determined at 520 nm, using a spectrophotometer (model uv-120-20, japan). cell membrane injury (cmi) cell membrane injury was calculated according to the method of blum and ebercon (1981). for the cmi, 20 samples of stressed and unstressed young leaves were washed with distilled water to remove the dust and injured cells from samples. the samples were then immersed in 20 ml distilled water at room temperature. after 24 h the conductivity of the solutions was read. the samples were autoclaved for 15 min, cooled to room temperature and the conductivity of the solutions was read again. the electrolyte leakage was measured with a conduct meter (644 conduct meter, metrohm, herisau, switzerland). cmi was estimated from the formula: id (drought injury index) = 1 −(1 −t1/t2)/(1 -c1/c2) × 100 where t1 and t2 are the first and second measurement of the conductivity of the solutions in which the table 1 the physiochemical characteristics of the soil used characteristics zn (ppm) 1.5 fe (ppm) 7.6 mn (ppm) 21.14 cu (ppm) 1.76 k (ppm) 400 p (ppm) 23.8 total nitrogen (%) 0.094 oc (%) 1.54 ph (%) 7.9 ec (%) 1.93 clay (%) 34.4 silt (%) 44.2 sand (%) 21.4 rahemi et al. physiological responses of olive cultivars to salinity stress 55 treated samples were immersed and c1 and c2 are the respective values for the conductivity of the solutions. soluble carbohydrate extraction to determine soluble carbohydrate concentration, 150 mg of dried leaf samples was extracted twice with 80% ethanol. the sample was centrifuged at 3500 rpm for 10 min and the volume of the supernatant was adjusted to 25 ml soluble carbohydrate c o n c e n t r a t i o n w a s m e a s u r e d a c c o r d i n g t o t h e method of buysee and merckx (1993). in summary, 1 ml of supernatant was transferred to a test tube and 1 ml phenol 18% and 5 ml sulfuric acid were added. the mixture was shaken immediately and its absorption was recorded at 490 nm using a spectrophotometer (model uv-120-20, japan). starch concentration starch concentration in the leaf samples was measured using anthron reagent (mccready, 1950). in this method, 5 ml of water (0°c) and 6.5 ml perchloric acid (52%) were added to the pellet used for sugar analysis and mixed for 15 min. about 20 ml water was then added and the sample was centrifuged. the supernatant was separated and the same procedure was repeated with the pellet for each leaf samples. the supernatants were combined and left for 30 min at 0°c. after filtration, the supernatant volume was adjusted to 100 ml. about 2.5 ml of cold 2% anthron solution was added, and the sample was heated at 100°c for 7.5 min. it was then transferred immediately to an ice bath and cooled to room temperature. absorption at 630 nm was recorded using a spectrophotometer (model uv-120-20, japan). statistical analysis the experiment was conducted as a complete randomized design with factorial arrangements. analysis of variance was performed using the spss software package and significant differences among mean values were compared by duncan multiple range test (dmrt) (p<0.05). 3. results effect of salinity on plant growth after 90 days of salinity treatment plant growth (total shoot length, number of branches and leaf number) significantly reduced in all cultivars (table 2, 3 and 4). the effect of salinity on these parameters showed a significant genotypic variation. as expected, the highest reduction of shoot length was found in 12 dsm-1 treatment; considering the mean over all treatments, conservalia and shiraz cultivars had the highest shoot length while ‘dakal’ showed the lowest one under each salinity stress conditions (table 2). the highest reduction in number of branches was found in 8 and 12 dsm-1 treatments on average, the lowest branch number was found in ‘conservalia’ whereas the ‘amigdalilolia’, ‘dakal’ and ‘tokhm-e* mean separation within columns and rows by dmrt, at 5% level. table 2 effect of interaction between salinity treatment and cultivar on total shoot length (in comparison to the beginning of the experiment table 3 effect of interaction between salinity treatment and cultivar on branch number of olive cultivars table 4 effect of interaction between salinity treatment and cultivar on leaf number of olive cultivar (calculated as: leaf number at the beginning of the stress-the leaf number at ending the stress) cultivar total shoot lenght (cm) ec (dsm-1 ) of irrigating water 0 4 8 12 mean ‘conservaliaʼ 158.4 ab* 158.7 a 131.2 a-e 106.3 def 138.6 a ‘shirazʼ 149.1 ab 137.7 a-d 137.7 a-d 124.1 a-f 138.8 a ‘tokhm-e-kabkiʼ 149.1 a-d 119.6 b-f 114.7 def 112.7 def 121.0 bc ‘dezfulʼ 159.9 abc 136.0 a-d 119.2 b-f 121.4 b-f 134.1 ab ‘zard’ 156.3 ab 124.7 a-f 127.3 a-f 118.0 c-f 131.6 ab ‘dakalʼ 154.4 ab 102.7 f 104.8 ef 103.9 ef 116.4 c ‘amigdaliloliaʼ 142.4 a-d 121.8 b-f 106.4 ef 115.7 c-f 120.2 bc mean 151.4 a 128.5 b 120.2 bc 141.6 c * mean separation within columns and rows by dmrt, at 5% level. cultivar branch number ec (dsm-1 ) of irrigating water 0 4 8 12 mean 'conservalia' 21.0 h-k * 24.3 f-j 8.3 l 8.0 l 15.4 d ‘shirazʼ 28.3 c-h 18.6 ijk 18.4 ijk 17.6 ijk 20.7 c ‘tokhm-e-kabkiʼ 40.0 a 35.6 a-d 25.2 e-i 30.2 b-g 32.7 a ‘dezfulʼ 29.8 b-h 28.5 c-h 22.9 g-k 20.9 h-k 25.3 b ‘zard’ 27.6 d-h 17.6 ijk 16.0 k 17.0 h-k 19.3 c ‘dakalʼ 29.3 c-g 31.0 b-f 32.0 b-f 37.6 ab 32.7 a ‘amigdaliloliaʼ 36.0 abc 34.6 a-d 33.0 a-e 22.6 g-k 31.4 a mean 30.3 a 28.0 b 21.8 c 22 c * mean separation within columns and rows by dmrt, at 5% level. cultivar leaf number ec (dsm-1 ) of irrigating water 0 4 8 12 mean 'conservalia' 151.0 a-f * 138.6 c-g 21.0 jk -56.0 l 66.3 d ‘shirazʼ 114.6 b-f 113.6 e-i 74.3 g-j -11.3 kl 80.3 c ‘tokhm-e-kabkiʼ 190.6 a-d 207.1 ab 100.0 f-i 73.6 g-j 142.8 b ‘dezfulʼ 224.6 a 217.6 a 135.0 c-g 113.3 e-i 172.6 ab ‘zard’ 160.0 a-f 96.0 f-i 65.6 g-j 62.6 hij 96.8 c ‘dakalʼ 218.3 a 218.6 a 200.6 abc 130 d-h 191.9 a ‘amigdaliloliaʼ 185.3 a-d 177.3 a-e 174.0 a-e 98 f-i 158.6 b mean 177.8 a 167.0 a 110.1 b 60.0 c adv. hort. sci., 2017 31(1): 53-59 56 kabki’ obtained the most one (table 3). leaf number was also significantly reduced by salinity treatments. at 12 dsm-1, the reduction in leaf number ranged from -56.0 for ‘conservalia’ to 130 for ‘dakal’ with respect to control plants. the greatest number of leaves was found in controls and 4 dsm-1 treatments (table 4). effect of salinity on cell membrane injury (cmi) cell membrane injury (cmi) was significantly increased in all studied cultivars from the treatment of 12 dsm-1 except in ‘amigdalilolia’, ‘zard’ and ‘dakal’. the highest percentage of cmi was obtained in ‘conservalia’ with respect to the control treatment (table 5). furthermore, the maximum and minimum cmi belonged to 4dsm-1 and control, respectively. therefore, cmi was influenced by both salinity treatment and type of cultivar (table 5). proline content leaf proline content of olive cultivars was significantly influenced by salinity treatment and type of cultivar (table 6). maximum proline content was obtained in the leaf tissue of ‘dezful’ while the minimum proline content was determined in the leaves of ‘conservalia’. in general, the highest proline content was observed in 8 dsm-1 treatments and the lowest one was observed in 4 dsm-1 treatments (table 6). soluble carbohydrate content the concentration of soluble carbohydrates of leaves under salinity stress was significantly changed in all studied cultivars and the highest content of soluble carbohydrates was found in shiraz and zard cultivars and the lowest was observed in the leaves of ‘amigdalilolia’ (table 7). the maximum soluble carbohydrate content was found in 4 dsm-1 treatment and the minimum content was observed in control and 12 dsm-1 treatments, respectively. starch concentration content the stored carbohydrate content of leaves was significantly affected by salinity treatments and type of cultivar. ‘conservalia’ had the highest stored carbohydrate content whereas ‘shiraz’ showed the lowest (table 8). plants grown at 12 dsm-1 had the highest stored leaf carbohydrate content and the lowest one was observed in 8 dsm-1 treatments (table 8). table 5 effect of interaction between salinity treatment and cultivar on cmi of olive leaf (%) * mean separation within columns and rows by dmrt, at 5% level. cultivar cell membrane injury (%) ec (dsm-1 ) of irrigating water 0 4 8 12 mean 'corservalia' 74.7 abc* 72.8 abc 67.9 b-f 49.1 g 66.1 c ‘shiraz’ 66.6 c-f 60.4 f 52.7 g 52.9 g 58.2 d ‘tokhm-e-kabki’ 72.4 abc 71.6 a-d 63.6 def 63.1 ef 67.7 bc ‘dezful’ 69.7 a-e 66.2 c-f 68.7 a-e 53.3 g 64.5 c ‘zard’ 69.6 a-e 73.0 abc 68.3 b-f 69.4 a-e 70.1 b ‘dakal’ 70.3 a-e 77.1 a 68.9 a-e 68.6 a-e 71.2 b ‘amigdalilolia’ 76.7 ab 75.2 ab 76.3 ab 76.0 ab 75.6 a mean 71.4 a 70.9 a 66.6 b 61.6 c table 6 effect of interaction between salinity treatment and cultivar on proline content of olive cultivar leaf (µm g dw-1) * mean separation within columns and rows by dmrt, at 5% level. cultivar proline content (µm g dw-1) ec (dsm-1 ) of irrigating water 0 4 8 12 mean 'conservalia' 29.9 b-e* 21.1 g 24.0 efg 22.1 fg 24.3 c ‘shirazʼ 36.7 ab 22.7 fg 26.8 d-g 23.0 efg 27.3 bc ‘tokhm-e-kabkiʼ 28.9 b-f 28.9 b-f 33.2 a-d 26.6 d-g 29.4 b ‘dezfulʼ 35.4 abc 32.7 a-d 40.7 a 37.8 ab 36.7 a ‘zard’ 26.6 d-g 26.5 d-g 35.7 abc 28.6 b-f 29.3 b ‘dakalʼ 24.1 e-g 29.9 b-e 33.8 a-d 29.9 c-g 28.7 bc ‘amigdaliloliaʼ 22.4 fg 25.9 d-g 26.5 d-g 34.0 a-d 27.2 bc mean 29.1 b 26.8 c 31.5 a 29.5 ab table 7 effect of interaction between salinity treatment and cultivar on soluble carbohydrate content of olive leaf ( %) * mean separation within columns and rows by dmrt, at 5% level. cultivar soluble carbohydrate content (%) ec (dsm-1 ) of irrigating water 0 4 8 12 mean 'conservalia' 30.6 abc* 28.3 c-g 26.9 d-h 27.2 d-h 28.3 ab ‘shirazʼ 31.5 a 28.7 a-f 29.9 a-e 28.7 b-f 29.7 a ‘tokhm-e-kabkiʼ 28.0 c-h 31.3 ab 29.0 a-f 26.8 e-h 29.0 ab ‘dezfulʼ 26.7 d-h 29.1 a-f 26.8 e-h 28.3 c-g 27.6 bc ‘zard’ 30.1 a-d 30.0 a-e 28.9 a-f 29.3 a-f 29.6 a ‘dakalʼ 23.5 h 31.2 ab 30.6 abc 28.6 b-f 28.7 ab ‘amigdaliloliaʼ 24.5 gh 25.7 fgh 28.4 c-g 27.3 d-h 26.5 c mean 27.9 b 29.4 a 28.6 ab 28.1 b table 8 effect of interaction between salinity treatment and cultivar on stored carbohydrate content of olive cultivar leaf (mg g dw-1) * mean separation within columns and rows by dmrt, at 5% level. cultivar stored carbohydrate content (mg g dw-1) ec (dsm-1 ) of irrigating water 0 4 8 12 mean 'corservalia' 279.6 a-e* 280.6 a-d 251.3 b-g 339.1 a 290.7 a ‘shirazʼ 221.5 d-g 164.0 h 191.0 gh 296.9 abc 220.2 d ‘tokhm-e-kabkiʼ 238.7 c-g 242.8 c-g 239.0 c-g 284.2 a-d 249.7 bc ‘dezfulʼ 221.6 d-g 277.0 a-d 244.9 c-g 261.8 b-e 251.3 b ‘zard’ 251.9 b-f 233.4 c-g 244.2 c-g 237.3 c-g 241.7 bcd ‘dakalʼ 254.6 b-f 229.4 c-g 211.5 e-h 198.6 fgh 221.6 cd ‘amigdaliloliaʼ 288.9 a-d 319.7 ab 233.4 c-g 229.6 c-g 269.1 ab mean 249.4 ab 249.1 ab 230.0 b 264.5 a rahemi et al. physiological responses of olive cultivars to salinity stress 57 chlorophyll content the highest chlorophyll content was observed in the ‘dakal’, ‘dezful’ and ‘amygdalolelia’ while the lowest chlorophyll content was determined in the leaves of ‘shiraz’. in general, the highest chlorophyll content was measured in plants grown with the 4dsm-1 treatment and the lowest was observed for 12dsm-1 treatment (table 9). 4. discussion and conclusions the purpose of this study was to evaluate the s a l i n i t y t o l e r a n c e o f s e v e n o l i v e c u l t i v a r s (‘conservalia’, ‘amigdalilolia’, ‘dakal’, ‘tokhm-ekabki’, ‘shiraz’, ‘dezful′ and ‘zard) based on the effect of salinity on growth characteristics, and on physiological and biochemical response of different cultivars, grown in south of iran. salt stress has been r e p o r t e d t o h a v e g e n o t y p i c l i n k e d r e s p o n s e (chartozoulakis, 2005). different parameters were used to indicate the response of seven cultivars to nacl stress. the main result concerns the reduced number of leaves in salt treated olive plant respect to control ones. such decrease was not only connected with the growth inhibition effects of salt but also to plants defoliation (karimi et al., 2009). the impact of salinity stress on reducing growth indices was clear and confirmed the response shown in previous research on different species and cultivars of olives (perica et al., 2004). it was clear that the decline in growth rate of olive trees under salinity stress was dependent on the duration of salt exposure, the concentration of salt and the potential of tolerance of cultivars. in the current study, olive cultivars showed different reactions to salinity stress. in general, vigorous cultivars were more susceptible to salinity stress than low growth cultivars. partial growth reduction of olive tree growth can be related to the osmotic stress resulting of elevated level of ions in soil solution and irrigation water (weissbein et al., 2008). because no visual signs of salt toxicity in the plants e.g. tip burn, necrosis and/or shoot die back (chartozoulakis, 2005) under low salinity level; in addition, monitoring leaf number changes indicated no sign leaf abscission due to salt toxicity or oxidative damages under low salt stress level. low osmotic potential of soil solution under salinity stress limits growth of plant by reducing water uptake, transpiration and stomatal conductance, which is associated with the reduced photosynthesis (benasher et al., 2006). hence it can be concluded that, osmotic stress is the primary reason of olive growth limitation under salinity stress. the cmi of leaf is the quantitative index which shows either health of plasma membrane or the rate of membrane disruption in plant leaf tissue under salinity stress. the reduction in cmi of olive cultivar was parallel to intensity of sodium chloride concentration in the irrigation water. under the severe salinity stress, the highest cmi rate was observed in the ‘amigdalilolia’, ‘dakal’ and ‘zard’ (101.8%, 99.4% a n d 9 7 . 6 % ) , r e s p e c t i v e l y a n d t h e l o w e s t o n e belonged to ‘conservalia’ (65.6%). furthermore, leaf tip burning and leaf abscission were observed that might be due to accumulation of specific ions in olive leaf under salinity stress. though the leaf proline content in ‘shiraz’ and ‘conservalia’ declined with the increase of salinity stress, it increased in the other cultivars. during the salinity treatment, proline content was induced to accumulate in plant leaf tissue by the accumulation of the sodium and chloride ions and water stress as a result of enhancing of salt in soil solution (delauney and verma, 1993). in this study, despite salinity stress injuries, the increase in proline content may have played an important role in protection of olive cultivars in stress situations. the data presented here agreed with previous studies which show the direct relationship between tolerance of salinity stress and proline accumulation. it has been demonstrated that proline can protect protein from denaturation and maintain cytoplasmic membrane in salinity stress (khedr et al., 2003; karimi et al., 2009). data were shown that soluble sugar content of leaves of tolerant olive cultivars (‘amigdalilolia’, ‘ d a k a l ’ ) w a s e n h a n c e d d u r i n g s a l i n i t y p e r i o d . table 9 effect of interaction between salinity treatment and cultivar on chlorophyll of olive cultivar * mean separation within columns and rows by dmrt, at 5% level. cultivar chlorophyll ec (dsm-1 ) of irrigating water 0 4 8 12 mean 'corservalia' 1.84 efg* 2.56 a 2.31 a-e 0.74 i 1.86 b ‘shirazʼ 2.10 a-e 1.61 fg 1.55 g 0.20 i 1.36 c ‘tokhm-e-kabkiʼ 2.23 a-e 2.30 a-e 2.12 a-e 1.02 h 1.97 b ‘dezfulʼ 2.52 a 2.47 abc 1.95 d-g 2.05 a-f 2.25 a ‘zard� 1.92 d-g 2.24 a-e 1.98 c-g 1.59 fg 1.93 b ‘dakalʼ 2.25 a-e 2.48 abc 2.42 a-d 2.01 b-g 2.29 a ‘amigdaliloliaʼ 2.51 ab 2.25 a-e 2.27 a-e 1.80 efg 2.21 a mean 2.19 ab 2.27 a 2.09 b 1.36 c adv. hort. sci., 2017 31(1): 53-59 58 however in semi-tolerant cultivars (‘dezful’, ‘zard’ and ‘tokhm-e-kabki’) the sugar content was not significantly changed and in sensitive cultivars (‘shiraz’ and ‘conservalia’), this factor in comparison to control treatment was reduced. soluble sugars play an important role in maintaining turgor pressure in osmotic stress. furthermore, they can protect the plasma membrane in the stress situation (sanchez et al., 1998). this study showed that among tolerant olive cultivars, the accumulation of sugars could be due to the reduced consumption of stored carbohydrate contents (chartzoulakis, 2002). among the sensitive olive cultivars in which the starch and soluble sugar content were limited, it can be concluded that this phenomenon was related to photosynthesis disruption and the subsequent consumption of stored carbohydrate. under severe salinity stress condition, starch accumulation in the tissue of sensitive cultivars, may be due to disruption of enzyme activity and plant metabolism which led to stored carbohydrate in the tissue. salinity stress decreased chlorophyll concentration in the olive cultivar leaf. tolerant cultivars were shown more chlorophyll concentration than susceptible ones. therefore, it can be concluded that leaf chlorophyll concentration might be the best index for evaluating of olive cultivars to tolerate salinity stress as reported by noble and rogers (1994). the same authors suggested that chloroplast dysfunctions and decrease in the number and volume of chloroplast were influenced by salinity stress, and as a result, a reduction in chlorophyll content was a main reaction of olive trees to salinity stress. winicov and seemann (1991) observed that a salt tolerant alfalfa cell line exhibited an 11-fold increase in chlorophyll content compared to the unadapted cell line. the increase in chlorophyll content in alfalfa was associated with large increase in the activity of ribulose-1, 5bisphosphate carboxylase (winicove and seemnann, 1991). in conclusion, the relative tolerance of olive cultivars to salinity stress was in the following order: tolerant cultivars were ‘amigdaliloliaʼ and ‘dakalʼ, semitolerant cultivars included ‘dezful’, ‘zardʼ and ‘tokhm-e-kabkiʼ and finally sensitive cultivars were ‘shirazʼ and ‘conservaliaʼ. acknowledgments authors gratefully thank fars organization of jahad agriculture for their financial support of this project. references aragues r., puy j., royo a., espada j.l., 2005 three year field response of young olive trees (olea europea l., cv. arbequina) to soil salinity: trunk growth and leaf ion accumulation. plant soil, 271: 265-273. ayers r.s., westcot d.w., 1976 water quality for agriculture. fao irrigation and drainage, fao, rome. bates l.s., waldren r.p., teare i.d., 1973 rapid determination of free proline for water-stress. plant and soil, 39: 205-207. ben-asher j., tsuyki i., bravdo b.a., sagih m., 2006 irrigation of grapevines with saline 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and molecular responses to high salinity. ann. rev. plant physiol. plant molec. biol., 51: 463-499. karimi s., rahemi m., maftoun m., eshghi s., tavallal i.v., 2009 effects of long-term salinity on growth and performance of two pistachio (pistacia vera l.) rootstocks. aust. j. basic .appl. sci., 3(3): 1630-1639. khedr a.h.a., abbas m.a., wahid a.a.a., quick w.p., abogadallah g.m., 2003 proline induces the expression of salt-stress-responsive proteins and may improve the adaptation of pancratium maritinum l. to saltstress. j. exper. bot., 54: 2553-2562. kozlowski t.t., pallardy s.g., 1997 growth control in woody plants. academic press, san diego, ca, usa. mccready r.m., guggolz j., silviera v., owens h.s., 1950 determination of starch and amylase in vegetables. analytical chemistry, 22: 1156-1158. melloni d.a., oliva m.a., martinez c.a., cambraia j., 2003 photosynthesis and activity of superoxide dismutase, peroxidase and glutathione reductase in cotton under salt stress. environ. exp. bot., 49: 69-76. munns r., 1993 physiological processes limiting plant rahemi et al. physiological responses of olive cultivars to salinity stress 59 growth in saline soils: some dogmas and hypotheses. plant, cell environ., 16: 15-24. munns r., 2002 comparative physiology of salt and water stress. plant, cell environ, 25: 239-250. noble c.l., rogers m. e., 1994 response of temperate forest legumes to waterlogging and salinity, pp. 473496. in: pessaraki m. (ed.) handbook of plant and crop stress. marcel dekker, new york, usa, pp. 720. perica s., brkljača m., goreta s., romić m., 2004 vegetative growth and salt accumulation of six olive cultivars under salt stress. acta horticulturae, 664: 555-560. saini r.s., sharma k.d., dhankhar o.p., kaushik r.a., 2001 laboratory manual of analytical techniques in horticulture. agrobios, jodhpur, india, pp. 134. sanchez f.j., manzanares m., andres e.f., tenorio j.l., ayerbe l., andres. e.f.,1998 turgor maintenance, osmotic adjustment and soluble sugar and proline accumulation in 49 pea cultivars in response to water stress. field crops res., 59: 225-235. therios i.n., misopolinos n.d., 1988 genotypic response to sodium chloride salinity of four major olive cultivars (olea europea l.). plant and soil., 106: 105111. weissbein s., wiesman z., ephrath y., silberbush m., 2008 vegetative and reproductive response of olive cultivars to moderate saline water irrigation. hort. sci., 43(2): 320-327. wiesman z., david y., bendom n., 2004 optimization of saline water level for sustainable barnea olive and oil production in desert conditions. sci. hort., 100: 257-266. winicov i., seemann j.r., 1991 expression of genes for photosynthesis and the relationship to salt tolerance of alfalfa (medicago sativa) cells. plant cell physiol., 31(8): 1155-1161. zhu j.k., 2001 plant salt tolerance. trends in plant sci., 6: 66-71. 3 1. introduction buriti (mauritia flexuosa l.) is a palm tree found from the atlantic forest to the cerrado (a vast tropical savanna ecoregion) of the brazilian north, northeast and mid-west in the state of minas gerais (manzi and coomes, 2009). it also extends to the state of mato grosso, as well as bolivia, colombia, ecuador and peru. there is debate between brazilian and peruvian scientists about its origin (cavalcante, 1991). female buriti palm trees produce four to eight infructescences, and each raceme bears 500-2000 fruits (goulding and smith, 2007). the fruit is a reddish-brown drupe, with a thin oily yellow-orange pulp that surrounds a relatively large seed (manzi and coomes, 2009). the oil contains tocopherols (de frança et al., 1999; albuquerque et al., 2005), carotenoids (mariath et al., 1989; silva et al., 2009) and pro-vitamin a (mariath et al., 1989; klemm et al., 2008). moreover, candy made from buriti is an effective treatment of xerophthalmia in children in northeastern brazil, but the fruit used to make it is not easily preserved. it is good for no more than two to three days under ambient conditions. the buriti fruit has a hard, red shell that covers an oily pulp that contains carotenoids and ascorbic acid (embrapa, 2007; silva et al., 2007). the local population collects the fruits when they are released from the mother plant and most of the fruits on the ground are near the desired state of maturity. a 55-kg bunch produces 40 kg of fruits. the local population collects the fruits that they are going to use from the ground. buriti and products made from it are widespread in the brazilian cerrado. the fruit pulp is used to make a flour for a porridge that helps meet the nutritional requirements of the locals (almeida and silva, 1994). the fruit is also a source of vegetable oil, as described by albuquerque et al. (2003). these authors obtained an ir spectrum of the oil that revealed the presence of triolein, the triglyceride of oleic acid, which could be used to control cholesterol in the blood. the oil has been reported to have a relatively high concentration of so-called monounsaturated fatty acids (de frança et al., 1999), although it really contains fatty acyls that are a part of mono-, diand triglycerides. that is, the oil had to be hydrolyzed to break down the mono-, diand triglycerides, forming glycerol plus non-volatile free fatty acids, which are then converted to volatile fatty acid methyl esters and analyzed by gas chromatography (de frança et al., 1999; aoac, 2003). in order for the fruits to be used, they must be preserved properly. this is discussed in the next paragraph. usually the fruits are collected in a form that is not easily preserved. the palm trees are cut to facilitate the removal of the fruits, drastically reducing the population of buriti trees in the amazonian region of peru. this forces the harvesters to travel long distances to gather a significant quantity. the management of buriti via extractavism is small compared to the demands of the regional market (manzi and coomes, 2009; horn et al., 2012). extractavism refers to natural tropical forest areas that are reserved for the extraction of potentially renewable commercial forest products. so, better storage methods are needed to meet the needs of the market and provide social benefits, which are described next. buriti offers social benefits to the local population as a source of wealth and employment in the manufacture of products such as licorice, wine, candy, juice and sorbets. refrigerated storage of the fruits of buriti (mauritia flexuosa l.) e. fujita*, r.l. vieites*, é.r. daiuto*, r.e. smith**(1) * unesp botucatu, faculty of agronomic sciences, unesp, c.p. 237, botucatu 18610307, sp, brasil. ** fda, 11510 w 80th street, lenexa, ks 66224, kansas, usa. key words: brazilian cerrado, buriti, mauritia flexuosa, post-harvest. abstract: the objective of this study was to evaluate different storage conditions to maximize the shelf-life of buriti fruits; under ambient conditions the fruits last only 2-3 days. buriti fruits were stored refrigerated at 10, 12 and 15oc with 85±5% relative humidity, and at room temperature (23±5°c) and 60±5% relative humidity. fruits were analyzed every three days over a 12-day period for weight loss, respiratory activity, soluble solids, ph, titratable acidity, lipids, protein and fiber. under the considered conditions, refrigeration at 15oc was found to give the best results. adv. hort. sci., 2014 28(1): 3-8 (1) corresponding author: robert.smith@fda.hhs.gov received for publication 16 september 2013 accepted for publication 10 december 2013 4 however, these activities have not been given the technical or scientific support needed to make them sustainable and more profitable. while an adequate storage method has not yet been well defined, it is known that the fruit must be scraped off the seeds and dehydrated at room temperature, followed by refrigeration for an undetermined period of time (almeida and silva, 1994). therefore, the objective of this study was to evaluate different storage conditions for buriti fruits in order to find the best way to maximize shelf-life with attention to the post-harvest behavior of the fruits so as to maintain their quality. 2. materials and methods the fruits used in the study were from the ecological preserve in jalapão, near the city of dianópolis in the state of tocantins at 11°37’40” south latitude and 46°49’14” west longitude at an altitude of 691 m above sea level, where the trees grow in sandy soil. the fruits were picked from the trees when they were 3-4 cm in diameter and dark yellow to brown. the trees were not cut. due to the height of the tree (10 m), a scaffolding was erected so that the bunches of fruit could be removed with the utmost caution, to prevent damage caused by falling. the bunches were placed in a ventilated polystyrene box to maintain a temperature of 16°c. after collection, the fruits were sent to the laboratório de frutas e hortaliças do departamento de gestão e tecnologia agroindustrial da faculdade de ciências agronômicas – unesp, botucatu, são paulo, where they arrived after two days. they were separated randomly into four lots, each containing ten fruits. three were stored under refrigeration at 10, 12 and 15°c and 85±5% relative humidity. the fourth lot (control) was stored under ambient conditions (23±5°c and 60±5% relative humidity). the following analyses were carried out on ten fruits for each storage condition: weight loss, respiratory activity, soluble solids, titratable acidity, and ph. analyses were carried out for fruits that were viable for commercialization. respiratory activity was determined by the release of co 2 in each package according to the method of bleinroth et al. (1976), using a saturated solution of barium hydroxide and 0.1 n koh (0.1 normal koh, which is the same as 1 mol/l koh) and using the formula: tco 2 = 2.2 (vo-v 1 ) 10 m t where: tco 2 is the rate of respiration (ml of co 2 kg-1h-1); vo = ml of hcl needed to titrate the koh as a standard before the absorption of co 2 ; v 1 = ml of hcl needed to titrate the koh after the absorption of co 2 ; m = mass of the fruits; t = respiration time; 2.2 = equivalent weight of co 2 (44/2), multiplied by the concentration of hcl; 10 = adjustment for the total amount of koh used. soluble solids, ph and titratable acidity were determined by the method of the instituto adolf lutz (ial, 2008). soluble solids were measured with a digital palette pr 32 refractometer (atago inc., bellevue, wa), equipped with automatic temperature compensation. results were expressed directly in obrix. the ph was measured with a ph meter and titratable acidity was measured by titrating the acidic fruits with 0.1 mol/l naoh. the amount of total sugars, lipids, proteins and fibers were determined using the method of somogyi (1945) and nelson (1944) by reacting samples with the somogyi reagent and measuring the absorbance at 535 nm using a micronal b382 spectrophotometer (micronal, são paulo, sp, brazil). in detail, samples were diluted sufficiently so that an absorbance between 0.2 and 0.8 was produced after reacting 1 ml of neutralized and filtered sample with 1 ml of somogyi reagent. after putting the samples in a boiling water bath for 10 min, they were cooled to room temperature. then, 1 ml of the nelson reagent and 7 ml of water were added. finally, the absorbance at 535 nm was read. the somogyi reagent is an arsenomolybdate complex formed by the reaction of ammonium molybdate with sodium arsenate. the nelson reagent was made of two parts: part a contained 2.5 g each of na 2 co 3 and potassium sodium tartrate, 2 g each of nahco 3 and na 2 so 4 in 100 ml water. part b contained 7.5 g cuso 4 ·5h 2 o per 100 ml water, acidified with a drop of conc h 2 so 4 . glucose was used to construct a calibration curve. total lipids were determined by performing a 2-h soxhlet extraction on 3 g of sample using 200 ml of petroleum ether, evaporating off the solvent and weighing the residue. protein was determined on 0.1 to 0.2 g of sample using the kjeldahl method using a conversion factor of 6.5 to convert percent nitrogen to percent protein. the experimental data were analyzed as a 4x5 matrix (temperature x time) by the sisvar 4.6 program. averages were evaluated by the tukey test at 5% probability (gomes, 1987). 3. results and discussion beginning on the third day of storage, a weight loss of >10% was found in all storage conditions. the fruits stored at 10 and 23oc had the greatest loss of mass after 12 days. the lowest loss was at 15oc (table 1). all weight losses were calculated by comparing weights to day zero. ten fruits were analyzed in each experiment. according to finger and vieira (2002) the weight loss of most fresh fruits should be 5-10% to avoid withering or wrinkling. thus, buriti fruits examined suffered a weight loss that reduced their commercial value. 5 buriti fruits demonstrated respiratory behavior that is characteristic of climacteric fruits, as shown in table 2. according to chitarra and chitarra (2005) they are characterized by a rapid increase in respiration and ethylene production during ripening. the apparent peak at day three of storage was probably due to an adaptation of the fruits to the storage conditions. the fruits that were kept at ambient temperature had their peak respiration on the sixth day of storage, while the fruits stored under other conditions had their peak respiration on the ninth day of storage. the lowest respiratory activity (lowest production of co 2 ) was found in fruits stored at 10ºc, i.e. with the lowest production of co 2 . therefore, a temperature of 15ºc proved to be the most effective for storing the fruits as it resulted in the lowest loss of weight and the latest peak in respiratory activity. the amounts of soluble solids, ph and titratable acidity are shown in table 3. table 1 weights of buriti when stored refrigerated temperature initial wt. (g) 3 days (g) 6 days (g) 9 days (g) 12 days (g) 10°c 263.03±7.81 a 238.43±5.74 a 214.50±6.78 a 197.95±3.98 a 191.60±2.45 b 12°c 256.32±6.82 a 237.31±4.81 a 222.10 ±4.91 c 212.56±4.89 c 208.59± 3.99 c 15°c 261.23±5.73 a 245.60±5.97 b 233.65±5.60 d 226.13±6.71 d 223.12± 4.58 d ambient 274.75±6.99 b 251.80±6.52 c 229.53±4.57 b 207.65± 5.86 b 194.47±4.98 a averages followed by the same lower case letter do not differ significantly from others in the same column and those followed by the same upper case letter do not differ significantly from others in the same row by the tukey test at 5% probability. table 2 respiratory activity in buriti fruits stored under refrigeration and 80 ± 5% relative humidity for 12 days respiratory activity temperature days of storage 0 3 6 9 12 10°c 24.5±0.64 35.1±1.90 18.5±0.99 35.5±0.42 13.8±0.64 12°c 24.5±0.64 12.1±0.14 17.3±0.42 58.6±2.62 44.2±3.11 15°c 24.5±0.64 31.4±0.35 17.9±0.78 70.9±0.28 36.8±0.64 ambient 24.5±0.64 54.2±0.28 55.6±0.07 50.8±0.35 averages followed by the same lower case letter do not differ significantly from others in the same column and those followed by the same upper case letter do not differ significantly from others in the same row by the tukey test at 5% probability. table 3 soluble solids (°brix), ph and titratable acidity (g of acid 100g-1 fresh weight) in buriti fruits under refrigerated storage (80±5% relative humidity) and ambient conditions (23±5ºc and 60±5% relative humidity) temperature days of storage 0 3 6 9 12 soluble solids 10°c 12.37±1.51 ab 14.67±0.58 aab 16.33±2.52 aa 15.00±1.73 aab 16.00±0.00 aa 12°c 12.37 ±1.51 aa 14.67±0,58 aa 14.00±0 aba 13.66±0.58 aa 14.67±1.53 aa 15°c 12.37 ±1.51 ab 14.67±1.53 aab 13.33 ±2.08 bcab 12.67± 0.58 ab 16.00 ±0.0 aa ambient 12.37±1.51 aa 10.33±0,58 bb 10.80±0.00 cb c.v. (%) 9.37 ph 10°c 3.83± 0.06 ac 3.77± 0.58 abc 4.33±0.15 aab 4.57±0.06 aa 4.1±0.00 abb 12°c 3.83 ±0.06ac abc 3.73±0.58 abc 4.07 ±0.15 bab 3.93± 0.23 bbc 4.23±0.25 aa 15°c 3.83± 0.06ac abc 3.67±1.53 bc 4.13±0.06 aba 4.00±0.10 bab 3.97±0.12b ab ambient 3.83±0.06ac ab 3.90±0.58 aab 4.10 ±0.0 ba c.v. (%) 2.63 titratable acidity 10°c 0.68±0.06 ab 0.84±0.02 aa 0.41±0.02 ac 0.47±0.07 bc 0.60±0.00 ab 12°c 0.68 ±0.06 ab 0.84±0.02 aa 0.51 ±0.03 ac 0.60 ±0.04 abc 0.52±0.05 ac 15°c 0.68 ±0.06 aab 0.76 ±0.02 aa 0.49±0.02 ac 0.58±0.04 abbc 0.60 ±0.01 aab ambient 0.68± 0.06 aa 0.63±0.02 ba 0.42±0.00 ab c.v. (%) 9.54 averages followed by the same lower case letter do not differ significantly from others in the same column and those followed by the same upper case letter do not differ significantly from others in the same row by the tukey test at 5% probability. 6 the amount of soluble solids did not show a significant difference when stored at 10, 12 or 15°c. the fruits stored under ambient conditions exhibited a decrease in soluble solids on day three, but it dropped no further on day six. on the ninth day, contamination by the fungus monilinia fructicola was observed and this made the fruits unsuitable for consumption. the fruits stored under ambient conditions maintained a relatively low amount of soluble solids during the experimental period, possibly due to the lower respiratory rate compared to refrigerated storage. the small increase in the concentration of soluble solids at the end of the experiment could be related to the weight loss. on the third day of storage, there was a tendency for the ph to increase, as shown in table 3. in the case of ambient storage, this can be due to the process of senescence (chitarra and chitarra, 2005). the increase in ph was seen throughout the experiment. when stored at 10°c, the ph was nearly constant on day three, but increased on days six and nine, followed by a decrease on day 12. when stored under ambient conditions, the ph increased until the last day of analysis. the titratable acidity increased on day three under all storage conditions, followed by a decrease on subsequent days. ambient storage presented the lowest amount of titratable acidity, probably due to infestation by pathogens that consumed acid in their metabolism (chitarra and chitarra, 2005; özcan and haciseferogullari, 2007). the data on total sugars, lipids and protein are presented in table 4. albuquerque et al. (2005) reported levels of total sugars in buriti that varied by about 2.10%, similar to what we found. moreover, hiane et al. (1992) reported values of 11.36% ± 1.81, which are higher than those found in the present experiment. the amount of total sugars increased from day zero to the third day of storage, followed by a decrease on the sixth day and increases on subsequent days. carbohydrates are oxidized by the respiratory process (chitarra and chitarra, 2005; rodriguez-guisado et al., 2009), causing the decrease. the increase in concentration on later days was probably related to the loss of weight. according to cavalcante (1991), buriti fruit contains a relatively large amount of lipids, which are an important source of energy. this was also reported by de frança et al. (1999), albuquerque et al. (2005), and silva et al. (2009), rodrigues et al. (2010). however, all these authors reported finding free fatty acids, when they were most likely fatty acyls as a part of mono-, diand triglycerides. table 4 amounts of total sugars, lipids, protein and fiber (%) in buriti fruits under refrigerated storage (80±5% relative humidity) and ambient conditions (23±5ºc and 60±5% relative humidity) for 12 days temperature storage days 0 3 6 9 12 total sugars 10°c 2.22 ab 3.25 aa 0.91 ac 1.29 bc 2.72 aab 12°c 2.22 ab 2.93 aa 0.80 ac 2.69 aab 2.42 aab 15°c 2.22 ab 3.09 aa 0.83 ac 2.40 ab 2.68 aab ambient 2.22 ab 3.07 aa 0.96 ac c.v. (%) 12.64 lipids 10°c 14.00 ac 18.67 ab 18.53 ab 14.80 cc 21.00 aa 12°c 14.00 ac 18.13 ab 17.30 abb 18.23 ab 21.33 aa 15°c 14.00 ac 15.67 bb 16.93 abb 16.70 bb 20.47 aa ambient 14.00 ab 13.30 cb 18.00 ba c.v. (%) 3.65 protein 10°c 0.26 ad 0.26 ab 0.35 ab 0.37 ac 0.33 aa 12°c 0.26 ac 0.25 ab 0.21 cb 0.29 bb 0.31 ba 15°c 0.26 ab 0.21 bb 0.35 ab 0.27 bb 0.26 ca ambient 0.26 ab 0.22 bb 0.29 ba c.v. (%) 2.75 fiber 10°c 10.43 ac 10.73 abc 10.10 bc 11.30 ab 13.00 aa 12°c 10.43 ac 9.60 bd 10.37 abcd 11.70 ab 12.67 aa 15°c 10.43 ac 8.50 cd 10.90 ac 11.53 aab 11.83 ba ambient 10.43 aa 8.70 cb 10.50 aba c.v. (%) 3.37 averages followed by the same lower case letter do not differ significantly from others in the same column and those followed by the same upper case letter do not differ significantly from others in the same row by the tukey test at 5% probability. 7 this is a common mistake which is made when fatty acyl amounts are determined by gas chromatography (gc) only after hydrolyzing the glycerides. this forms free fatty acids, which are then esterified to form fatty acid methyl esters (fames) that are volatile enough to be analyzed by gc. therefore, a clever marketing or sales representative from producers of a competing company could say that all these studies demonstrate that buriti fruits rapidly turn rancid, since free fatty acids were supposedly found. when oils (glycerides) turn rancid, it is due to partial hydrolysis of glycerides to form malodorous free fatty acids. the article by silva et al. (2009) is especially confusing, since it reports actually finding 3.1% free fatty acids in buriti oil, based on their separation by size exclusion chromatography: it is not clear whether the authors really meant free fatty acids or fatty acyls that are part of triglycerides. they go on to report the profile of free fatty acids, but it is based on gc analysis of fames, so it refers to the fatty acyl profile of mono-, diand triglycerides. vegetable oils contain mono-, diand triglycerides, and therefore the analysis method requires that they be hydrolyzed into free fatty acids and glycerol, followed by forming volatile fatty acid methyl esters (fames), which can be analyzed by gas chromatography (mannina et al., 1999; aoac, 2003). it is not clear whether the 3.1% free fatty acids separated by size exclusion chromatography were part of glycerides or if they were truly free fatty acids, caused by the oil turning rancid. however, the present experiment truly measured total lipids and found more than the 2.5 to 5.5% reported by hiane et al. (1992) and donadio et al. (2002). albuquerque et al. (2005) reported finding 11.24% lipids. carneiro and carneiro (2011) found 18.16% lipids in buriti pulp. the amount of lipids increased starting on the third day of storage and stayed almost constant throughout the 12-day experiment (table 4). on the third day, the fruits stored at 10 and 12°c showed no significant differences, but more lipids were found when stored at 15°c. the fruits stored under ambient conditions had fewer lipids that the fruits stored under refrigeration. on day 12 there were no significant differences in lipid content in the refrigerated samples. the increase in lipid concentration with time could be related to the weight loss that occurred. according to donadio et al. (2002) buriti fruits had about 2.3 to 5.5% protein, while hiane et al. (1992) found 2.12%, carneiro and carneiro (2011) found 1.30% and darnet et al. (2011) found 3.7%. in the present study, there were no significant differences in protein concentrations in fruits stored at 10 and 12°c, but these values were higher than those of fruits stored at 15°c and under ambient conditions, which were not statistically different from each other. on the sixth day of storage, there was an increase in protein concentration, with the exception of those stored at 12°c. on the ninth day, the concentration of protein increased in the fruits stored at 10 and 12°c, while those stored at 15°c showed a decrease. donadio et al. (2002) found that the fiber content in buriti fruits varied from 10.4 to 27.5%. hiane et al. (1992) reported about 12.31% fiber. darnet et al. (2011) found 22.8% dietary fiber in buriti fruits from the amazon. the concentrations of fiber found in the current study ranged from 8.5 to 13.0%: on the third day, the fruits stored at 10°c had the highest concentration of fiber, followed by those stored at 12°c; those stored at 15°c and under ambient conditions had the lowest concentrations and were not significantly different from each other. starting on the third day there was an increase in the concentration of fiber which then continued slowly throughout the 12-day experiment. the increase could be simply due to the loss of weight, so the total amounts were about the same. 4. conclusions refrigeration was effective in extending the shelf-life of buriti fruits, increasing it by at least three days. the data presented regarding the decrease in weight and respiratory activity demonstrate that a temperature of 15°c was the most effective in maintaining the quality of buriti fruits. this work should not be taken as reflecting fda 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a.j.a., 2009 characterization of oil extracted from buriti fruit (mauritia flexuosa) grown in the brazilian amazon region. j. amer. oil chem. soc., 86(7): 611-616. somogyi m., 1945 determination of blood sugar. j. biol. chem., 160: 69-73. 105 1. introduction in recent years, malignant neoplasm (cancer), cerebrovascular disease and heart disease have become major causes of death among japanese people, with approximately 75% of deaths in japan caused by these diseases (japanese ministry of health, labour and welfare, 2009 a). such diseases are generally called lifestyle diseases because they are thought to be strongly related to a lack of exercise and high intake of fat and salt. therefore, emphasis has shifted from early diagnosis (secondary prevention) to lifestyle improvement (primary prevention) (japanese ministry of health, labour and welfare, 2009 b). in this context, research on foods that have pharmacological effects or physiological functions, such as disease prevention and health maintenance, has become more important (namba, 1999; tokui et al., 2003). regarding the pharmacological effects of food, there is a form of medicinal cooking called “yakuzen” which is based on the philosophy of oriental medicine and is intended to maintain good health and improve physical condition. the preparation of yakuzen dishes draws from the theory of “yaku-shoku dou-gen”, which means that the same principle underlies the daily diet and medical treatment, and on the yin-yang theory, the five-phase theory in oriental medicine. as a result, yakuzen has attracted considerable attention for the prevention of lifestyle diseases. to prepare yakuzen dishes, in-season materials with appropriate properties and tastes are selected according to the health status and constitution of each person (namba, 1995; lan et al., 2002; tokui et al., 2003). these properties and tastes are called “sei-mi” in yakuzen theory. sei-mi consists of four properties (making the human body hot, warm, cool, or cold) and five tastes (salty, bitter, sweet, pungent, and sour), and each is considered to have its own function in the human body (namba, 1999; tokui et al., 2003). if the concept of sei-mi can be applied to vegetables, a cultivar that has a strong flavor and a high content of functional constituents related to the properties and taste is considered to have strong sei-mi and is suitable for yakuzen dishes. however, the inherent flavor of vegetable cultivars has been weakened by breeding because priority has been given to ease of consumption for consumers or ease of production for growers. in the case of eggplant (solanum melongena l.), popular cultivars in japan today are f 1 (first filial generation) cultivars derived from a parental line with oval fruit, a deep purple pericarp, and high yield; these cultivars have improved fruit quality with less unpleasant or harsh taste. evaluation of local eggplant cultivars in terms of the suitability as materials for “yakuzen” dishes s. tsukagoshi* (1), k. kuroda**, m. hohjo*, f. ikegami*, n. kunisaki***, t. hanamura***, k. yamada***, t. hagiwara*** * center for environment, health and field sciences, chiba university, 6-2-1 kashiwanoha, kashiwa, chiba 277-0882, japan. ** faculty of horticulture, chiba university, 648 matsudo, matsudo, chiba 271-8510, japan. *** research and development department, nichirei foods inc., 9 shinminato, mihama-ku, chiba 2610002, japan. key words: functionality, local cultivars, property and taste, solanum melongena. abstract: “yakuzen” is a form of medicinal cooking based on the theories of oriental medicine. to prepare yakuzen dishes, in-season materials with appropriate properties and tastes, “sei-mi,” are selected according to the health status and constitution of each person. in this study, the suitability of eggplant (solanum melongena l.) cultivars for yakuzen was evaluated by sensory tests and by analysis of the functional constituents considered to be closely related to the taste and functionality of yakuzen dishes. twenty-two eggplant cultivars including 21 japanese and 1 italian cultivar, and a thai species (solanum xanthocarpum schrad & wendl.) were evaluated. principal component analysis (pca) was used for comprehensive evaluation among the cultivars. from the pca, many of the cultivars with round or oval fruit were characterized as juicy and sweet and considered easy to eat; most of the long-fruit cultivars were characterized as having higher specific amino acid contents. the small and round fruit cultivar dewako and the thai species (makhuea pro) were considered to contain many functional ingredients, such as ash, polyphenols, and specific amino acids, and to have higher suitability for yakuzen dishes. adv. hort. sci., 2014 28(2): 105-110 (1) corresponding author: tsukag@faculty.chiba-u.jp received for publication 31 march 2014 accepted for publication 07 july 2014 106 however, eggplant was introduced into japan more than 1,260 years ago, and it is mentioned in nara-period documents (the shõsõin documents) edited in 750 a.d. (yoshida, 2010). therefore, many cultivars have been developed over the long history of cultivation, and even today there are many cultivars with local origins. the chemical composition, flavor, and texture of the edible parts of plants vary among cultivars. local cultivars grown for many years may have retained more flavor, and some of them may be more suitable for yakuzen dishes than the currently popular cultivars. regarding the functionality of foods, many studies have focused on certain ingredients and have discussed the relationship between the amounts of those ingredients and the functionality of the food. however, it is important to consider the functionality of foods comprehensively in yakuzen theory. in previous studies, we used principal component analysis (pca) to comprehensively evaluate the functionality of local cultivars, and we demonstrated that two local cultivars of japanese radish (raphanus sativus l.), two of carrot (daucus carota l.), and one of bitter melon (momordica charantia l.) were more suitable for yakuzen dishes than the widely used f 1 cultivar (saito et al., 2010; tsukagoshi, et al., 2011 a, b). in this study, two f 1 cultivars and 21 local cultivars of eggplant were evaluated for their suitability as materials for yakuzen dishes according to their taste and content of functional constituents. 2. materials and methods plant materials and growing condition twenty-one japanese eggplant cultivars with different fruit shapes were selected for this study (table 1). most of them were purebred cultivars, but two f 1 cultivars were included. in addition, an italian cultivar and a thai species (solanum xanthocarpum schrad & wendl.) were used. all cultivars were grown at the center for environment, health and field sciences, chiba university. seeds of all cultivars were sown in 9-cm plastic pots filled with upland soil on 17 april 2012, and the seedlings were raised in a glasshouse. on 5 june seedlings were transplanted to the open field at a spacing of 50 cm between plants and 100 cm between rows. fruits were harvested when they reached the regular size for each cultivar. harvest began on 31 july and ended on 14 september. all other management was carried out according to the conventional methods in japan (chino, 2001). sensory test fruits were harvested on 7 and 9 august and the sensory test was conducted the following day. the fruits were washed, cut to a size of 1.5×1.5×3.0 cm, and steamed at 100°c for 5 min. the characteristics of steamed samples were evaluated by six panelists. the panelists passed recognition tests for five tastes (sweetness, umami, saltiness, table 1 eggplant cultivars used for the experiment fruit shape (country) cultivar abbreviation fruit length, weight and color remark small, round (japan) dewako de 3-8 cm, 10-15 g, deep purple minden min 3-8 cm, 10-15 g, deep purple round (japan) aizu maru am 8-10 cm, 200-300g, deep purple   kamo nasu kn 12-15 cm, 200-350 g, deep purple tonosama to approx. 15 cm, 300-450 g, deep purple yamatoyo maru ym 10-12 cm, 250-350g, deep purple money pouch (japan) nagaoka kinchaku nk 8-10 cm, 300-350g, deep purple   (swelling toward the bottom) saitama ao daimaru sa approx. 15 cm, 300-450 g, green oval (japan) heta murasaki hm approx. 5 cm, 30 g, deep purple   senryo 2 gou se2 10-12 cm, 80-90 g, deep purple f 1 , control cultivar in this experiment se2 (control) sk 10-11 cm, 150-180 g, deep purple wase shinkuro ws 10-12cm, 80-120 g, deep purple yamashina ya 10-12cm, 80-120 g, deep purple long (japan) chikuyou chi 20-25 cm, 120-150 g, deep purple f 1 hakata naga hak 40-45 cm, 200-300 g, deep purple hhogo naga hho 17-18 cm, 85-90 g, deep purple himo nasu hn 25-30 cm, 150-200 g, pale purple kitta chunaga kc 10-12cm, 80-120 g, deep purple kurume oh naga ko 30-35 cm, 250-300 g, deep purple shikon sendai naga ss 8-10 cm, 20-30 g, bluish purple shin nagasaki naga sn 35-40 cm, approx. 250 g, deep purple big, oval (italy) zebra ze 20-25 cm, 300-400 g, purple and white stripes small, round (thailand) makhuea pro mp 4-5 cm, approx.10 g, green solanum xanthocarpum 107 bitterness, and sourness) and the discrimination tests for four solutes (sucrose, sodium chloride, tartaric acid, and sodium glutamate). in addition, they had more than one year experience in evaluating vegetables and were classified as expert assessors (japanese society for sensory evaluation, 2009). the characteristics listed in table 2 were evaluated on a scale of -5 (weaker) to 5 (stronger) compared to se2, the control cultivar in this study. taste sensor analysis and amino acid content approximately 50 g of fresh fruit was homogenized in 100 ml of water in an ice bath, then filtered through cotton cloth. although filtrates were prepared separately from three or four fruit samples, the filtrates were mixed to obtain the quantity necessary for measurements. bitterness, astringency, acridity, and pungency of the filtrates were measured using a taste sensor system (sa402b, intelligent sensor technology, kanagawa, japan). each value was expressed relative to the control cultivar (se2), which was set at zero. a portion of each filtrate was filtered again through a 0.45μm filter (dismic-25cs, advantec, tokyo, japan) and the amino acid content was measured using an amino acid analyzer (jlc-500/v, jeol, tokyo, japan). soluble solids content fresh fruit was cut into small pieces and pressed in gauze to extract the juice. the soluble solids content of the juice was measured using a refractometer (pal-1, atago, tokyo, japan) and expressed as percent brix. ash and polyphenol contents the harvested fruit was stored at -30°c until use. fresh-frozen fruit samples were freeze-dried and ground into a fine powder. ash content was determined using the dry ashing method. briefly, 0.3 g of the powder was put in a crucible and ashed at 550°c for 24-48 hr. after cooling, the weight of the residue was measured. polyphenol content was determined using iron tartrate spectrophotometry. first, 0.2 g of the powder was mixed with 10 ml of distilled water, and shaken for 10 min at 80°c. after cooling, the sample was centrifuged at 3,000 rpm for 15 min. then, 3.2 ml of the supernatant was mixed with 1.6 ml of iron tartrate reagent (0.1% (w/v) ferrous sulfate and 0.5% (w/v) potassium sodium tartrate) and 3.2 ml of phosphate buffer (0.1 m, ph7.5). the absorbance at 540 nm was then measured using a spectrophotometer (u-2000, hitachi, tokyo, japan). the polyphenol content was calculated from a table 2 evaluation of the taste of eggplant cultivars by sensory test (z) fruit shape cultivar aroma   softness juiciness sweetness bitterness astringency     good grassy   pericarp flesh       & acridity small, round de (y) 0 (x) 0 -1 0 0 0 1 1 min 1 1   0 -1 0 -1 2 1 round am 0 1 0 -1 0 0 0 1 kn 1 1 0 -1 0 0 0 0 to 0 0 -1 0 0 0 1 0 ym 0 0   0 1 1 1 -1 0 money pouch nk 0 2 -3 -2 -1 -1 0 0   sa 0 2   -4 0 -1 0 0 1 oval hm 0 0 0 1 0 0 0 0 se2 (control) 0 0 0 0 0 0 0 0 sk 0 2 0 1 2 1 0 0 ws 0 0 -1 0 -1 -1 0 0 ya 1 0   -1 0 0 0 0 0 long chi 0 0 0 1 0 -1 0 0 hak 0 0 1 1 -1 0 -1 0 hho 0 0 0 0 0 0 0 0 hn 0 1 -1 1 -1 -1 0 0 kc 0 0 -2 1 0 -1 0 1 ko 0 0 -2 1 -1 -1 0 0 ss 0 1 -1 0 0 -1 0 1 sn 0 2 0 1 -2 0 -1 0 big, oval ze 2 2   0 -2 0 -1 0 2 small, round mp 0 3   0 -1 0 -1 0 1 (z) eggplant fruit was cut to the size of 1.5 x 1.5 x 3.0 cm, then steamed at 100 degree c for 5 min before the test. (y) amino acid which is considered to be important for the functionality of eggplant in yakuzen theory. (x) tastes were evaluated on a scale of -5 (weaker) to 5 (stronger) as compared to senryo 2 gou (se2). 108 standard curve of ethyl gallate. principal component analysis and characterization of cultivars data were analyzed by principal component analysis (spss for windows version 13), and the characteristics of cultivars were comprehensively evaluated to determine the suitability of the cultivars as materials for yakuzen dishes. 3. results and discussion cultivars nk and sa (with money-pouch fruit shape) tended to have harder fruits, and long-fruit cultivars tended to be less sweet (table 2). the aroma of the italian cultivar, ze was characterized as both “good” and “grassy”. however, most cultivars were very similar to se2 (the control cultivar in this study) in the sensory test. taste sensor analysis showed that the local cultivars tended to have a less unpleasant taste than se2 (table 3). this result did not correspond to the results of the sensory test, and the difference may be due to the heating of samples before the sensory test but not before the taste sensor analysis. nevertheless, we can conclude that the local cultivars were not unpalatable compared with the commonly used cultivar. min, ym and some other cultivars tended to have higher soluble solids contents, but there was no significant difference between cultivars (table 4). “mi” (the taste) of eggplant is “kan” (sweet). in yakuzen theory, mi means not only the taste on the tongue but also specific functions in the human body (tokui et al., 2003). in this study, we could not discern differences of kan characteristics from the results of sensory test and soluble solids contents among cultivars; therefore, the suitability of cultivars was evaluated on the basis of other characteristics. ash content was higher in cultivars de and min (both of which have small, round fruit) and lower in cultivar ws, and tended to be lower in cultivars with oval fruit. “sei” (the property) of eggplant is to cool the human body. potassium accounts for most of the ash of eggplant fruit (usda, 2013), and the function of this mineral is to release heat inducing diuresis. this function is closely related to the property of eggplant, and higher ash content may be related to greater suitability of cultivars for yakuzen dishes. polyphenol content was also higher in de and min. eggplant contains polyphenols such as chlorogenic acid and nasunin, which are considered to have antioxidant activity, and to suppress lipid peroxidation, aging, various lifestyle diseases, and cancer (kimura et al., 1999; noda et al., 2000; kitsuda et al., 2005; singh et al. 2009). das et al. (2011) reported that grilled eggplant had a higher polyphenol content, though the cardioprotective ability was not different. the high polyphenol content in de and min may increase the pharmacological value of these cultivars. as mentioned above, mi (the taste) also encompasses specific functions in the human body, and mi of eggplant is kan (sweet). kan is considered to have functions such as supplying nutrition and energy, promoting relaxation, etc. some amino acids are considered to have kan functions. for example, glutamine is an energy source for digestion and plays an important role in the maintenance and improvement of immunity and the repair of organs (ajinomoto co. inc., 2003 a, b), and this may correspond to a kan function. alanine has a sweet taste and supplies sugars to the body, and it is also considered to have a kan function. the amino acids strongly related to the kan of eggplant include alanine, citrulline, glycine, glutamine, proline and serine. therefore, these amino acid contents were summed to give specific amino acid content (table 4): it was highest in se2 and sn and tended to be higher in the long-fruit cultivars and lower in the round-fruit cultivars. total amino acid content also tended to be higher in the long-fruit cultivars, especially in sn. however, no other trends in amino acid content were observed. the higher content of specific and total amino acids in sn would indicate greater suitability of this cultivar for yakuzen dishes. table 3 evaluation of the taste of eggplant cultivars by taste sensor (z) fruit shape cultivar bitterness astringency acridity pungency small, round de (y) -0.27 (x) 0.06 -1.74 -0.69 min -0.38 0.04 -2.27 -0.96 round am -0.23 0.20 -1.14 -0.48 kn -0.11 0.21 -2.11 -0.94 to -0.11 0.13 -2.18 -1.18 ym -0.16 -0.02 -1.33 -1.30 money pouch nk -0.26 0.27 -1.78 -0.64   sa -0.07 0.30 -2.30 -1.07 oval hm 0.22 0.13 -1.83 -0.99 se2 (control) 0.00 0.00 0.00 0.00 sk -0.28 0.05 -1.51 -0.76 ws -0.06 0.23 -2.26 -1.17 ya -0.09 0.08 -2.57 -1.30 long chi -0.33 0.12 -1.39 -0.61 hak -0.28 -0.09 -2.55 -1.40 hho -0.15 0.05 -1.61 -0.86 hn -0.01 0.15 -2.87 -1.45 kc -0.12 0.11 -2.24 -1.28 ko -0.43 0.07 -0.52 -0.15 ss -0.26 0.04 -2.03 -1.00 sn -0.47 -0.05 -2.27 -1.09 big. oval ze -0.32 -0.02 -2.73 -1.23 small. round mp -0.34 0.19 -2.15 -0.75 (z) taste sensor was prepared to express the value of se2 was zero. (y) amino acid which is considered to be important for the functionality of eggplant in yakuzen theory. (x) positive and negative value means the taste was stronger and weaker than se2, respectively. 109 from the pca, 23 principal components (pcs) were derived and 14 pcs were considered to be meaningful (contribution rate>1). these 14 meaningful pcs accounted for 96.6% of the total rate (data not shown). although the first two pcs accounted for only 41.3% of the total rate, a twodimensional scatter diagram of factor loading was constructed. specific and total amino acid contents were in the positive direction along the x-axis, and juiciness, sweetness, percent brix and some unpleasant taste were in the negative direction. ash, polyphenol content, and bitterness were in the positive direction along the y-axis, and softness was in the negative direction (data not shown). a two-dimensional scatter diagram constructed from the pc1 and pc2 scores of each cultivar enabled classification of the cultivars (fig. 1). many of the common f 1 cultivars of eggplant used for commercial production in recent years have been developed to improve ease of consumption and cultivation. se2 is one of the common cultivars in japan. it was characterized as juicy and sweet by the pca and is considered easy to eat. the other cultivars with round or oval fruit were also characterized as juicy and sweet. on the other hand, most of the long-fruit cultivars were distinct from the round-fruit and oval-fruit table 4 soluble solid, ash, polyphenol and amino acid content of eggplant cultivars fruit shape cultivar soluble solids (% brix) ash (g 100 g-1 fw) polyphenol (z) (mg 100 g-1 fw) specific amino acid (y) (mg 100 g-1 fw) total amino acid (mg 100 g-1 fw) small. round de (x) 5.1 a (w) 0.60 a (w) 673.4 a (w) 120.6 284.9 min 5.2 a 0.60 a 434.0 a 71.1 186.0 round am 4.8 a 0.45 ab 169.0 b 37.2 151.2 kn 5.4v 0.53 (v) 150.0v 48.8 203.1 to 4.8 a 0.39 b 253.7 ab 52.5 196.6 ym 5.3 a 0.50 ab 255.0 ab 36.3 144.3 money pouch nk 5.0 a 0.45 ab 172.9 b 63.1 197.8 sa 4.6 a 0.50 ab 247.3 ab 39.5 167.3 oval hm 4.7 a 0.50 ab 300.6 ab 64.5 203.1 se2 (control) 4.6 a 0.44 ab 129.1 b 145.6 302.0 sk 4.3 a 0.39 b 158.4 b 48.0 170.6 ws 5.1 a 0.37 b 181.9 b 52.8 199.1 ya 4.3 a 0.40 b 248.5 ab 58.1 185.4 long chi 4.5 a 0.42 ab 188.1 b 34.1 161.2 hak 4.3 a 0.38 b 127.9 b 116.3 286.5 hho 4.8 a 0.52 ab 166.2 b 76.8 253.3 hn 4.5 a 0.52 ab 144.1 b 83.6 288.6 kc 4.4 a 0.48 ab 236.6 ab 50.8 211.0 ko 5.1 a 0.45 ab 135.1 b 93.6 314.2 ss 4.1 a 0.38 b 250.6 ab 106.1 281.8 sn 4.5 a 0.41 ab 119.2 b 139.2 342.0 big. oval ze 3.8 a 0.46 ab 124.0 b 95.1 308.9 small. round mp 5.1 a 0.56 ab 245.9 ab 63.9 196.7 (z) polyphenol content was expressed as ethyl gallate equivalent. (y) amino acid strongly related to kan of eggplant. (x) abbreviations as in table 1. (w) different letter within the row indicates significant difference by tukey’s multiple range test at 5% level (n=5). (v) number of harvested fruit was not enough for statistical analysis. fig. 1 two dimensional scatter diagram of the principal component score of eggplant cultivars ■: small, round ◆: round ●: monry pouch ▲: oval : long : italy : thailand. abbreviations as in table 1. 110 cultivars. they were characterized as having higher specific amino acid contents and little unpleasant taste or sweetness. the small and round fruit cultivar de and the thai species (mp) were considered to contain many functional ingredients, such as ash, polyphenols, and specific amino acids, and to have greater suitability for yakuzen dishes. 4. conclusions among the local japanese cultivars used in this study, cv. de is highly suitable for yakuzen dishes because it contains many ingredients associated with the properties and taste (sei-mi) of eggplant in yakuzen theory. in addition, the italian cultivar (ze) and the thai species were highly distinct. different results may have been obtained had we grown the cultivars in another area or under different conditions. nevertheless, we have demonstrated that some local eggplant cultivars have stronger sei-mi than current f 1 cultivars. these characteristics could add value to the local cultivars and lead to regional development. references ajinomoto co. inc., 2003 a amino san no hataraki (function of amino acid). amino acid handbook. ajinomoto co. inc., kogyo chousa-kai publishing, tokyo, japan, pp. 20-63. ajinomoto co. inc., 2003 b hinmoku betsu kakuron (detailed explanation of each amino acid). amino acid handbook. ajinomoto co. inc., kogyo chousa-kai publishing, tokyo, japan, pp. 147-174. chino k., 2001 nasu no roji saibai (cultivation method of eggplant in open field). yasai tsukuri no jissai -ka sai 1(practical method of vegetable cultivation fruit vegetables 1). rural culture association japan, tokyo, japan, pp. 80-90. das s., raychaudhuri u., falchi m., bertelli a., braga p.c., das d.k., 2011 cardioprotective properties of raw and cooked eggplant. food funct., 2: 395-399. japanese ministry of health, labour and welfare, 2009 a annual changes in death rate for leading causes of death. health, labour and welfare report 20082009 part 1. japanese ministry of health, labour and welfare, no. 10. japanese ministry of health, labour and welfare, 2009 b changes in national health promotion measures, health, labour and welfare report 2008-2009 part 2. japanese ministry of health, labour and welfare, no. 62. japanese society for sensory evaluation, 2009 panel. kannou hyouka shi text (textbook for sensory evaluator). japanese society for sensory evaluation, kenpakush, tokyo, japan, pp. 49-53. kimura y., araki y., takenaka a., igarashi k., 1999 protective effects of dietary nasunin on paraquat-induced oxidative stress in rats. biosci. biotechnol. biochem., 63: 799-804. kitsuda k., nakamura t., morita n., imahori y., suzuki t., ikeda h., 2005 antioxidative activity in eggplant ‘mizu-nasu’ fruit and its enhancement after injury. hort. res. (japan), 4: 229-232. lan s., sakai e., tanaka t., 2002 the role of “ishokudougen” in japan. a study of “yaku-zen” which is both old and new meal science. a. proc. gifu pharm. univ., 51: 47-53. namba t., 1995 yakuzen to kampo (yakuzen and oriental medicine). farumashia, 31: 19-21. namba t., 1999 yakuzen no genri to shoku/yaku zai no kouyou (1) [principes of yakuzen, and the effect of food and medicine (1)]. j. cookery sci. japan, 32: 374-379. noda y., kaneyuki t., igarashi k., mori a., packer l., 2000 antioxidant activity of nasunin, an anthocyanin in eggplant peels. toxicology, 148: 119-123. saito y., hohjo m., tsukagoshi s., ikegami f., nakao c., hanamura t., yamada k., hagiwara t., 2010 study on the adequacy of vegetables as materials for medicinal foods. 5. comprehensive evaluation of pre-selected momordica charantia l. cultivars by principal component analysis. hort. res., 10 (suppl.1): 481. singh a.p., luthria d., wilson t., vorsa n., singh v., banuelos g.s., pasakdee s., 2009 polyphenols content and antioxidant capacity of eggplant pulp. food chemistry, 114: 955-961. tokui n., minari y., zhang z.l., guo x., 2003 yakuzen no gaiyou (outline of yakuzen). yakuzen to chu-igaku (yakuzen and chinese medicine). kenpakusha, tokyo, japam, pp. 2-32. tsukagoshi s., inuzuka s., hohjo m., ikegami f., takenaga s., nakao c., uryu n., hagiwara t., aoki h., hanamura t., 2011 a evaluation of japanese radish cultivars in terms of medicinal properties for “yakuzen” dishes. hortresearch, 65: 81-86. tsukagoshi s., takano a., hohjo m., ikegami f., hagiwara t., nakao c., yamada k, hanamura t., takenaga s., aoki h., 2011 b evaluation of some local cultivars of carrot in terms of the suitability as materials for “yakuzen” dishes. j. trad. med., 28: 106-114. united states department of agriculture, 2013 national nutrient database for standard reference, release 26. united states department of agriculture, http://ndb.nal. usda.gov/ndb/search/list. yoshida t., 2010 gensan to raireki (origin and history of eggplant). nougyo gijutsu taikei yasai hen (compendium of agriculture, vegetables edition). vol. 5. kisohen (basis). rural culture association japan, tokyo, japan, pp. 3-7. 115 1. introduction mangosteen (garcinia mangostana l.), the queen of tropical fruits (almeda and martin, 1976; ibpgr, 1986; kusumo and verheij, 1994), is a very important crop of the warm humid tropics. the crop like other polyaxial tropical and sub tropical trees such as rubber, mango, cashew and citrus, exhibits a rhythmic growth habit under the relatively constant environmental conditions of the tropics (alvim, 1964; borchert, 1973). it bears profusely and fits very well as a component of home gardens of kerala. though it is a fruit crop with immense potential, both as a monocrop and as a mixed crop in coconut gardens with very high domestic and foreign demand, its cultivation on a commercial basis is limited by its long gestation phase (wiebel et al., 1992 b). the apomictic mangosteen (richards, 1990; normah et al., 1992) seedlings used for commercial planting are extremely slow growing, both at nursery stage and in orchards (hume, 1947; almeda and martin, 1976; wiebel et al., 1991, 1992 a). the slow growth rate and consequent long pre-bearing phase have been a cause for concern wherever mangosteen is grown. though the long gestation period, ranging from 10-15 years (lim, 1984; richards, 1990; wiebel et al., 1995), can be reduced by resorting to vegetative propagation, the problem of slow growth becomes all the more conspicuous (wiebel et al., 1992 b, 1995). this crop lacks root hair (richards, 1990) and this reduced vital link responsible for absorption of nutrients and water may be one of the prime reasons for slow growth. low carbon acquisition capacity and prolonged dormancy of buds at the apex have also been listed as probable causes of the slow growth rate (downton et al., 1990). a critical review of the literature has revealed that only very few references to the crop exist. selection index studies are available in palms but the aims differ as the thrust of the present work is directed toward the growth rate. this study was undertaken with the objectives of identifying some of the basic reasons for the slow growth rate and, secondly, to address this problem by identifying the mother plant, fruit and seed characters in relation to seedling growth and formulating the selection indices at these three basic levels. accelerating the growth rate of mangosteen trees and thereby reducing the gestation period is one of the pre-requisites for an extensive commercialization of this crop. preliminary studies on selection indices for activating seedling growth in mangosteen (garcinia mangostana l.) l.m. yusuf, s. kurien(*) department of pomology and floriculture, kerala agricultural university, kau post, thrissur, kerala, 680656 india. key words: d2 values, dendrogram, discriminant function analysis, multiple regression analysis, principal component analysis, selection indices, garcinia mangostana. abstract: studies on selection indices for activating seedling growth in mangosteen were conducted in the central orchard at the main campus of kerala agricultural university. the present investigation was undertaken with the main aim of identifying some of the basic reasons for slow growth in mangosteen, and to address this problem by developing and identifying criteria to select the age of the mother plant, fruit, seed and seedling characters or direct selection indices at all four stages with respect to seedling growth. mother plants of four distinct age groups were used in the study. variables were generated using all fruit, seed and seedling characters such as fruit index, seed index and seedling index by principal component analysis (pca). using pca and multiple regression analysis, prediction models was fitted for the three indices. major fruit, seed and seedling characters were identified by stepwise regression. hierarchial analysis was performed based on euclidean distance to find the similarities between the four age groups. discriminant function analysis was performed and six discriminant functions were fitted with corresponding d2 values to discriminate the six pairs involving the four age groups of the mother plants. for practical purposes, selection indices and best age group of mother plants are described in the work. adv. hort. sci., 2012 26(3-4): 115-130 (*) corresponding author: sajanalice@gmail.com. received for publication 21 february 2012 accepted for publication 12 september 2012 116 2. materials and methods the materials and methodologies used in this investigation are presented separately. identification of mother plants belonging to various age groups the first experiment on fixation of a selection index of mangosteen (garcinia mangostana l.) was based on the age of the mother plant and on fruit, seed and seedling characters. hence a preliminary survey was conducted in the kerala mangosteen growing tracts during the bearing phase ending with the onset of southwest monsoon. the main aim was to identify plants belonging to the different age groups as envisaged in the study. the required number of plants belonging to different age groups could have been located in different regions, but the study was restricted to pariyaram village of chalakudi taluk as age groups and numbers were available in a compact area. hence the mother plants and fruit collection were centered on this area. furthermore this approach aided in eliminating possible errors from an ecological point of view to the minimum. location of mother trees (pariyaram village) pariyaram village is located at 10° 20’ n latitude and 76° 26’ e longitude, at an altitude of 3.25 m above mean sea level and 5 km (east) of the chalakudi railway station, thrissur district. climate and soil conditions of the area the area receives an average rainfall of 2150 mm distributed over a period of a year. the mean maximum temperature ranges 28-36°c with a mean of 32°c. the mean minimum temperature ranges 12.8 to 20.6°c with a mean of 16.7°c. the relative humidity ranges from 90 to 98% with a mean of 94%. the soil of the area where the mother trees are located is sandy alluvium with ph 5.5 -5.8 and belonging to the order of entisol. the soil is low in available n and p 2 o 5 and high in available k 2 o. once fruit collection and observations were completed, the studies on seed germination and observations on seedling characters were carried out in the central orchard attached to the department of pomology and floriculture, the college of horticulture in the main campus of kerala agricultural university, vellanikkara, thrissur district, kerala. the experiment site experiences a warm humid tropical monsoon climate. it is situated at 12° 32’n latitude and at 74° 20’e longitude at an altitude of 22.5 m above mean sea level. soil type is a typical sandy clay loam with ph 5.4, ec 1.25 dsm-1, and belonging to the order ultisols. mother plant characters trees belonging to various age groups were identified and categorized into the four age groups as envisaged in this investigation: 1. trees having an age of less than 25 years; 2. trees having an age of 25-50 years; 3. trees having an age of 51-75 years; 4. trees having an age of more than 75 years. within each age group, ten fruits per tree were collected from five mother plants randomly, so that each age group had a total of fifty fruits. care was taken to see that the mother plants under selection fell near the mid value of each age group and so that the five plants in a group were more or less uniform in growth characters (height and spread). fruits were labeled individually and seeds were extracted from the fruits. the seeds were sown separately in black polythene bags (45x30 cm) filled with potting mixture made up of farmyard manure, sand and cow dung in the ratio of 2:2:1. the following observations on various fruit, seed and seedling characters of the four age groups (table 1) were recorded separately as presented below. fruit characters fruit weight, fruit girth, fruit volume, pulp weight, rind weight, number of segments, number of seedless fruits, number of one-seeded fruits, number of two-seeded fruits, number of threeor more seeded fruits, number of seedlings produced per fruit and seed specific gravity were recorded. seed characters observations on seed weight, seed length, seed thickness at centre, seed volume, seed specific gravity, number of seeds per fruit, number of seedlings per seed, number of ungerminated seeds, number of seeds producing one seedling, number of seeds producing two or more than two seedlings (table 2), number of days taken to germination, and germination rate were recorded based on ista guidelines (ista, 2003). seedling characters height, girth at collar, total number of leaves, and survival rate of the seedlings at three months, six months, nine months and one-year stage after germination were recorded. increment in height, girth, and total number of leaves at quarterly intervals up to one year were computed. number of new flushes per year, number of leaves/flush, and total leaf area were limited to a twelve-month stage. shoot and root characters shoot and root weight, dry weight, root to shoot dry weight ratio, length of longest root, number of primary, secondary and tertiary roots, and total number of roots were recorded. based on the fruit index, seed index and seedling index the selection indices were determined. biochemical analysis the following biochemical characters were estimated in seeds and leaves of the mother plants and seedlings of the four age groups. however biochemical analyses were restricted to the treatments showing the growth of best, in117 termediate and least categories, in the experiments of activation of seedling growth using growth regulators and a m fungi separately. 1. nitrogen seeds and leaves 2. phosphorus seeds and leaves 3. potassium seeds and leaves 4. protein seeds and leaves 5. sodium seeds and leaves 6. chlorophyll leaves only 7. total sugar content seeds only 8. total carbohydrates seeds and leaves 9. total phenols seeds and leaves 10. abscisic acid seeds and leaves selection index for this experiment, fully matured (dark purple colour) fruits were harvested randomly and the following observations were taken. fruit characters individual fruits were labeled immediately after harvest. fruit weight was then recorded using an electronic balance (contech precision balance) and the average expressed in grams. girth of the fruit was measured using a thread and its length measured using a metre scale and the average expressed in centimeters. fruit volume was measured by water displacement method and the average volume expressed in milliliters. the fruit hull was carefully removed and the weight of the white-segmented pulp (aril) of each fruit with seeds was measured using an electronic balance. seeds were extracted and their weight recorded and the average expressed in grams. pulp weight of each individual fruit alone was calculated using the formula: pulp weight alone (g) = pulp weight with seed (g) seed weight alone (g) the rind weight of each individual fruit was calculated using the following formula and the average expressed in grams: rind weight (g) = total fruit weight (g) pulp weight with seed (g) numbers of white juicy segments were counted immediately after the fruits were opened and the average table 1 latent vectors and variance of the principal component analysis performed for generating fruit, seed and seedling indices separately in mangosteen (garcinia mangostana l.) sl. no. characters principle component 1 principle component 2 fruit characters 1 fruit weight 0.62 0. 14 2 fruit volume 0.62 0 .14 3 fruit girth 0.02 0.00 4 no. of segments 0.00 0.00 5 pulp weight 0.15 0.16 6 rind weight 0.47 -0.62 7 fruit specific gravity 0.00 0.00 8 number of seedless fruits 0.00 0.00 9 number of one seeded fruits 0.00 -0.01 10 number of two seeded fruits 0.00 0.0 1 11 number of three and multi seeded fruits 0.00 0.01 12 number of seedlings/fruit 0.00 0.04 cumulative variance 96.27 98.68 seed characters 1 seed weight 0.00 -0.07 2 seed length 0.00 -0.04 3 seed thickness at center 0.00 -0.03 4 volume 0.00 -0.08 5 seed specific gravity -0.01 0.1 i 6 number of seeds 0.00 -0.04 7 number of seedling/seed 0.00 0.00 8 number of days to germination 0.02 0.99 9 seed with one seedling 0.00 0.00 10 seed with twin and multiple seedlings 0.00 0.00 11 germination percentage l .00 -0.01 cumulative variance 97.63 99.50 seedling characters 1 height 0.03 -0.01 2 girth at collar 0.00 0.00 3 total number of leaves 0.03 0.00 4 number of flushes/year 0.00 0.00 5 total leaf area 0.98 -0.21 6 survival rate at twelth month (%) 0.04 0.18 7 shoot fresh weight 0.01 0.06 8 root fresh weight 0.01 0.03 9 total fresh weight 0.02 0.09 10 shoot dry weight 0.01 0.03 11 root dry weight 0.00 0.01 12 total dry weight 0.01 0.04 13 root to shoot dry weight ratio 0.00 0.00 14 root length (longest root) 0.03 0.1 8 15 total number of roots 0.20 0.94 cumulative variance 70.05 95.77 table 2 average fruit index, seed index and seedling index values of four age groups in mangosteen (garcinia mangostana l.) age groups (years) fruit index seed index seedling index <25 90.55 80.54 93.69 25-50 88.62 88.36 101.17 51-75 132.90 84.47 65.20 >75 110.42 62.12 67.17 average 102.16 81.58 86.21 118 expressed as a number. numbers of seeds per fruit were counted and categorized as one, two, three and more in three-seeded and seedless fruits and the average expressed as a number. total number of seedlings obtained from individual fruits was counted irrespective of the number of seeds per fruit and the average was expressed numerically. the specific gravity fruits was calculated using the following formula and the average expressed as grams/milliliter: fruit specific gravity = (fruit weight / fruit volume) seed character the seeds were extracted from the pulp and the weight of individual seeds in fruits was measured using an electronic balance; the average seed weight was expressed in grams. seed length and thickness at the centre were also measured, with the aid of a meter scale, and averages expressed in centimeters. seed volume was measured by water displacement method and the average expressed in milliliters. seed specific gravity was calculated using the formula seed specific gravity = (seed weight / seed volume) and the average expressed as grams/milliliter. total number of seeds present in individual fruits, total number of seedlings produced by individual seeds and total number of ungerminated seeds were counted and averages expressed as a number. the number of seedlings produced by an individual age group was calculated and the average expressed as a number. the number of seedlings produced by individual seeds was counted and categorized as seeds producing one, two and more than two seedlings and the average expressed as a number. the number of days from date of sowing to seed germination was counted and the average expressed as number of days. germination rate was calculated for each individual age group and the average expressed as a percentage. germination % = (total number of seeds germinated / total number of seeds sown) x 100 seedling characters seedling height was measured from the collar region to the tip of the main stem using a meter scale and expressed in centimeters. seedling girth was measured at the collar using a thread; thread length was measured using a meter scale and averages expressed in centimeters. total number of leaves produced by an individual seedling was counted and the average expressed as a number. the increments were calculated by computing the difference between two consecutive values of the particular interval and the average expressed in centimeters. the number of flushes and number of leaves/flush produced by an individual seedling were counted and the average expressed as a number. leaf area was calculated by multiplying the length, the breadth and the factor (0.6727) and the average expressed as cm2. the factor was pre-standardized, for this purpose, by measuring the length and breadth of 100 leaves: the leaf area of the corresponding leaf was measured by leaf area meter to work out the factor value. thus, the factor value (0.6727) was derived using the formula: factor = (leaf area / length x breadth) survival rate was determined from the number of established plants as a percentage of the total number of seedlings observed after germination. survival % = (total number of seedings observed at each interval / total number of seedlings observed immediately after germination) x 100 shoot and root characters seedlings were uprooted one year after germination. the plants were immediately cut and separated into shoots and roots. fresh shoot and root weights were recorded separately using an electronic balance and the average expressed in grams. the samples collected for the fresh weight were dried in an oven at 60°c till the weight of the samples remained constant. dry weights were recorded separately and averages expressed in grams. dry weight ratio of root to shoot was calculated by the formula: dry weight ratio = (root dry weight / shoot dry weight) length of the longest root (taproot) was measured from the collar region to the growing tip of the taproot using a meter scale and expressed in centimeters. after carefully removing the potting mixture using water spray, the number of primary, secondary, tertiary roots, and total number of roots were counted and their averages expressed as the number of roots. biochemical studies leaf samples from seedlings were collected one year after germination; leaf samples from mother plants were also collected. the third leaf from the tip was collected and oven dried at 60°c, ground and used to estimate the content of n, p, k and na. the total nitrogen content of leaf samples was determined by microkjeldhal method (jackson, 1973) and the average expressed as percentage. the phosphorus content of the samples were determined using the di-acid extract method (jackson, 1973). a spectrophotometer was used to determine colour intensity developed by vanado-molybdo phosphoric yellow colour method and readings were taken at 420nm wavelength. phosphorous content was cal119 culated using a standard graph and the average expressed as a percentage. the potassium and sodium contents of samples were determined with di-acid extract (jackson, 1973) and read in an eel flame photometer, at 548 nm and 598 nm respectively, and averages expressed as a percentage. nitrogen content was estimated by microkjeldhal method (jackson, 1973) and the value of nitrogen content was multiplied with the factor 6.25 to get the crude protein content and the average expressed as a percentage. chlorophyll content (total chlorophyll, chlorophyll a and chlorophyll b) was estimated in leaf samples by arnon’s acetone method (sadasivam and manickam, 1996) and the average expressed in milligrams. total sugars were estimated using standard procedure (a.o.a.c., 1980), total carbohydrates were estimated using anthrone method (dubois et al., 1951), and averages expressed in milligrams; total phenol content was estimated using the folin-ciocalteau method (sadasivam and manickam, 1996) and expressed in milligrams. procedure adopted for quantification of abscisic acid the procedure adopted for quantification of abscisic acid was a modification of the standard method of little et al. (1972). the modification became imperative as bands were not obtained. the procedure was standardized and bands were obtained corresponding to the standard abscisic acid. further quantification was done using a u-v spectrophotometer and standards of known concentration from which a standard graph was obtained. generation of new varieties as index variability in morphology characters (38 characters) were recorded. principal component analysis (pca) was carried out independently for 12 fruit, 11 seed and 15 seedling characters in order to establish a list of minimum descriptors (a.o.a.c., 1980) making it possible to identify the best age group of mother plants. the first principal component which accounted for maximum possible variance was selected. this is supported by the work of manzano et al. (2001). estimation of correlation associations between the various characters were made using the karl pearsons product movement correlation coefficient (r). correlations between age of mother plant, fruit characters, seed characters, seedling characters, fruit index, seed index and seedling index were calculated according to the method of searle (1961). principal component analysis the observations of 12 fruit, 11 seed and 18 seedling characters of the four age groups and their corresponding index values (i.e. fruit, seed and seedling) of the 135 plants were used for the study. the volume of the data had to be reduced first for the sake of simplicity. this was based on the fact that principal component analysis is one of the variabledirected techniques aimed at reducing dimensionality of the problem and which finds new variables that make the data easier to understand (chatfield and collins, 1980). principal component analysis (pca) was used to determine the relationship among the fruit characters, seed characters, seedling characters and four age groups (meilgard et al., 1991) and was performed in xlstat version 5.1v2 package. it was also used to provide a graphical description of the characteristics. factor scores were calculated for each fruit, seed and seedling characteristics using the formula: factor scores= each attribute factor loading x the original attribute mean score although the characteristic factor scores were calculated from a single analysis, they were plotted on separate figures to facilitate interpretation. these figures then provided a visual representation of the dominant age groups and characters for each fruit, seed and seedling character (dever et al., 1996). step-wise regression to facilitate the prediction of indices reduction in number of variables is imperative. hence the step-wise regression procedure was adopted to identify major characters, which can be used to predict the fruit, seed and seedling indices. furthermore, adoption of step-wise regression has been scientifically established to reduce multi-collinearity among the independent variables and to arrive at the best subset of variables (draper and smith, 1966). cluster analysis cluster analysis was performed to find the similarities (or dissimilarities) between the four age groups based on absolute square euclidean distance (johnson and wichern, 1998). the clusters and square euclidean distances (chatfield and collins, 1980) were graphically represented by dendrograms (manzano et al., 2001). the euclidean distance was calculated using the formula p drs : {∑ (x ri – x si )2 }to ½ and the variable were the principal component co-ordinates. where drs distance from individual r to individual s, x rj value of individual r x sj value of individuals. selection index selection index or total index values were worked out using principal component analysis. principal component analysis was performed on all the fruit, seed and seedling characters collectively and the first principal component was taken as the index value for selection, as in the method described above. regression analysis was performed for each age group separately to find the best age group with equal weightage of all fruit, seed and seedling characters. then the best age group or selection index (total index) 120 was predicted using the regression equation with the fruit, seed and seedling indices as the explanatory variables. the predictor equation was found to be y=a 1 f1 + a 2 s1 + a 3 sdli + c where a 1 , a 2 , a 3 were regression coefficient or weightage of f1, s1 and sdli, which are fruit index, seed index and seedling c the intercept constant (manoj, 1992). 3. results the results of the study have been divided into broad aspects for presentation. fruit, seed and seedling indices the numbers of independent variables were large in the present study. hence, to reduce the number of descriptors, principal component analysis was performed using the deviation squares and products matrix of the 12 fruit, 11 seed, and 15 seedling characters independently in order to establish a list of minimum descriptors (i.e. fruit index, seed index and seedling index), thereby enabling identification of the best age group of mother plants, as well as reducing the complexity of the statistical analysis. the first principal components of fruit, seed and seedling characters, which accounted for the cumulative variance of 96.27, 97.63 and 70.05% respectively, are given in table 1. the computed average fruit, seed and seedling index values of each age group are presented in table 2 and figure 1. fruit index the highest fruit index value was in the age group of 51-75 years of mother plants, followed by the age group of more than 75 years. seed index the highest seed index value was recorded in the 25-50 year age group, followed by 51-75 years. the lowest seed index values were observed in the more than 75 years age group. seedling index the highest seedling index was observed in the age group 25-50 years, followed by the age group of less than 25 years. an important observation was made: the difference between the best group (25-50 years) and the two age groups of more than 50 years was near to one and a half times more. index values observed for fruit, seed and seedling characters differed but the trend was the same for the seed and seedling characters. however, equal importance was given to each of the three index values and to the age group for finally determining the best age group of mother plant. principal component analysis principal component analysis was performed using the deviation squared and product matrix of the 14 fruit characters, 13 seed characters, 20 seedling characters, the age of the mother plants and all the three fruit, seed and seedling indices separately. the first two principal components, which accounted for the cumulative variance of fruit, seed and seedling characters, were found to be 88.78, 92.94 and 92.49% respectively. the cumulative variance, factor scores and contribution of variation of each character are presented in table 3 and the same for each age group in table 4. the fruit index among the fruit characters, seed index among the seed characters and seedling index among the seedling characters were predicted using the regression equation with the first two principle components as the explanatory variables. the predictor equations were found to be y = 0.0695 p 1 – 0.1283 p 2 0.0197 with an r2 of 0.999 where y 1 p 1 and p 2 are the fruit index first and second principal components respectively; y= 0.3198 p 1 + 0.6307 p 2 0.0135 with an r2 of 1.00 where y 1 p 1 and p 2 are the seed index, first and second principal components respectively; y= 0.7003 p 1 0.0703 p 2 + 3.263 with an r2 of 0.999 where y 1 , p 1 and p 2 are the seedling index first and second principal components respectively. also the relationship and dominance of the fruit, seed and seedling characters including four age groups of the mother plants and three (fruit, seed and seedling) indices separately were determined using factor scores. the depiction of the various characters of factor i showed negative scores for characters such as number of seeds per fruit, specific gravity and selection index, whereas factor ii revealed positive scores only for seedlessness and seed specific gravity (table 3). distinct variations in the dominant fig. 1 fruit, seed and seedling index of four age groups in mangosteen (garcinia mangostana l.). 121 characters were observed between the various age groups. in the case of fruit characters, fruit index, fruit weight, fruit girth, fruit volume, pulp weight, number of segments and rind weight characters of age group three (51-75 years) were dominant. on the other hand, the number of seeds per fruit was observed as the dominant characters in the table 3 eigen vectors, factors loadings and contribution of variations by each character in fruit, seed and seedling characters in mangosteen (garcinia mangostana l.) characters eigenvectors factor loadings contributions of variation (%) f1 f2 f1 f2 f1 f2 fruit characters fruit weight 0.30 -0. 17 0.96 -0.28 9.27 3.01 girth 0.21 -0.07 0.66 -0.11 4.44 0.44 volume 0.29 -0.24 0.92 -0.39 8.50 5.84 pulp weight 0.21 -0.46 0.66 -0.73 4.39 20.90 rind weight 0.31 -0.05 0.99 -0.08 9.86 0.25 segments 0.30 -0.14 0.95 -4.22 9.14 1.94 seeds -0.28 -0.20 -0.87 -0.32 7.70 3.97 seedless fruits 0.29 0.27 0.90 0.43 8.20 7.08 one seeded fruits -0.27 -0.24 -0.84 -0.39 7.22 5.98 two seeded fruits -0.21 -0.18 -0.65 -0.29 4,29 3.37 three and >three seeded fruits -0.27 -0.31 -0.84 -0.49 7.16 9.32 specific gravity -0. 11 0.58 -0.34 0.94 1.16 34.01 fruit index 0.31 -0.06 0.97 -0.10 9.54 0.41 selection index -0.30 -0.19 -0.95 -0.30 9.13 3.49 cumulative variance 70.37 88.79 seed characters seed weight 0.29 -0.28 0.82 -0.55 8.29 7.81 seed length 0.34 -0.02 0.98 -0.04 11.68 0.03 thickness at center 0.31 0.06 0.89 0.l l 9.68 0.31 seed volume 0.29 -0.27 0.84 -0.54 8.69 7.40 specific gravity -0.28 0.29 -0.80 0.57 7.81 8.17 number of seedlings / seed 0.32 0.07 0.90 0.14 9.94 0.49 ungerminated seeds -0.30 -0.22 -0.87 -0.44 9.30 5.04 seeds producing one seedlings 0.26 0.27 0.74 0.53 6.69 7.23 seeds producing 2 and >2 seedlings 0.33 0.10 0.95 0.19 11.05 0.94 days to germination -0.24 0.31 -0.68 0.62 5.72 9.81 germination % 0.24 0.37 0.68 0.73 5.59 13.73 seed index 0.20 0.41 0.57 0.82 4.01 17.04 selection index -0.12 0.47 -0.35 0.93 1.54 21.99 cumulative variance 62.79 92.94 seedling characters height 0.26 0.09 0.99 0.15 6.85 0.88 girth 0.26 -0.09 0.97 -0.13 6.62 0.74 total leaves 0.26 0.08 0.97 0.12 6.60 0.59 new flushes / year 0.10 0,55 0.39 0.85 1.09 29.76 total leaf area 0.25 0.19 0.95 0.30 6.35 3.63 survival rate 0.2l -0.36 0.78 -0.55 4.27 12.70 shoot fresh weight 0.23 0.06 0.87 0.09 5.34 0.33 root fresh weight 0.26 0.05 0.99 0.08 6.87 0.23 shoot dry weight 0.24 -0.04 0.90 -0.06 5.64 0. 14 root dry weight 0.26 0.03 0.99 0.04 6.89 0.08 root to shoot dry weight ratio -0.14 0.47 -0.54 0.72 2.08 21.65 root length 0.23 -0.16 0.86 -0.25 5.23 2.68 primary roots 0.25 -0.15 0.94 -0.23 6.18 2.29 secondary roots 0.26 -0.01 1.00 -0.02 7.01 0.02 tertiary roots 0.14 0.49 0.55 0.76 2.09 24.22 total roots 0.26 0.01 1.00 0.02 7.01 0.02 seedling index 0.26 -0.01 0.99 -0.02 6.93 0.02 selection index 0.26 -0.01 0.99 -0.02 6.93 0.02 cumulative variance 79.13 92.49 122 less two age groups (i.e. <25 and 25-50 years age groups). interestingly, in age group four (>75 years), the most dominant character was observed to be seedlessness (fig. 2). the depiction of the various seed characters of factor i showed negative scores for seed specific gravity, ungerminated seeds, days to germination and selection index. in the case of factor ii the negative scores were found for seed weight, seed length, seed volume and ungerminated seeds. all other characters showed positive factor scores. in the case of seed characters, also variations in dominant characters were observed with respect to age groups. the seed index, selection index, germination percentage, seeds producing one seedling and number of seedlings per seed were the dominant characters observed in the second age group (25-50 years). in age group one (<25 years) the number of days taken to germination and seed specific gravity were noted to be the dominant characters. in age group three, seed weight, seed volume and seed length were the dominant characters, whereas in age group four, the dominant character was the number of ungerminated seeds (fig. 3). in the case of seedling characters, negative scores were observed only for the root to shoot dry weight ratio in factor i; in factor ii negative scores were found for girth, survival rate, shoot dry weight, root length and number of roots (primary, secondary and tertiary), seedling index and selection index. all other characters showed positive factor scores. a study of the dominant characters of seedlings revealed that the major characters (seedling index seedling girth, total number of roots, number of primary, secondary roots, survival rate, shoot, fresh weight, dry weight, root dry weight, height of the seedlings and selection index were the dominant characters in age group two. in the case of age group one characters such as the root fresh fig. 2 plot showing the percentage of variation contributed by each fruit character and the relationship between age of the mother plant and fruit characters in mangosteen (garcinia mangostana l.). table 4 factor scores and contribution of variations by each age group based on the fruit, seed and seedling characters in mangosteen (garcinia mangostana l.) characters factor scores contributions of age groups {%} f1 f2 f1 f2 fruit characters age 1 -1.74 -0.62 6.14 2.99 age 2 -4.34 -0.66 38.27 3.39 age 3 4.96 -l .76 49.91 24.02 age 4 1.60 3.00 5.21 69.59 mean -0.48 0.05 0.47 0.02 seed characters age 1 -1.64 1.04 6.59 5.53 age 2 -0.64 2.87 0.99 41 .98 age 3 5.26 -1.13 67.89 6.55 age 4 -3.16 -2.99 24.46 45.70 mean 0.17 0.22 0.07 0.24 seedling characters age 1 3.35 0.92 15.73 7.10 age 2 4.93 -0.91 34.14 6.95 age 3 -4.52 -2.27 28.75 42.95 age 4 -3.90 2.27 21 .35 43.00 mean 0. l5 -0.01 0.03 0.00 123 weight, total number of leaves, number of tertiary roots and new flushes per shoot were the dominant characters. in the higher two age groups, no specific dominance were observed (fig. 4). principal component analysis reduces only the dimensionality, but not the number of variables involved. hence, to reduce the number of variables and to identify the major fig. 3 plot showing the percentage of variation contributed by each seed character and the relationship between age of the mother plant and seed characters in mangosteen (garcinia mangostana l.). fig. 4 plot showing the percentage of variation contributed by each seedling character and the relationship between age of the mother plant and seedling characters in mangosteen (garcinia mangostana l.). 124 variables that contribute greater variation to the fruit, seed and seedling index, a step-wise regression was performed. step-wise regression step-down regression of the fruit index on 13 fruit characters, seed index on the same 13 fruit characters, seed index on the 12 seed characters separately and all the 25 fruit and seed characters collectively, and seedling index on 13 fruit characters, 12 seed characters, 19 seed characters including age separately and also collectively (all the 42 fruit, seed, seedling characters and age) was carried out and the following regression equations with major variable, to each, indices on each group of characters, were observed. fruit index on fruit characters alone y = 1.295 x 7 + 1.7 l5x 8 + 1.3 19 x 14 4.17 with an r2 at 0.993 where y fruit index, x 7 pulp weight, x 8 rind weight, x 14 number of seedlings per fruit. seed index on fruit characters alone y = -a.282 x2 + 0.427 x3 + 14.416 x5 + 22.368 x10+ 126.73 with an r2 at 0.203. where y – seed index, x2 – age of the mother plant, x 3 fruit weight, x 5 fruit girth, x 10 number of seedless fruits. seed index on seed characters alone y= 0.007 x15 0.004 x16 0.012 x17 + 0.009 x180-008 x19 + 0.002 x20 +0.004 x21 + 0.015 x22 + 0.002 x24 + x25 + 0’01 with an r2 at 1.00 where y seed index, x 15 seed weight, x 16 seed length, x 17 seed thickness at centre, x 18 seed volume, x 19 seed specific gravity, x 20 number of seeds per fruit, x 21 number of seedling per seed, x 22 number of days to germination, x 24 number of seeds producing two or more than two seedlings, x 25 – germination percentage. seed index on fruit and seed index collectively y = -0.002 x 11 0.006 x 15 0.006 x 17 + 0.006 x 18 0.007 x 19 + 0.005 x 21 + 0.015 x 22 – 0.002 x 23 + x 25 + 0.01 with an r2 at 1.00 where y seed index, x 11 – number of one seeded fruits, x 15 seed weight, x 17 seed thickness at centre x 13 seed volume, x 19 seed specific gravity, x 21 number of seedling per seed, x 22 number of days to germination, x 23 seed with one seedling x 25 germination percentage. seedling index on fruit characters including age y =-0.481 x2 + 107.15 with an r2 of 0.0388 where, y seedling index, x 2 age of the mother plant. seedling index on seed characters y =136.67 x17 1083 with an r2 of 0.1087 where, y seedling index, x 17 seed thickness at centre. seedling index on seedling characters y= 0.979 x30 + 0.037 x31 + 0.042 x32+ 0.027 x33 + 0.031 x39+ 0.202 x40 + 0 .36 with an r2 of 1.00 where, y seedling index, x 30 total leaf area per seedling, x 3l survival rate at l2-months stage, x 32 – shoot fresh weight, x 33 root fresh weight, x 39 foot length, x 40 total number of roots. seedling index on fruit, seed and seedling characters collectively including age of mother plant y= 0.026 x26 + 0.030 x28+ 0.977 x30 + 0.037 x31 + 0.0034 x320.029 x33+ 0.017 x35 + 0.009 x36 + 0.031 x 39 + 0.031 x 39 + 0.201 x 40 with an r2 of 1.00 where, y seedling index, x 26 height of the seedling, x 28 total number of leaves, x 30 total leaf area, x 31 survival rate at twelve-months stage, x 32 shoot fresh weight, x 33 root fresh weight, x 35 shoot dry weight, root dry weight, x 36 root dry weight, x 39 root length, x 40 total number of roots. cluster analysis cluster analysis was performed to highlight the similarities and differences based on the fruit, seed and seedling characters separately and all characters collectively among the four age groups. absolute euclidean distances between the four age groups were calculated based on the fruit, seed and seedling characters and are presented in table 5. figure 5 illustrates the dendrogram showing the clusters (or similarities) based on the fruit, seed and seedling characters among the four age groups. considering fruit characters, closest distance was observed between age groups of more than 25 years and 2550 years, followed by the age group of 25-50 years and more than 75 years; in all the remaining combinations greater distances were observed, indicating that the age group of more than 25 years and 25-50 years have more similar fruit characters. with regard to seed characters, closest distance was again observed between age groups of more than 25 years and 25-50 years, followed instead by the age group of more than 25 years and more than 75 years. with regard to all the remaining combinations greater distances were observed, indicating that the age group of more than 25 years and 25-50 years have more similar seed characters. for seedling characters, closest distance followed the same trend (i.e. age group of more than 25 years and 25-50 years). all the remaining combinations were observed at greater distances, indicating that also for seedling charac125 ters the age group of more than 25 years and 25-50 years are more similar. when the fruit, seed and seedling characters were taken collectively, the closest distance was observed between age group of more than 25 years and 25-50 years, followed by the age group of more than 25 years and more than 75 years; in all the remaining combinations greater distances were observed. finally it may be confirmed that, based on all four dendrograms, maximum similarity for all the characters was between the age group of less than 25 years and 25-50 years. discriminant function analysis fruit index, seed index and seedling index of the four age groups were discriminated using discriminant functional analysis. discriminant functions were fitted for discrimination pairs of these age groups. the functions derived were as follows. for groups 1 and 2 z = 0.0004 fi 0.0547 si 0.0004 sdli with d2 = 0.4313 for groups 1 and 3 z = -0.0483 fi 0.0313 si + 0.0075 sdli with d2 = 2.381 for groups 1 and 4 z = -0.0183 fi 0.0680 si + 0.0047 sdli with d2 = 1.739 for groups 2 and 3 z = -0.0406 fi + 0. 1264 si + 0.0137 sdli with d2 = 2.782 for groups 2 and 4 z = -0.0265 fi + 0.3508 si + 0.0150 sdli with d2 = 10.29 for groups 3 and 4 z = 0.0024 fi + 0.2152 si + 0.0004 sdli with d2 = 4.064 where fi the fruit index, si the seed index and sdli the seedling index are the explanatory variables. the average values of fruit index, seed index and seedling index for each age group were fitted in the equation and the discriminant values calculated for each pair of age groups. the direction of association of the discriminant coefficients in each age group is given in table 6. in this maximum number of positive directions were observed in the age group of 25-50 years, when discriminated with other three table 5 proximity matrix showing the absolute squared euclidean distance between four age groups based on fruit, seed, seedling characters separately and collectively in mangosteen (garcinia mangostana l.) characters and age groups absolute squared euclidean distance 1 2 3 4 fruit 1 0.00 14.41 39.25 20.57 2 14.41 0.00 3 1.60 15.6 3 39.25 31.60 0.00 22.57 4 24.57 15.60 22.57 0.00 seed 1 0.00 13.14 30.09 13.24 2 13.14 0.00 21.58 20.37 3 30.09 21.58 0.00 33.59 4 13.24 20.37 33.59 0.00 seedling 1 0.00 5.65 29.97 35.44 2 5.65 0.00 26.48 34.63 3 29.97 26.48 0.00 11.83 4 35.44 34.63 11.83 0.00 all characters 1 0.00 39.06 109.58 77.77 2 39.06 0.00 100.31 90.23 3 109.58 100.31 0.00 75.05 4 77.77 90.23 75.05 0.00 fig. 5 dendrogram showing the similarity and proximity of the four age groups based on the fruit, seed and seedling characters individually and collectively in mangosteen (garcinia mangostana l.). table 6 direction of association of six discriminant function coefficients involving the four age groups in mangosteen (garcinia mangostana l.) pairs of compared age groups magnitude of the values fruit index seed index seedling index 1 and 2 + 1 and 3 + 1 and 4 + + 2 and 3 + + 2 and 4 + + 3 and 4 + + 126 age groups (table 7). the corresponding discriminant values of each age group (criteria) and the mid values of each pair of discriminant values were taken to draw the conclusion that, if the values is below the mid values it falls in the group i, if not it falls in the group ii. the criterion and criteria are presented in table 8. selection index principal component analysis was performed on all the fruit, seed and seedling characters collectively and first principal component was taken as the index value for selection (fig. 6). the selection index was predicted using the multiple regression equation constant for each age group independently and also the average of all age groups. the predictor equation was found to be for the less than 25 years group y = -0.0402 fi + 0.0513 si + 0.9960 sdli – 0.4317 (adj. r2= 0.99) table 7 direction of association of six discriminant function coefficients when used to compare each age group with the remaining three age groups in mangosteen (garcinia mangostana l.) discriminating pairs of age groups magnitude of the values total number of positive values age group i age group ii fruit index seed index seedling index 1 2 + 1 1 3 + 1 1 4 + + 2 total positive values for age group 1 4 2 1 + + 2 2 3 + + 2 2 4 + + 2 total positive values for age group 2 6 3 1 + + 2 3 2 + 1 3 4 + + 2 total positive values for age group 3 5 4 1 + 1 4 2 + 1 4 3 + 1 total positive values for age group 4 3 table 8 criteria and criterion for discrimination of age groups in mangosteen (garcinia mangostana l.) sl. no age groups comparisons criteria 1 criteria 2 criterion conclusion 1 <25 and 25-50 -4.41 -4.84 -4.62 if the value is >-4.62 it falls in group i, if not group ii 2 <25 and 51-75 -6. 19 -8.57 -7.38 if the value is >-7.38 it falls in group i, if not group ii 3 <25 and >75 4.26 2.52 3.39 if the value is > 3.39 it falls in group i, if not group ii 4 25-50 and 5 1-75 8.96 6.17 7.57 if the value is > 7.57 it falls in group i, if not group ii 5 25-50 and >7 5 30.17 19.87 25.02 if the value is >25 .02 it falls in group i, if not group ii 6 51-75 and >75 18.47 13.61 16.04 if the value is >16.04 it falls in group i, if not group ii fig. 6 selection index or total index values of all the four age groups in mangosteen (garcinia mangostana l.). 127 for group 25-50 years y= -0.0562 fi + 0.0129 si + 0.9979 sdli + 4.331(adj. r2= 0.99) for the 51-75 years group y= -0.0479 fi + 0.0115 si + 0.9967 sdli + 3.070 (adj. r2 = 0.99) for the more than 75 years group y= -0.0495 fi + 0.0191 si + 0.9955 sdli + 2.247 (adj. r2 = 0.99) for the average of all age groups y= -0.0527 fi + 0.0446 si + 0.9978 sdli + 1.056 (adj. r2 = 0.99) where fifruit index, siseed index and sdliseedling index. the average of fruit index, seed index and seedling index values for each age group were fitted in the equation independently and the selection index values were determined. the averages of all the age groups were recorded and are given in table 9. table 9 mean selection index or total index values of four age groups in mangosteen (garcinia mangostana l.) age groups (years) selection index values less than 25 years 93.38 25 50 years 101 .45 51 75 years 62.66 more than 75 years 64.84 mean 80.64 index values the highest index value was found in the 25-50 years age group of mother plant, which was concluded to be the best age group for selection of the mother plants. this was followed by the age group of less than 25 years. the mean values of the important characters in the best age group (25-50 years) were fixed on the basis of the corresponding character for selection. classification was made as above average if positively correlated and below average, if negatively correlated. 4. discussion and conclusions presentation of the results of the studies are broadly discussed and organized under subheadings to make them more accessible to the reader. association of morphological characters of fruit, seed and seedling the fruit index was positively correlated with most of the fruit and seed characters, except fruit specific gravity, number of one-seeded fruits, seed thickness at centre, seed specific gravity, number of seedlings per fruit, and germination rate; all the seedling characters were negatively correlated. an analysis of the age group variations revealed that the maximum values of all the morphological characters of fruit and seed were recorded in the 51-75 years group and the lowest values were recorded in the 25-50 years group. as fruit index is a function, which is derived from all the morphological characters of fruit, the characters ought to be naturally correlated. however the negative correlations observed were due to the lowest values of fruit characters recorded in the 25-50 years age group, which in turn gave maximum seeded fruits, maximum germination of seeds, maximum survival rate and the best quality seedlings as revealed by the improved morphological characters. seed index was observed to be negatively correlated with all the fruit characters. although the pattern of gradation observed in the values of seed characters in the various age groups was the same as that observed for fruit characters in the same age group, a corresponding size or weight of the seed was not observed. the relative differences observed at seed level are not as explicit as in the case of fruits and this could be the reason for the negative correlation observed between seed index and fruit characters. this is more evidenced if a ratio of the seed to fruit weight is computed. the second age group registered the highest ratio values, which again reveals that seed weight expressed as a fraction of the fruit weight is a more important character. similarly, the differences are most clear in seed thickness to fruit girth ratio, where the second age group showed a very high ratio in spite of the fact that for both of the above characters the first age group had the highest values individually. the positive correlations of seed index with all seedling characters once again confirms that the seedling characters are more governed by both the individual as well as collective characters of the seed. while in the standard methodology that was adopted, equal importance (weightage) was given to seed, fruit, and seedling characters and age of the mother plant, the results confirm that it is the seed characters which play a more determining role for better seedling characters. even though identical reports are not available in mangosteen, the studies of reddy (1997) revealed that the size of the seeds was highly variable, the difference in seed weight brought about the variation in germination and that the ability to germinate and grow successfully is related to the amount of food stored in the seed. another author suggested that it is better to establish new plantings only from the larger seeds weighing 1 g and above in florida (campbell, 1966). seedling index, which was computed as a total variation of all the seedling characters collectively, was negatively correlated with number of seedlings per seed, number of days to germination, and seeds producing more than two seedlings. this is basically due to the fact that as a consequence of an increase in the number of seedlings per seed, the growth characters of the seedling are affected. the number of days taken to germinate was negatively correlated with seedling index because the seed characters 128 were positively correlated with seedling characters and the age groups that recorded the highest values for seed morphological characters also recorded the least number of days to germination. the number of seeds producing more than two seedlings were negatively correlated with the seedling index as the positive aspect of more seedlings produced per seed was negated by the character of comparatively slower growth. another positive aspect in this case is that normally seedlings with exposed food storage or haustorial cotyledons are very vulnerable, and this attractive food source is prone to attack by rodents when it is present above ground. however in the garcinia type, the food reserve is stored in the hypocotyl, which is protected by the envelopments and situated at, or below, soil level making it less vulnerable (vogel, 1980). in general, most of the fruit characters are positively correlated with seed characters and negatively correlated with seedling characters; most seed characters are positively correlated with seedling characters. generally this is because the values of fruit characters increase with mother plant age group, but this increase is not matched by a corresponding increase in seed characters. in the case of seedlings, the age groups with increased fruit characters produce only weaker seedlings, as revealed by the lower seedling index. the increase generally observed in the seed characters leads to increased seedling growth and, hence, over emphasizes the absolute command of seed characters in determining the quality attributes of seedlings. principal component analysis. fruit characters and index a figurative plot of the principal components of the fruit characters is presented in figure 6, showing very narrow angles among the fruit characters such as rind weight, pulp weight, fruit weight, fruit index, number of segments, fruit girth and fruit volume and describing actually the high positive correlation that exist between them. the principal component analysis of fruit characters and age of the mother plant showed that factor i and factor ii account for 88.76% of the cumulative variance. a critical analysis of the plot reveals that for all major characters of the fruit, age group three showed narrow angles, underlining that most of the improved fruit morphological characters are observed in this group. on the other hand, seedlessness showed narrow angles to age groups three and four, confirming that it is a factor that goes hand in hand with increased age. seedlessness in fruits and seediness (one-seeded, two-seeded and more than three-seeded) are in opposite directions, illustrating that they are negatively correlated. a similar positioning is observed in the case of specific gravity and pulp weight, which reconfirms the negative correlation. seed characters and index principal component analysis of the seed characters and age of the mother plant showed that factors i and ii account for 92-94% of the cumulative variance. a critical perusal of the plot (fig. 3) of the seed characters revealed that maximum seed characters (seed index, selection index, germination percentage, seed producing one seedling, number of seedlings per seed and seed thickness at centre) were in close proximity and with very narrow angles to age group two, which clearly establishes the distinct superiority of this age group. the number of ungerminated seeds was in close proximity to age group four and positioned opposite germination percentage, showing the negative correlation. similarly, seed weight and volume are positioned at opposite ends with respect to the days to germination and specific gravity, confirming the highly significant negative correlation observed in the study. seedling characters and index principal component analysis of the fruit characters and age of the mother plant showed that factors i and ii account for 92.49% of the cumulative variance. the most important or major characters of the seedling and selection index are in close proximity to age group two (fig. 4), which clearly establishes that seedlings of this age group are superior in all characters. the narrow angles between the various characters and this age group also establish the high positive correlations of the characters with this age group. the placement of survival rate and root to shoot dry weight ratio at opposite ends not only reveals the negative correlation but, more notably, the importance of roots which are critical in the case of mangosteen. step-wise regression step-wise regression was carried out to identify the variables contributing maximum variations and to reduce the number of variables. there was reduction in the number of variables influencing the fruit characters. with regard to seed characters, the thickness of the seed at the centre was found to be the most important factor. this is supported by the highest seed specific gravity found to be maximum in the best age group. for seedling index, the important characters were height of seedling, total number of leaves, total leaf area, survival rate, shoot and root fresh weight and dry weight, root length and total number of roots. as for seedlings, root characters were logically found to be important as the root system in mangosteen is magnolioid but cannot be recommended as an index for selection as they are underground. mangosteen plants are very sensitive and even removal of bits of leaves for chemical analysis normally resulted in death of the plant. similarities between age groups based on fruit, seed and seedling characters a critical analysis of the various age groups points to a very important conclusion: for most of the fruit characters, age groups one and two gave comparatively lesser values, whereas the older groups gave maximum values and have higher fruit index. likewise in the case of seeds, the older groups had better size but the younger age groups gave more seeded fruits and gave better germination and more 129 seedlings per seed. these characters though were highest in the age group two were equally high in the youngest age group and thus the similarity between these two younger age groups were higher resulting in more closer distance in the dendrogram showing hierarchical clusters based on the euclidean distance. in the case of seedling characters, a comparative study reveals more similarity and also when all fruit, seed and seedling characters were collectively taken. there was more similarity between the younger two age groups and this should be the reason for the very close distance between these age groups (less than 25 years and 25 50 years) and the close proximity in the dendrogram (fig. 5). discriminant function further extrapolation of the coefficients (table 7) used in discrimination of various groups were critically analysed and are presented in table 8. the directions of coefficients were used to prepare the dendrogram presented in figure 7, showing both the positive and negative direction of the coefficients of fruit index, seed index and seedling index. it is again obvious from the table and the dendrogram that maximum coefficients were positively linked in the case of age group two when two specific pairs of age groups were discriminated as envisaged in this study. this was actually a reflection of the higher seed and seedling characters in age group two, compared to the other age groups, which reconfirms that this group is the best age group for selection as mother plants. selection index based upon the equation, the elaborated selection index revealed that the 25-50 years age group was the best. this is actually more a reflection of the improved seedling characters, particularly the germination percentage, survival rate and other morphological characters. the seed characters as revealed by the seed index were also highest in this particular age group. in the selection index equation, the seed characters were given equal weightage with all characters and hence this age group, which showed better seed characters, gave higher index values. although the fruit characters were better in the older groups, seedlessness, low germination percentage and poor survival rate were the main reasons for the lower selection index values. root characters cannot be taken as indices for selection as they are below ground, and hence not visible. furthermore, any attempt to lift the bare seedlings normally results in death of the plants, as they are highly sensitive. as the root characters are positively correlated with shoot characters, selection based on the shoot characters reflects root characters as well. having identified the best age group, the mid value of the dominant characters were chosen as viable indices for the selection. the mid values of seedling characters were fixed so that destructive procedures could be avoided. considering the complex of results obtained in the study, the following characters and mid values were selected as indices for selection. the selection should be such that the fruits from mother plants of the age group of 25-50 years should have fruit weight of not less than 58 g, fruit girth of not less than 5 cm, fruit volume of not less than 51 ml, pulp weight of not less than 16 g, rind weight of not less than 41 g, and number of segments less than 5. seeds obtained from the fruits with the above characters should have a seed weight of not less than 0.66 g, seed length of not less than 1.61 cm, seed thickness at centre of not less than 0.66 cm, seed volume of not less than 0.44 ml, seed specific gravity of not less than 1.50, days to germination not less than 21, germination percentage not less than 87% and preferably have more than one seed per fruit. furthermore, seedlings at the one-year stage from this age group with the identified fruit and seed characters should have a height of not less than 10 cm, seedling girth at collar region of not less than 2 cm, total number of leaves per seedling not less than 9, number of new flushes per year more than one, total leaf area per seedling not less than 72 cm2 and survival rate not less than 85%. in conclusion, it can be said that the present study has generated results of immense practical relevance which will directly aid in selection or act as a powerful tool for the selection of mother plants, fruit, seed and seedling characters for improved seedling growth. acknowledgements the authors acknowledge the assistance provided by the associate dean, college of horticulture, kerala agricultural university during the survey of mother plants, and for the infrastructural support received and lab facilities available for the study. fig. 7 dendrogram showing the directions of discriminant coefficients of fruit, seed and seedling indices of each age group when compared to the remaining three 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five samples chipped well after cold storage with reconditioning, while good chippers were not identified after cold storage without reconditioning. the use of an arbitrary index calculated for each clone is proposed to assist the selection of materials with a good trait combination. 1. introduction potato, solanum tuberosum, is the most cultivated not cereal crop in the world, ranking fourth after rice, wheat and corn (fao statistics, 2012). it represents an important component of human diet, because tubers are able to supply several nutrients, such as essential amino acids, vitamins (as vitamin c) and minerals. it can be consumed either as fresh product or as processed ready-to-eat food (i.e. chips and french fries) (carputo et al., 2005). overall, the number of potato varieties cultivated for processing purposes is increasing and more than 50% of potato yield is driven to food industries. in order to follow the food market trends, new varieties with a specific combination of processing-related traits are continuously requested. a traditional potato breeding program begins with sexual hybridization of tetraploid varieties or élite clones, in order to generate biparental families. progenies are then evaluated for quality traits and the (*) corresponding author: smelito@uniss.it citation: melito s., d’amelia v., garramone r., villano c., carputo d., 2017 tuber yield and processing traits of potato advanced selections. adv. hort. sci., 31(3): 151-156. copyright: © 2017 melito s., d’amelia v., garramone r., villano c., carputo d. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/ index.php/ahs/) and distribuited under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 18 january 2017 accepted for publication 5 may 2017 ahs advances in horticultural science adv. hort. sci., 2017 31(3): 151-156 152 most promising clones are then selected for further evaluations (mackay, 2005; rak and palta, 2015; melito et al., 2017). because of the autotetraploid nature of s. tuberosum and its heterozigosity level, the f1 presents trait segregation; as a consequence, vegetative propagation and field selection are needed for several years to identify superior clones (haynes et al., 2012). tuber traits considered by breeders in developing new varieties are different. among them, tuber yield and tuber quality characteristics, including both external (skin color, tuber size and shape, eye depth) and internal (dry matter content, chip ability) traits (carputo and frusciante, 2011) are deeply evaluated. external traits play an important role in potato fresh market, because they influence consumers’ choice. on the counterpart, internal traits mainly affect tuber processing. one of the principal goals of potato breeding programs is the selection of varieties with a good chipping ability following cold storage. indeed, following harvest, potato are normally stored at low temperatures (<8°c) to keep an interrupted supply used by the processing industries, to prevent sprouting, to reduce bacteria soft rot attack and to contain the loss of dry matter (malone et al., 2006; zhao et al., 2013). unfortunately, in response to cold storage most potato varieties convert tuber starch to reducing sugars (glucose and fructose). this reaction is known as “cold-induced sweetening” and is recognized as a serious problem for potato processing industry (dale and bradshaw, 2003). the accumulated reducing sugars undergo the non enzymatic maillard reaction with free amino acids when potatoes are fried in hot oil, resulting in dark unacceptable chips. furthermore, mottram et al. (2002) and stadler et al. (2002) reported high level of acrylamide because of the maillard reaction, which is potentially damaging for human health. equally important for the potato processing industry is the availability of varieties with high tuber specific gravity. tubers with specific gravity higher than 1.080 are generally considered suitable for processing. the main aim of this study was to test the performance of 27 potato advanced clones belonging to 7 families with different genetic background. tuber yield, specific gravity, chipping ability and earliness were determined to select the most promising clones. finally, an evaluation index useful for selection of genotypes with a good combination of traits was calculated. 2. materials and methods genetic material and experimental design the material used in this study derived from a conventional program of clonal selection started in 2009 in a single hill plot. from 2010 to 2013 selected clones were cultivated in larger unreplicated plots with spaced plants. clones with undesired characteristics (i.e. long stolons, deep tuber eyes and tuber defects) were discarded and the number of seed tubers was increased. three commercial varieties (spunta, adora and agria,) were used as control. the field trials were conducted at marigliano (district of naples) (lat. 40.927759°, long. 14.451370°). tuber production and quality evaluation control varieties and experimental clones were planted in a randomized complete block design with 3 replications. for each clone, 10 tubers were planted in a single row with spacing of 30 cm between plants and 70 cm between rows. plants were grown following the standard cultural practices of the geographic area. tubers were planted in march and harvested in july, when plants start senescing (roughly 120 days after plantation). total yield (ty) was evaluated at harvest. chips were produced by frying 10 longitudinally cut tuber slices from the center of each tuber and 2 tubers for each clone were used. the chips color was evaluated in 3 consequent times: at harvest, at three months of cold storage (7°c) and at 2 weeks of reconditioning (20 24°c) after cold storage. to optimize the chipping test, slices were washed in water before frying in soybean oil. tubers were considered completely fried when oil end to bubble. a colorimetric scale, from 1 (very light) to 10 (very dark) was used to determinate chipping ability. based on carputo et al. (2002), clones with a score ≤ 4.5 were considered suitable for chipping. the specific gravity of tubers (tsg) of each clone was estimated on 1 kg of tuber sample, evaluating the ratio weight in air/weight in water (woolfe, 1987). foliage earliness was evaluated at 90 days after planting. an earliness score (from 1= very late to 5= very early) was associated to each clone comparing foliage senescence to the control variety spunta (earliness score= 3). evaluation index to assist the selection of clones, an evaluation index (ei) was elaborated, associating an arbitrary score to each trait based on the value obtained: ty melito et al. tuber yield and processing traits of potato advanced selections 153 (kg/plant): 1= <0.5; 2= 0.51-1 ; 3= 1.1-1.5; 4= 1.51-2 ; 5= >2.1. tsg: 1= ≤1.080 (not suitable for processing); 2= 1.081-1.085; 3= 1.086-1.090; 4= >1.091. chipping color: 1= >4.5 at each test after cold storage; 2= <4.5 at least in one test after cold storage; 3= <4.5 at both tests after cold storage. average earliness score: 1= ≤1.0; 2= 1.1-2.0; 3= 2.13.0; 4=> 3.1. the ei was calculated summing the scores for each trait: the higher index values, the more desirable genotypes. the ei was calculated only for clones for which all the evaluation data were available. statistical analysis one-way anova was run using jmp 7 software (sas institute, cary, nc, usa). when a significant f was found (p<0.05), data were compared using tukey’s post hoc multiple comparison test. each trait was used to compare the mean values among clones and varieties and to varieties individually. 3. results and discussion the development of new potato varieties addressing production efficiency and sustainability requires a well-planned breeding program. the accumulation of multiple traits in a single variety is one of the main goal of potato breeding programs. among the available different strategies, those based on sexual hybridization between tetraploid varieties or clones, followed by selection, still represent a successfully and widely used option. in this research, 27 advanced clones were selected and evaluated for yield and processing traits. they belong to seven families obtained from crosses involving ten cultivated varieties (spunta, victoria, jenny, blondy, agria, bolesta, sandy, majestic, alcmaria, primura,) and one breeding clone (mc 329). as reported in table 1, clones under evaluation showed differences in terms of flesh color and tuber shape, whereas skin color, eye table 1 tuber characteristics of the potato clones under selection. for each clone, pedigree, tuber skin color (tsc), tuber flesh color (tfc), tuber shape (ts), eye and stolon characteristics are reported. families pedigree tsc tfc ts eyes stolons ‘spunta’ x ‘victoria’ s04-2-10 yellow yellow oblong superficial short s04-2-17 yellow yellow round superficial short s04-2-18 yellow yellow long superficial short s04-2-28 yellow yellow oblong superficial short s04-2-34 yellow yellow oblong superficial short s04-2-40 yellow yellow oblong superficial short s04-2-53 yellow yellow long superficial short s04-2-55 yellow yellow long superficial short s05-7-15 yellow light yellow oblong superficial short s05-7-4 yellow yellow long superficial short ‘jenny’ x ‘mc 329’ s04-7-2 yellow white round superficial short s04-7-6 yellow white round superficial short s04-7-27 yellow white round superficial short ‘blondy’ x ‘victoria’ s05-1-2 yellow yellow long superficial short s05-1-25 yellow white long superficial short ‘bolesta’ x ‘mc 329’ s05-2-10 yellow white round superficial short s05-2-11 yellow light yellow round superficial short s05-2-15 yellow yellow round hallowed short s05-2-18 yellow yellow round superficial short s05-2-23 yellow yellow round superficial short s05-2-3 yellow yellow round superficial short s05-2-4 yellow yellow oblong superficial short s05-8-5 yellow yellow round superficial short ‘agria’ x ‘sandy’ s04-5-32 yellow yellow round superficial short ‘majestic’ x ‘alcmaria’ s04-6-2 yellow white oblong superficial short ‘primura’ x ‘alcmaria’ s05-4-2 yellow yellow oblong superficial short solanum tuberosum adora light yellow yellow round superficial short agria yellow yellow oblong superficial short spunta light yellow yellow oblong superficial short adv. hort. sci., 2017 31(3): 151-156 154 depth and stolon length were uniform. this is the result of the previous selection pressure aimed at discarding clones with undesired characteristics (e.g. deep eyes). tuber yield, specific gravity, and chipping ability of material under evaluation are reported in table 2. data are summarized on a family basis. overall, significant differences in ty were found among families. the average ty was 1.2 kg/plant, ranging from 0.43 to 3.00 kg, detected in a clone from ‘spunta’ x ‘victoria’ and ‘majestic’ x ‘alcmaria’, respectively. by contrast, the comparison between the mean ty of clones and that of control varieties r e v e a l e d n o t s i g n i f i c a n t d i f f e r e n c e ( t a b l e 3 ) . however, analysis of ranges within each family revealed the presence of clones with good yield performances. in this research, 12 very promising clones for tuber yield were identified, belonging to ‘spunta’ x ‘victoria’ (5), ‘majestic’ x ‘alcmaria’ (1), ‘jenny’ x ‘mc 329’ (1), ‘blondy’ x ‘victoria’ (1), ‘bolestra’ x ‘mc 329’ (4) (not shown). this may outline the occurrence of allelic combinations providing satisfactory diversity, a prerequisite for heterosis in yield (mendoza and haynes, 1974). potato quality evaluation does not include only tuber yield, but also several qualitative and quantitative traits. among them, tuber dry matter and chipping ability are fundamental for processing. in particular, the chipping ability is a quality parameter highly important for food industries because it influences not only yield of the processed product, but also oil absorption rate in fried products (asmamaw et al., 2010). on the other hand, tuber specific gravity (tsg) is commonly accepted as measure of the dry matter content and it provides the suitability of potato varieties for processing (kabira and berga, 2003). based on this finding, fitzpatrick et al., (1964) identified three classes of tsg: low (less than 1.077), intermediate (between 1.077 and 1.086), and high (more than 1.086). furthermore, kabira and berga (2003) reported that tuber should have a specific gravity higher table 2 tuber yield (kg of tubers per plant) (ty), tuber specific gravity (tsg), chip category color and evaluation index (ei) of 27 potato advanced clones. chip category color was evaluated at harvest and after 90 days of cold storage at 7°c, with and without reconditioning at room temperature for two weeks (respectively + rec; rec) (see materials and methods). for each trait, the average family value (range) is reported material no. of clones ty * tsg * chip category colour * ei * direct cold storage rec cold storage + rec families ‘spunta’ x ‘victoria’ 10 1.09 (0.43-1.58) b 1.081 (1.073-1.088) bc 3.8 (2.0-7.0) 8.5 (7.0-10.0) b 6.1 (2.0-7.0) abc 10.1 (6.0-14.0) c ‘jenny’ x ‘mc 329’ 3 0.95 (0.48-1.51) b 1.095 (1.086-1.104) a 2.0 (2.0-2.0) 10.0 (10.0-10.0 a 4.3 (2.0-7.0) bc 13.0 (11.0-15.0) ab ‘blondy’ x ‘victoria’ 2 1.20 (1.13-1.28) b 1.087 (1.086-1.089) ab 2.5 (2.0-3.0) 8.5 (8.0-9.0) ab 6.0 (5.0-7.0) abc 11.5 (11.0-12.0) abc ‘bolesta’ x ‘mc 329’ 9 1.20 (0.98-1.45) b 1.086 (1.079-1.091) b 4.0 (7.0-2.0) 8.9 (7.0-10.0) ab 6.7 (3.0-8.0) a 10.6 (7.0-13.0) bc ‘agria’ x ‘sandy’ 1 0.76 b 1.080 bc 2 10.0 ab 8.0 ab 7.0 c ‘majestic’ x ‘alcmaria’ 1 3.00 a 1.079 bc 3 7.0 b 3.0 c 15.0 a ‘primura’ x ‘alcmaria’ 1 1.02 b 1.070 c 3 10.0 ab 8.0 ab 7.0 c varieties adora 1 0.80 b 1.071 c 2 9.0 ab 5.0 abc 8.0 c agria 1 1.18 b 1.075 bc 5 10.0 ab 5.0 abc 11.0 abc spunta 1 1.10 b 1.078 bc 4 8.0 ab 7.0 abc 10.0 abc f-ratio 13.52 8.92 ns 3.03 3.56 5.42 p-value <0.0001 <0.0001 0.0036 0.0009 <0.0001 table 3 means, ranges and comparisons between clones and control varieties (spunta, adora, agria) obtained evaluating tuber yield (ty), tuber specific gravity (tsg), chipping ability (direct, after cold storage, ± reconditioning, rec) and the evaluation index (ei) (z) number of clones with a significantly better score compared to the control. ns, *, ** indicates that means are not different or statistically different at p<0.05 and p<0.01, respectively (lsd 0.05). family ty (kg) (z) tsg (z) chip category color (z) ei (z) direct cold storage rec cold storage + rec selections 1.16 (0.43-3.00) 1.083 (1.070-1.104) 3.4 (2.0-7.0) 8.8 (7.0-10.0) 6.1 (2.0-8.0) 10.5 (6.0-15.0) cultivars 1.03 (0.80-1.18) 1.075 (1.071-1.078) 3.3 (2.0-4.0) 8.3 (8.0-9.0) 6.3 (5.0-7.0) 9.7 (8.0-11.0) comparisons selection vs cultivar ns 11 ** ns ns ns ns selection vs adora ns 13 ** ns ns ns ns selection vs agria ns 7 ** ns ns ns ns selection vs spunta ns 4 ** ns ns ns ns * means comparison using tukey’s test. levels not connected by the same letter are significantly different (p<0.05). ns indicates not statistically significant data. melito et al. tuber yield and processing traits of potato advanced selections 155 than 1.080 to ensure good processing ability. in this research, tsg significantly varied among families studied (table 2). on average, the specific gravity was 1.083, higher than the three control varieties used (1.075). it ranged from 1.070 (‘primura’ x ‘alcmaria’) to 1.104 (a clone from ‘jenny’ x ‘mc 329’). nineteen clones (70%) showed a specific gravity higher than 1.080; and 4 clones, belonging to ‘bolesta’ x ‘mc 329’ (2) and ‘jenny’ x ‘mc 329’ (2), revealed a tsg higher than 1.090 (table 2). the mean specific gravity of the studied potato clones was significantly higher than that of the controls, with 11 clones showing a higher specific gravity than the mean of the controls (table 3). the chipping ability of the selected clones was evaluated at three times (at harvest and 90 days of cold storage at 7°c, with and without reconditioning at room temperature), based on the req u i remen t o f th e p o tato p ro cessi n g market. indeed, cold storage allows potato industries to process tubers when fresh product is not available, preventing sprouting and diseases (malone et al., 2006). meanwhile, cold storage induces degradation of starch, conversion of sucrose in glucose and fructose causing an accumulation of reducing sugars. this process is extremely disadvantageous for the potato processing industry because it induces browning of chips (dale and bradshaw, 2003). during frying, high t e m p e r a t u r e s o n r e d u c i n g s u g a r s a c t i v a t e t h e maillard reaction on chips, which became dark colored and bitter, and so not marketable (kumar et al., 2004). variability in chipping ability was found among families, with significant differences after cold storage with or without reconditioning (table 2). by contrast, no significant contrast between the mean chipping value of clones and that of control varieties fried at harvest and after cold storage was found (table 3). however, the analysis of the chipping score ranges of clones indicated that at harvest and after cold storage with reconditioning, 11 and 5 clones respectively, presented a chipping score lower than 4.5 (not shown). therefore, they were all good chippers. by contrast, good chippers were not identified after chips were fried directly out of cold storage. these data were expected considering that during cold storage two principal phenomena occur. the first is called “reversion” and is caused by reducing sugar accumulation: potatoes that generally show a good chipping ability after harvest, give dark, not acceptable chips (oltmans and novy, 2002). the second event occurs when stored potatoes are subjected to a warm (room temperature) period. it is called “reconditioning” and it induces a decrease of reducing sugars in the tuber. this phenomenon happens because during the warming period about 80% of reducing sugars (glucose and fructose) are converted b a c k t o s t a r c h ( o l t m a n s a n d n o v y , 2 0 0 2 ) . consequently, tuber cold storage may produce lighter colored chips compared to the chips produced without a warming period. in order to support the selection of clones with interesting trait combination, an arbitrary evaluation index (ei) was estimated (melito et al., 2017) (table 2). the average family ei was 10.90, ranging from 7 in ‘spunta’ x ‘victoria’ (1) and ‘primura’ x ‘alcmaria’ (1) to 16 in ‘majestic’ x ‘alcmaria’ (1). the best control variety was agria, with an ei of 11. in general, high variability and significant differences in ei were observed among the seven families studied (table 2). analysis of ranges revealed that in almost all families clones with ei higher than that of control varieties were present. the development of a score to evaluate the performance of breeding materials can be used to summarize the analysis of multiple traits, providing a synthetic parameter to support and simplify the practical selection of potato: high ei could indicate interesting genotypes that can be further analyzed deeper for additional traits, while low ei could be discarded as inferior genotypes. additional experiments will be carried out to evaluate the most promising genotypes for new traits, with the purpose t o e i t h e r p r o d u c e n e w v a r i e t y / s u s e f u l i n mediterranean environmental conditions or select parental lines for further breeding. references asmamaw y., tekalign t., workneh t.s., 2010 specific gravity, dry matter concentration, ph, and crisp-making potential of ethiopian potato (solanum tuberosum l.) cultivars as influenced by growing environment and length of storage under ambient conditions. potato res., 53: 95-109. carputo d., aversano r., frusciante l., 2005 breeding potato for quality traits. acta horticulturae, 684: 55-64. carputo d., frusciante l., 2011 clasfsical genetics and traditional breeding, pp. 20-40. in: bradeen j.m., and k. chittaranjan (eds.) 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j., berga l., 2003 potato processing: quality evaluation procedure for research and food industry applications in east and central africa. kenya agric. res. inst. nairobi, kenya. kumar d., singh b., kumar p., 2004 an overview of the factors affecting sugar content of potatoes. ann. of appl. biol., 145: 247-256. mackay g.r., 2005 propagation by traditional breeding methods, pp. 65-81. in: razdan m.k., and a.k. matto (eds.) genetic improvement of solanaceous crops. volume 1. potato. science publishers inc., enfield, usa, pp. 476. malone j.g., mittova v., ratcliffe r.g., kruger n.j., 2006 the response of carbohydrate metabolism in potato tubers to low temperature. plant cell physiol., 47: 1309-1322. melito s., garramone r., villano c., carputo d., 2017 chipping ability, specific gravity and resistance to pectobacterium carotovorum in advanced potato selections. new zeal. j. crop hort. sci., 45(2): 81-90. mendoza h.a., haynes f.l., 1974 genetic basis of heterosis for yield in the autotetraploid potato. theor. appl. genet., 45: 21-25. mottram d.s., wedzicha b.l., dodson a.t., 2002 acrylamide is formed in the maillard reaction. nature, 419: 448-449. oltmans s.m., novy r.g., 2002 identification of potato (solanum tuberosum l.) haploid x wild species hybrids with the capacity to cold-chip. am. j potato res., 79: 263-268. rak k., palta j.p., 2015 influence of mating structure on agronomic performance, chip fry color, and genetic distance among biparental tetraploid families. am. j potato res., 92: 518-535. stadler r.i., blank n., varga f., robert j., hau p., guy m., robert m., riediker s., 2002 acrylamide from maillard reaction products. nature, 419: 449-450. woolfe j.a., 1987 the potato in the human diet. cambridge university press, cambridge, uk, pp. 231. zhao q.x., zhao b.x., zhang q.q., yu b., cheng l.x., jin r., wang y.p., zhang j.l., wang d., zhang f., 2013 screening for chip-processing potato line from introgression of wild species germplasms with post-harvest storage and chip qualities. amer. j. pot. res., 90(5), 425-439. 43 1. introduction the world’s cultivated lands are increasingly affected by drought and salinity (barrett-lennard, 2000). halophytes growing in highly saline soils could thus serve as a resource for the identification and development of new crop systems for marginal saline soils (debez et al., 2011; ben hamed et al., 2013). the actual yield of halophytes remains largely unknown since their domestication is still limited, yet the economic potential of some of these plants has been positively assessed by various groups (aronson, 1989; ashour and thalooth, 1993; abdelly et al., 2006). numerous brassicaceae species are current and emerging biodiesel crops: in addition to the oil-rich seed, the ability of brassicaceae species to grow on marginal land with minimal inputs make them particularly attractive and potentially viable for this application. among halophyte species studied for their potential as oleaginous plants, the brassicaceae cakile edentula (o’leary et al., 1985), crambe abysinnica (mandal et al., 2002) or cakile maritima (ghars et al., 2005) have been reported to contain high amounts of oil. cakile maritima, the sea rocket, is an annual succulent halophyte frequently found from the black sea coasts to the mediterranean region, and from the atlantic coasts of north africa to the north of europe (clausing et al., 2000). tunisian accessions of c. maritima contain up to 40% seed-oil (ghars et al., 2005). plant growth, harvest index, silique number and seeds produced per fruit segment is maximal at 100 mm nacl (debez et al., 2008) but c. maritima can survive at up to 800 mm nacl (ellouzi et al., 2013) and successfully reproduces till 500 mm nacl salinity (debez et al., 2004). seed-oil content did not seem to be affected by salinity, although erucic acid level could increase (debez et al., 2006). these facts highlight the need to better understand the basis of adaptation to saline environments, as well as traits associated with oil production itself. in previous works we described some aspects of the response of c. maritima to salt stress (debez et al., 2004, 2006, 2008; ellouzi et al., 2011) and our data point out that c. maritima adopts a complex survival strategy at high salinity, however numerous data concerning the cellular mechanisms allowing this plant to resist salinity are still lacking. suspension culture cells offer an in vitro system that is widely used in plant biology as a convenient tool to investigate a wide range of phenomena. it consists in a model system as suspension culture provide a ready source of a homogenous cell type and avoids the complications of multicellular tissue types in planta (moscatiello et al., 2013). suspension culture cells were recently used in various studies and models concerning plant responses to salinity, such as proteomic studies (chen et al., 2012; liu et al., 2013), metabolomic studies (liu et al., 2013) or establishment of a cell suspension culture of the halophyte cakile maritima i. ben hamed*, **, b. biligui*, d. arbelet-bonnin*, c. abdelly**, k. ben hamed**, f. bouteau*(1) * université paris diderot, sorbonne paris cité, institut des energies de demain, umr 8632, paris, france. ** laboratoire des plantes extrêmophiles, centre de biotechnologie de borj cedria, university of carthage-tunis, bp 901, 2050 hammam lif, tunisia. keys words: biofuel, cakile maritima, cell suspension, halophytes, oil. abstract: cakile maritima is a member of the brassicaceae family also known as sea rocket. it is an annual succulent halophyte frequent in coastal dune vegetation in mediterranean regions and atlantic coasts from north africa to the north of europe. this halophyte presents a complex survival strategy at high salinity and its seeds contain up to 40% of an oil which could be suitable for biofuel production and other industrial applications. however, data concerning the cellular mechanisms allowing this plant to resist salinity are still lacking. cell suspension cultures offer an in vitro system convenient for cell biology studies and biotechnological methods are still not developed for this putative crop. the present paper reports initiation of c. maritima cell suspension cultures from callus obtained from aerial parts of seedlings. the establishment of a suspension culture which preserves its salt resistance provides an opportunity to gain insights into c. maritima biology. adv. hort. sci., 2014 28(1): 43-48 (1) corresponding author: francois.bouteau@univ-paris-dodierot.fr received for publication 17 march 2014 accepted for publication 4 june 2014 44 transcriptomic studies (matsuura et al., 2010; bae et al., 2012). it is also a convenient means to study transport system regulations and oxidative responses to various biotic and abiotic constraints (kadono et al., 2010; baz et al., 2012; yukihiro et al., 2012; tran et al., 2013), comprising salinity (cessna et al., 2007; wang et al., 2010; pons et al., 2011; queirós et al., 2011). here, we report the development of a cell suspension culture of c. maritime; growth performance was evaluated on control and nacl cultured cells to validate the biological system. these suspension culture cells could be a valuable tool to gain further insights into halophyte studies and their potential applications. 2. materials and methods establishment of callus of cakile maritima in this study we used cakile maritima seeds harvested in the raoued region in the north of tunisia. callus cultures were initiated from the aerial part of 14-day-old, light-grown seedlings. seeds were submerged in 70% ethanol for 1 min, then rinsed with sterile distilled water, submerged in chlorine bleach for 10 min and then rinsed three times (5 min each) with sterile distilled water. the seeds were placed in petri-dishes containing murashige and skoog medium including vitamins (ms) (murashige and skoog, 1962), supplemented with 30 g.l-1 sucrose, 8 g.l-1 agar. the ph was adjusted to 5.8 with koh. stem segments were finely cut and then placed on a solid callus-inducing medium (cim) containing 6.2 g.l-1 gamborg b5 (gamborg et al., 1968) supplemented with 20 g.l-1 glucose, 8 g.l-1 agar and with growth regulators 9.06 µm of 2.4 d and 0.46 µm of kinetin. the ph was adjusted to 5.7 with koh. after two to three weeks, callus appeared on the sides of the segments. when the size of callus became larger than 1 cm, they were divided and transferred to a new medium. establishment cell suspension cultures of cakile maritima approximately 5 g of callus were transferred to 125 ml flasks containing fresh gamborg b5 medium supplemented with 30 g.l-1 glucose, 0.2 µm 2,4-d and 0.45 µm kinetin. the ph was adjusted to 5.7 with koh. flasks were incubated on a rotary shaker at 120 rpm and maintained at 22°c in the dark. feeding of the cultures with fresh medium was done at 10-14 day intervals during which time the suspensions were allowed to settle under agitation (rotary shaker at 120 rpm). this procedure was repeated for about eight weeks. then, the suspension was subcultured every seven days by transferring 20 ml of the culture into 50 ml of fresh medium in 250 ml erlenmeyer flasks. arabidopsis thaliana cell suspension culture conditions arabidopsis thaliana l. cell suspensions were prepared from calluses of the cell line t87 generated from the ecotype columbia plant as previously described (tran et al., 2013). the suspension cells were obtained after about two months and five to six subcultures in 1 l round-bottom flasks containing 350 ml liquid gamborg b5 culture medium (ph 5.8). cell suspensions were sub-cultured weekly using a 1:10 dilution. growth evaluation growth of the culture was evaluated by measuring the fresh weight of cells and the density of cells using a nageotte cell. cell viability cell viability was assayed using the vital dye neutral red. cells (100 µl) were incubated for 5 min in 400 µl phosphate buffer ph 7 with neutral red to a final concentration of 0.001% (w/v). cells that did not accumulate neutral red were considered dead. at least 500 cells were counted for each replicate and the procedure was repeated at least three times for each treatment. external ph measurements measurements of extracellular ph were performed with ph-sensitive electrodes every 24 h for six days from 5 ml of cultured medium cells. the procedure was repeated on three independent subcultures. cell size cell images were recorded with a camera (kappa cf11dsp) on a light microscope (labophot-2 nikon) and sizes were measured using image analysis software kappaimagebase-2.2sp2-metreo (kappa optoelectronics gmbh, gleichen, germany). protoplast isolation protoplasts were isolated from suspension cultures six days after subculture. fifteen ml of suspension cells were used. after cell sedimentation, the supernatant was removed and replaced by 5 ml of gamborg b5 fresh medium containing 0.1 g cellulysin, 0.05 g macerase and 0.3 m sorbitol. the digestion was carried out under shaking at 120 rpm at 22°c for 30 min. after incubation, protoplasts were collected by centrifugation at 300 rpm for 3 min and re-suspended in 5 ml of gamborg b5 fresh medium supplemented with 0.6 m sorbitol or 0.3 m sorbitol. 3. results and discussion the different steps in the development of cakile maritima callus from stem segments are reported in figure 1. callus appeared on the sides of the discs after two to three weeks. although produced in light, callus became non-chlorophyllian and pale yellow in color (fig. 1c, d). thus cakile maritima loses its power of chlorophyll synthesis during its passage from plant stage to callus stage. to generate cell suspension, approximately 5 g of calluses were transferred in gamborg medium. these calluses progressively disintegrated to the smallest cell aggregates in the liquid medium. 45 large quantities of cells could be obtained after two months feeding (fig. 2a, b). at this stage suspension cells were subcultured every week in fresh gamborg medium and after an additional 1.5 months the culture became more homogeneous as the cell aggregates became smaller and the cell size increased (fig. 2c, d). the level of dead cells in the culture also progressively decreased reaching about 10% after four months of culture, in the same range as observed for tobacco or a. thaliana suspension cells (baz et al., 2012; yukihiro et al., 2012; tran et al., 2013). using four-month-old c. maritima culture, growth curves were established by measuring the fresh weight of cells (fig. 3a) and the cell density (fig. 3b). both curves showed a typical sigmoidal shape with a latency period of 24 h, then an exponential phase lasting about four days prior to a plateau phase (fig. 3a, b) supposed to be due to nutrient depletion. we monitored the medium ph during the culture procedure (fig. 3c). after a slight acidification at the beginning of the exponential phase, the ph became more alkaline, reaching 6.5 at the end of the culture (fig. 3c). this alkalization could be involved in the decrease in biomass production as ling et al. (2008) pointed out that ph of 6.7 lowered the growth of suspension culture of ficus deltoidea. however, the ph variations recorded during the culture should remain suitable for nitrate uptake as reported for ipomoea suspension cells (martin and rose, 1976). histological analysis on six-day-old suspension cells revealed distinct morphological features of the cultured cells (fig. 4). although the cell aggregate size diminished during culture establishment, a few large aggregates (>20 cells) remained. however the largest part of the culture consisted of compact small groups of less than 20 cells, 60% corresponding to groups of three to 10 cells, single cells representing about 10% of the population (fig. 4a). most of the cells were rounded in shape (fig. 4b, left and center); less than 2% were elongated (fig. 4b, right). interestingly, as observed during the establishment of the culture (fig. 2b, d), the size of the cells seemed to be dependent on the size of the groups, the isolated cells being the largest (fig. 4c). it is known that protoplasts can be used for transient expression, trafficking assay or ion homeostasis analysis, notably in studies on plant resistance to salinity (laohavisit et al., 2012; haro et al., 2013; morgan et al., 2013; mottaleb et al., 2013; son et al., 2013). thus, we evaluated the usefulness of the cell cultures for the isolation of protoplasts. protoplasts were isolated from six-day-old suspension cultures. based on the different cell sizes measured (fig. 4b, c) we observed protoplasts of different sizes. most of these protoplasts maintained in 0.6 m sorbitol apfig. 1 establishment of cakile maritima calluses. (a) three-weekold c. maritima seedling grown from sterilized seed. (b) slices of leaf and rod were finely cut for callus generation. (c) onemonth-old calluses derived from rod and leaf slices. (d) magnification of a callus. fig. 2 establishment of cakile maritima cell suspensions. (a) dense suspension cells grown in flask after two months feeding and (b) corresponding cell aggregates magnified 385x. (c) dense suspension cells grown in flask after 1.5 months of subculture after feeding and (d) corresponding cell aggregates magnified 385x. fig. 3 growth pattern of cakile maritima cell suspension determined by fresh weight (a) or cell density measurements (b). evolution of the medium ph during cell suspension growth (c). bars indicate mean ± sd of at least three experiments. 46 peared plasmolyzed and numerous protoplasts seemed to be shrunken (fig. 5a). we then used 0.3 m sorbitol and obtained rounded protoplasts with large vacuoled evidenced by neutral red staining (fig. 5b). finally, cakile maritima being a halophyte, we checked the salinity resistance of the suspension cells. suspension cells were subcultured in gamborg medium complemented with 100, 400 or 800 mm nacl. sigmoidal growth curves were obtained and no significant differences were observed between the control and the suspension cells growing in presence of 100 mm nacl when 400 mm and 800 mm strongly decreased the growth of the suspension cells (fig. 6a). these data are in accordance with what was described for seedlings, c. maritima even requiring the presence of a moderate salt concentration (50-100 mm nacl) to maintain a significant growth activity and plant development (debez et al., 2004, 2008). although strongly reduced at 400 mm or 800 mm nacl, the growth of the suspension cells was also in accordance with previous data indicating that c. maritima can survive up to 800 mm fig. 4 morphology of six-day-old cakile maritima suspension cells. (a) frequency of cell groups in the culture. (b) different morphologies of cells. (c) sizes of the cells according to the size of the cell groups. at least 250 cells were analyzed; bars indicate mean ± sd. fig. 5 protoplasts derived from six-day-old cakile maritima cell suspension maintained in 0.6 m sorbitol (a) or 0.3 m sorbitol (b). fig. 6 evaluation of cakile maritima cell suspension resistance to salinity. (a) growth pattern of cell suspensions grown in presence of 100, 400 or 800 mm nacl determined by fresh weight measurement (b). comparison of cell death extents of c. maritima and arabidopsis thaliana cell suspensions treated 6 h with nacl concentrations ranging from 100 to 800 mm. bars indicate mean ± sd of three experiments. 47 nacl (ellouzi et al., 2013) and successfully reproduces till 500 mm nacl salinity even if the biomass was reduced (debez et al., 2004). we further compared the extent of cell death induced 6 h after the addition of various nacl concentrations on c. maritima and a. thaliana suspension cells. cell death began to increase from 100 mm nacl to almost 100% at 600 mm for a. thaliana (fig. 6b). for c. maritima, the increase in cell death was significant only with 400 mm nacl and reached only 60% at 800 mm, remaining largely inferior when compared to a. thaliana (fig. 6b). it is worth noting that 40% of surviving cells probably go on dividing, which explains the growth of the culture, although reduced, at 800 mm nacl (fig. 6a). as a whole, these data demonstrate that c. maritima suspension cells preserve their ability to resist salinity. 4. conclusions the present study reports the development of c. maritima cell suspension cultures which maintained their salt resistance, offering greater understanding about adaptation to saline environments, as well as traits associated with biofuel production. acknowledgements ibh was supported by fellowships from tunisian higher education and scientific research ministry and université paris-sud. references abdelly c., barhoumi z., ghnaya t., debez a., hamed ben k., ksouri r., talbi o., zribi f., ouerghi z., smaoui a., huchzermeyer b., grignon c., 2006 potential utilization of halophytes for the rehabilitation and valorisation of salt-affected areas in tunisia, pp. 163-172. in: özturk m., y. waisel, m.a. khan, and g. görk (eds.) biosaline agriculture and salinity tolerance in plants. birkhäuser verlag, switzerland, pp. 205. aronson j.a., 1989 haloph: a database of salt tolerant plants of the world. in: whitehead e. 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bouteau f., kadono t., kawano t., 2012 peroxyacetyl nitrateinduced oxidative and calcium signaling events leading to cell death in ozone-sensitive tobacco cell-line. plant signal. behav., 7: 113-120. impaginato 47 1. introduction the potato (solanum tuberosum l.) has been considered one of the most important food crops, along with rice, wheat, and maize (ross, 1986; douches et al., 2004). potatoes grow in a variety of geo-environmental conditions. developing countries cultivate potato to add nutritional balance to their food basket (douches et al., 2004; navarre et al., 2009). in syria, more than 29,000 ha were planted with potato, prod u c i n g a b o u t 6 0 9 , 0 0 0 t o f t u b e r s i n 2 0 0 5 (alammouri, 2008). however, severe damage may occur to potato crops at storage periods particularly in developing countries. within the lepidoptera order, potato tuber moth phthorimaea operculella (zeller) belongs to the gelechiidae family and it has been reported in more than 90 countries, making it a cosmopolitan pest (visser, 2005; golizadeh and esmaeili, 2012). it damages potato throughout the growing season by mining stems, petioles, leaves and tubers by larvae, with the latter considered the typical damage. the procedure of potato damage begins when larvae penetrate the foliage, including leaves and stems. this insect can infest potato tubers stored and in field or it may develop on plants remaining in the field including tomatoes, aubergine or other solanaceous plants (gilboa and podoler, 1995; coll et al., 2000; alvarez et al., 2005). farmers depend broadly on the use of insecticides and other varieties of farming practices (clough et al., 2008); insecticides are widely used to control this pest. however, insecticides are costly, nonselective, unfriendly to the environment, and affective for only a short period of time (simmons et al., 2006). additionally, the phenomenon of resistance to insecticides in lepidoptera has increased significantly (gonzalez and trevathan, 2001). plant resistance, together with appropriate biological and farming practices in combination with insecticides may provide the best management options (rondon, 2010). plants defeat pathogens through their active defense mechanisms that can be stimulated in some cases by plant growth-promoting rhizobacteria (pgpr) which ultimately reduce disease and render the host plant more resistant to any foreseeable pathogen attacks (pieterse et al., 2002). induction of such enhanced defensive capacity is systemic as root adv. hort. sci., 2016 30(1): 47-52 doi: 10.13128/ahs-18701 induced resistance in potato plants by a non-pathogenic pseudomonas putida btp1 against potato tuber moth (phthorimaea operculella zeller) a. adam (*), i. idris, n. khalil, k. houssian department of molecular biology and biotechnology, atomic energy commission of syria (aecs), p.o. box 6091, damascus, syria. key words: biocontrol, plant resistance, potato tuber moth, p. putida btp1, rhizobacteria. abstract: pseudomonas putida strain btp1 is able to promote induced systemic resistance (isr) in a wide spectrum of pathosystems. in this study, we investigated induced resistance in potato plants against potato tuber moth (phthorimaea operculella zeller) by non-pathogenic p. putida btp1. several physiological indicators in the life cycle of the potato tuber moth, such as survival rate, mean weight of pupae, and sex ratio were studied to assess the protective effect of p. putida btp1. our results showed that treatment of potato tubers by bacterial suspension of p. putida btp1 caused evident disturbance to the development of p. operculella in potato plants. survival rate of larvae feeding on treated plant leaves and mean weight of pupae decreased significantly. in addition, a clear deviation in the sex ratio in moths, in favor of males, resulted from larvae fed on bacteria-treated plants. this study preliminarily reports the ability of btp1 to induce resistance in potato plants against potato tuber moth. consequently, p. putida strain btp1 could be a promising approach for potato tuber moth biocontrol. (*) corresponding author: ascientific@aec.org.sy received for publication 9 august 2015 accepted for publication 8 february 2016 copyright: © 2016 author(s). this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. adv. hort. sci., 2016 30(1): 47-52 48 treatment with a pgpr was shown to trigger protective effects on above-ground plant parts. these reactions are thought to typically result from the activation of latent defense mechanisms that are overexpressed upon subsequent pathogen challenge (van loon et al., 1998; ongena et al., 2002; bakker et al., 2007). this induced systemic resistance (isr) can be the basis for integrated plant disease management strategies (ramamoorthy et al., 2001; zehnder et al., 2001; saravanakumar et al., 2007). induced resistance in plants by non-pathogenic rhizobacteria against pests is a very important additional factor for the protection of agricultural crops. several studies have indicated the ability of many strains of rhizobacteria (pgpr) to induce systemic resistance against a large number of insect pests (racke and sikora, 1992; zehnder et al., 1997). for example, in cucumber against striped cucumber beetle acalymm avittatum (zehnder et al., 1997, 2001), in cotton against american boll worm h e l i c o v e r p a a r m i g e r a (vijayasamundeeswari et al., 2009), in tomato against whitefly bemisia tabaci (valenzuela-soto et al., 2010) and also in cucumber against spider mites tetranychus urticae (tomczyk, 2006). a non-pathogenic btp1 showed enhancement of resistance level in many plants including bean, cucumber, and tomato against fungal pathogens (ongena et al., 2002, 2004; adam et al., 2008). in a previous study performed in vitro on grapevine rootstocks, we demonstrated the influence of p. putida btp1 on reproduction and development of grapevine phylloxera (adam et al., 2013). however, to our knowledge no studies have been performed yet to assess the effects of pgpr on p. operculella in potato plants. therefore, the present work aims to demonstrate the protective effect triggered by p. putida strain btp1 against p. operculella in potato plants. the larvae survival rate, the mean weight of pupae and the sex ratio were studied as a biometers to detect the induced resistance. 2. materials and methods establishment of the potato tuber moth colony insects used in the experiments were reared on waxed potato slices as described by rahalakar et al. (1985). the experiments were conducted at a constant temperature of 25±1˚c with 70±5% rh, and a photoperiod of 12:12 (l:d) h. microbial strain and inoculum preparation psudomonas putida strain btp1, isolated from barley roots, was originally selected for its specific features regarding pyoverdine-mediated iron transport (jacques et al., 1995; ongena et al., 2002). it was maintained and prepared for use in the isr assays as previously described by ongena et al. (2002). for the bioassays, btp1 strain was grown in erlenmeyer flasks (250 ml) containing 100 ml of casamino acids medium (caa) for 24 h on a rotary shaker (150 rpm) at 28 oc. cells were removed by centrifugation at 16500 g for 15 min at 4°c and washed in sterile nacl (5g l–1). the final pellet was resuspended in an adequate volume of sterile distilled water to obtain a bacterial suspension at 108 cfu ml-1. assays for induced resistance “draja” potato tubers were washed in sterile water, dipped separately in a suspension of p. putida strain btp1 for 30 min, and air-dried, while control tubers were treated with sterile water. the tubers were then planted in 10 l plastic pots containing autoclaved, moistened soil (three tubers/pot) to exclude any microorganism could affect btp1. the pots were placed in a greenhouse at 25±1°c (day) and 23±1°c (night) with daylight of 16 h and relative humidity of 85-95%. both control and treated plants were under the same watering and fertilizing conditions during the planting period. fresh leaves excised from potato plants (six to seven weeks old) were used for feeding the newly hatched larvae (24 h). for each treatment, 120 larvae in 10 (18 x 12 x 8 cm) plastic boxes (12 larvae/box) were fed on leaves until they reached the pupal stage. the boxes were resealed with parafilm to keep the larvae from escaping, and were then incubated at 25±1°c with daylight of 12 h and relative humidity of 70%. each four-day-old pupae was weighed and placed separately within a small plastic tube. the pupae were classified into three groups according to their weights: small pupae (<5 mg), medium pupae (6-7 mg) and large pupae (>8 mg) to determine the larger sex. the number of pupae and the number of emerging moths (males or females) were recorded in order to calculate the survival rate of larvae and the sex ratio (the number of male/the number of female). the experiment was repeated three times. isolation of bacteria from potato plant leaves small leaf samples were taken from different parts of the potato plants treated with p. putida adam et al. potato plant resistant to potato tuber moth by a non-pathogenic pseudomonas putida 49 btp1. the samples were sterilized with sodium hypochlorite solution (5%) for 3 min and washed three times for 3 min. samples were left to dry on sterile paper. they were then grown on petri dishes containing the casamino acid (caa) medium. the dishes were incubated at 30±1°c for 72 h. statistical analysis statistical analyses were performed using statistic program version 6 (statsoft, inc. 2003) at 5% level (p = 0.05). data were subjected to analysis of variance (anova) for the determination of differences between means. differences between means of pupal weight were tested for significance using tukey hsd test. ratio analysis test (z-test) was used to compare the percentages of larval survival rate. 3. results isolation of bacteria from potato plant leaves the isolation of bacteria test on treated-potato plant leaves showed no bacterial colonies grew in the petri dishes, indicating that p. putida btp1 did not migrate through the plant (from the tubers to the leaves). there was no direct contact between bacteria and larvae. effect p. putida btp1 on potato plants against potato tuber moth larvae survival rate. induced resistance experiments showed the death of large numbers of larvae of p. operculella in different ages of development, particularly in p. putida btp1-treated potato plants (fig. 1.1). the larval survival rate decreased significantly (35%) when the larvae were fed on the excised leaves from p. putida btp1-treated plants compared with the control plants (fig. 1.2). significant differences between btp1 and control were observed in all experiments. effect on pupal weight. the results of three independent experiments showed that there was a negative impact on mean pupal weight in p. putida btp1treated potato plants. where mean of pupal weight was 7.77±0.12 mg in the control potato plants, it decreased significantly in p. putida btp1-treated plants to 6.24±0.15 mg (fig. 2). this implies an approximately 20% weight reduction of pupae in potato plants pre-inoculated with p. putida btp1 as compared with the control. significant differences between btp1 and control were observed in all experiments. with regard to pupae weight, we observed that the pupae, which were classified into three groups according to their weight (small, <5 mg; medium, pupae 6-7 mg; large >8 mg), in control plants were mostly large pupae (approximately 61%) while the fig. 1 example of potato leaves infested by potato tuber moth showing larvae feeding on p. putida btp1-treated plant leaves (1a), and control plant leaves (1b). (1c): profile of the dead larvae in different stages because of malnutrition. (2): influence of potato tuber treatment by the bacterial suspension of p. putida btp1 on the survival rate of larvae of the potato tuber moth. three separate experiments were carried out (120 larvae per treatment and per experiment were used). data were subjected to anova and the differences between means were tested for significance using tukey hsd test (values with different letters are significantly different at p<0.001). adv. hort. sci., 2016 30(1): 47-52 50 small pupae was almost absent (3%) (fig. 3). in contrast, in btp1-treated plants, the percentage of the large pupae decreased significantly to reach (36%), while the percentage of the small pupae increased significantly to reach (27%) (fig. 3). sex ratio of p. operculella the sex ratio of moths (number of males/number of females) emerged from control (180 moths) and p. putida btp1-treated potato plants (91 moths) was calculated. a clear deviation was observed in the sex ratio in favor of males from feeding on btp1-treated plants: 1.84:1, compared to 1:1 in control plants. 4. discussion and conclusions our study has shown that the larval survival rate and the mean of pupal weight were significantly decreased in btp1-treated potato plants compared to control. these results are consistent with previous studies conducted on whitefly, which showed a significant decrease in survival rate (number of nymphs which are able to develop and reach the adult stage) in tomato plants treated by rhizobacteria (valenzuela-soto et al., 2010). in addition, similar results were found in pgpr-treated cotton bolls with mortality of larval, malformation of pupal and adult with decreased adult emergence of american bollworm h. armigera (vijayasamundeeswari et al., 2009). moreover, changes in dietary behavior of the rice leaf roller (cnaphalocrocis medinalis) was observed, and there was a decrease in the larval and pupal weight in treated rice leaves by rhizobacteria (radjacommare, 2002). the reduction of larval survival rate and pupal weight in btp1-treated potato could be attributed to the inability of larvae to feed on treated plant leaves. it is well known that the growth of phytophagous larvae is affected indirectly by chemical or physical conditions or even both which characterize their host plants. for instance, pgpr-treated plants may have a decrease in essential nutrients or have compounds that inhibit growth, or both (reese and field, 1986; bong and sikorowski, 1991; yaman et al., 1999). on the other hand, treatment of potato tubers with p. putida btp1 also caused a clear deviation in sex ratio in favor of males. quezada-garcia et al. (2014) proved that nutritional variation causes differential mortality to the larger sex and the most sensitive to nutritional stress (female) in spruce budworm (choristo neural fumiferana (clemens); lepidoptera). in contrast, house et al. (2011) demonstrated that offspring mortality in the dung beetle (onthopha g u s t a u r u s; coleoptera) vitally depends on the amount of resources that females have provisionally. in addition, they also showed that males have greater nutritional demands than females during development, which ultimately leads to higher mortality in the male population (the larger sex and the fig. 2 influence of potato tuber treatment with bacterial suspension of p. putida btp1 on mean of pupal weight of the potato tuber moth. three separate experiments were carried out. each column represents the weight mean of 25 pupae. data were subjected to anova and the differences between means were tested for significance using tukey hsd test (values with different letters are significantly different at p<0.001). fig. 3 influence of potato tuber treatment with bacterial suspension of p. putida btp1 on percentages of pupal weight of the potato tuber moth (75 pupae/treatment). data were subjected to test ratio analysis (z-test) to determine the significant differences at p<0.05 (values with different letters are significantly different). adam et al. potato plant resistant to potato tuber moth by a non-pathogenic pseudomonas putida 51 most sensitive to nutritional stress) (house et al., 2011; quezada-garcia et al., 2014). these findings are consistent with our results which showed that females were larger (16.74%) than males. in conclusion, understanding the mechanisms of induced defense by p. putida btp1 is very important to enhance the resistance in potato plants. the current study provides evidence that p. putida strain btp1 has a protective effect in potato plants against potato tuber moth. based on similar studies that illustrated that the accumulation of some toxic phenolic compounds in the cells of resistant plants led to an increase in the death rate in insects, we believe that the treatment of potato tubers with p. putida btp1 leads to secondary metabolic changes in treated plant cells which elicit the production of defense compounds (lattanzio et al., 2000; zehnder et al., 2001; arimura e t a l . , 2005; melvin and muthukumaran, 2008). this study preliminarily reports the ability of p. putida strain btp1 to induce resistance in potato plants against potato tuber moth. this bacterial strain could be a promising agent for potato tuber moth biocontrol. however, the controlled environment (plastic pots, sterile soil, and humidity) may lead to different results compared to farm applications due to competitors, t/hr condition, dispersion of inoculum, etc. consequently, more research is needed to determine the mechanisms of defense induced in potato plants. acknowledgements the authors thank prof. i. othman (atomic energy commission of syria) and dr. n. mirali (department of biotechnology) for their help. we thank prof. p. thonart and dr. m. ongena of the university of liege, who provided us with the p. putida btp1 strain. references adam a., idris i., ayyoubi z., 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for induced resistance. eur. j. plant pathol., 107(1): 39-50. impaginato 23 1. introduction food security is a central theme of the new millennium and it must be faced at national and international levels, taking into consideration the multidisciplinary nature of the field which involves socio-cultural, political and environmental, as well as agronomic and economic aspects (deaton and paxson, 1998). from an economic and environmental point of view, food security is defined as a situation in which people have safe and appropriate food with nutritional requirements, for an active and healthy life (wfs plan of action, 1996). food security is based on three pillars: food availability, food access, and f o o d u s e s a s r e p o r t e d i n t h e f a o g u i d e l i n e s (matushke, 2009). food security is a priority in all developing countries, in particular in the urban areas of africa with the egyptian situation and the conditions of its capital city, cairo, being critical. despite the underestimation of risks, the population of the slum areas is affected by malnutrition. in 2005 the egyptian demographic and health survey stated that 18% of egyptian youth and 16% of the residents of urban areas were affected by malnutrition. in a city like cairo, the improvement of agricultural hydroponic systems might be a possible solution to this problem, in particular for informal urban settlements. the development of an efficient and productive growing protocol such as simplified hydroponic systems could be used to grow horticultural produce and increase the availability of natural and safe food for the poorest classes of the population (dresher, 2004). food insecurity is a global problem because it is caused by growth of the demand by the world population for secondary food like meat, making, basic resources, such as vegetables and cereals, less available for the poor parts of population (godfray et al., 2010). the world’s urban population is expected to double in the next 30 years, meaning there will be a growing number of urban poor people. according to the united nations human settlement programme (un-habitat), urban population expansion will be more pronounced in developing countries as a result adv. hort. sci., 2016 30(1): 23-29 doi: 10.13128/ahs-18698 vegetable production using a simplified hydroponics system inside city of dead (cairo) a. giro 1 (*), s. ciappellano 2, a. ferrante 1 1 dipartimento di scienze agrarie e ambientali produzione, territorio, agroenergia, università degli studi di milano, via celoria, 2, 20133 milano, italy. 2 dipartimento di scienze per gli alimenti, la nutrizione e l'ambiente, università degli studi di milano, via celoria, 2, 20133 milano, italy. key words: food security, heavy metals, middle east, sustainable agriculture. abstract: this research work was performed in the poorest urban area of cairo (egypt) in a slum area called al-quarafa. the aim was to develop and evaluate a simplified hydroponics system (hs) to grow vegetables for the local inhabitants who live in an extreme status of food unsafety. in the hydroponic growing system the tomato plants were cultivated and two substrates were compared: peat:perlite (70/30 w/w) and sand:coir (50:50 w/w). this technique guarantees high levels of production and low contamination by avoiding the use of polluted urban soils which are often common in slum areas. macro and microelements in tomato fruits were analysed by icp-ms. results showed low concentrations of heavy metals (cd, sr, as, cr, mo) with all heavy metals under the levels set by european community laws. lack of food safety in slums is more closely linked with malnutrition than starvation, so it is also important to understand mineral availability (both micro and macro) and nutritional significance for health. for this reason micro and macro elements (k, ca, na, fe, mn, mg, cu, zn) were analysed in the harvested fruits. this study showed that hs is a valid method to grow vegetables in urban areas to improve food security in middle east cities. (*) corresponding author: andrea.giro@unimi.it received for publication 19 october 2015 accepted for publication 28 january 2016 copyright: © 2016 author(s). this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. adv. hort. sci., 2016 30(1): 23-29 24 of high birth rates and immigration from rural areas as people flock to cities in search of food, employment and security. population growth will lead to an increase in urban slum areas, with high levels of unemployment, lack of food safety, and malnutrition. the increase of urbanization in developing countries enhances food insecurity of large cities. the poorest people move from rural to urban areas in order to improve their life with hopes of finding a job. unfortunately, in many cases people do not find employment and are obliged to live in the slum areas, increasing the food demand. for this reason, they become more vulnerable in their new position as citizens and consumers. by 2030, it is estimated that approximately 800 million people in developing countries will live in big cities, instead of in rural areas. this prospective is shocking for many nations, in particular for the social and economic relationships among citizens; this phenomenon is unescapable due to economic development and it could create a negative impact on the food security of populations of cities and, in particular, megacities (cohen and garrett, 2010) (fig. 1). in order to counteract the reduction of food availability in african cities, urban people started growing vegetables in urban and peri-urban areas. studies showed a considerable degree of self-sufficiency in the production of fresh vegetables, poultry production and the raising of other animals in many large cities of developing countries (armar-klemesu, 2000). for example, dakar produces 60% of its vegetable consumption and produces poultry for 65-70% of the national demand (mubvami and mushamba, 2004). accra produces 90% of the fresh vegetables consumed in the city (cencosad, 1994; maxwell et al., 2000). in dar es salaam, more than 90% of leafy vegetables in the markets come from the open spaces adjacent to the city or in garden houses (lee-smith, 2010). urban agriculture improves the nutritional state of vulnerable communities as evidenced by studies carried out by mwangi (1995) in nairobi. however, one of the main problems of urban agriculture is the competition for spaces within the city itself between people, vehicles, and animals. another important issue is the agricultural knowledge of the people. many of them do not know how to grow plants, therefore very simple growing systems must be developed. simplified hydroponic (sh) systems allow cost-effective agricultural production in confined spaces, such as internal urban spaces, particularly in the cities of developing areas (seikh, 2006). the sh technique allows higher yield, safer food, and at lower costs. the costs of the entire sh system are fundamental for sustainability of the production of goods; for this reason local materials, best if recycled, were used to construct low budget hydroponic systems. horticulture works well in urban and peri-urban zones because it is highly labour-intensive, involving perishable products and short-cycle, productive, high-value crops, which require less land and water per unit of product than other food crops. hydroponic systems are growing techniques that do not use soil, making them suitable in urban areas to cultivate horticultural commodities (seikh, 2006). it is a relatively young technique used in commercial fields over the past 40 years (grewal et al., 2011). the potential of sh is underestimated: it can be a solution for marginal areas, in developing countries with malnutrition problems. sh technique has the objective of reducing the costs of traditional hydroponic techniques, thus making it available and easy to manage for the poor and people without agriculture skills living in slums, like al-quarafa. for the aims of this study, we used local materials to construct sh greenhouses in order to diminish the cost and to make the technique reproducible for the local population. one of the main costs of sh is the substrate, so it is important to understand which substrate is best in which environment. standardisation of an efficient protocol of sh is important in order to cultivate horticultural products within the urban context (santos and ocampo, 2005). nowadays, many examples of hydroponics are available in developing countries but they are not usually well set up and tend to be expensive. hence, the new challenge is represented by the inception fig. 1 rural and urban index of food energy. international food policy research institute (ifpri). giro et al. vegetable production using a simplified hydroponics system 25 and enhancement of a hydroponic system sustainable in all aspects, from production to management. 2. materials and methods simplified hydroponic systems and plant cultivation boxes made of wood (100x50x20 cm) ensure the best conditions for plant growth and for transportation which are fundamental aspects for the socioenvironmental context (iwasa and roughgarden, 1984). tomato plants (solanum lycopersicum l.) require a minimum depth of 20-30 cm for root system development (pardossi et al., 2005). for the present study, plant density was 3 plants/m2. individual modules of locally manufactured palm wood boxes (50x50x25 cm) were used to reduce the cost of materials, planting one tomato plant per box. the ratio between the surface and the plant was chosen following the standard for tomato crops in open field. all boxes were plastic-coated with a black polyethylene film in order to maintain the moisture of the substrate, preventing excessive evaporation (hassanwassef, 2004). during the summer season, high temperatures in cairo cause rapid water evaporation, therefore to reduce this affect, the cultivation boxes were covered with white mulching. evaluation of substrate performance was carried out by comparing the production of 24 tomato plants. for each substrate, plant growth and fruit quality were determined. substrates comparison perlite, peat or coconut fibre, and sand were compared. the substrates were mixed and the final composition was 50% sand + 50% coir (s+c) and 70% peat + 30% perlite (p+p). three steps followed: fertilization and irrigation up to saturation, mulching and planting, and covering with net-shading to reduce sun intensity. the fertigation strategy used was based on an initial fertilization with 25 g of 20-20-20 npk fertilizer containing fe 5 g/kg, zn 5 g/kg, and mg 1 g/kg during the preparation of substrate with the addition of 1 g microelements (table 1). the peat used had the following characteristics: ph 3.5; ec 10 ms/m; hydric retention 70% vol. peat ph was adjusted to 5.5 by adding 200 g caco3. the simplified weekly fertigation schedule provided irrigation with 20 l/week of water containing 3 g ca(no3)2 for each plant. nitrogen was provided as 13.7% n-no3 and 1.5% n-nh4, with a total of 15.2%. the addition of 25 g npk and 1 g of micronutrients was carried monthly for each box. assessment of tomato growth and fruit quality plant growth was evaluated monthly and the following parameters were recorded: height, time of flowering, and number of flowers for plants in the different substrate conditions. fruit quality evaluations were carried out on dried fruits and comprised the determination of macro (k, na, ca) and microelements, which are important for a balanced human diet. the concentrations of heavy metals (cd, pb, sr, cr, mo, as), as possible contaminants, were also measured. analytical determinations were carried out using an icp-ms (inductivity coupled plasmamass spectroscopy). to better understand the potential of the cultivation technique related to the lack of food safety in the slum community, we also analysed the quality of tomatoes bought in local markets of the cemetery area of al-quarafa. to evaluate the quality, comparisons were made with standard dried tomato analysed by the fda (food and drug administration). dry materials were ground and digested with nitric acid (7:0.1). mineral compounds (fe, mn, zn, and cu) were measured using inductively coupled plasma mass spectrometry (icp-ms). values were reported as means and standard errors (n=3). data were subjected to anova analysis and differences among means were determined using bonferroni’s post-test. 3. results plants growth and productivity plant growth was monitored in sand and coir or peat and perlite in the two growth containers during cultivation. higher values were found in plants grown in the s+c substrate in both palm boxes (fig. 2a) and woody boxes (fig. 2b). however, statistical differtable 1 eu heavy metal security limits (ec 420/2011) elements concentration cr 0.050 mg/kg pb 0.100 mg/kg mo 0.025 mg/kg as 3.500 mg/kg cd 0.050 mg/kg adv. hort. sci., 2016 30(1): 23-29 26 ences were found only in woody boxes. in the s+c substrate the plants reached an average height of 50 cm after three months. in the p+p substrate the plants ranged from 35 to 38 cm. in june, in both substrates, plants reached a plateau (fig. 2). food quality, nutrients and heavy metals content the results revealed that simplified hydroponics guarantee the safety of food produced, even in heavy metals-polluted environments. the main mineral nutrient contents were determined in ripe tomato fruits and compared with those sold in the local markets. the sodium (na) content was lower in fruits harvested from plants grown in the s+c substrate (1.7 mg/kg dw) compared with the other samples. the fruits purchased from local markets had a higher na amount: 7.5 mg/kg dw on average (fig. 3a). the magnesium (mg) content in fruits grown in sh was similar without significant differences between the two substrates and ranged from 2.7 to 3.4 mg/kg dw (fig. 3a). in fruits obtained from local markets, the mg content was almost double (fig. 3a). analogous results were observed for potassium (k) and calcium (ca). the k in fruits harvested from plants grown in the two different substrates ranged from 65.5 to 68.0 mg/kg dw, while for fruits purchased from the markets the values were higher, 110.0 mg/kg dw on average (fig. 3). the ca content was similar to mg, double the content was found in the commercial tomato fruits compared with those harvested from the sh system (fig. 3a). considering the heavy metals microelements, different concentrations were found in the tomato fruits. copper (cu) in the analysed fruits ranged from 0.6 to 0.8 mg/100 g dw. the highest values were found in the p+p substrate (fig. 3). manganese (mn) was similar in distribution in the different treatments, but the concentration was higher: from 0.7 to 4.4. mg/100 g dw. the highest value was found in the fruits harvested from plants grown in the p+p substrate (fig. 3). on the contrary, zn content was higher in the sh cultivated fruits compared with those obtained from local markets. the values ranged from 1.3 to 2.0 mg/100 g dw. iron (fe) was fig. 3 nutritional elements expressed as mg/100 g dried weight. a) na, mg, k and ca. b) cu, mn, zn and fe in tomato fruits harvested from plants grown in sand and coir (s+c), peat and perlite (p+p) or purchased at the local market (m values are means with standard errors. data were subjected to two-way anova analysis and differences among means were determined using bonferroni’s post-test. different letters indicate statistical differences for <0.05. the heavy metals in fruits were under the safety thresholds established by the european union. fig. 2 plant height of tomato plants grown in palm boxes (a) or woody boxes (b). values are means with standard errors. data were subjected to two-way anova. differences among means were determined using bonferroni’s post-test. giro et al. vegetable production using a simplified hydroponics system 27 higher in fruits obtained from plants grown in the p+p substrates and the mean was 13.0 mg/100 g dw. half the amount was found in fruits harvested from the s+c grown plants and purchased from local markets (fig. 3). heavy metals were found in trace amounts and the values were below those set by eu regulations. the as content was higher in local market fruits (6.03 µg/kg dw), while in the sh system the as content was 0.63 µg/kg dw in fruits harvested from plants grown on the s+c substrate and 2.58 µg/kg dw in those obtained from plants grown on p+p substrate. strontium (sr) in the sh system ranged from 17.97 to 20.7 µg/kg dw, while in tomatoes purchased from the local market the concentration was 44.6 µg/kg dw on average (fig. 4). molybdenum (mo) was 10fold higher in fruits harvested from plants grown in the p+p substrate compared with s+c and local market tomato samples. cadmium (cd) was higher in fruits sold in local markets compared with those obtained from the sh system. the lowest value, 1.6 µg/kg dw, was found in fruits harvested from plants grown on the p+p substrate. analogous results were observed for lead (pb); the highest value found in fruits purchased from the market was 28.97 µg/kg dw (fig. 4). on the contrary, higher cr values were found in the fruits harvested from the sh system (21.03-45.67 µg/kg dw) compared with 16.83 µg/kg dw measured in the fruits purchased at the market (fig. 4). 4. discussion and conclusions the cultivation of vegetables in highly polluted environments where there is poor soil quality can only be achieved using soilless systems. most people living in the slum areas do not have skills or knowledge about agricultural practises, therefore urban agricultural systems must be simple and easy to manage. unfortunately, high density urban and periurban areas can also be subjected to heavy metal pollution. contamination can also come from airborne trace elements such as cd and mo (francini et al., 2010) that are deposited on the surface of fruits. heavy metal accumulation in vegetables is closely correlated with the pollution level of the area (antisari et al., 2015). sh is a good strategy to improve food security for urban slum populations and, in particular, it is linked to an increase of food availability (orsini et al., 2013). however, the food situation could also improve through direct sale of the products if the cultivated surface were enough to have a surplus of fresh produce. for these reasons it is important when choosing the cultivar to consider the most profitable ones in order to improve saleability of the products. the sh technique makes it possible to cultivate safe vegetables, in small spaces, and with low-tech instruments. the results of the current study reveal that local materials for substrate and palm boxes were a good compromise between cost and production capability. the community of al-quarafa follows a typical mediterranean diet based on cereals and legumes, however they do not consume fresh vegetables because they are very expensive and perishable (arenas and vavrina, 2002; hamza and mason, 2004). thus, the application of sh represents an interesting method to combat food insecurity in al-quarafa, a symbol of all cairo slums. moreover, the use of the substrates in the study avoided the accumulation of heavy metals in vegetables and allowed enrichment of some of the micronutrients that improve human health. sh have been used in africa for 10 years to cultivate fresh products in urban and peri-urban settlements. for example in dakar, in 1999 a fao pilot project for family farming employed sh. the dakar models, however, were adapted to tropical temperature and climate. the substrates were 40% peanut shells, 40% rice husks, and 20% perlite, suitable for leafy vegetable production because they are harvested at a young stage and do not have vertical growth. fig. 4 heavy metals expressed as µg/kg dw determined in tomato fruits harvested from plants grown in sand and coir (s+c), peat+perlite (p+p) or purchased from the local market (m). values are means with standard errors. data were subjected to two-way anova analysis and differences among means were determined using bonferroni’s post-test. different letters indicate statistical differences for <0.05. adv. hort. sci., 2016 30(1): 23-29 28 unfortunately, they are not able to support the growth of plants such as tomatoes. in fact, tomatoes prefer more stable substrates, from a mechanical point of view, such as peat/perlite or coir/sand (esposito, 2010). also the depth (14-20 cm) of the recycled-pallet boxes used in dakar was less than that required by cultivated varieties that need greater volume for adequate root development to favour production (ghehsareh et al., 2011). the irrigation schedule in the dakar project provided 2 l/m2 two to three times a day (ghehsareh et al., 2011). the nutritional elements were recollected in a small container under the boxes. this daily schedule means that plants must be managed three times a day and this is one of the differences between the dakar project and the method applied in the current study tested in cairo in which an irrigation schedule was followed of once a week, thanks to the water capacity (wc) of the peat and coir. the combination of 50% coir and 50% sand provided the best growth performance for two reasons: 1) because using a weekly irrigation schedule the coir had greater wc than peat (abad, 2002), and 2) because the weight of sand simplified the growth of the vegetative part of the plants, offering mechanical stability. it is also important to consider the ph of the substrate used: the combination between coir and sand naturally had a sub acid ph (arenas and vavrina, 2002), with is the naturally ideal ph for tomato growth. this aspect favoured the availability of the micro and macro nutrients (ghehsareh et al., 2011). from a productive point of view, the tomato plants produced on average 0.4-0.5 kg per plant in the current investigation; the best results were achieved with tomatoes grown in sand/coir. considering two plants per box, these values translate to a productive capability of 0.8-1 kg/m2 of tomatoes on average. these productivity ranges consider the stressed conditions of the climate in cairo which allows two growing cycles per year (september to november, march to june). in terms of nutrition, considering the nutritional level of macro and micro elements of tomatoes analysed by the fda, the tomatoes grown in sh in the current study had similar nutritional levels and sometimes better than the tomatoes the fda considered as standard. social agriculture and future strategy this sh growing system has been developed for poor urban communities with very limited agricultural knowledge. these cultivation systems are suitable for urban and social agriculture as reported in fao guidelines. moreover, for production in highly populated areas, sh systems also play a social role as they can be meeting points for the families within the community. therefore, these growing systems should not be considered only as vegetable production tools. these systems must be easy to manage especially for water and nutrients supply. the management of the cultivation is often carried out by women and children, who are the most susceptible to food insecurity. in slum areas like al-quarafa, people are depressed, marginalized and unemployed. hence, the growing areas improve food production, as well as the physic and psychological health of the inhabitants. collaborations among the people living the community improve relationships between families and create opportunities to establish networks. these networks are essential to sharing information on crop cultivation in sh systems and to organising selling points in local market. in the future, the “cairo model” of the sh system may be exported to other depressed urban areas in africa and in the middle east. this new, low-cost and low-tech growing system for vegetable production provides a great opportunity for poor populations who want to redeem themselves within society. acknowledgements this study was logistically supported by ngo “liveinslums” and financially supported by municipality of milan. references abad m., 2002 physico-chemical and chemical properties of some coconuts dust for use as a peat substrate for containerised ornamental plants. bio-resource technology, 82: 241-245. antisari l.v., carbone s., gatti a., vianello g., nannipieri p., 2015 uptake and translocation of metals and nutrients in tomato grown in soil polluted with metal oxide (ceo2, fe3o4, sno2, tio2) or metallic (ag, co, ni) engineered nanoparticles.environ. sci. pollut. res., 22(3): 1841-1853. arenas m., vavrina c.s., 2002 coir as an alternative to peat in media for tomato trasplant production. hortscience, 37(2): 309-312. armar-klemesu m., 2000 urban agriculture and food security, nutrition and health, pp 99-118. in: bakker n., dubbeling m., gundel s., sabel-koschella s., giro et al. vegetable production using a simplified hydroponics system 29 de zeeuw h., (eds.) growing cities, growing food: urban agriculture on the policy agenda. a reader on urban agriculture. gtz/dse, germany. cencosad, 1994 urban market gardens in accra. centre for community studies, action and development and the mega cities project, accra, ghana. cohen m.j., garrett j.l., 2010 the food price crisis and urban food (in) security. environment & urbanization, 22(2): 467-482. deaton a., paxson c., 1998 economies of scale, household size, and the demand for food. journal of political economy, 106(5): 897-930. dresher a.w., 2004 food for cities: urban agriculture in developing countries. acta horticulturae, 643: 227-231. esposito t., 2010 agriculure urbaine et periurbaine pour la securite alimantaire en afrique de 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international forum on food security under water scarcity in the middle east: problems and solutions. como, italy, 24-27 nov, pp. 12. hassan-wassef h., 2004 food habits of the egyptians: newly emerging trends. east mediterr health j, 10(6): 898-915. iwasa y., roughgarden r., 1984 shoot/root balance of plants: optimal growth of a system with many vegetative organs. theoretical population biology, (25): 78-105. lee-smith d., 2010 cities feeding people: an update on urban agriculture in equatorial africa. environment and urbanization, 22(2): 483-499. matushke i., 2009 rapid urbanization and food security: using food density maps to identify future food security hotspots. fao contributed paper for international association of agriculture economist conference, beijing, china, august 16-22, pp. 15. maxwell d., levin c., armar-klemesu m., ruel m.t., morris s.s., ahiadeke c., 2000 urban livelihoods and food and nutrition security in greater accra. the international food policy research institute research report 112. international food policy research institute, washington, d.c., usa. mubvami t., mushamba s., 2004 mainstreaming gender in urban agriculture: application of gender analysis tools: a case study of musikavanhu project, harare. m u n i c i p a l d e v e l o p m e n t p a r t n e r s h i p , h a r a r e zimbabwe. women feeding cities, accra, ghana, sept. 20-23, pp. 42-43. mwangi a.m., 1995 the role of urban agriculture for food security in low income areas in nairobi report 54/1995, ministry of planning and national development, nairobi, and african studies centre, leiden, kenya, pp. 42-44. orsini f., kahane r., nono-womdim r., gianquinto g., 2013 urban agriculture in the developing world: a review. agron. sustain. dev., 33: 695-720. pardossi a., malorgio f., incrocci l., tognoni f., 2005 hydroponic technologies for greenhouse crops, pp. 360-378. in: ramdane d. (ed.) crops: quality, growth and biotechnology. wfl publisher science & tecnology, helsinki, pp. 1356. santos p.j., ocampo e.t., 2005 snap hydroponics: development & potential for urban vegetable production. philippine journal of crop science, 30(2): 3-11. seikh b.a., 2006 hydroponics: key to sustain agriculture in water stressed and urban environment. pak. j. agric., agril. eng., vet. sci., 22: 53-57. 100 1. introduction open-field elevation cultivation of strawberries (fragaria x ananassa duch.) is being developed for fruit production in tropical regions (lópez et al., 2002; pirlak et al., 2002; riyaphan et al., 2005; pádua et al., 2009) and also in temperate areas (faedi, 2010; gambardella, 2010; rowley et al., 2010). nevertheless, little information is available on the quality of strawberries obtained from plants grown in mountain areas. a strong variability of horticultural characteristics and antioxidant profiles were observed among 12 cultivars grown at 730 m asl in the trentino region (italian alps), the most relevant attributes limiting quality being represented by poor taste and low flesh firmness (giongo et al., 2006). in the same area, a study by faedi et al. (2009) reported that ‘elsanta’ showed the best qualitative results in the first season crop among the tested cultivars. two commercial, ordinary june-bearing strawberry cultivars, ‘elsanta’ and ‘marmolada®onebor’, were selected for an elevation cultivation trial in the area of abetone mountain (apennine) in italy. the present paper reports the results observed on the quality of berries of these cultivars grown in open field mountain area. two cultivation strategies are also comparatively discussed one based on “first season crop” (fsc), carried out in 2008, and the subsequent year (2009) “second season crop” (ssc) as well as some aspects related to the ecological environment. 2. materials and methods location and environmental characteristics three experimental fields (ef1, ef2 and ef3) were established in three different locations around the abetone pass (apennine mountains) in tuscany (italy); details of the sites are reported in table 1. the three experimental fields had good exposure to light; all of them laid on humic umbrisols soils (lamma, 2012) with similar chemico-physical characteristics. the yearly average rainfall for the area is 2,000 mm; snow is present from late november until late spring (may). historical average of air temperature and humidity data and rainfall for the period 2008-2009 are reported in figure 1. from an ecological point of view, the selected sites for the experiment are represented by scarcely anthropized natural grasslands, highly biodiverse in terms of flora being constituted mainly by festuca pucinellii, trifolium thalii, plantago alpina, poa alpina, brachypodium genuense and nardus stricta, and surrounded by forests of european beech (fagus silvatica), european silver fir (abies alba mill.) and elder (sambucus racemosa l.) often consociated with blueberry (vaccinium myrtillus l.) (dondini and vergari, 2009). production of strawberries (fragaria x ananassa duch.) in mountain areas: a comparative evaluation of berries from two june-bearing cultivars e. giordani, w.a. petrucci, d. morelli, a. ferri dipartimento di scienze delle produzioni vegetali, del suolo e dell’ambiente agroforestale, università degli studi di firenze, viale delle idee, 30, 50019 sesto fiorentino (fi), italy. key words: elevation cultivation, ‘elsanta’, garden strawberry, ‘marmolada®onebor’. abstract: two uniferous strawberry cultivars, ‘marmolada®onebor’ and ‘elsanta’, were tested in three experimental fields located at 1,200 m asl; phenological phases and fruit quality of first and second season crops were compared. harvesting time in the first year crop occurred 40 days later than in the second season crop. cultivar significantly affected fruit skin brightness and chroma index, flesh firmness, titratable acidity and yield in the first season crop; the experimental field location exerted a significant effect also on fruit weight and diameter, but not on total acidity. fruit weight was higher in the second season crop, and ‘marmolada®onebor’ fruits (22.7 g) resulted heavier than those of ‘elsanta’ (19.7 g). total solid soluble content ranged from 7.4 to 8°brix in ‘elsanta’, compared to 6.2 to 7.4°brix in ‘marmolada®onebor’. neither diseases nor arthropod attacks were noticed on plants and fruits. adv. hort. sci., 2012 26(2): 100-109 received for publication 17 february 2012. accepted for publication 4 june 2012. 101 soil management and plant spacing in may 2008 the soil area destined for the three experimental fields (500 m2 each) was ploughed 30 cm deep and mature manure was distributed uniformly at the rate of 80 t/ha; soil was not fumigated. the strawberries were planted in parallel hill rows 110 cm apart with a distance of 30 cm between plants within the row, with a density of 4 plants/m2; the plants of each cultivar were distributed in an alternate four-row scheme for a total of eight rows for each experimental field. a black polyethylene film was used along the rows for mulching. each plot was surrounded by guard rows. plant material, planting and management one-crown homogeneous-size certified refrigerated plants of ‘elsanta’ and ‘marmolada®onebor’ were supplied by a qualified commercial nursery. in early may 2008 strawberries were planted in the experimental fields, from which the fsc was obtained; the same open field unprotected, overwintered and not de-crowned strawberry plants generated the ssc in 2009. plants were drip irrigated with 1,000 l/d average per experimental field, from flowering time up to the end of the productive season; no treatment was adopted for pest and disease control. weeds were manually removed. data collection during the two years of study (2008 and 2009), observations regarded phenological, morphological and chemical characteristics. flowering and ripening time were recorded on one sample every five plants and reported as days from the beginning of the year. morphological data were measured on four replicated sets of 25 ripened fruits each for every experimental unit (cultivar/location) during the period of maximum productive peak. fruits were harvested and promptly characterized for weight and maximum diameter, skin colour, flesh firmness (two sides), fruit shape and presence of internal cavity. weight (g) was measured with a precision balance (sartorius te 150/2s sartorius) and diameter (mm) with hand calliper; colour (l, a, b coordinates) was determined with a minolta chromameter cr200 konica minolta electronic colorimeter and the a and b coordinates were transformed in the chroma index (a2 + b2)1/2; flesh firmness (g) was measured with a 6 mm diameter plunger hand penetrometer tr 53200. fruit shape and presence of an internal cavity were visually assessed following the upov descriptor for strawberry (upov, 2008). chemical parameters such as ph, titratable acidity and total solid soluble (tss) content were assessed on samples (four replicates per experimental unit) of juice extracted from 10 fruits. titratable acidity (meq malic acid/100 g fresh weight) was determined at ph 8 with a 0.1 n solution of naoh, adopting a basic 20 crison ph meter; total soluble solid content (°brix) was quantified with a hand refractometer (atago n1 atago co., ltd). fruits were tasted by five experts to assess sensorial quality and flavour quality was rated on a five-score scale (1 very weak; 2 medium weak; 3 medium; 4 good; 5 very good), while for persistence of taste 3 min after ingestion and scent intensity, a three-score scale (1 weak; 2 medium; 3 strong) was adopted. experimental design and statistical analysis each of the three experimental fields was split into 16 rows (eight alternate rows per cultivar); every row held 125 plants. a two-way analysis of variance was employed to test the significance of the effects due to cultivar (‘eltable 1 description of experimental fields (ef): altitude, coordinates, soil texture, n concentration (‰), c concentration (%), ph, field capacity (f.c.) and wilting point (w.p.) experimental fields altitude (m a.s.l.) coordinates (n/w) soil texture n‰ c% ph f.c. (-0,33 bar) w.p. (-15 bar) ef 1 1,213 n 44°08.650’ sandy-loam 0.3 3.5 5.2 20.1 11.6 w 10°41.412’ ef 2 1,250 n 44°08.517’ loam 0.1 3.6 6.2 18.4 11.1 w 10°42.043’ ef 3 1,250 n 44°07.810’ sandy-loam 0.1 3.7 5.9 23.6 16.5 w 10°45.336’ fig. 1 mean air temperature (°c), relative humidity (%) and rainfall (mm) of the trial location of years 2008, 2009 and historical series (data elaborated from lamma). 102 santa’ and ‘marmolada®onebor’) and experimental field location (efl1, efl2 and efl3), both considered as independent factors, and their interaction. duncan’s test was applied for mean separation; the averages were reported with standard errors. two-tail chi2 test was applied to qualitative and scale-scored parameters exposed as frequencies. spss statistics 17.0 software was used for analyses; differences at p < 0.05 and p < 0.01 were considered significant and very significant, respectively. 3. results the anova statistical significance of the different factors on the studied parameters are reported in table 2; each of them will be discussed in the following sections. first season crop (fsc) both elsanta and marmolada®onebor cultivars showed the same pattern of phenological stages in the three locations; in detail the full blooming started on 8 july 2008 (day 190) and portrayed until day 203 for a total of 13 days. the first fruits of both cultivars ripened 24 days later (day 214), and harvest time closed on day 234; the period of maximum peak of ripened strawberries fell in the period 4-11 august 2008 (days 217-224) (fig. 2). average fruit weight resulted identical for both cultivars (15.0±0.4 g)(anova; p = 0.69) (table 2). field location and its interaction with cultivar exerted a statistically significant effect on this parameter (anova; p < 0.01), with the highest and the lowest weights of 19.1±0.7 g and 11.8±0.7 for ‘marmolada®onebor’ obtained in ef1 and ef3, respectively; for ‘elsanta’ the maximum and minimum values were 15.9±0.7 g in ef1 and 13.8±0.7 g in ef3 (fig. 3a). as expected, the fruit maximum diameter (average 32.9±6.1 mm in ‘elsanta’ and 32.04±6.2 mm in ‘marmolada®onebor’) followed a behaviour similar to that observed for fruit weight, and it was affected significantly by the experimental field and its interaction with cultivar (fig. 3a). skin colour was analysed taking into account brightness (l) and chroma index. l values ranged from 30.9 to 47.6 for ‘elsanta’ and 24.7 to 47.8 for ‘marmolada®onebor’, with averages of 38±0.3 and 35.2±0.3, respectively (anova; p <0.01). fruit skin brightness showed differences of 2 units (≈ 35 against ≈ 37) due to the effect of the experimental field location (anova; p < 0.01). analogous results were observed for chroma index, with average values of 40.3±0.4 and 37.1±0.4 for ‘elsanta’ and ‘marmolada®onebor’ (anova; p < 0.01); similarly to skin brightness, ef exerted a very significant influence on chroma index, with a variation of ≈ 2 units. flesh firmness resulted statistically different (anova; p<0.01) between the cultivars; the highest average value was observed in ‘marmolada®onebor’ (473.1±9 g), which is about 33% higher than the firmness of ‘elsanta’ fruits (355.6±7.9 g). the experimental field location exerted a significant effect on fruit firmness (p<0.05); a 5% difference between the maximum and the minimum average values was observed. no relevant differences were found in ph of juice, showing a value of 3.6 for both cultivars, and no effect was attributable to the location of the experimental fields. different results were obtained for titratable acidity, which was higher in ‘elsanta’ fruits (12.6±0.8 meq/100 g fw) than in ‘marmolada®onebor’ (9.6±0.4 meq/100 g fw) (anova; p<0.05); location did not affect this parameter. taking into account the total solid soluble content of strawberry juice, the cultivar elsanta showed an average value of 7.4±0.3 °brix, against 6.2±0.3 °brix of fig. 2 blooming and fruit ripening time in years 2008 (fsc) and 2009 (ssc). table 2 significance of cultivar, experimental field location and their interactions on the quantitative parameters analyzed on strawberry fruits resulted from analysis of variance (anova) attribute crop cultivar cv experimental field ef cv*ef replicates fruit weight fsc ns ** ** ns ssc ** ** ** ns fruit diameter fsc ns ** * ns ssc ns ** ns ns fruit brightness (l) fsc ** ** * ns ssc ns ** ** ns chroma index fsc ** ** ** ns ssc ns ** ** ns flesh firmness fsc ** * ** ns ssc ** ** ** ns fruit ph fsc ns ns ns ns ssc ns ns ns ns titratable acidity fsc * ns ns ns ssc ** ** ns ns tss content fsc ns ns ns ns ssc * ns ns ns ns= non significant; * = 0.05<p<0.01; **= p<0.01. 103 ‘marmolada®onebor’; such differences were not statistically significant (anova; p>0.05). the distribution of fruit shape resulted similar among cultivars; no statistical differences were observed between cultivars for conical and globose-conic classes, the most frequent fruit shapes. significant differences (chi2; p< 0.01) were found in the percentages (40% and 27%) of globose-conic fruits of ‘elsanta’ and ‘marmolada®onebor’, respectively (fig. 4). the internal cavity was present in approximately 81% of fruits of both cultivars; a significant statistical difference (chi2; p<0.01) was observed taking into account the experimental field (ef factor), since almost 100% of fruits from ef2 showed internal cavity, against about 75% of those obtained in the other two sites. the distribution of fruits for each cultivar taking into account the quality of taste, resulted as an average value of five taste assessors, is reported in figure 5a. ‘elsanta’ showed a percentage (86%) of fruits of acceptable quality (namely the sum of the “medium”, “good” and “very good” classes) which was higher than the one observed in ‘marmolada®onebor’ (63%); such difference resulted non significant (chi2; p>0.05). on the other hand, no relevant effect was exerted by the experimental field location. the fruits of ‘elsanta’ showed a higher percentage of fruits with medium (69%) and strong (21%) persistence of taste 3 min after ingestion, against 61% and 3% observed on ‘marmolada®onebor’ fruits. the effect of the location of the experimental field was not statistically significant for this parameter, but the best results were obtained in ef2. a similar situation was observed for scent intensity, which resulted superior in ‘elsanta’ fruits compared to those of ‘marmolada®onebor’, with a higher percentage of strongly scented fruits (26%) against (8%)(chi2; p <0.01)(fig. 6a); the location producing more fruits with strong scent was ef2. second season crop (ssc) full blooming of ‘elsanta’ and ‘marmolada®onebor’ was observed in the three plots on 29 may (day 150) and it finished 14 days later (day 164). the first fruits ripened at day 177 (26 june 2009) and the last ripened on day 209 (28 july 2009), with the maximum peak of ripened fruits between days 186 and 192 (fig. 2). the highest fruit weight was observed for ‘marmolada®onebor’ (average 22.4±0.5 g) while a lower mean value was found in ‘elsanta’ fruits (19.7±0.5 g) fig. 3 fruit weight and diameter for ‘elsanta’ and ‘marmolada®onebor’ in the first (a) and second (b) season crop observed in each experimental field (ef). bars with different letters are statistically different (duncan’s test; p<0.05). fig. 4 fruit shape distribution (%) of ‘elsanta’ and ‘marmolda® onebor’ for years 2008 (fsc) and 2009 (ssc). chi2 statistical differences within columns: ns= not significant; * significant (p<0.05). ns ns ns 104 (anova; p< 0.01). also the experimental field location affected fruit weight very significantly, with the highest mean values observed in ef3 (28.7±0.7 g); a reduction of about 40% of this parameter was observed in ef1 and ef2. the effect of the interaction cultivar by location on fruit weight was also very significant (anova; p<0.01) with the highest mean values found in fruits of ‘marmolada®onebor’ grown in ef3 (31.5±0.9 g) and the lowest for those of ‘elsanta’ grown in ef1 (15.3±1.0 g). fruit diameter resulted practically identical between the cultivars (37.2±0.4 mm and 36.1±0.4 mm for ‘marmolada®onebor’ and ‘elsanta’, respectively); in acfig. 5 fruit taste: distribution (%) of the first (a) and second (b) season crop strawberries for different quality classes of ‘elsanta’ and ‘marmolada®onebor’. acceptable fruit is the sum of medium, good and very good classes. bars with different letters are statistically different (chi2; p<0.05). fig. 6 fruit scent intensity: distribution (%) of strawberries of elsanta and marmolada®onebor cultivars for the first season crop (a) and the second season crop (b). bars with different letters are statistically different (chi2; p<0.05). 105 cordance with fruit weight, the greatest diameters were observed in ef3 for fruits of ‘marmolada®onebor’ (fig. 3b). skin brightness (l coordinate) was similar between the cultivars, ranging from 29.3 to 47.8 for ‘elsanta’ and 29.2 to 47.1 for ‘marmolada®onebor’, with averages of 37.7±0.3 and 37.3±0.3 respectively. only field location exerted a strong effect on this parameter (anova; p<0.01); skin brightness reached the highest value (39.7±0.4) in ef2, followed by ef3 (36.8±0.4) and ef1 (35.9±0.3). similarly, chroma index of skin colour was influenced by the location of experimental field (anova; p<0.01) with the highest values in fruits from ef2, decreasing then in those obtained in ef3 and ef1. the chroma index average value for fruits of ‘elsanta’ was 34.3±0.5 while for ‘marmolada®onebor’ it was 33.8±0.5. the interaction of cultivar and location affected very significantly these two colour parameters (table 2). taking into account fruit flesh firmness, ‘elsanta’ showed the highest average (490±0.5 g) compared to that of ‘marmolada®onebor’ fruits (460.2±4.7 g)(p<0.01). the experimental field location and its interaction with cultivar exerted a very significant effect on fruit firmness (p<0.01), with differences of around 13% between the maximum and the minimum average value found in ef3 and ef1, respectively. the juice of ‘elsanta’ and ‘marmolada®onebor’ fruits showed the same ph value (3.6), while titratable acidity was affected by the cultivar and location of cultivation. ‘elsanta’ showed a higher titratable acidity (average 12.8±0.6 meq/100 g fw) than that observed in ‘marmolada®onebor’ fruits (10.7±0.5 meq/100 g fw) (anova; p< 0.01). analogously, the location of cultivation exerted a very significant effect on this parameter; the highest mean value (13.5 meq/100 g fw) was found in berries obtained in ef2, against 10.9 meq/100 g fw of those harvested in ef1. the cultivar elsanta showed an average value of 8.1±0.7 °brix for total solid soluble content of juice, against 7.4±0.8 °brix of ‘marmolada®onebor’ (anova; p <0.05). no significant differences were observed due to the location of the experimental field; in any case, the highest value was observed in ‘elsanta’ cultivated in ef2 (8.6±0.3 °brix); ‘marmolada®onebor’ showed the maximum value of total solid soluble content in ef1 (8±0.3°brix). the most frequent fruit shapes were conical and globose-conic (fig. 4). the differences between the cultivars resulted statistically very significant (p<0.01) for both shapes; ‘elsanta’ presented 68% and 26% of fruits belonging to the globose-conic and conical shapes, while in ‘marmolada®onebor’ the frequencies were of 36% and 58%, respectively (fig. 4). the difference between cultivars for the presence of internal cavity was non significant, with values of 35% and 45% of cases in ‘elsanta’ and ‘marmolada®onebor’ respectively. the location of cultivation exerted a significant effect (chi2; p<0.05) on this characteristic; the lower percentage of fruits showing internal cavity was observed in ef1. the distribution of cases for fruit taste is illustrated for both cultivars in figure 5b. taking into account the percentage of acceptable fruits in terms of quality of taste, 59% and 46% for ‘elsanta’ and ‘marmolada®onebor’ respectively, the cultivar effect resulted statistically not significant. on the contrary, the effect of the experimental field location on fruit taste was very significant: ef1 showed 72% of fruits with acceptable taste, while these values were of 44% and 18% for ef2 and ef3, respectively (chi2; p<0.01). the persistence of taste after ingestion resulted identical for both cultivars and the location did not exert a relevant effect on this parameter. none of the cultivars produced fruits with strong scent; ‘elsanta’ showed higher percentages of fruits belonging to the medium class (59%) against 22% of ‘marmolada®onebor’ (chi2; p<0.01) (fig. 6b). taking into account the location of the experimental field, ef3 showed the worst result, with the highest percentage of fruits with weak scent (80.7%), against ≈ 55% observed in the other two fields. 4. discussion and conclusions no differences were observed for flowering and fruit ripening time between cultivars and experimental fields. in 2008, the interval between planting date and flowering time corresponded to 24 days for both cultivars, which is almost half the time indicated by perez de camacaro et al. (2004) for ‘elsanta’ plants grown in the uk; conversely the interval flowering-ripening time (36 days) coincided with that found by perez de camacaro et al. (2004). in the second season crop, this interval was eight days shorter for the strawberries grown in tuscany (28 days), and the same (36 days) as in the trial reported by perez de camacaro et al. (2004). the fsc ripening time for ‘elsanta’ and ‘marmolada®onebor’ cultivars showed a delay of 40 days in relation to the second year crop. this finding confirms the possibility of scheduling the strawberry harvesting time offered by elevation cultivation, as indicated by faedi et al. (2009). the average weight of fsc fruits was about 15 g for both cultivars, but experimental field and its interaction with the cultivar exerted a strong effect on this parameter. the heaviest fruits (average 19.1 g) were obtained from the plants of ‘marmolada®onebor’ grown in ef1; they weighed about 7 g more than the fruits collected in ef3. a lower magnitude difference was observed in ‘elsanta’ fruits. in the second season, crop fruit weight was on average 25% higher than the value for 2008; furthermore the cultivar factor was more relevant, since ‘marmolada®onebor’ fruits (average 22.7 g) were about 3 g heavier than those of ‘elsanta’ (19.7 g). a similar situation was observed by coman et al. (2002) for the same cultivars grown on the north-eastern coast of the usa (19 and 14 g/fruit for ‘marmolada®onebor’ and ‘elsanta’, respectively). the values obtained for fruit weight of ‘elsan106 ta’ in the second season crop are similar to those indicated by fitogest (2011) and much higher than those observed by giongo et al. (2006) in similar growing conditions and by palha et al. (2009) in soilless culture. on the other hand, the fruit weight of ‘marmolada®onebor’ fruits was very close to the value indicated by fitogest (2011). slightly lower average weights (17.2 g) were found in a set of cultivars grown in brazil at 1,370 m asl by pádua et al. (2009), while a higher value (26.3 g/fruit) was observed for ‘marmolada®onebor’ in cesena (italy) on oneyear-old planted strawberries (tagliavini et al., 2005). the observations confirm that average fruit weight is lower in the first season crop than in the second, which is in accordance with the results of previous studies on ‘elsanta’ and ‘bolero’ (perez de camacaro et al., 2004). also the experimental field influenced fruit weight with heavier fruits obtained in ef3. the opposite influence of ef3 on fruit weight in the second year crop can be attributed, more than to the intrinsic characteristics of the location (e.g. soil chemico-physical properties), to a predominant effect of the cultivar due to cumulate effects associated to the changes in vegetative growth of the second year. in strawberry, the predominant sink is represented by fruits (olsen et al., 1985); fruiting affects dry-matter partitioning, hence the changes in vegetative development may significantly interfere with fruit production as suggested by perez de camacaro et al. (2004). fsc strawberries were underweight by a few grams when taking into account the optimal market values indicated by lovati (2010). conversely this parameter was acceptable for the second year crop. fruit diameter resulted highly correlated with fruit weight (r2= 0.87; p<0.01), nevertheless it was less influenced than fruit weight by cultivar effect. again, the observed values of ‘elsanta’ fruit diameter are in accordance to those indicated by giongo et al. (2006). taking into account fruit colour, brightness resulted close or superior to the acceptable threshold of 37, as indicated by lovati (2010) for both cultivars and for the two crops; on the other hand, chroma index was lower than the limit value (40). colour parameters were less affected by cultivar in the second season crop; conversely experimental field was found to be a more relevant factor. during 2008 the fruits obtained in f1 showed the highest values of brightness and chroma index, while in 2009 the most attractive fruits in terms of brightness and chroma index were grown in ef2. brightness values in both years were almost the same: chroma index was 4 points higher in 2008 than in 2009. flesh firmness of fruits was significantly affected by the studied factors and by their interactions. the optimal range of flesh firmness for harvesting and handling strawberries is indicated as 300-400 g (lovati, 2010), about 20% lower than that observed for first and second season crops for both cultivars under study. the average firmness found for ‘elsanta’, regardless of year and experimental field, was 423 g, approximately 25% higher than that found by giongo et al. (2006) in plants grown at 730 m asl, demonstrating a good texture of the fruits at ripening time, and hence better suitability to handling and transport. fruit juice ph was not affected by any studied factor, resulting substantially stable with a value of 3.6 in both fsc and ssc. titratable acidity was significantly influenced by the cultivar in both fsc and ssc, with average values of about 12.7 and 10.2 meq/100 g fw for ‘elsanta’ and ‘marmolada®onebor’ respectively. such values are within the range of 10-15 meq/100 g fw indicated as optimal for strawberries by roudellac and trajkovsky (2003) and lovati (2010). giongo et al. (2006), observed a higher value (13.6 meq/100 g fw) in ‘elsanta’ fruits obtained in similar growing conditions; in a trial conducted in slovenia, ‘elsanta’ showed a total acid content higher than the one found for ‘marmolada®onebor’ (sturm et al., 2003), which is in accordance with the results of this research. titratable acidity was lower (7 meq/100 g fw) in the juice of ‘marmolada®onebor’ fruits collected in cesena (italy) from one-year-old plants (tagliavini et al., 2005); location seems to have a strong effect on this parameter, as confirmed in our experiment for the second season crop where location exerted a significant influence with a maximum variation of about 30% of the amount of titratable acidity. taking into account the total solid soluble content of strawberry juice, the cultivar ‘elsanta’ showed an average value of 7.4 and 8°brix, against 6.2 and 7.4°brix of ‘marmolada®onebor’, in 2008 and 2009 respectively. these results, even if higher in value, show a similar trend to those found for ‘elsanta’ (about 5.8°brix) and ‘marmolada®onebor’ (about 5.2°brix) grown under identical conditions in slovenia by sturm et al. (2003). similar results were obtained by radajewska and dejworborowiak (2002) on both cultivars grown in poland. nevertheless, different values of total solid soluble content of ‘elsanta’ fruits have been found by different authors and for plants growing in diverse areas and cultivation systems. kovačević et al. (2008) noted a value of 7.2°brix in fruits grown under organic and conventional cultivation. palha et al. (2009) found values ranging from around 7.7 up to 10 °brix on strawberries in soil less cultivation, depending on planting date, tray and bare-rooted plant type, and similar values were observed by voća et al. (2007) in croatia for the same cultivar. giongo et al. (2006) recorded an average value of 8.4°brix in fruits obtained from cultivation in buckets at 730 m asl. lower mean values of solid soluble content (7.2°brix) were observed by pádua et al. (2009) on a different set of cultivars grown in brazil at 1,370 m asl. the values observed for ‘marmolada®onebor’ were higher than those shown by the same cultivar grown in flat areas (4.7°brix) (tagliavini et al., 2005), thus confirming the positive influence of environmental conditions of mountain areas on fruit quality. no statistical differences were observed taking into account the experimental fields and their interaction with the cultivar, nevertheless it is worth noting that ‘elsanta’ fruits obtained in ef1 reached 8.1°brix, which is in accordance with the values found by giongo et al. (2006). however the results obtained in the present study are higher than the threshold of 7°brix, 107 adopted as a standard quality parameter for strawberries (roudeillac and trajkosky, 2003). the results here obtained for ‘marmolada®onebor’ are higher than those reported by kovačević et al. (2008) and plantgest (2012) of 5.7-6.0 and 5.7 °brix respectively. in this study total solid soluble content was associated with taste quality, even if this sensorial parameter is influenced also by many other factors such as acidity, flavours, flesh texture, etc. ‘elsanta’ fruits resulted better in taste quality than those of ‘marmolada®onebor’ for the two years of observation (fig. 5) and in taste persistence in the fsc. similarly ‘elsanta’ fruits showed a higher percentage of fruits in the superior class of scent intensity (fig. 6). these results are in accordance with those found by coman et al. (2002) in fruits of ‘marmolada®onebor’ and ‘elsanta’ grown on the north-eastern coast of the usa and with those of radajewska and dejwor-borowiak (2002) in a trial conducted in poland. fruits of the fsc resulted better than those obtained as ssc for all the analysed sensorial attributes (taste, taste persistence and scent intensity); this result may be associated to the about 15% higher total soluble content and slightly lower titratable acidity observed in the juice of strawberries derived from the fsc, with respect to those of the ssc. most of consumers prefer conical, or slightly roundconical strawberries (tirelli, 2010). the predominant fruit shapes found in the present study were conical and globose-conic; a cultivar effect was noted, but location did not seem to exert any influence on this parameter. the most frequent class of fruit shape in the fsc was conical while conversely globose-conic was predominant for the second season crop. the variation from one class to the other was more evident in ‘elsanta’ fruits (fig. 4). the internal cavity is a morphological attribute that does not have a relevant effect on quality but may modify the shape and volume of fruits. this parameter was not cultivar-dependent but location had a significant influence on the percentage of fruits with an evident internal cavity. furthermore, the growing system (fsc or ssc) seemed to exert a similar effect, since during the fsc around 80% of fruits showed this attribute compared to 40% in the second year crop. ‘elsanta’ is considered highly susceptible to soil-born diseases and powdery mildew, while ‘marmolada®onebor’ is deemed to be less susceptible to fungal diseases (baruzzi et al., 2009). nevertheless, no disease or arthropod attacks were observed in this trial on either of the cultivars in both years of experimentation. conversely łabanowska et al. (2004) observed heavy symptoms caused by powdery mildew and strong attacks of mites (tetranychus urticae and phytonemus pallidus ssp. fragariae) on ‘elsanta’ plants, while ‘marmolada®onebor’ was strongly infected by leaf spot (mycosphaerella fragariae) and attacked by strawberry blossom weevil (anthonomus rubi). analogously, hietaranta et al. (2004) evaluated ‘elsanta’ as insufficient for resistance to pest and diseases in nordic european countries. this means that the environmental characteristics of the area chosen for the trial exert a positive effect on plant health. this is not a secondary aspect in strawberry production, since the practices adopted to reach high yields in flat area cultivation (including a massive use of pesticides, soil fumigants and fertilizers) are in contrast with the market demand oriented towards healthy, organic and high quality strawberries (gengotti et al., 2008; mennone et al., 2008). furthermore, the demand by consumers for healthy fresh fruits, rich in antioxidants, produced locally and in unpolluted environments is noticeably increasing (tulipani et al., 2008). strawberry high-altitude cultivation opens a new scenario since the ecological conditions (climate, soil, and spontaneous flora and fauna, associated with a generally low anthropized environment), seem to positively influence the nutritional properties of berries, for example increasing their polyphenol content as reported by szajdek and borowska (2008), and above all exert a strong influence on the amount and spectrum of harmful pests and diseases and hence on the cultural practices needed for production. guerena and born (2007) found that spontaneous flora alongside strawberry fields represented a shelter and a source of pollen and nectar to predators and parasites of insect pests, thus reducing the amount of damaged plants and strawberries. furthermore, in almost natural conditions and sustainable farming systems the soils are rich in arbuscular mycorrhizal fungi which strongly contribute to counter-balance the appraisal and diffusion of soil-born diseases, such as fungi like fusarium, phytophthora, pythium, rhizoctonia and verticillium, and nematodes (branzanti et al., 2002; harrier and watson, 2004). additionally, naturally fertile soils, namely in terms of organic matter content, are typical of many mountain areas. in this regard, gonzalez and acuña (2009) found that in strawberry cultivation a soil rich in organic matter may supply fertilization at least for the first year of establishment, hence reducing the chemical inputs which are a source of pollution to adjacent ecosystems (tagliavini et al., 2005). strawberry high-elevation cultivation is feasible also in temperate areas as shown by this study. the main factors affecting fruit quality and productivity are related to cultivar and to location, this latter being a relevant aspect even under highly homogeneous environmental conditions. the first season crop, showing higher fruit quality than the second season crop, is particularly adequate for scheduling ripening time, hence filling the gaps in product offer by flat-area cultivation during specific periods. furthermore, mountain cultivation in marginal and low anthropized areas allows production of high quality marketable strawberries. further comparative tests are needed to study in detail the effect of climate, soil, and especially of the ecological background of mountain areas on strawberry cultivation. acknowledgements research funded by arsia tuscany region and comunità montana appennino pistoiese. 108 references baruzzi g., capriolo g., lucchi p., rocco m., sbrighi p., faedi w., 2009 liste varietali fragola. terra e vita, 49: 24-35. branzanti m.b., gentili m., perini r., neri d., cozzolino e., 2002 the micorrhizal status of strawberry plants under different pre-planting and cultivation techniques in the field. acta horticulturae, 567: 495-497. coman m., faedi w., nourse t., 2002 field performance of some italian strawberry genotypes under conditions of the northeastern coast of usa. acta horticulturae, 567: 191-193. dondini g., vergari s., 2009 natura sull’appennino pistoiese (toscana settentrionale). felici editore, pisa, italy, pp. 190. faedi w., 2010 fragola nel mondo, pp. 358-361. in: angelini r. 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(ed.) la fragola. art servizi editoriali spa, bologna, tulipani s., mezzetti b., capocasa f., bompadre s., beekwilder j., ric de vos c.h., capanoglu e., bovy a., battino m., 2008 phenolic compound and nutritional quality of different strawberry genotypes. j. agric. food chem., 56: 696-704. upov, 2008 guidelines for the conduct of tests for distinctness, uniformity and stability. strawberry. http://www. upov.int/en/publications/tg_rom/tg_index.html. voća s., dobričević n., skendrović babojelić m., družić j., duralija b., levačić j., 2007 differences in fruit quality of strawberry cv. elsanta depending on cultivation system and harvest time. agriculturae conspectus scientificus, 72(4): 285-288. impaginato 135 1. introduction in animal cells, the role of plasma membrane behavior is deeply investigated, and it may reflect health status. cellular defense against stress is a key function in which the plasma membrane redox system is involved. moreover, some of the plasma membrane redox system enzymes produce reactive oxygen species as well as play a protective role against them. moreover, evidences underline as redox state of the cell and, accordingly, plasma membrane electron transport contribute to control cell growth, development and apoptosis (ly and lawen, 2003). in animal cells, virus infection can lead to variation in membrane potential (akeson et al., 1992). these effects may occur even during early pathogen recogn i t i o n e v e n t s i n p l a n t m i c r o b e i n t e r a c t i o n . a s reviewed by elmore and coaker (2011), plasma membrane h+-atpases are dynamically regulated during plant immune responses and quantitative proteomics studies suggest complex spatial and temporal modulation of plasma membrane h+-atpase activity during early pathogen recognition events, even if no data relative to virus were available. anyway, an important role is played by h+-atpases in bacterial infect i o n , w h e r e t h e e n z y m e c o o p e r a t e w i t h p l a n t immune signaling protein (namely rin4) to regulate stomatal apertures during pest invasion of leaf tissue. these enzymes are ubiquitous and are involved in plant immune responses. moreover, they may are targeted by pathogens to increase plant susceptibility, even if these evidences are related to fungi or bacteria. thus shabala et al. (2010) affirm that little is known about the ion fluxes generated during plantvirus interactions, despite significant losses caused by viruses to agricultural crops. viruses represent a major threat to agricultural production, mainly due to the lack of effectiveness treatments, and preventative measures represent the main tools available to protect plants. while it is widely believed that in many seed-propagated crops virus the threat is of limited importance, the health status of plants subjected to vegetative propagation is critical. although the viruses that infect woody plants raised more adv. hort. sci., 2016 30(3): 135-139 doi: 10.13128/ahs-20249 insight on trans-plasma membrane behavior of virus-infected plant cells a. luvisi 1 (*), e. rinaldelli 2 1 dipartimento di scienze e tecnologie biologiche ed ambientali, università del salento, via provinciale lecce monteroni, 73100 lecce, italy. 2 dipartimento di scienze delle produzioni agroalimentari e dell’ambiente, università degli studi di firenze, viale delle idee, 30, 50019 sesto fiorentino (fi), italy. key words: grapevine, potassium channel, tobacco, tmv. abstract: little is known about the ion fluxes generated during plant-virus interactions, despite significant losses caused by viruses to agricultural crops. changes in average ion currents were identifying an early event in the signal transduction pathway related to virus/host interaction. while significant decrease in the average inward currents, mainly due to ca2+ moving into the cell was observed, the role of potassium may be significant. host specific k+ efflux with a concomitant decrease in the intracellular k+ was observed in tobacco plants during the early minutes after infection, suggesting many hypothesis about the role of potassium in host-virus interaction. in the last years, trans-plasma membrane potential was evaluated for some viruses, observing as effect on membrane was different in relation to virus infection and host. conversely, settle virus infection generally lead to an increase of activity in trans-plasma membrane electron transport. (*) corresponding author: andrea.luvisi@unisalento.it received for publication 21 june 2016 accepted for publication 8 july 2016 copyright: © 2016 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2016 30(3): 135-139 136 health concerns, trials are longer and more difficult to manage compared to test with herbaceous plants. 2. viruses and hosts: a poorly investigated binomial in trans-plasma membrane events few study investigated the trans-plasma membrane behavior of virus-infected plants, thus few viruses were evaluated in trials, mainly focused on herbaceous plant-related virus. tobacco mosaic virus (tmv) is a widespread virus difficult to eradicate or control from plant and soils (luvisi et al., 2012 a; panattoni et al., 2013 a; luvisi et al., 2015 a). tmv was frequently chosen as “model virus” in host-virus interaction tests. it was also chosen in first studies in t r a n s p l a s m a m e m b r a n e b e h a v i o r o f p l a n t s (schvarzstein, 1997). cucumber mosaic virus (cmv) (rinaldelli et al., 2012), papaya mosaic virus (pmv) (schvarzstein, 1997), potato virus x (pvx) (shabala et al., 2010) and tobacco ringspot virus (trsv) (stack and tattar, 1978) were also investigated. among virus affecting woody plants, grapevine viruses (rizzo et al., 2012, 2015) were the most investigated. grapevine leafroll associated virus 1 or 3 (glrav-1, glrav-3), grapevine fanleaf nepovirus ( g f l v ) , g r a p e v i n e f l e c k m a c u l a v i r u s ( g f k v ) , grapevine vitivirus a (gva) and arabis mosaic virus (armv) were evaluated during antiviral tests or in settle infections (panattoni et al., 2013 b; rinaldelli et al., 2014). 3. trans-plasma membrane behavior in early virus infection events few data are available for early events in plantvirus interaction. stack and tattar (1978) observed as trsv-infected cells of cowpea showed altered transmembrane electropotentials and responded differently from healthy cells when subjected to the metabolic inhibitor sodium azide in their ability to recover. changes in average ion currents were also observed in protoplast of gomphrena globosa artificially infected by pmv and tmv, identifying an early event in the signal transduction pathway related to virus/host interaction (schvarzstein, 1997). in g. globosa, necrotic lesions surrounded by a chlorotic ring developed in inoculated leaves are thought to be the results of a non-host hypersensitive response (hr) that ultimately defend the plants from virus infection. schvarzstein (1997), using patch-clamp techniques, elucidated the early events occurring during pmv infection. whole cell recording indicate a significant decrease in the average inward currents, mainly due to ca2+ moving into the cell or gluconate moving out of the cell. in the same time, an increase in the outward currents was also observed, due to clmoving into the cell and k+ moving out of the cell. the outward currents were correlated to pmv concentration, and a similar behavior was observed during tmv infection. the studies of schvarzstein were the first one in which ion fluxes were characterized during virus-plant interaction and unfortunately, very few studies following. the phenomenon of rapid alteration of ion fluxes in host cells during the early phase of viral infection was reported in chlorella (neupartl et al., 2008). chlorella viruses tests suggested a key-role of potassium, due to an observed host specific k+ efflux with a concomitant decrease in the intracellular k+. the use of blockers of the viral-encoded k(+) channel (kcv) reduced this k+ efflux, suggesting as depolarization and k+ efflux are at least partially mediated by kcv. the k+ efflux seems virus-triggered and it occurs in the same time frame as host cell wall degradation and ejection of viral dna. therefore, neupartl et al. (2008) supposed that loss of k+ and associated water fluxes from the host lower the pressure barrier to aid ejection of dna from the virus particles into the host. even if chlorella shared some metabolic pathway with plants, this interesting findings cannot directly t r a n s f e r r e d t o p l a n t s w i t h o u t f u r t h e r s t u d i e s . m o r e o v e r , d n a b a s e d v i r u s e s a r e u n c o m m o n pathogens in plants. anyway, the key-role of potassium in plant-virus interaction was confirmed by shabala et al. (2010), moving the focus from calcium. the role of ion fluxes in plant defense signaling is well documented and elicitor-induced transient ca2+ influx from the external environment into the cytosol has always been named as a key element of the signaling cascade and appears to be crucial in the induction of plant defense against pathogens (scheel, 1998; zimmermann et al., 1999; blume et al., 2000; grant et al., 2000), but this studies are not related to viruses. shabala et al. (2010) observed as an addition of the purified pvx to the mesophyll tissue of tobacco plants caused no changes in the rate of ca2+ transport across the plasma membrane. these evidences were supported by no significant changes in concentration of ca2+ in cytosol for at least 50 min after pvx treatment. this behavior indicated that ca2+ release luvisi and rinaldelli trans-plasma membrane behavior of virus-infected plant cells 137 from internal stores was also not a part of the signal transduction mechanism in plant-viral interaction. a u t h o r s s u g g e s t t h a t , c o n t r a r y t o b a c t e r i a l pathogens, rapid ca2+ signaling may not be essential for the viral perception and initiation of downstream transduction pathway. conversely, massive k+ efflux was measured as early as 10 min after virus inoculation and this behavior in host-related. prolonged exposure virus caused lower concentration of ca2+ in cytosol compared with control plants, suggesting of the role of ca2+ efflux systems in downstream cascades of the plant responses to viral infection. k+ efflux was partially reduced by blockers of kcv channels such as cs+ (shabala et al., 2010), similarly to chlorella tests. the antiviral drug mycophenolic acid (mpa) (panattoni et al., 2014; guazzelli et al., 2015) was also indicated as interferent with katp channel activity in grapevine, as well as inhibiting activity of the inward-rectifier potassium ion channel which could be mediated by guanosine depletion induced by mpa (luvisi et al., 2015 b). nowadays, the physiological role of the observed viral-induced k+ efflux was not known. neupartl et al. (2008) suggest that the efflux of k+ and the associated water efflux from the chlorella cell may be needed to reduce turgor and lower the pressure barrier, to aid ejection of dna from the virus particles into the host, but adding several known blockers of k+ efflux channels to the buffer media during pvx inoculation in tobacco plants did not ameliorate infection symptoms, probably because the blocking effect of pharmacological agents was only partial (shabala et al., 2010). further hypothesis may be related to role of potassium homeostasis in plant adaptive responses to environment (shabala et al., 2007), including programmed cell death (shabala, 2009). interestingly, the observed viral-induced activation of k+ efflux systems appears to be a highly hostspecific process, indicating that the observed k+ fluxes may be linked to the plant’s ability to recognize compatible viral infection and activate defense pathways (shabala et al., 2010). 4. trans-plasma membrane behavior in settle virus infection membrane potential membrane potential of grapevine cells were monitored during antiviral drugs treatments (luvisi et al., 2012 b). the complex of results obtained in this research highlights, first of all, that membrane electrical response of the tested antiviral drugs is supported by the metabolism of plant cell, and no differences in δ em were found glrav-1-infected grapevine leaves compared to virus-free leaves. however the behavior of membrane of plants treated with antiviral drugs was different in other hosts, as reported in tobacco plants infected by cmv, where trans-plasma membrane potential activity seemed to be influenced by virus presence, acting differently in infected or healthy samples during drug uptake by cells (rinaldelli et al., 2012). anyway, drugs may hindered or enhance virus effects of membrane behavior and specific trials in settle virus infection were carried out in grapevine (rinaldelli et al., 2014). samples infected by leafroll viruses showed no difference in membrane potential values compared to healthy samples, confirming the similar behavior of glrav-1 or -3 infected samples and healthy ones observed during antiviral drug treatments. gflv, or armv infected tissues led to plasma membrane hyperpolarization with higher values compared to healthy samples, while gfkv and gva infected tissues showed plasma membrane depolarization significantly lower than the control (rinaldelli et al., 2014). according to the role of h+atpase activity in generating trans-plasma membrane potential gradient (sondergaard et al., 2004), gflv and armv infected cells could be considered more energized compared to others. conversely, gfkv and gva infection was also associated to increasing difficulty of cell membrane measurements, as occurs under stress conditions (rawyler et al., 2002). electron transport rubinstein and luster (1993) indicated that that trans-plasma membrane electron transport (t-pmet) occurs in all types of organisms, including plants. tpmet allows reduction of extracellular oxidants at the expense of intracellular reducing equivalents that may derive from nadh or nadph (del principe et al., 2011). the t-pmet enzymes have been investigated based on their ability to reduce external artificial impermeant electron acceptors. ferricyanide (fe3+) has been commonly used as electron acceptor in assays performed with intact cells; ferricyanide is converted to ferrocyanide (fe2+), and the rate of this reduction can be monitored. using a carbon fibre microelectrode (cfme) it is possible to map oxidoreductase activity using impermeant electron acceptors or donors (taylor and chow, 2001). this techniques adv. hort. sci., 2016 30(3): 135-139 138 were also used to evaluate in vivo estimation of the inosine monophosphate dehydrogenase inhibition caused by antiviral drugs (panattoni et al., 2015). the t-pmet was observed during antiviral treatments in cmv-infected tobacco plants (rinaldelli et al., 2012). infected samples were less sensitive to antiviral treatments considering t-pmet. this effect may be due to the concurrent entry of drug within the symplast that, as indicated by membrane potential, was lower in infected samples and that can lead to lower inhibition of nad+/nadh conversion by drug and to the following increase of fe3+ conversion. this virus-related behavior was confirmed in glrav-1 and -3 tests (panattoni et al., 2013 b). virusinfected samples exhibited elevated t-pmet activity compared to healthy samples. the [fe2+] in healthy samples was set at 26 µm, while glrav-1and glrav-3-infected samples showed 34-49 µm [fe2+]. these data can be linked to the nadh content in glrav-1 and glrav-3 samples, that was set at 1.2 times higher than healthy samples. this trans-plasma membrane behavior was partially confirmed for other grapevine viruses (rinaldelli et al., 2014). in virus-infected samples, while the [fe2+] produced by gfkv and gva infected tissues were similar to healthy tissues, the samples infected by gflv, armv and leafroll viruses showed higher t-pmet activity. the higher nadh content due to virus infection was used differently by infected samples during their tpmet activity. samples whose infectious status did not interfere negatively with membrane potential, such as armv, gflv, glrav-1, and -3 showed higher t-pmet activity compared to healthy samples, in agreement with the higher nadh availability. 5. discussion the high level of specialization attained by many viruses due to their replication and pathogenetic m e c h a n i s m s t o w a r d s t h e h o s t m a k e t h e m a n extremely variable and complex target. this complexity is the background upon which a defensive strategy can be optimized. therefore, the outcome of therapeutic action is strongly influenced by the ontological properties of the virus to be eliminated as well as the characteristics expressed by the plant as well as the trans-membrane transport of drugs. in human pathology, trans-membrane 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vivo. plant cell environ., 24: 749-754. zimmermann s., ehrhardt t., plesch g., mullerrober b., 1999 ion channels in plant signaling. cell. mol. life sci., 55: 183-203. impaginato 207 1. introduction almond [prunus dulcis (mill.) d.a. webb] presumably originated in mountain and desert areas of central asia and the middle east (martínez-gómez et al., 2007; gradziel, 2011; zeinalabedini et al., 2012). after its domestication, which possibly occurred during the third millenium bc, it spread towards western areas via seeds carried by caravans along the old silk route (fernandez i martì et al., 2015). in afghanistan, a geographical area criss-crossed by ancient trade routes and a cultural bridge between east and west, almond is called by its persian name ‘‘bādām’’, similarly to iran, india, kashmir, pakistan, tajikistan, tibet, and turkey, or “badam-e-shirin”. almond is considered a species belonging to afghan flora (alam, 2011) and its cultivation has spread widely from ancient times. the presence of bitter tasting seeds is considered a relic of the domestication process (rigoldi et al., 2015), hence the ancient presence of almond in afghanistan can be corroborated by the occurrence of many bitter tasting seed specimens s c a t t e r e d t h r o u g h o u t t h e a f g h a n t e r r i t o r y . furthermore, recent studies on almond evolution showed the existence of an unambiguous gene flow of wild almonds from the centre of origin to iran (fernandez i martì et al., 2015), neighbouring to afghanistan. on the other hand, amygdalus species, among which a. zabulica, growing spontaneously in a restricted area of afghanistan, was not considered by ladizinsky (1999) as a possible almond progenitor because of its fleshy fruits, as well as a. kuramica, another species found only in afghanistan and north west pakistan; nevertheless kester et al. (1991) reported that this species may bring sweet and “paper shell” fruit types, the latter trait being quite common in afghan almond germplasm. although it has a cold, harsh continental climate, with hot dry summers and cold winters, afghanistan is an important almond producing and exporting country; about 53,000 t of almonds in shell have been produced yearly in the last five years, representing about 1.8% of total world production (faostat, 2016). almond kernels are an important source of nutritional and nutraceutical compounds for local populations, as well as a significant income for the adv. hort. sci., 2016 30(4): 207-216 doi: 10.13128/ahs-20346 phenotypic characterisation of almond accessions collected in afghanistan e. giordani 1 (*), m. berti 2, m. rauf yaqubi 3 1 dipartimento di scienze delle produzioni agroforestali e dell’ambiente, università degli studi di firenze, viale delle idee, 30, 50019 sesto fiorentino (fi), italy. 2 independent consultant 3 afghanistan national horticulture development organisation (anhdo), kabul, afghanistan. key words: genetic resources, germplasm, kernel, prunus dulcis. abstract: almond [prunus dulcis (mill.) d.a. webb] accessions of afghanistan have been surveyed, propagated, and grown in ex situ collections. trees, leaves, flowers, and fruits were characterised following standardized procedures taking into account 48 phenotypic traits. the national collection of varieties of almonds of afghanistan showed a significant variability in terms of morphological traits, with a predominance of early flowering time accessions. among the 56 accessions, sattarbai, a unique group of cultivar typology characterized by crescent dry fruit, soft and thin “paper shell” and high kernel/dry fruit weight ratio (>0.65) was clearly represented by cluster analysis. other accessions resulted closer to the international cultivars lauranne, carmel, ferraduel and ferragnes, considered as reference. (*) corresponding author: edgardo.giordani@unifi.it received for publication 23 july 2015 accepted for publication 3 february 2016 copyright: © 2016 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2016 30(4): 207-216 208 country since a substantial percentage of almond nuts are exported to india and other countries of the region (kaska et al., 2006; aaido, 2015). the main production areas of export quality almonds are located in the regions of samangan, kunduz, takhar, and balkh (aaido, personal communication), and almonds are also traditionally grown in the “bagh” (home garden) and cultivated in orchards in almost all the valleys and flatlands of the country. the specific orography of the country, rich in narrow and isolated valleys, the multi-ethnic composition of the afghan population, together with a primitive traditional agriculture, based in ancient times mostly on sexual reproduction of trees which is a suitable method of propagation for a species with a short juvenile period like almond, were factors that fostered the occurrence of a rich and differentiated local almond germplasm. furthermore, almond is considered the most diverse species belonging to the genus prunus (socias i company and felipe, 1992) due to its self-incompatibility system (socias i company, 1990), the use of open pollinated seedlings in traditional fruitculture, and its adaptability to more diverse microclimates (fernandez i martì et al., 2015). w i t h i n t h e f r a m e w o r k o f t h e p e r e n n i a l horticulture development project (phdp phase i), several almond producing areas of the country were surveyed during the period 2007-2008 in order to collect the most valuable varieties (giordani et al., 2014). this was achieved by following the indications of local fruit growers, scientists, and agronomists. the in situ collection was comprehensive of 82 local accessions which were propagated by budding. the obtained saplings were planted in ex situ replicated repositories in kunduz and mazar-i-sharif, representing the national collection of fruits and nut of afghanistan. usually the primary evaluation of germplasm accessions of fruit tree species, including almond, is based on the observation and standardized description of phenotypic traits of the tree and its main organs, with the adoption of specific descriptor lists (gülcan, 1985; talhouk et al., 2000; de giorgio et al., 2007; chalak et al., 2006; upov, 2011; rigoldi et al., 2015) and suitable uniand multivariate statistical analyses to study the morphological variability, the relationships among the studied accessions and the correlation between the described traits (lansari et al., 1994; khadivi-khub and etemadi-khah, 2015). such approach is essential for germplasm characterisation and protection against bio-piracy, and it is a suitable method for the phenotypic distinction of collected varieties, a basic step for the further development of the almond industry given the high occurrence of synonyms and homonyms among the accessions indicated for commercial propagation and production purposes. the present study aims to describe and analyse the morphological variability of a set of 56 accessions of almond described in the national collection of varieties of fruits and nuts of afghanistan almond (ministry of agriculture, irrigation and livestock of afghanistan, 2014), in relation to the results achieved by similar approaches on other almond gene pools. 2. materials and methods plant material flowers (n. 10), fully expanded leaves (n. 10), and fruits (n. 20) were randomly collected for two consecutive years from trees (n. 6) of the same accessions grown following traditional cultural practices in the orchards of the national collection of fruits and nuts located in kunduz (latitude 36.70893 n; longitude 68.86145 e; altitude 405 m a.s.l.) and mazar-elshariff (latitude 36.73232 n; longitude 67.02216 e; altitude 353 m a.s.l.) in afghanistan. trees, grafted on almond seedlings, were four to five years old, productive and free of harmful viral diseases; off-type trees observed in the six planting plots were not included in the trial. data collection morphological and pomological characterisation o f g e n o t y p e s w e r e p e r f o r m e d f o l l o w i n g t h e perennial horticulture development project procedures, based on dus-upov and ipgri guidelines for almond (gülcan, 1985; upov, 2011). data related to tree characteristics were collected in the field; measurements and descriptions of the other organs were performed in the laboratory. descriptive traits were determined based on rating and coding according to the adopted almond descriptor. data were entered in microsoft excel® sheets, designed for the purpose with multiple choice notes for qualitative traits or open fields depending on the descriptor typology. quantitative variables were measured and weighed adopting a manual calliper and a precision (0.01 g) electronic balance, respectively. colour parameters w e r e v i s u a l l y d e t e r m i n e d u s i n g a s p e c i f i c a l l y designed colour chart; other qualitative characteristics such as organ shape were attributed by using illustrated charts. giordani et al. phenotypic characterisation of almond accessions collected in afghanistan 209 data processing and statistical analysis average and mode were calculated for all quantitative and qualitative parameters respectively; such values were used for the attribution of each accession to one class, regardless of the year and place of data collection. classes and relative notes were defined for each measured quantitative trait. after mean values standardization, multivariate principal component analysis and hierarchical cluster analysis (euclidean distance and ward’s agglomeration method) were performed on xls stat software. 3. results and discussion studied accessions the 56 studied accessions (table 1) were initially collected in stands (orchards or scattered plants in home gardens) located in different areas distributed throughout the country, within latitudes 31° 36’ 42.8400’’ n 36° 43’ 42.9276’’ and longitudes 65° 40’ 51.9600’’ e 71° 9’ 10.0008’’ e. altitudes ranged from 287 m to 1022 m a.s.l. most of the accessions (>70%) were collected in various districts of northern provinces of kunduz, samangan and balkh, conside r e d t h e t y p i c a l a r e a s o f a l m o n d p r o d u c t i o n , although all accessions were susceptible in varying degrees to the risk of frost damage during the flowering time common in these locations. there were no late flowering varieties equivalent in flowering time t o c u l t i v a r s s u c h a s l a u r a n n e , f e r r a g n e s a n d ferraduel. morphologic and phenological trait variation minimum, maximum, mean and standard deviation values for each analysed trait for the whole set of studied accessions is reported in table 2. among 140 possible notes attributable to 46 different internationally agreed morphological descriptors related to tree, leaf, flower, shell and kernel, only 17 (≈12 %) of them showed no cases, indicating a wide variability for almost all traits. the sole descriptor showing no variation is related to the number of pistils (always 1 in 100% of cases); colour of tip of flower, sepals and petals showed low variability as well. among fruit traits, a special note, not present in the international standardised descriptor lists, was included to describe the “crescent” shape of green fruits and kernels, which showed a frequency of 1% and 16% of cases respectively for the whole collection. the set of described accessions was characterised mainly by vigorous, open trees with dense foliage; medium size, mostly dark green leaves with short petiole and crenate margin. flowers very frequently showed red brown sepals, broad elliptic white petals, stigmas above the anthers, and reddish stamen filaments. the predominant dry fruit traits were elongated and crescent shape, pointed apex, thick but medium-low resistant to cracking endocarps. kernels were characterised by elliptic-narrow elliptic and crescent shapes, red brown colour, medium-weak rugosity and small-medium size. kernel weight (1.1±0.4 g) was lower than the mean value (2.4 g) reported for a germplasm collection of iranian, european and north american accessions, and similar to those observed in different almond collections of the persian and middle-east areas (talhouk et al., 2000; chalak et al., 2006; zeinalabedini et al., 2012; khadivi-khub and etemadi-khah, 2015) and afghanistan (kaska et al., 2006). a distinctive trait of afghan almonds is related to shell cracking resistance, a characteristic with relevant implications in value-added products (ledbetter, 2008), which was found to be very low or low for 30% of accessions, indicating a consistent occurrence of “paper shell” and soft dry fruits. conversely, a similar percentage of accessions with hard and very hard shell (28%) was observed. kernel weight/dry fruit (nut + shell) weight ratio, sometimes indicated as shelling percentage or kernel yield, is another important commercial trait. afghan accessions in the present study showed higher mean value (0.51±0.12) for this characteristic than those found by other authors on almond germplasm of the same geographic area ( b e t w e e n 0 . 3 1 a n d 0 . 3 5 f o r t h e p e r s i a n a r e a ) (sorkheh et al., 2010; zeinalabedini et al., 2012; k h a d i v i k h u b a n d e t e m a d i k h a h , 2 0 1 5 ) , f r o m lebanon (from 0.30 to 0.41)(talhouk et al., 2000; chalak et al., 2006), and similar to the mean value (0.50±0.11) found in a previous study carried out on 17 local varieties of northern afghanistan (kaska et al., 2006). lower mean values of kernel/dry fruit ratio were observed also on moroccan almond germplasm (0.38)(lansari et al., 1994), on european germplasm collections (godini, 1984; cordeiro et al., 2001; de giorgio and polignano, 2001), and on newly released cultivars (vargas et al., 2008). with regard to other fruit morphological traits linked to market value, the percentage of double kernel, a negative trait for international standardised trade but not very influent on domestic and subregional commerce, resulted absent or very low (0-2% adv. hort. sci., 2016 30(4): 207-216 210 national collection code cultivar name area of collection (province/locality) remarks afg0153 abdul wahidi balkh khulm good quality afg0144 belabai balkh khulm slight bitterness afg0167 carmel kunduz char darah (i) international cultivar, later flowering than any native afghan almond afg0527 changaki kandahar kandahar low market value afg1008 du maghza kulula kunduz char darah low market value afg1007 du maghza spin kunduz char darah afg6308 ferraduel kunar asad bad (i) international cultivar afg6206 ferragnes nangarhar jalalabad (i) international cultivar afg0793 kaf samangan aybak medium market value afg0773 kafmal kunduz char darah afg4048 kaghazi du posta herat guzara low market value afg4016 kaghazi gerd herat guzara medium market value afg0739 kaghazi herati herat enjil low market value (exceptionally large kernel) afg0532 kaghazi kalan kandahar kandahar valuable for kernel size (afg0739 has 15% bigger kernels and half the percentage of doubles afg6040 kaghazi maida nangarhar muhmand dara low market value; small kernel afg0535 kaghazi sia dana kandahar kandahar good quality afg0847 kajak samangan samangan aybak good market quality afg0778 kelk arus kunduz char darah afg0146 khairodini balkh khulm moderately hard shellafg6309 lauranne kunar asad bad (i) international cultivar afg0173 mahali kunduz kunduz char darah low market value; hard shell afg0147 majidi balkh balkh khulm small kernel afg2010 majidi samangan samangan aybak small kernel afg0166 marawaja doum kunduz char darah low market value afg0165 marawaja du maghdza kunduz char darah afg0775 marawaja maida dana kunduz char darah low market value afg0534 marawaja safid post kandahar kandahar hard shell afg0151 pista badam balkh khulm low market value afg0160 qaharbai samangan aybak afg1003 qaharbai allah mir kunduz char darah afg1006 qaharbai aykhanum kunduz char darah afg0780 qaharbai hazratan kunduz char darah afg0143 qambari balkh khulm afg0142 qambari 142 kunduz char darah afg0772 qambari kunduz kunduz char darah afg0158 sangak dahum samangan aybak low market value afg0740 sangak haftum herat enjil low market value afg0519 sangak hashtum kandahar kandahar low market value afg0530 sangak nohum kandahar kandahar low market value afg0531 sangak shashum kandahar kandahar low market value afg0380 sangi du maghza kalan kandahar kandahar low market in shell afg0774 satarbai yaqubi kunduz char darah afg0159 sattarbai bakhmali samangan aybak high market value afg1002 sattarbai doum kunduz char darah high market value afg0157 sattarbai guldar samangan aybak high market value afg0168 sattarbai mumtaz kunduz char darah highest market value, intrinsically low yielding afg0154 sattarbai no.4 balkh khulm high market valueafg2011 sattarbai sais aybak samangan aybak high market value afg0164 sattarbai sais kunduz kunduz char darah high market value afg0156 sattarbai sais talkhak samangan aybak afg0777 sattarbai sais zuhrabi samangan aybak high market value afg0145 sattarbai sufi balkh khulm afg1005 shakh-i-buz kunduz char darah afg0155 shakh-i-buz safid balkh khulm afg0162 shokorbai samangan aybak acceptable market value afg0148 zang kafter balkh khulm table 1 list of the described almond [prunus dulcis (mill.) d.a. webb] accessions from afghanistan, area of in situ collection, altitude and remarks on their commercial value giordani et al. phenotypic characterisation of almond accessions collected in afghanistan 211 of fruit with double kernel) and low (3-5% of fruits with double kernel) in 29% and 20.5% of cases, respectively, with a general average of 4% of fruits with double kernels. this value is similar to the average value (4.5%) found by sorkheh and colleagues (2010) in a germplasm collection composed of iranian and european and north american accessions, but much lower than the findings of kaska (kaska et al., 2006) on a set of almond fruit collected mainly in northern provinces of afghanistan (≈ 46 %) and those observed on persian (39%)(khadivi-khub and etemadi-khah, 2015) and lebanese (≈ 43%) germplasm (chalak et al., 2006). time of flowering showed a high variation, with table 2 list of phenotypic traits, codes, states, classes and descriptive statistics adopted to describe the set of 56 accessions from the national collection of almond from afghanistan. in brackets the percentage of cases observed for each note/descriptor no. trait classes (% of cases) min. max. mean sd 1 juvenile tree vigour 1 weak (0); 2 medium (45); 3 strong (55) 2 3 2.55 0.81 2 tree habit 1 up-right (3); 2 slightly open (19); 3 open (52); 4 spreading (24); 5 drooping (2) 1 5 3.00 0.63 3 foliage density 1 loose (4); 2 medium (48); 3 dense (48) 1 3 2.41 0.29 4 leaf blade length 1 short (12); 2 medium (88); 3 long (0) 1 2 1.91 0.42 5 leaf blade width 1 narrow (9); 2 medium (91); 3 broad (0) 1 2 1.84 0.60 6 leaf blade – ratio length/width ratio 1 small (17); 2 medium (81); 3 large (2) 1 2 2.54 0.45 7 leaf blade colour 1 light green (5); 2 medium green (36); 3 dark green (59) 1 3 1.73 0.47 8 leaf blade incisions of margin 1 serrate (26); 2 crenate (74) 1 2 1.32 0.50 9 leaf petiole length 1 short (67); 2 medium (33); 3 long (0) 1 2 1.41 0.36 10 flower buds: shape 1 conical (59); 2 ovoid (41); 3 rounded (0) 1 2 2.02 0.69 11 flower bud colour of tip of petals 1 white (3); 2 pink white (93); 3 pale pink (2); 4 pink (2); 5 carmine (0); 6 white, with carmine tip (0) 1 4 2.82 0.86 12 flower bud colour of sepals 1 green (0); 2 brown green (34); 3 red brown (50); 4 dark red (16) 2 4 2.34 0.64 13 flower bud hairiness of sepals 1 absent or very weak (24); 2 weak (22); 3 medium (52); 4 strong (2); 5 very strong (0) 1 4 1.80 0.82 14 flower size 1 small (31); 2 medium (57); 3 large (12) 1 3 2.27 0.40 15 flower shape of petals 1 narrow elliptic (24); 2 elliptic (26); 3 broad elliptic (50) 1 3 1.20 0.65 16 flower colour of petals 1 white (83); 2 pink white (17); 3 pink (0); 4 dark pink (0) 1 3 1.18 0.39 17 flower number of stamens 1 few (31); 2 medium (55); 3 many (14) 1 3 1.84 0.53 18 flower number of pistils 1 always 1 (100); 2 sometimes 2 (0); 3 frequently two (0); 4 fewmedium (0); 5 medium many (0) 1 1 1.00 0.00 19 flower position of stigmas compared to anthers 1 below (2); 2 same level (36); 3 above (62) 1 3 2.61 0.41 20 stamen anthocyanin colouration of filaments 1 absent (22); 2 present (78) 1 2 1.79 0.81 21 stigma size 1 small (26); 2 medium (65); 3 large (9) 1 3 1.73 0.63 22 green fruit length 1 small (9); 2 medium (72); 3 large (19) 1 3 2.11 0.53 23 green fruit shape 1 rounded (2); 2 ovate (9); 3 elliptic (2); 4 pointed (74); 5 crescent (1) 1 5 3.84 0.78 33 green fruit pubescence 1 slight (29); 2 medium (59); 3 much (12) 1 3 1.82 0.64 34 dry fruit shape 1 type 1 (5); 2 type 2 (0); 3 type 3 (48); 4 type 4 (28); 5 type 5 (19); 1 5 3.59 0.99 35 dry fruit shape of apex 1 flat (7); 2 rounded (9); 3 pointed (84) 1 3 2.77 0.57 36 dry fruit thickness of endocarp 1 thin (28); 2 medium (34); 3 thick (38) 1 3 2.09 0.82 37 dry fruit resistance to cracking 1 very low (9); 2 low (21); 3 medium (42); 4 high (19); 5 very high (9) 1 5 3.00 1.08 38 dry fruit keel development 1 absent or very weak (9); 2 weak (28); 3 medium (37); 4 strong (24); 5 very strong (2) 1 5 2.79 0.97 39 kernel shape 1 crescent (16); 2 narrow elliptic (31); 3 elliptic (34); 4 broad elliptic (17); 5 very broad elliptic (2) 1 5 2.57 1.02 40 kernel size 1 small (64); 2 medium (36); 3 large (0) 1 3 1.36 0.48 41 kernel thickness 1 thin (24); 2 medium (71); 3 thick (5) 1 3 1.82 0.51 42 kernel main colour 1 yellow (2); 2 yellow brown (26); 3 light brown (16); 4 red brown (49); 5 dark chestnut brown (7) 1 5 3.32 1.01 43 kernel intensity of colour 1 light (9); 2 .medium (74); 3 dark (17) 1 3 2.07 0.50 44 kernel rugosity 1 very weak (3); 2 weak (34); 3 medium (58); 4 strong (5) 1 4 2.63 0.65 45 kernel percentage of double kernel 1 absent/very low (23); 2 low (17); 3 medium (30); 4 high (22); 5 very high (8) 1 4 2.66 1.21 46 kernel/dry fruit weight percentage 1 very low (0); 2 low (8); 3 medium-low (15); 4 medium (23); 5 medium-high (33); 6 high (21); 7 very high (0) 1 6 4.48 1.19 47 time of flowering 1 very early (48); 2 early (31); 3 medium (14); 4 late (5); 5 very late (2) 1 5 1.88 1.03 48 ripening time (harvesting) 1 very early (0); 2 early (62); 3 medium (33); 4 late (5); 5 very late (0) 1 4 2.38 0.49 adv. hort. sci., 2016 30(4): 207-216 212 cultivars flowering from very early (before february 26; 49% of cases, among which many “sattarbai” and “sangak” type accessions) to very late (after march 12; ≈ 2% of cases, represented by the international cultivar lauranne), which clearly indicates a prevalence of early flowering accessions in the almond g e r m p l a s m f r o m a f g h a n i s t a n . c o n v e r s e l y , t h e national collection of almond lacks very early (before july 14) and very late (after 29 august) ripening accessions. the range of phenological and measured morphologic traits are shown in table 3. relationship between the described accessions individual descriptive sheets were published in 2014 as the national collection of varieties of fruits and nuts of afghanistan almond and are available online on the internet (http://afghanistanhorticulture.org/pages/germplasm.aspx), while the results of multivariate pca and cluster analysis are reported in table 4 and figure 1. principal component analysis traits scales leaf blade length (mm): short (< 58); medium (58-83); long (>83) leaf blade width (mm): narrow (<20); medium (20-29); broad (>29) leaf blade ratio length/width ratio: small (<2.5); medium (2.5-3.5); large (>3.5) leaf petiole length (mm): short (<11); medium short (11-19); medium (>19-28); medium long (>28-36); long (>36) flower size (mm): small (<3); medium (3-4); large (>4) green fruit length (mm): small (<32); medium (32-50); large (>50) kernel size (g): small (<0.7); medium (0.7-1.9); large (>1.9) kernel thickness (mm): thin (<5.5); medium (5.5-10.5); thick (>10.5) double kernel (%) very low (<15); low (15-27); medium-low (28-39; medium (40-51); medium-high (52-63); high (64-75); very high (>75) time of flowering very early (before february 26); early (february 27 march 2); medium (march 3-6); late (march 7-11); very late (after march 11) time of maturity very early (before july 14); early (july 14-29); medium (july 30 august 13); late (august 14-28); very late (after august 28) table 3 scales defined for quantitative and phenological traits observed on the almond accessions from afghanistan principal component f1 f2 f3 f4 f5 f6 f7 eigenvalue 5.3 3.0 2.6 2.4 2.2 2.0 1.7 variability (%) 14.0 7.9 6.8 6.5 5.9 5.3 4.6 cumulative variability % 14.0 21.9 28.8 35.3 41.3 46.6 51.3 variable correlation juvenile tree vigour 0.19 0.13 -0.27 0.05 0.12 0.12 0.25 tree habit -0.28 -0.01 -0.03 0.01 -0.50 -0.11 -0.43 foliage density -0.01 -0.34 0.07 0.04 0.55 0.16 -0.07 leaf blade length -0.01 0.44 0.47 0.19 0.28 0.25 0.09 leaf blade width 0.07 0.30 -0.57 0.03 0.16 -0.31 -0.21 leaf blade ratio length/width 0.07 0.36 0.62 0.19 -0.03 0.19 0.25 leaf blade colour 0.01 0.07 0.14 0.50 -0.23 -0.13 -0.03 leaf blade incisions margin -0.03 0.27 0.41 0.34 -0.15 0.41 -0.13 leaf petiole length 0.03 0.42 0.26 -0.36 0.10 -0.33 -0.17 flower bud shape 0.27 0.21 -0.22 -0.17 0.31 -0.20 0.26 flower bud colour of tip of petals 0.21 0.17 -0.13 0.46 -0.26 -0.25 0.16 flower bud colour of sepals 0.23 0.10 0.36 0.06 0.08 -0.26 -0.13 flower bud hairiness of sepals -0.04 0.51 0.05 -0.37 -0.08 0.06 -0.16 time of beginning of flowering -0.08 0.39 -0.33 0.21 -0.07 0.38 -0.07 flower size 0.19 0.11 0.49 -0.38 0.13 -0.19 -0.10 flower shape of petals 0.24 0.05 0.28 -0.24 0.22 0.11 0.13 flower colour of petals 0.11 -0.23 0.14 0.16 0.34 -0.10 -0.29 flower number of stamens 0.08 0.55 -0.13 -0.30 0.03 -0.11 -0.09 flower position of stigmas compared to anthers -0.31 0.32 -0.23 0.19 0.39 0.06 -0.43 stamen anthocyanin colouration of filaments -0.09 0.09 0.12 -0.38 -0.07 0.48 -0.18 stigma size 0.23 0.36 0.11 -0.14 0.33 -0.18 0.29 green fruit size -0.66 -0.15 -0.10 0.19 0.25 -0.08 0.15 green fruit shape -0.62 -0.01 0.26 0.34 0.07 -0.39 0.08 green fruit pubescence -0.04 0.12 -0.28 0.09 0.23 0.47 0.34 time of maturity 0.26 0.26 0.01 0.20 -0.38 0.09 -0.10 dry fruit shape -0.76 -0.06 0.26 0.09 0.10 -0.31 0.04 dry fruit shape of apex -0.50 0.18 0.01 0.10 -0.17 0.09 0.48 dry fruit thickness of endocarp 0.71 -0.16 0.14 0.23 0.03 -0.03 0.04 dry fruit resistance to cracking 0.81 -0.38 0.08 0.09 0.09 -0.10 0.04 dry fruit keel development -0.27 0.45 -0.06 -0.05 -0.07 -0.14 0.21 fruit percentage of double kernels -0.07 -0.30 -0.01 -0.36 0.19 0.01 -0.12 kernel shape 0.74 0.07 -0.11 0.09 -0.07 0.25 -0.25 kernel size -0.09 0.01 -0.44 0.18 0.28 -0.01 0.06 kernel thickness 0.63 0.24 -0.20 -0.26 -0.06 -0.10 0.26 kernel main colour 0.35 0.31 -0.01 0.32 0.20 -0.27 0.01 kernel intensity of colour 0.28 0.41 -0.03 0.46 0.31 -0.05 -0.35 kernel rugosity -0.16 -0.13 0.11 0.16 0.48 0.32 -0.11 kernel dry/fruit ratio -0.73 0.31 -0.09 -0.21 0.08 0.07 -0.14 table 4 eigenvalues, expressed variability and correlations of the first seven principal components (accounting for 51.3% of variability) obtained from the whole accession/variable data set. the highest absolute correlation of each variable is in bold giordani et al. phenotypic characterisation of almond accessions collected in afghanistan 213 based on the averages of the discrete scores of the 48 morphological and phenological variables for each accession indicated a low correlation between the utilized variables, with 15 pcas with eigenvalue higher than 1, accounting for 75.8% of variability within the whole set. such result can be attributed to the adoption of a multi-state discrete scoring methodology but also to a generalized low correlation between variables. the average euclidean distance of the generated proximity matrix between the studied accessions resulted equal to 8.46±1.21, ranging from 4.94 (minimum value) observed in the pair “kaghazi kalan kaghazi herati” (collected from kandahar dehe kochi village and herat province, enjil district, kahdestan village, respectively) up to 12.59 (maximum value) for the couple “sattarbai n.4 marawaja safid post”. the dendrogram generated by cluster analysis is reported in figure 1, where three main significant clusters (c1, c2 and c3) can be observed. the first cluster c1 (7.35±0.92 average distance; 5.43 minimum distance and 9.53 maximum distance for p a i r s “ s a t t a r b a i s u f i s a t t a r b a i m u m t a z ” a n d “sattarbai n.4 sattarbai doum”, respectively) is almost completely composed by “sattarbai” denominated accessions (10 of 14 accessions), while only one “sattarbai” type (“sattarbai sais talkhak”) belongs to a different cluster (c1). table 5 reports the representative characteristics of the “sattarbai” almond type, considered the best in terms of fruit quality and market value, as resulted from the analysis of the 48 morphological and phenological variables of the 10 “sattarbai” accessions included in cluster c1. even if a certain degree of variation within single traits of leaves (colour), flowers (petal shape), dry fruits (shape, thickness of endocarp, keel development and percentage of double kernel) and kernels (shape and main colour) was observed, as a whole, these accessions are very closely related and they constitute a clearly defined special subset within the afghan almond germplasm (fig. 1). the accession sattarbai sais talkhak, the only “sattarbai” named cultivar outside cluster c1, showed six characteristics (namely leaf colour, flower size, shell resistance to cracking, dry fruit shape and kernel main colour) with significant differences and 16 traits slightly differing from the “sattarbai” standard type; this fact substantiates its location in a different cluster. on the other hand, qambari, qambari kunduz, qambari 142 are varieties often confused with “sattarbai” type also by expert traders, which conf i r m s t h e h i g h p h e n o t y p i c s i m i l i t u d e b e t w e e n “qambari” and “sattarbai” groups and justifies their presence in cluster c1. conversely, belabai, albeit showing many traits of the “qambari/qaharbai” type, differentiates for the distinctive and sometimes bitter flavour of the kernel which detracts from its otherwise medium value. cluster c2 is formed by the four international cultivars (lauranne, carmel, ferragnes and ferraduel) belonging to the national collection and by 21 local accessions, among which all “sangak” (sangak meaning “stone” in local languages) accessions, a part of “kaghazi” types and other unique varieties. broad elliptic sepals, type 3 dry fruit shape, thick endocarp, fig. 1 dendrogram (euclidean distance; ward's agglomerating method) obtained by analyzing 48 multi-state, discrete morphological variables of 56 almond accessions belonging to the national collection of fruits and nuts of afghanistan (bold cursive: international cultivars; bold: accessions belonging to c1 cluster "sattarbai group"). adv. hort. sci., 2016 30(4): 207-216 214 medium resistance to cracking, red brown kernel, medium percentage of double kernels and medium kernel/dry fruit weight ratio (average 0.45, about 30% less that c1 mean value) were the traits differentiating this group of varieties from those of cluster c1. the last cluster (c3) held 17 local accessions mostly belonging to “qaharbai”, “marawaja” and “majidi” types and other unique varieties; such accessions resulted characterised by medium size flowers, dry fruits with weak keel development, high percentage of double kernels, and medium high kernel/dry fruit weight ratio (average 0.53). table 5 morphological and phenological characteristics of "sattarbai" almond type no. trait note (mode) range 1 juvenile tree vigour medium medium-strong 2 tree habit open open-spreading 3 foliage density dense medium-dense 4 leaf blade length medium medium-long 5 leaf blade width medium medium 6 leaf blade ratio length/width medium small medium 7 leaf blade colour medium green medium green dark green 8 leaf blade incisions margin crenate crenate serrate 9 leaf petiole length short short-medium 10 flower bud shape conical conical ovoid 11 flower bud colour of tip of petals pink white pink white 12 flower bud colour of sepals red brown red brown brown green 13 flower bud hairiness of sepals medium medium weak 14 flower size small small medium 15 flower shape of petals narrow elliptic narrow elliptic elliptic 16 flower colour of petals white white 17 flower number of stamens few few medium 18 number of pistils always 1 always 1 19 flower position of stigmas compared to anthers above above same level 20 stamen anthocyanin colouration of filaments present present ansent 21 stigma size small small medium 22 green fruit size large large medium 23 green fruit shape pointed pointed crescent 33 green fruit pubescence medium medium slight 34 dry fruit shape type 5 crescent type 5 type 4 35 dry fruit shape of apex pointed pointed 36 dry fruit thickness of endocarp thin thin medium 37 dry fruit resistance to cracking very low very low low 38 dry fruit keel development strong medium strong 39 kernel shape narrow elliptic narrow elliptic crescent 40 kernel size small small medium 41 kernel thickness thin thin medium 42 kernel main colour yellow brown yellow brown light brown 43 kernel intensity of colour medium medium light 44 kernel rugosity medium medium weak 45 fruit percentage of double kernels medium high absent very lowhigh 46 kernel dry/fruit ratio high (0.64) high (0.58-0.72) 47 time of beginning of flowering very early very early early 48 ripening time (harvesting) early early medium giordani et al. phenotypic characterisation of almond accessions collected in afghanistan 215 4. conclusions this first comprehensive study of morphological and phenological traits of almond accessions from afghanistan clearly reveals an important level of variation among traits of the local germplasm. analogous findings were reported for iranian and lebanese germplasm (talhouk et al., 2000; chalak et al., 2006; zeinalabedini et al., 2012; khadivi-khub and etemadikhan, 2015). furthermore, a special subset of local varieties, to the best of our knowledge not yet reported by scientific international literature and unique in its kind because of dry fruit and kernel crescent shape and very soft “paper shell” endocarp, denominated “sattarbai” type, has been clearly highlighted. the best sattarbai types are extremely crescent shaped, with very narrow, elongated kernels and a very clear fissure in the “paper shell” along its length. the qambari types have less exaggerated length and crescent shape nuts, but clearly have “paper shell”. qaharbai types are less elongated and tend to have more thickening of the semi “paper shells”. shakhe buz (=”goat’s horn”) has an exaggerated “s” shape. other kaghazi or “paper shell” types may be downgraded because of untypical shape, but the accession kaghazi herati afg0739 has kernels some 40-50% heavier than the typical sattarbai or qambari kernel. the sattarbai, qambari, qaharbai and some other types of almond were found exclusively in the north of afghanistan, indicating a probable connection to the central asian germplasm, particularly to the famous almond growing areas of the fergana valley. nevertheless, a very early flowering time represents a limit to the expansion of these cultivars, and breeding programs are already being developed to overcome this obstacle. specific morphological studies associated to dna fingerprinting would be the next step in order to better clarify the identity of individual local accessions, to reveal the relationship of the afghan almond germplasm with prunus dulcis genetic resources of other areas, and to study the role of afghanistan, an intersectionbetween east and west, in the flow of almond around the world. acknowledgements activity founded by the european commission europeaid program. a special appreciation to the staff of the ministry of agriculture, irrigation and livestock of afghanistan for the availability and support. references aaido afghan almond industry development organization, 2015. http://www.aaido.af. personal communication. alam m., 2011 trees and shrubs of afghanistan. a dendrological guide. rossolis, musée botanique 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and population structure in a worldwide almond [prunus dulcis (mill.) d.a. webb syn. p. amygdalus batsch] pool assessed by microsatellite markers. genet. resour. crop. evol., 62: 205-219. giordani e., cullen g., degl’innocenti p., masini g., 2014 local fruits and nuts as a tool for the development of afghanistan. journal of universities and international development cooperation, 1: 627-635. godini a., 1984 the influence of fresh pericarp on the kernel production in almond. grempa, colloque 1983, p a r i s , f r a n c e . c i h e a m c a h i e r s o p t i o n s méditerranéennes, 11: 57-61. gradziel t.m., 2011 origin and dissemination of almond. horticultural reviews, 38: 23-81. gülcan r., 1985 descriptor list for almond (prunus amygdalus). upov, geneva, switzerland. kaska n., kafkas s., padulosi s., wassimi n., ak b.e., 2006 characterization of nut species of afghanistan: i almond. acta horticulturae, 726: 147-152. k e s t e r d . e . , g r a d z i e l t . m . , g r a s s e l l y c . , 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(ed.) fruits and nuts. genome mapping and molecular breeding in plants. springer berlin heidelberg, germany, pp. 370. ministry of agriculture, irrigation and livestock of afghanistan, 2014 national collection of varieties of fruits and nuts of afghanistan. almond. ministry of agriculture, irrigation and livestock, kabul, afghanistan, pp. 194. rigoldi m.p., rapposelli e., de giorgio d., resta p., porceddu a., 2015 genetic diversity in two italian a l m o n d c o l l e c t i o n s . e l e c t r o n i c j o u r n a l o f biotechnology, 18: 40-45. socias i company r., 1990 breeding self-compatible almonds. plant breeding reviews, 8: 313-338. socias i company r., felipe a.j., 1992 almond: a diverse germplasm. hortscience, 27(7): 817 and 863. sorkheh k., shiran b., khodambashi m., moradi h., gradziel t.m., martínez p., 2010 correlations between quantitative tree and fruit almond traits and their implications for breeding. scientia horticulturae, 125(3): 323-331. talhouk s.n., lubani r.t., baalbaki r., zurayk r., alkhatib a., parmaksizian l., jaradat a.a., 2000 phenotypic diversity and morphological characterization of amygdalus l. species in lebanon. genetic resources and crop evolution, 47(1): 93-104. upov, 2011 almond. guidelines for the conduct of tests for distinctness, uniformity and stability. upov, geneva, switzerland. vargas f., romero m., clavè j., vergès j.s., battle i., 2008 ‘vayro’, ‘marinada’, ‘constantı´’, and ‘tarraco’ almonds. hortscience, 43(2): 535-537. zeinalabedini m., sohrabi s., nikoumanesh k., imani a., mardi m., 2012 phenotypic and molecular variability and genetic structure of iranian almond cultivars. plant. syst. evol., 298: 1917-1929. 138 1. introduction in tunisia water resources (36 km3 annually) are mostly used to irrigate about 400 000 ha of annual and perennial crops. these amounts fulfill on average 75% of the crop water requirements (hamza, 2009). in the north and center of the country, priority is always given to fruit trees and vegetables, although olive is considered the most important species and it is cultivated over a large area (1 700 000 ha). over the two last decades, the amount of available water has decreased consistently, thus the imbalance between water supply and water demand has intensified. this situation has given rise to much attention from the relevant authorities and the general public in terms of the average and long term water uses. obviously, water should be used judiciously with reasonable amounts to meet water needs and without any wastage. determination of crop water needs, i.e. crop evapotranspiration (etc), is therefore necessary to efficiently manage irrigation at the field level. however, it involves a highly complex set of processes which are influenced by watering conditions and tree and land cover characteristics. we have published in recent years technical papers (masmoudi-charfi, 2006; masmoudi-charfi et al., 2006) presenting the water requirements of olive trees for different cultivation areas, according to age, soil coverage and growth stages based on the climatic method of the fao (allen et al., 1998; habaieb and masmoudi-charfi, 2003; masmoudi-charfi et al., 2004). however, during the calculation procedure, we were confronted with a lack of information about the crop coefficient. in addition, long term climatic data were not available for all sites. the lysimetric measurements give more precise information on water use, but is hard to carry out and expensive (deidda et al., 1990). estimates of actual evapotranspiration for adult olive trees were published in tunisia and elsewhere for different environments (ozyilmaz and ozkara, 1989; cohen, 1991; pastor et al., 1998; michelakis, 2000; musters and bouten, 2000; palomo et al., 2002; bandino and dettori, 2003). these estimates require regular measurements of soil water content, which are essential to calibrate models estimating the vertical distribution of root water uptakes (hazrat et al., 2000; palese et al., 2000). gravimetry is amongst the devices used to reliably measure soil water content (hv) in the field. however, it is more useful for calibrating other devices than for scheduling irrigation because it takes a full day to dry samples and irrigation may be needed before the results of the measurements are obtained. the time domain reflectometer (tdr) is easy to use and reliable but the number of sites for measurements is limited. the neutron-scattering method was extensively used in field studies for measuring soil storage and its changes over quantitative analysis of soil water content in young drip-irrigated olive orchards c. masmoudi charfi olive tree institute, 17 avenue ahmed tlili, 1004 el menzah v (carnoy), tunisie. key words: evapotranspiration, neutron probe calibration, root density, stage of development. abstract: the present work was carried out in northern tunisia (36°40’ n, 10°16’ e) during a single growing season in order to examine how water is distributed within young drip-irrigated olive orchards on the basis of distance to trunk and depth. soil water content was measured by using a time domain reflectometer (tdr) and a neutron probe calibrated by concurrently measuring soil water content gravimetrically. measurements were made below the canopies, along the line of drippers and out of the projected canopy area, at distances of 1.4 m, 2.2 m, 2.8 m and 4.2 m from trunks (compartments g1, g2, g3 and g4), taking into account the heterogeneous distribution of roots. results showed significant and positive correlations between the series of data collected simultaneously with the pvc and aluminium access tubes and those collected using the different methods and apparatus, demonstrating that any device may be employed, depending only on their availability. results showed significant changes in soil water contents and stocks according to the season, depth and distance to trunk. during the rainy period, the stocks of water increased homogeneously within all soil compartments, but varied consistently during the dry season with lower values recorded within the upper soil layers. the area situated at 4.2 m from trunks was the driest in summer but it was the wettest during the rainy period. no roots were found at this distance while maximum root densities were observed at 0.4 m from trunk within the upper layers. the lack of water recorded after june affected tree height and fruit growth rates, although irrigation application was sufficient to meet the seasonal crop water needs. adv. hort. sci., 2012 26(3-4): 138-147 received for publication 22 june 2012 accepted for publication 21 december 2012 139 time (vachaud et al., 1977; evans et al., 1996; tarara and ham, 1997; xiong and guo, 1999). with this apparatus, measurements can be made at different depths and sites, but these ‘measurements’ represent a property of the soil that can be related to soil-water content and are, therefore, indirect estimates (hewlett et al., 1964; rana and katerji, 2000). on the other hand, a survey of literature (hewlett et al., 1964; vachaud et al., 1977; sinclair and williams, 1979; haverkamp et al., 1984; vauclin et al., 1984; villagra et al., 1995) shows that little attention has been paid to the associated errors and uncertainties resulting from the definition of the calibration curve itself, when calibrating the apparatus. instrumentation, timing and location variances are identified as the different components of the total variance of an individual water content estimate. sinclair and williams (1979) reported a comprehensive analysis of the contribution of instrument calibration and location variances to the variance of mean water content values and their changes in time. implicitly they assumed that all the observations were independent of one another. the present work illustrates, with results from a single growing season of a young olive orchard cv. chétoui aged six years and cultivated in northern tunisia, how water is distributed in such orchards taking into account time (stage of development) and root distribution. our approach is based on estimating water content at different distances from trunks. data were analyzed considering both spatial and temporal variability within different soil reservoirs, throughout the campaign and on some typical days. specifically, the aim was to highlight the main difficulties found when measuring soil water content in dripirrigated orchards characterized by discontinuous and low soil coverage. 2. materials and methods experiment site the study was performed during a single growing season (2003) on a young olive orchard located at the experimental farm of the institut national agronomique de tunisie, northern tunisia (36°40’ n, 10°16’ e). the area is characterized by a mediterranean climate with average annual water deficit of 750 mm and reference evapotranspiration (et0) of 1200 mm. weather variables were recorded continuously in a nearby automatic weather station. daily average values were used for et0 calculation (table 1) following the penman monteith equation (allen et al., 1998). table 1 annual and seasonal (march september) weather variables recorded in 2003 weather variables value rainfall (mm/year) 790 seasonal rainfall (mm) 346 et 0 (mm/year) 1211 seasonal et 0 (mm) 982 the year of experiment was rainy and hot with annual and seasonal effective rainfall amounts of 546 mm and 239 mm, respectively. these values were estimated following the usda method (fao, 1976). average maximum and minimum temperatures (24.9°c and 14.9°c, respectively) showed an increase of 3 and 7%, respectively, with regard to the average values recorded during the 25 previous years. rising temperatures were noted during the three first months of the year, resulting in an increase of the growth degree day (gdd) of about 300 days. olive orchard three six-year-old olive trees (cv. chétoui), representative of the whole orchard, were used in this experiment. they were planted at 6 m x 6 m spacing and stand on a textured clay-loamy soil of about 2 m depth. soil characteristics were determined at the beginning of the experiment for each trench of soil to 1 m depth (table 2). average bulk density (d a ), soil water contents at field capacity (θ cc measured at -0.3 mpa) and at wilting point (θ pfp measured at -1.5 mpa) were 1.6 g/cm3, 0.50 m3/m3 (50%) and 0.26 m3/m3 (26%), respectively. table 2 soil characteristics of the olive orchard horizon (cm) 0-20 20-40 40-60 60-80 80-100 average ecartype clay % 39 34 28 22 20 29 7.1 loam % 50 52 48 48 46 49 2.0 sand % 11 14 24 32 34 23 9.3 d a (g/cm3) 1.55 1.64 1.60 1.60 1.68 1.61 0.04 θ cc (%) 48 50 50 51 50 50 0.98 θ pfp (%) 25 26 27 26 25 26 0.75 trees were intentionally chosen of the same variety, with similar shape. leaf area (la) was determined after pruning by computing the number of leaves on representative branches and their specific area by planimetry (fernandez and moreno, 1999); individual average value of la was 14 m2. soil coverage was low, rarely exceeding 35%. at the end of the campaign, mean tree height and canopy diameter reached 4.9 m and 4.0 m, respectively. water requirements and irrigation management average weather variables published in local papers (masmoudi-charfi, 2006) were used to estimate daily et0 values. crop evapotranspiration (etc) was then determined following the fao method for non-standard conditions (allen et al., 1998) as et c = et o x k c x k r , with a crop coefficient k c = 0.5 (six-year-old trees) and kr = 0.75 (coi, 1997) accounting for an average soil coverage of about 33% (masmoudi-charfi, 2008). trees were irrigated from 15 may to 5 september with amounts ranging between 0.333 m3/tree and 1.098 m3/tree according to the stage of growth. the seasonal irrigation amount was 5.4 m3/tree. periods and doses of irrigation are 140 reported in table 3. water was supplied using four emitters per tree with a total discharge of 16 l/h. fresh water was provided alternatively from the ‘medjerda’ canal, the main river of northern tunisia and nearby wells. table 3 irrigation supply periods and amounts (m3/tree) 1 2 3 4 5 6 7 irrigation period 15-20/5 2-3/6 30/6-2/7 10-15/7 21-30/7 5-10/8 28/8-5/9 irrigation amount 0.823 0.333 0.549 0.843 0.902 0.902 1.098 field monitoring experimental protocol and soil water content measurements. this work was carried out in order to highlight the difficulties met at field level when elaborating protocols concerning irrigated olive orchards, characterized by heterogeneous distribution of light, soil coverage, roots and water application. difficulties concerned mainly the choice of measurement sites and the right measuring device, particularly: at which depths, frequency and distances from trunks measurements should be taken? which kind of apparatus and access tubes should be used for easy and precise soil moisture monitoring? is there any relationship between measurements taken with different apparatus? how many repetitions (trees) are necessary to get significant results? what precautions should be taken when preparing and installing the access tubes and when calibrating the neutron probe? taking all these questions in mind, but also the results obtained for this same orchard relative to the root distribution (masmoudi-charfi and ben mechlia, 2011), a specific diagram was built in which the area surrounding the three olive trees was instrumented with access tubes covering all soil occupation cases. figure 1 shows a series of 28 access tubes implemented vertically in the soil at distances from trunks ranging between 1.4 m and 4.2 m. measurements of volumetric water contents (h v ,%) were carried out within this area from april to october at depths ranging from 0.20 to 1.20 m using a neutron probe (solo 25, nardeux, france). two types of tubes were experimented and compared. a correlation was then established between measurements made simultaneously with aluminum and pvc-polyamide tubes, which were locally assembled (4 cm inside diameter and 170 cm long). specific glue was used to seal the components of the pvc-polyamide tubes in order to assure their tightness and impermeability to water. neutron probe countings were coupled with routine observations of h v made at the limit of the canopy (2 m from the trunk) on the eastern side of the medium tree, about 0.60-0.70 m from the emitters and 0.20, 0.40, 0.60, 0.80 and 1.0 m depth by using a time domain reflectometer (tdr) (fig. 1). fig. 1 distribution of access tubes for neutron probe measurements taking into account root distribution, soil humectation and soil coverage. tubes were set around three olive trees of cultivar chétoui at 1.5 m depth at distances of 1.4 m, 2.2 m, 2.8 m and 4.2 m from trunks. the heterogeneous distribution of roots and discontinuity of the soil coverage make the interpretation of our measurements difficult. for this reason, four soil compartments designated g 1 , g 2 , g 3 and g 4 were considered according to the distance to trunk. the groups g 1 and g 2 include measurements of soil water content made below the canopy at 1.4 m and 2.2 m, respectively, with three and two replications. observations made along the line of drippers at 2.8 m and out of the projected canopy area at 4.2 m belong to groups g 3 and g 4 , respectively. probe calibration. calibration of the neutron probe consists in relaying the count ratio (n/n water ) and the soil water content values determined for all depths exceeding 0.20 m by concurrently monitoring the probe countings and the gravimetric soil moisture. measurements were made at the same sites, weekly, in dry and humid conditions to cover all potential values, and more frequently during the irrigation period. before and after sampling, the counting was sampled in a water medium in order to control the possible drift in the electronic device provided by the probe itself. the average value was used to adjust the measurements made on the same day. also, we have considered for each trench of soil a specific value of the bulk density (d a ) instead of using an average value for all soil layers, which may increase the error intervals. finally, the series of data were correlated considering each trench of soil separately. the correlative equations were used to translate the counting values obtained during all the campaign into h v estimates. soil water storage. water stored in the soil was determined for each trench of soil and then for the whole profile to a depth of 1.2 m using the following equation: s (mm) = 10 x h v x d, where d = is the layer depth (0.20 m), assuming a standard error of 0.02 m3m-3 on h v measurements. analyses of results. soil water content values determined for each compartment (groups g 1 , g 2 , g 3 and 141 g 4 ) were analyzed separately considering two temporal scales. the first analysis was made during the campaign and the second concerned some typical days representing the main physiological processes that evolved during the growing season. the first date (29/5/2003), designated (s), coincided with the rapid fruit growth stage and it was dry, without any rain or irrigation supplies. the second date (16/7/2003) was chosen during an irrigation episode, corresponding to the stage of flower induction, designated (i). the third date (23/09/2003) was the period of fruit enlargement, designated (p). it corresponds to a high soil moisture period and was chosen after the first heavy rains (90 mm) of that autumn. through measurements taken on these dates, we analyzed soil behavior under welland low-watered conditions and different climatic demand. 3. results calibration of the neutron probe count ratios (n/n water ) and soil water content values (h v ) obtained gravimetrically were positively correlated with r correlative coefficients (r) ranging between 0.69 and 0.83 for the portion of soil from 0.20 to 1.20 m depth (fig. 2). the curves established for the four medium layers (20-40 cm, 40-60 cm, 60-80 cm, 80-100 cm), drawn with either the same or different values of bulk density, provided the same coefficient of correlation (r= 0.83, r= 0.76, r= 0.70 and r= 0.73 for 0.20-0.40 m, 0.40-0.60 m, 0.600.80 m, 0.80-1.00 m, respectively) for each soil layer. the trench of soil from 1.00 to 1.20 m showed some deviation with r= 0.69 when using a specific da value, and r= 0.66 when using an average value of da. these differences are due to transition between the clay-loamy and clay-sandy soil layers. relationships between tdr, neutron probe and gravimetric soil water content measurements measurements of h v values taken using aluminum and pvc-polyamide access tubes were inter correlated, showing a positive and significant correlation curve with r = 0.73 (fig. 3). the tdr-measurements were also correlated to the neutron probe estimates and to the gravimetric observations with high correlative coefficients of 0.87 and 0.79, respectively (fig. 4). these results are of practical interest as they allow use of any apparatus or method with confidence depending on their availability. however, it is important to take into account the representativety of the measurements which could not be taken simultaneously every time at each location (the case of tdr). fig. 3 relationship between h v (%) measurements made simultanuously with the aluminium and pvc-polyamide access tubes. fig. 2 probe calibration curves determined for the neutron solo 25 for different soil layers. gravimetric measurements (h v ) were correlated to the counting ratio n*= n/n water , where n and n water referred to counting made into the soil and water, respectively. fig. 4 relationships between h v (%) measurements made simultanuously with tdr, neutron probe and gravimetry. 142 spatio-temporal variability of soil water content soil water contents measured with tdr fluctuated during the growing season by 15 to 46% depending on depth, season and watering conditions (fig. 5). during the irrigation period (beginning from 15 may), h v -values ranged from 25 to 39% with maximum and minimum values observed within the medium depths and at the top soil layer, respectively. resumption of irrigation at the end of august provided a significant increase of h v values which decreased rapidly after mid october. the lowest values were recorded at the end of the year under low soil evaporation conditions and root activity, and specially in the upper layer. fig. 5 evolution of the tdr-soil water content measurements according to depth in 2003. soil water content also varied according to the distance to trunk (fig. 6). this was observed through estimates of water stocks determined throughout the campaign for all soil compartments (g 1 , g 2 , g 3 and g 4 ). considerable variability was observed between these reservoirs with regard to soil humectation events (i.e. rainfall and irrigation supplies) with values between 225 and 400 mm. the stock of water recorded at the beginning of the campaign (april) was close to 350 mm with little variability between soil compartments. it then decreased during the first decade of may in response to the increasing climatic demand and plant activity. the beginning of irrigation on 15 may marked the start of a consistent increase of the stocks around the emitters, enhancing the disparities between compartments. values subsequently evolved continuously with a downward trend following water application. from mid july to the end of august, irrigation was interrupted, leading to a significant decline of these stocks, to reach their lowest value of 225 mm in g 4 . rainfall received in the autumn increased and homogenized the soil water status. the highest stocks were recorded in this case for g 1 , while reservoirs g 3 and g 2 provided intermediate values; these areas were not subject to irrigation but they were partially shaded during the diurnal period (reduction of soil evaporation). reservoir g4 showed the lowest stocks in summer, but it gave the highest values during the rainy period. these results indicate that soil water status mainly depends on depth, water application and distance to trunk, but it may vary depending on plant activity. soil water content and tree response root growth and canopy relationship. roots extended rapidly during the growing season to reach in may the limit of the canopy at 2.12 m from trunks. maximum root number was observed at 0.40-0.60 m depth (table 4), i.e. at depths characterized by high soil water contents (fig. 5), while maximum root densities (dr) were recorded in the top soil layers at 0.40 m from the trunks (fig. 7). the highest value of dr (0.67 cm/cm3) was observed in g 1 . then, as distance to trunk increased, root densities decreased, as did the stocks of water. at a greater distance from trunks (80 cm), maximum root densities ranged between 0.15 cm/cm3 (deeper layers) and 0.35 cm/cm3 (upper layers). during this same period, the canopy diameter increased at similar rates leading to equilibrium between the above and underground areas, just a few years after planting. we recorded at the end of the campaign an optimum la/l r (leaf area/root length) value of 2.3 km/m2 while the ratio s r /s c (root area/projected canopy area) approximated the unit (table 5). this result indicates that as leaf area increased, the amount of carbohydrates increased allowfig. 6 soil water stocks (mm) determined for all the profile (0-100 cm depth) at 1.4 m (g 1 ), 2.0 m (g 2 ), 2.8 m (g 3 ) and 4.2 m (g 4 ) from trunks. table 4 root distribution, number and diameter observed during the experimental year for six-year-old chétoui olive trees compared with measurements made on five-year-old tree soil layer (cm)/age 5-year-old tree 6-year-old tree distribution of roots 0-20 9 51 20-40 5 91 40-60 3 116 60-80 5 97 80-100 3 81 100-120 0 36 total number of roots 25 472 maximum root diameter (mm) 24 27 volume of the rooting system (cm3) 5.3 11.2 maximum distance to trunk (cm) 150 212 143 ing good development of roots and fruits. this hypothesis is analyzed in the following section through simultaneous monitoring of fruit growth and tree height. watering conditions, tree growth and fruit development. growth patterns relative to tree height and fruit diameter observed in 2003 were different from those recorded for the previous year (fig. 8). in 2002, tree height and fruit diameter increased with irregular rates, but continuously from april till october, peaking at 1.4 cm/day (105 doy) and 0.22 mm/day (142 doy), respectively. in the following year, we did not record any peak values for tree height but rather a low and constant rate of about 0.100.15 cm/ day. from april to june, fruits grew with increasing rates to peak at 0.29 mm/day on 155 doy. soil water contents recorded during this period of cell division and early fruit growth (10/4-10/6, 100-160 doy) (fig. 9) were apparently sufficient to assure suitable fruit development. fig. 8 growth patterns of olive tree height (cm/day) and fruit diameter (mm/day) recorded in 2003 compared to tree height and fruit development curves recorded in 2002. values are averages of 96 tree height and 480 fruit diameter measurements made on chétoui olive trees. tree height and fruit growth decreased significantly in july-august most likely because of interruption of irrigation and the decrease of soil water content values (fig. 9). after this period of a lack of water, hv-values increased particularly in the deeper depths, enhancing the ultimate fruit development (284 doy, 0.1 mm/day). soil profiles established in may, july and september showed different behavior depending on the watering conditions and the climatic demand. for measurements made at 1.4 m from trunk (g 1 ), soil water content values ranged fig. 9 soil water content curves observed at different stages of fruit development. fig. 7 average root densities (cm/cm3) recorded for fiveand six-year-old olive trees at 0.40, 0.80, and 1.20 m distance from trunks. measurements were made in both east and south directions at different depths (0-20 cm, 20-40 cm, 40-60 cm, 60-80 cm and 80-100 cm). table 5 characteristics of the rooting system of the six-year-old-tree of cultivar chétoui compared with those recorded for a fiveyear-old tree soil layer (cm)/age 5-year-old tree 6-year-old tree root area (m2) 7.10 13.8 projected canopy area (m2) 8.04 11.94 root area (sr, m 2) / projected canopy area (sc, m 2) 0.9 1.2 maximum canopy radius (m) 1.60 1.95 average root density (cm/cm3) 0.13 0.30 length of the rooting system (km) 7.05 33.94 144 between 30 and 42% (fig. 10), providing distinguishable profiles with constant differences between the lowest and the highest values within each trench of soil. however, minimum and maximum soil water contents were not observed during the driest (s) and the wettest period (p), respectively. minimum values of h v were recorded in the first 40 cm in may (s) and at deeper depths (0.40-1.20 m) in july (i), while maximum values were recorded in july in the superficial top layer, in september for the 0.20-0.60 m layer and in may at deeper depths (0.60-1.20 m). fig. 10 soil water content (h v , %) measured within the first reservoir (g 1 ). measurements were made to 1.2 m depth at a distance of 1.4 m from trunks on may (29-05-2003) after a 20-day period of dryness (g 1 -s), in july (16-07-2003) during an irrigation period (g 1 i) and in september (23-09-03) following the first heavy autumn rains (g1-p). groups g 2 , g 3 and g 4 showed important variations between h v values within the top soil layers (0-0.40 m) and small differences at deeper depths despite their distances from trunks (2.0 m, 2.8 m and 4.2 m) (fig. 11). minimum values (20%) were recorded in may and july despite the different climatic conditions, while the highest values of soil water content were about 40 % at all depths. 4. discussion and conclusions this case study demonstrates the potential of using the neutron probe, tdr and gravimetric measurements to determine soil water content in a young olive orchard, taking into account the heterogeneous distribution of roots, localized irrigation and low canopy shade. values of h v were obtained by using the probe calibration curves established specifically for the solo 25 apparatus for each trench of soil. these curves cannot be used in any other situation. data collected with the different methods and apparatus showed positive correlations between soil water content measurements. this result is of practical interest because it indicates that any of these apparatus or methods can be used depending only on their availability. for example, the relationship developed between the two types of access tubes allowed us to use with confidence the pvc-polyamide tubes which were assembled locally with a lower cost. regarding the apparatus, the tdr with probes installed vertically has proven to be fast, accurate and non-destructive, but it allowed measurements in one location only. this is a major disadvantage. on the contrary, gravimetric measurements can be made at any location and the method is suitable for calibrating other methods, although it is destructive and hard to carry out. measurements made with the neutron probe are difficult. the apparatus requires specific calibration and some care should be taken when using such radioactive probes. with regard to these ‘constraints’, uncertainties arise because soil water content monitoring is incomplete as it is impossible to do measurements at all depths and in all directions. for the neutron probe, it is important to know if the observed variance of water content measurements is really due to errors associated with the location, which can be randomly distributed or spatially structured, or to errors arising from the use of a neutron probe itself. indeed, uncertainty intervals are also influenced by the charge of the probe battery, the number of access tubes used, their placement and the depths at which they were installed. the number of access tubes should be determined depending on the heterogeneity of the orfig. 11 soil water content (%) measured within the second (g 2 ), third (g 3 ) and fourth (g 3 ) reservoirs. measurements were made to 1.2 m depth at distances of 2.0, 2.8 and 4.2 m from trunks, respectively, in may (29-05-2003) after a 20-day-long period of dryness (g 1 -s), in july (16-072003) during an irrigation period (g 1 -i) and in september (23-09-2003) following the first heavy autumn rains (g 1 -p). 145 chard in order to avoid situations where sample data size is not sufficiently distributed in the field. about 25 locations would be necessary to obtain a mean value with a relatively high precision; observations are thus considered independent of one another regardless of their location in the field. it is also obvious that when installing soil-water measuring devices in the plant row in the irrigated field, at least one device should be located in each of the major soil types to take into account all soil occupation cases. additional care should be taken with regard to possible drift in the electronic device: (1) counting in a water medium before and after profiles are sampled, (2) using the apparatus with a fully charged battery and (3) the use of a specific d a value for each trench of soil rather than an average value for all soil layers when the soil texture is variable. however in light of recent concepts introduced in soil physics studies, and as previously stated, it is obvious that the auto-correlation between measurements in estimating the variance of the mean must be taken into account. soil water contents varied consistently according to the proximity of measurements to trunk and depth. maximum variations were observed in the first top soil layers at 0.40 m from trunks as a result of the heterogeneous distribution of roots. there was a massive presence of roots near the trunk, mainly confined to the canopy projected area (masmoudi et al., 2007; masmoudi-charfi et al., 2011). these results are concordant with those reported by bonachela et al., (1999), fernandez and moreno (1999), palese et al., (2000), fernandez et al., (2003) and connor and fereres (2005) regarding high root densities in these areas. some roots were also found at greater distances from the trunk, outside the canopies, i.e. within the reservoirs g 2 and g 3 but with lower densities in comparison to values obtained within the first compartment. this indicates that root uptakes are still possible in these areas, and these roots may have a significant role in enhancing root absorption and water transfer. furthermore, results showed that the zones where roots develop behave differently, even below the canopy, involving different processes of water uptake and depletion. unfortunately it was not possible to separate these processes in the present study to determine if these roots are more active than those located near the trunk or not. fernandez and moreno (1999) and connor and fereres (2005) explain that root densities are necessarily higher in the area of irrigation but roots may be less active. on the contrary, roots far from the trunk may be larger with numerous fine roots and thus they are more active. in another study carried out during the same year, abid-karray (2006) reported the presence of lateral water transfers within an olive orchard cultivated in central tunisia under complementary irrigation. high water depletion was observed in that olive orchard and others cultivated under semi-arid and arid climates due to advective transfers of heat in soil (fernandez et al., 1990; fernandez et al., 1991; villagra et al., 1995; bonachela et al., 1999; granier et al., 2000; fernandez et al., 2003). this makes the situation more complex because roots situated outside of the projected canopy limit may contribute significantly to supply other reservoirs. their role is however, highly dependent on the distance from the point of water but also the stage of development. we have published in previous papers (masmoudi-charfi and ben mechlia, 2007 and 2008) that under irrigated conditions young olive trees cultivated in this same location continued to grow even during the winter months, under different watering conditions (extreme rainy and rainless years), however with relatively low rates. variability of soil water content is also dependent on soil coverage, which varied consistently from year to year, following the season, the severity of pruning and measurement site, thus modifying significantly the relative importance of the evaporative processes involved within each soil reservoir (dichio et al., 2002; masmoudi et al., 2004; 2007). this is because the contribution of the different processes of water uptake and water depletion depend on the amount of solar radiation intercepted by the tree canopy, which is the most important factor controlling water losses and extension of the leaf area. water applied during the growing season seemed to be sufficient to meet the overall tree water needs although some water shortage was observed during the fruit set-maturation period. stocks of water recorded at the beginning of the growing season (in may) were apparently insufficient to insure suitable growth of the tree, but high enough to assure early fruit growth. however, the lack of water observed from midjuly to end of august under high evaporative demand, reduced both tree height and fruit size (-10%), fruit weight (-26%) and also fruit number through an important fruit drop observed early september. comparative results between the year of study and the preceding year showed a significant reduction of yield at harvest. six-year-old olive trees yielded 1.9 t/ha (2003 was normally an ‘on’ year), while yield exceeded 2.0 t/ha in 2002 (‘off’ year). these different responses may be inheritent to other exogenous factors like soil type, pruning, and fertilizer schedules (masmoudi-charfi and ben mechlia, 2009) or to some endogenous parameiters which have an impact on how irrigation changes affect the production and growth levels. in september, although water was abundant and trees were loaded with fruits, water uptakes seem to be reduced due to the decrease of climatic demand. these results are supported by observations of sap fluxes recorded during the same period (masmoudi-charfi et al., 2011; 2013), showing that when water is available, sap flux measurements increased in correlation with the increasing climatic demand. this experiment, concerning the choice of devices used to measure soil water content, despite the observed constraints, has practical interest. the positive correlations developed between soil water content measurements made with the different methods and apparatus indicate that any of these devices can be used indifferently, depending only on their availability and ease. particularly, the relationship developed between the two types of access tubes allowed us to use with confidence the pvc-polyamide tubes assembled locally with a lower cost. this study has shown also that soil water content consistently affects tree height 146 and fruit growth rates and varies depending on the depth and distance from the trunk. this spatio-temporal variability makes it difficult to provide proper assessment of the components of the water balance, if it is used for water consumption estimation in young orchards. therefore, associating methods should be used that make it possible to distinguish between soil evaporation and water lost by transpiration like sap flux measurements. additional measurements of root activity are also necessary to confine intervals in simulated uptake distributions. nevertheless, these methods and estimations remain useful tools to decide when and how to irrigate, how much water is stored in the soil for plant use (soil water logging capacity) and to determine allowable water depletion. acknowledgements author gratefully acknowledge prof 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r.l., reichardt k., 1995 difficulties of estimating evapotranspiration from the water balance equation. agricultural and forest meteorology, 72: 317-325. xiong l., guo s., 1999 a two-parameter monthly water balance model and its application. journal of hydrology, 216: 111-123. impaginato 225 1. introduction the market of pomegranate fruits is noticeably e x p a n d i n g w o r l d w i d e . t h e i n c r e a s i n g i n t e r e s t towards this photophilous and moderately xerophytic species relies essentially on it nutritional and nutraceutical properties, being considered a functional product of relevant benefit for the prevention of human diseases (martinez et al., 2012; kotsiou and tesseromatis, 2015). punica granatum l. is native to iran (teixeira da silva et al., 2013), nevertheless a western area (which includes part of anatolia, the caucasus and iran) and an eastern area (southeastern turkmenistan, southern tajikistan, afghanistan, northern pakistan, kashmir, northern india and nepal) are generally recognized (alam, 2011). due to its hardiness pomegranate naturalizes quite easily and many local landraces and varieties can be found in the area of the mediterranean basin, in middle east and in the arabic peninsula (al-sadi et al., 2015). iran embrace a rich germplasm of pomegranate, with 760 specimens, 24 genotypes and various commercially important cultivars listed in different studies, n e v e r t h e l e s s a l s o i n d i a , i s r a e l , e g y p t , t u n i s i a , morocco, spain and italy hold valuable genetic resources of punica granatum (holland et al., 2009). more than 20 varieties of pomegranate (locally called ‘anar’), among which ‘bedana’ (soft seeded), ‘khog dandar’, ‘tarwah’, ‘boocha’, ‘pastakai’, and ‘soor’, were grown in afghanistan during the second half of the xx century (alam, 2011). more recently, 48 cultivars of pomegranate from various provinces of afghanistan were described and characterized in relation to fruit colour, flavour and seed hardiness adv. hort. sci., 2016 30(4): 225-230 doi: 10.13128/ahs-20348 short note selected pomegranate germplasm from afghanistan: morphological variability and relationship among collected accessions e. giordani 1, m. berti 2, m. rauf yaqubi 3, s. stanikzai 3, a. amad 3, b. zadran3, a. saeedi 3, m. ghous3 1 dipartimento di scienze delle produzioni agroforestali e dell’ambiente, università degli studi di firenze, viale delle idee, 30, 50019 sesto fiorentino (fi), italy. 2 indipendent consultant. 3 afghanistan national horticulture development organisation, kabul, afghanistan. key words: genetic resources, phenotype, punica granatum. abstract: during the period 2008-09 and in the frame of phdp 58 afghan and 18 foreign pomegranate accessions have been collected and grown under homogeneous environmental and cultivation practice conditions in ex situ collections. standardized procedures were adopted to describe mature trees, leaves, flowers and fruits for a total of 30 phenotypic traits. within the national collection of pomegranate of afghanistan, coefficients of variability ranged from 8.8 to 31.7% for fruit diameter and weight of non edible part, respectively. principal component analysis revealed the absence of correlated variables among different organs. the whole set of accessions resulted discriminated on the basis of the studied morphological parameters and all the accessions were grouped into 3 sub-sets by hierarchical cluster analysis. local accessions resulted distributed in the 3 clusters, nevertheless the largest one held all the foreign varieties while the second one included all the accessions collected under the putative name of ‘bedana’. the adopted morphological studies allowed to identify one true-to-type ‘bedana’ accession, considered the best afghan variety for fresh consume due to its very soft seed, and to solve the cases of homonymy. analogously, various accessions originally collected from different regions of afghanistan under the name of ‘kandahari’ were identified and renamed. (*) corresponding author: edgardo.giordani@unifi.it received for publication 11 january 2016 accepted for publication 27 july 2016 copyright: © 2016 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2016 30(4): 225-230 226 ( s a m a d i , 2 0 0 8 ) ; a m o n g t h e m , ‘ k a n d a h a r i ’ a n d ‘bedana’ were considered excellent cultivars (glozer and fergusson, 2008). i n t h e f r a m e o f t h e p e r e n n i a l h o r t i c u l t u r e development project (phdp phase i), several pomegranate producing areas of afghanistan were surveyed during the period 2007-2008 and a set of the most valuable varieties was collected (giordani et al., 2014). the in situ pomegranate collection resulted comprehensive of 59 local accessions which were propagated by cuttings. the obtained saplings were planted in ex situ collections, together with 19 imported cultivars in kandahar and jalalabad, thus representing the national collection of fruits and nut of afghanistan (saeedi et al., 2012). m o r p h o l o g i c a l c h a r a c t e r i s a t i o n o f n a t i v e germplasm accessions is the first step for their rational introduction in modern and efficient production chains and a standardized description of phenotypic traits based on the adoption of specific descriptor lists is a pre-requisite for this purpose (frankel, 1970). t h e p r e s e n t s t u d y a i m s a t d e s c r i b i n g a n d analysing the pomegranate accessions belonging to the national collection of varieties of fruits and nuts of afghanistan. 2. materials and methods plant material data were collected during 2013 and 2014 from self-rooted 4 years old trees grown in the repository of the national collection of fruits and nuts of afghanistan located in kandahar (latitude 31°36’ n and longitude 65°42′ e; altitude 1009 m a.s.l.). for each accession flowers (10 samples), fully expanded leaves (10 samples) and fruits (20 samples) were randomly collected for two consecutive years (20132014) from trees (6 samples) grown following standardized cultural practices. data collection traits were described by rating and coding proced u r e s b a s e d o n t h e e c 2 9 g e n r e s p r o j e c t pomegranate descriptor list and guidelines (genres, 1998; bellini et al., 2008). quantitative variables were measured and weighed adopting a manual calliper and a precision (0.01 g) electronic balance, respectively. colour parameters were visually determined using a specifically designed colour chart; other qualitative characteristics were attributed by using illustrated charts. data of 13 qualitative characteristics (tree habit, vigour and suckering tendency; shoot thorniness; leaf apex shape; number of flowering periods; flower calyx and corolla colour; colour change of calyx after petal fall; fruit juiciness and fruit skin colour and thickness; seed colour) and 17 biometrical variables (table 1) were collected for all the studied accessions. data processing and statistical analysis average and mode were calculated for all qualitative and quantitative parameters respectively; such values were used for the attribution of each accession to one class, regardless the year of data collection. classes and relative notes were defined for each quantitative measured trait. after average value standardization, multivariate principal component analysis and hierarchical cluster analysis (euclidean distance and ward’s agglomeration method) were performed on xls stat software. 3. results and discussion studied accessions the survey allowed to collect 58 afghan accessions (fig. 1) in stands represented by orchards and home gardens distributed in the eastern (kapisa, nangarhar; 29 accessions), southern (kandahar and farah; 16 accessions) and northern (takahr, balkh and sar-e-pul; 13 accessions) areas, at altitudes ranging from 422 m a.s.l. to 1513 m a.s.l. such accessions variable mean range cv (%) standard deviation leaf total length (mm) 68.4 52.6-86.2 11.5 7.9 leaf blade length (mm) 63.2 48.0-78.5 11.5 7.3 leaf blade width (mm) 20.7 16.2-26.7 11.7 2.4 flower petal length (mm) 26.1 19.2-30.8 9.1 2.4 flower petal width (mm) 19.9 14.3-30.2 13.4 2.7 flower length of calyx (mm) 38.5 29.5-46.7 10.2 3.9 flowerwidth of calyx (mm) 13.8 9.1-24.3 14.4 2.0 fruit equatorial diameter (mm) 84.5 64.9-99.2 8.1 6.8 fruit diameter of calyx (mm) 21.1 11.6-35.3 19.6 4.1 fruit height without calyx (mm) 76.4 56.7-94.0 8.8 6.7 fruit calyx height (mm) 23.2 14.8-34.6 18.6 4.3 fruit total weight (g) 309.3 134.8-490.1 24.3 75.1 fruit weight of non edible part (g) 144.6 76.6-323.1 31.7 45.8 fruit edible/total weight (%) 53.8 18.7-70.8 13.5 7.3 seed weight (g) 0.3 0.2-0.4 18.8 0.1 seed length (g) 10.3 8.0-13.6 8.9 0.9 seed width (mm) 6.8 4.9-8.4 12.2 0.8 table 1 mean values, ranges and coefficients of variation for 17 quantitative traits of pomegranate accessions of the national fruit and nuts collection of afghanistan giordani et al. afghanistan selected pomegranate germplasm. morphological variability and relationship 227 were grown together with 19 imported varieties i n c l u d i n g t h e i n t e r n a t i o n a l r e f e r e n c e c u l t i v a r ‘wonderful’. morphologic and phenological trait variation descriptive statistical values for continuous biometric data of the whole set of 77 accessions are reported in table 1. leaf size showed a relatively low variation (cv% ≈ 11.5 for total leaf length, length and width blade), as well as flower biometric traits, while higher values of variations were observed in fruit and seed traits. namely, minimum and maximum mean of fresh fruit weight (≈ 134-490 g) fall within the range of analogous values observed by okatan et al. (2015), caliskan and bayazit (2013) in pomegranate collections from turkey and by mars and marrakchi (1999) in a set of 16 accessions collected in tunisia. on the other hand, orhan et al. (2014), mondragòn jacobo (2012), bartual et al. (2012), ferrara et al. (2012), russo et al. (2012), and legua et al. (2012) observed higher mean values of fruit weight in accessions collected in coruh valley (turkey), in mexico, in elche (spain), in apulia (italy) and in morocco, respectively. a narrower range of variability for this parameter (204-288 g) was observed in a study on iranian accessions (theranifar et al., 2010). seed (aril) fresh weight (mean 0.33 g; range 0.17-0.33 g), resulted higher than the values reported by okatan et al. (2015) (0.28 g; range 0.14-0.41 g) for turkish accessions and by mondragòn jacobo (2012)(0.20 g; range 0.12-0.26 g) for mexican accessions, but lower than those found out by caliskan and bayazit (2013) in turkish pomegranate accessions (0.42 g; range 17-67 g) and by ferrara et al. (2012) (0.38 g; range 0.270.51) in apulian pomegranates in italy. the ratio between the edible part of the fruit (arils) and the total fresh fruit weight ranged from 18.7% to 70.8%, with an average of 53.8%, lower than the value (55.6%) found by legua et al. (2012) in accessions from morocco. the beginning of flowering time ranged from 104 to 115 days from the beginning of the year while day 253 resulted the earliest date of the beginning of ripening time (fig.1 a, b). the range of flowering and ripening time of the investigated accessions resulted of 11 and 44 days, with a discontinuous frequency for ripening time between days 269 and 284 (fig. 1 b). t h e r i p e n i n g t i m e p e r i o d ( e n d o f s e p t e m b e r october) resulted similar to the spanish and turkish one; no significant correlation has been found between fruit size and ripening time, a pattern observed by different authors in the germplasm of other areas (holland et al., 2008; martínez et al., 2012; caliskan and bayazit, 2013). fig. 1 distribution (%) of the collected pomegranate accessions in relation to: a) beginning of flowering time (day of the year); b) beginning of ripening time (day of the year); c) fruit skin color and d) fleshy part of seed (aril) color. adv. hort. sci., 2016 30(4): 225-230 228 attaining to colour fruit traits, which are important quality attributes in pomegranate marketing (mena et al., 2011), most of accessions presented fruits with yellowish skin (53.1%), followed by reddish colouration (45.7%), and only 1.2% of accessions showed totally black skin colour. fleshy part of seeds was in most cases white (39.2% of accessions), while p i n ki s h a n d red wh i te co l o u r s h a red th e s a me percentage (≈30%) of accessions and only 1.4 % of varieties showed dark red arils (fig. 1 c, d). relationship between the described accessions principal component analysis based on the averages of the discrete scores of the morphological qualitative and quantitative variables showed a low correlation between the utilized variables, with 12 principal components with eigenvalue higher than 1, all together accounting only for 76.8% of the variability. taking into account the sub-set of 19 biometric continuous variables regarding leaves, fruits, and seeds, the 6 principal components with eigenvalue>1 accounted for 78% of the whole variability, confirming a very low correlation among variables for the same set of accessions. higher levels of correlation among the biometric variables have been reported for different groups of turkish (caliskan and bayazit, 2013; okatan et al., 2015) and tunisian (mars and marrakchi, 1999) local varieties. the average euclidean distance of the generated proximity matrix based on the 17 biometric variables resulted equal to 5.96±1.21, ranging from 1.99 o b served i n th e p ai r ‘bed an a s amash kel i jan mohammadi’ up to 11.67 for the couple ‘sorkhaksakerdze’. the dendrogram generated by the cluster analysis is reported in figure 2, where three main significant clusters (c1, c2 and c3) can be observed. cluster c1 resulted the largest with 35 accessions, 19 of which represented by foreign non afghan access i o n s , i n c l u d i n g t h e i n t e r n a t i o n a l c u l t i v a r ‘wonderful’. cluster c2 was constituted by 19 accessions, 6 of which were originally collected as ‘bedana’, a typical afghan variety appreciated by the presence of very soft seeds. albeit this easily appreciable and discriminating trait, the standardized characterisation of the presumed ‘bedana’ accessions showed that only one accession could be identified as belonging to the true ‘bedana’ type. the accession collected in kandahar and entered in the collection as ‘bedana-afg0383’ showed heavy and hard seeds and other relevant differences from the ‘bedana’ type, such as big calyx, pale skin colour and low juiciness, and it has been fig. 2 dendrogram (euclidean distance; ward's agglomerating method) obtained by analyzing 30 multi-state discrete morphological variables of 58 pomegranate accessions of afghan origin and 19 imported varieties belonging to the national collection of fruits and nuts of afghanistan (bold: imported cultivars; bold cursive: putative ‘bedana’ type; cursive: putative ‘kandahari’ type). giordani et al. afghanistan selected pomegranate germplasm. morphological variability and relationship 229 identified as ‘mirwais khani’ (fig. 2). similarly the accession originally denominated ‘bedana-afg0762’, brought medium hard seeds, white-pink arils, and was renamed as ‘abasi’. also ‘bedana-afg5018’, collected in tabag district of kapisa province, and ‘bedana afg6061’, originated from sherzad district of jalalabad province, did not show the typical characteristics of the true-to-type ‘bedana’, and were renamed as ‘ghani kheli’ and ‘jan mohammadi’ respectively. finally, the presumed ‘bedana’ type ‘afg6053’, originated in the district of jalalabad, notwithstanding its high morphological similarity with the true-to-type ‘bedana’ (soft seeds, juicy and good taste), showed low productivity and was denominated ‘bedana samaskheli’. cluster c3, holding 23 local accessions, is not evidencing any special relationship among previously known cultivars. during the survey, a part from ‘bedana’ name, another recurrent denomination attributed by farme r s t o l o c a l a c c e s s i o n s w a s ‘ k a n d a h a r i ’ ( f r o m kandahar). indeed ‘kandahari’ accessions were collected from different provinces of afghanistan and they showed different and unique phenotypic traits during the characterisation hence they have been considered as different varieties. for instance, ‘kandahari afg6066’ originated from sher zad district of nangarhar province, had thin fruit skin and hard seeds, and it was renamed as ‘rahmani’; ‘saifi’ was the accepted name for the former ‘kandahari a f g 5 0 1 9 ’ , c o l l e c t e d i n t a g a b d i s t r i c t ( k a p i s a province) producing fruits with hard seeds, reddish arils and low juiciness. ‘mukhtari’ was originally ‘kandahari afg0862’ from khulam district (balkh province) with medium hard seeds and white arils. actually ‘kandahari’ name was kept only for the accession collected as ‘kandahari afg6057’ originated from tagab district (kapisa province) and producing large fruits with medium hard skin, medium hard seeds and red arils. 4. conclusions this study was the first of its kind carried on for pomegranate in afghanistan in terms of standardized methodology; comparative analysis with previous data collected from local afghan varieties of pomegranate trees grown in different environmental conditions and as unique individuals resulted not completely reliable. the results revealed a wide variability of morphological traits within the afghan pomegranate collected accessions; this finding is consistent with similar researches carried on local pomegranate accessions in countries where p. granatum is present since ancient time. such variation can be considered as a result of the combined effect of sexual reproduction and asexual propagation. on the other hand, many cases of homonyms have been observed, hence confirming a common trend in the denomination of varieties by farmers of countries where perennial species are grown traditionally. more specific morphological studies associated to the evaluation of tree productivity and fruit biochemical and commercial traits would be the next step in order to better classify the afghan pomegranate accessions in relation to their main suitability for different uses (fresh fruit consume, juice production, food supplements, etc.). acknowledgements activity founded by the european commission europeaid program. a special appreciation to the staff of the ministry of agriculture, irrigation and livestock of afghanistan for the availability and support. an unique everlasting memory for mohammad ghous and monica berti. references alam m., 2011 trees and shrubs of afghanistan. a dendrological guide. rossolis, musée botanique cantonal lausanne, switzerland, pp. 512. al-sadi a.m., al-fahdi a.r., al-yahyai r.a, al-ghaithi a.g., al-said f.a, soleiman m.j., 2015 genetic analysis suggests a shared origin of punica granatum cultivars in oman with 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hort., 26: 180-185. 64 1. introduction seedlessness is a desirable characteristic for both fresh and processed citrus markets (vardi et al., 2008). in fact, major citrus cultivars on a global level such as ‘valencia,’ navel orange [citrus sinensis (l.) osbeck] and ‘marsh’ grapefruit (c. paradisi macfad.) are seedless (reuther, 1988). although a major early-maturing citrus, satsuma mandarin (c. unshiu marcow.), is seedless, almost all mid and late-maturing citruses, such as natsudaidai (c. natsudaidai hayata), iyo (c. iyo hort. ex tanaka), and hassaku (c. hassaku hort. ex tanaka), are seedy in japan (iwamasa, 1988). thus, breeding new seedless cultivars is an urgent issue to develop the japanese citrus industry. to develop seedless citrus at the diploid (2x=18) level, utilization of sterility is essential (iwamasa, 1966). sterility can be divided into three types: male sterility, female sterility, and self-incompatibility. in addition, selection of triploid (3x=27) individuals is useful for breeding seedless cultivars (ollitrault et al., 2007). therefore, various kinds of investigations on the mechanism and genetic factors underlying seedlessness in citrus have been conducted and many breeding works have been carried out to develop new seedless cultivars, many of them carried out in japan over the last few decades. in this review, the progress on studies for seedless breeding of citrus in japan, with regard to 1) seedlessness at the diploid level and 2) ploidy manipulation for seedless breeding, is discussed. 2. seedlessness at the diploid level male sterility the degree of male (pollen) sterility is variable in citrus, and usually pollen sterile accessions produce seedless or low-seedy fruits when cultivated in solid blocks. male sterility couples with parthenocarpy to produce seedless fruits when cross-pollination is prevented. even in mixed planting with pollen fertile accessions, male sterility reduces seed production and increases the percentage of seedless fruits because those accessions have a smaller chance of fertilization than male fertile ones (yamamoto et al., 1993, 1995). iwamasa (1966) and ollitrault et al. (2007) summarized the various levels of male sterility at the diploid level in citrus (table 1). chromosome aberration was one of the most important phenomena causing pollen sterility. asynapsis in ‘mukaku yuzu’ (c. junos sirbold ex tanaka) is genetically controlled, while that in ‘eureka’ lemon (c. limon (l.) burm. f.) and ‘mexican’ lime (c. aurantifolia (cristm.) swingle) is induced by low temperature (nakamura, 1943; iwamasa and iwasaki, 1962; iwamasa, 1966). reciprocal translocation is found to cause pollen sterility of ‘valencia’ orange (c. sinensis) (iwamasa, 1966). inversion is the cause of partial pollen sterility of ‘mexican’ lime (c. aurantifolia) (iwamasa, 1966). male sterility that is not caused by chromosome aberration is also well known. anther aborprogress on studies for seedless breeding of citrus in japan m. yamamoto(*) faculty of agriculture, kagoshima university, korimoto, kagoshima 890-0065, japan. key words: female sterility, male sterility, protoplast fusion, self-incompatibility, triploid. abstract: seedlessness is a desirable characteristic for both fresh and processed citrus markets and one of the most important breeding objectives. in this paper, progress on studies for seedless breeding of citrus in japan is reviewed. among the several types of male sterility, anther abortion is the strictest male sterility in citrus and was shown to be controlled by both nuclear and cytoplasmic genes. several seedless cultivars with male sterility have been developed. the mechanism and inheritance of the strictest female sterility derived from ‘mukaku kishu’ (citrus kinokuni hort. ex tanaka) were clarified and seedless cultivar and parental lines with this female sterility have been released. some self-incompatible cultivars show seedlessness when coupled with parthenocarpy. s (self-incompatibility) genotypes of several cultivars have been estimated. tetraploid plants, as parents of triploid offspring, were obtained from nucellar seedlings, and by ploidy mutation, colchicine treatment, and protoplast fusion. triploid plants were produced from the combination of not only tetraploid and diploid crosses but also diploid and diploid crosses. new triploid seedless cultivars were bred by programmed cross-breeding and protoplast fusion. adv. hort. sci., 2014 28(2): 64-72 (*) corresponding author: yamasa@agri.kagoshima-u.ac.jp received for publication 31 march 2014 accepted for publication 17 june 2014 review paper 65 tion in satsuma mandarin (c. unshiu) hybrids is the strictest male sterility in citrus (iwamasa, 1966). the sterile stamen appears only as the filament, and no pollen grains are produced (fig. 1). male sterility of ‘washington’ navel (c. sinensis), ‘tahiti’ lime (c. latifolia tanaka), and some other hybrids is due to early degeneration of pollen mother cells (pmcs) (osawa, 1912; uphof, 1931; frost, 1948; iwamasa, 1966). pollen sterility of satsuma mandarin (c. unshiu) is caused by plural sterility such as abnormal behavior and degeneration of pollen grains (nakamura, 1943; yang and nakagawa, 1969, 1970). to develop new seedless cultivars efficiently, genetic analysis of male sterility has been conducted. among these studies, genetic analysis of anther abortion has progressed remarkably. this male sterility is due to genecytoplasmic interaction [satsuma mandarin (c. unshiu), ‘encore’ mandarin (c. nobilis lour. × c. deliciosa ten.), yuzu (c. junos), and lemon (c. limon) possess sterile cytoplasm] and is probably controlled by more than one major gene (iwamasa, 1966; yamamoto et al., 1992 a, b, 1997; nakano et al., 2001; dewi et al., 2013 a). dewi et al. (2013 a) postulated that a dominant nuclear fertilityrestoring gene system comprising one epistatic gene and two complementary genes controls the restoration of male table 1 diagramatic representation of various kinds of the male-sterility in citrus, according to the sequential order of development (modified from iwamasa, 1966) developmental stage nature of sterility cultivar or hybrid reference initiation of anther development anther abortion satsuma mandarin × sweet orange, etc. iwamasa, 1966 archesporial stage resting stage degeneration of pmcs washington navel tahiti lime lemon × valencia, etc. satsuma mandarin × trifoliate orange osawa, 1912 uphof, 1931 frost, 1948 iwamasa, 1966 meiosis i-division asynapsis (genic) asynapsis (by low temp.) translocation inversion mukaku yuzu eureka lemon mexican lime c. assamensis valencia orange c. assamensis, etc mexican lime iwamasa, 1966 nakamura, 1943 iwamasa and iwasaki., 1962 naithani and raghuvanshi, 1958 iwamasa, 1966 raghuvanshi, 1962 a iwamasa, 1966 ii-division failure of spindle marsh grapefruit raghuvanshi, 1962 b liberation from tetrad degeneration jaffa orange oppenheim and frankel, 1929 mitotic division degeneration satsuma mandarin nakamura, 1943 mature pollen grain fig. 1 flowers of male-sterile (aborted anthers, ms) and male-fertile (normal anthers, mf) citrus. 66 fertility and male-sterile anther size in citrus plants with sterile cytoplasm. nakano et al. (2000) found dna markers linked to aborted anther for juvenile screening of malesterile plants. male-sterile (aborted anther) progenies were also determined at the early period of seedling growth to exhibit precocious flowering, a phenomenon in which very young seedlings have flowers (dewi et al., 2013 b). another type of male sterility, inheritance of pollen fertility/sterility, was also studied (ueno, 1986). some pollensterile progenies arose from two pollen fertile parents. in citrus, not only somatic hybrids but also cybrids were produced by symmetric protoplast fusion (saito et al., 1993; moriguchi et al., 1996; 1997; tokunaga et al., 1999). yamamoto and kobayashi (1995) produced a cybrid having the sweet orange (c. sinensis) nuclear genome and satsuma mandarin (c. unshiu) cytoplasmic genome by fusion between satsuma mandarin protoplasts isolated from embryogenic callus and sweet orange mesophyll protoplasts. the cybrid is useful for seedless breeding because it has sterile cytoplasm derived from satsuma mandarin. several new seedless cultivars with male sterility were released in japan (nishiura et al., 1983; okudai et al., 1991; matsumoto et al., 1991, 2003; yoshida et al., 2005 c) (fig. 2, table 2). all cultivars with aborted anther possess cytoplasm derived from satsuma mandarin (c. unshiu). female sterility female sterility is a very important trait which is closely related to seedlessness. yamamoto et al. (1995) reported that the degree of female fertility/sterility is rated on the basis of the average number of seeds per fruit obtained through hand pollination. a high positive correlation (r = 0.93**) was found between the number of seeds of handpollinated fruits and that of open-pollinated fruit. this result indicated that female sterility is directly related to seediness. female sterility estimated by the above-mentioned method was revealed to be a heritable characteristic (yamamoto et al., 2001). table 2 seedless cultivars and parental lines of citrus released in japan cultivar or parental line cross combination note male sterility kiyomi miyagawa wase (citrus unshiu) × trovita (c. sinensis) seiho kiyomi × minneola (c. paradisi × c. tangerina) tsunokaori kiyomi × okitsu wase (c. unshiu) amaka kiyomi × encore (c. nobilis × c. deliciosa) setoka (kiyomi × encore) no. 2 × murcott (probably tangor) harehime e647 (kiyomi × osceola) × miyagawa wase (c. unshiu) tsunokagayaki (kiyomi × okitsu wase) no. 14 × encore (c. nobilis × c. deliciosa) female sterility southern yellow tanikawa buntan (c. maxima) × mukaku kishu (c. kinokuni) citrus parental line norin no. 5 lee (clementine × orlando) × mukaku kishu (c. kinokuni) citrus parental line norin no. 6 king mandarin (c. nobilis) × mukaku kishu (c. kinokuni) self-incompatibility ariake seike navel (c. sinensis) × clementine (c. clementina) triploid puchimaru oval kumquat (fortunella margarita) × tetraploid meiwa kumquat (c. crassifolia) white love cybrid having lemon (c. limon) somatic hybrid nuclear and sudachi (c. sudachi) cytoplasmic genome + haploid clementine (c. clementina) tokushima 3x no. 1 tetraploid sudachi hs4 (c. sudachi) × sudachi ryokuko-kei (c. sudachi) yellow bell open-pollinated seedling of diploid michitani-line villafranca (c. limon) fig. 2 male-sterile seedless citrus ‘setoka’. 67 ‘mukaku kishu’, a bud variant of the seedy kinokuni mandarin (citrus kinokuni hort. ex tanaka), is completely seedless and considered to have the strictest female sterility in citrus. yamasaki et al. (2007, 2009) studied the mechanism of expression of seedlessness derived from ‘mukaku kishu’. in fruits of ‘mukaku kishu’, specific very small and swollen seeds called “type a seeds” were observed. the expression of ‘mukaku kishu’-type seedlessness is characterized by formation of “type a seed” with an immature seed coat and an embryo arrested at an early stage. however, arrested embryo development in the “ type a seed” is not caused by endosperm abortion. this female sterility is controlled by two major genes: sterility and fertility are dominant and recessive, respectively (nesumi et al., 2001). new seedless cultivar and parental lines with this sterility were bred in japan (yoshida et al., 2005 a, b) (fig. 3, table 2). navel orange (c. sinensis) and satsuma mandarin (c. unshiu) have strong female sterility; only a few seeds were developed when they were hand-pollinated (miki, 1921; nagai and tanikawa, 1926; nishiura and iwasaki, 1963; yamamoto et al., 1995). osawa (1912) observed degeneration of the embryo sac in both navel orange and satsuma mandarin. nesumi et al. (2000) assumed that the female sterility of satsuma mandarin is controlled by two major genes: sterility and fertility are recessive and dominant, respectively, and they were mapped on a linkage map (omura et al., 2000). self-incompatibility self-incompatibility is a genetically controlled phenomenon preventing seed set in self-pollinated plants producing functional gametes. self-incompatibility in citrus is a very important trait for fruit production. without parthenocarpy, it requires cross pollination to achieve stable fruit production (nagai and tanikawa, 1926; miwa, 1951). however, its coupling with parthenocarpy could produce seedless fruit (iwamasa and oba, 1980; yamamoto et al., 1995; yamamoto and tominaga, 2002). thus, much research has been conducted to determine self-incompatibility of many accessions (nagai and tanikawa, 1926; miwa, 1951; nishiura and iwasaki, 1963; iwamasa and oba, 1980; yamamoto and tominaga, 2002; yamamoto et al., 2006, 2012). the incompatibility system of citrus is of the gametophytic type and soost (1965, 1969) proposed s (self-incompatibility) genotypes of some accessions. since then, the source of s genotypes has been less well elucidated. recently, however, research has progressed remarkably via certain methods. pollen tube growth was strongly inhibited in incompatible pollination; those pollen tubes exhibited abnormal behaviors, namely twisted and heavy and irregular callose deposition. on the other hand, in compatible pollination, many normal pollen tubes penetrated into the style (ngo et al., 2001). self-incompatibility s genotypes of several citrus cultivars were estimated by the observation of pollen tube behavior in the styles after controlled pollination with a restricted number of pollen grains on their stigmas (ngo et al., 2010). cross-incompatible, cross-semi-compatible, and cross-full-compatible relationships were clarified based on the results of the number of pollen tubes that reached the base of the style. from these results, s genotypes of several cultivars were estimated. the s genotype could be estimated with the aid of allozymes produced by the glutamate oxaloacetate transaminase isozyme gene (got-3), which appeared to be linked to the s gene (ngo et al., 2011). the most efficient way to determine the s genotype is considered to be pollination with pollen homozygous for the s genotype. kim et al. (2010, 2011) revealed the s genotype of some cultivars by pollination of homozygous s 1 seedlings of ‘hirado buntan’ [c. maxima (burm.) merr.] and ‘banpeiyu’ (c. maxima). there are no differences in estimated s genotypes among the three above-mentioned methods. table 3 shows the estimated s genotypes of several accessions. incompatibility s alleles are distributed widely, not only in self-incompatible accessions but also self-compatible ones such as satsuma mandarin (c. unshiu), grapefruit (c. paradisi), and ‘dancy’ (c. tangerina hort. ex tanaka) (soost, 1965, 1969; vardi et al., 2000). thus, self-incompatible individuals can be produced from cross combinations between two selffig. 3 female-sterile seedless citrus ‘southern yellow’. table 3 estimated s genotype of citrus accessions (kim et al., 2011; ngo et al., 2010, 2011) accession latin name estimated s genotype (z) banpeiyu citrus maxima (burm.) merr. s 1 s 2 tosa buntan c. maxima (burm.) merr. s 1 s 3 iriki buntan c. maxima (burm.) merr. s 1 s 2 kaopang c. maxima (burm.) merr. s 1 s 2 soyu c. maxima (burm.) merr. s 1 s 2 hassaku c. hassaku hort. ex tanaka s 4 s 5 yuge-hyokan c. yuge-hyohan hort. ex yu. tanaka s 6 s 7 shishiyuzu c. pseudogulgul hort. ex shirai s 1 s 6 hyuganatsu c. tamurana hort. ex tanaka s 1 s 8 tachibana no. 1 c. tachibana (makino) tanaka sfs 8 rough lemon c. jambhiri lush. sfs 1 zadaida c. aurantium l. sfs 1 kinukawa c. glaberima hort. ex tanaka sfs 2 kawano natsudaidai c. natsudaidai hort. ex tanaka sfs 2 (z) sf: self-compatible. 68 compatible parents, for example, ‘orlando’ and ‘minneola’ arose from ‘duncan’ grapefruit and ‘dancy’ combination (swingle et al., 1931). in japan as well, self-incompatible seedless ‘ariake’ was bred by crossing self-compatible ‘seike’ navel orange (c. sinensis (l.) osbeck) and self-incompatible clementine (c. clementina hort. tanaka) (yamada et al., 1995; yamamoto et al., 2006) (fig. 4, table 2). 3. ploidy manipulation for seedless cultivar breeding although spontaneous triploid (3x=27) accessions were very rare in citrus (krug, 1943; krug and bacchi, 1943; noro and kajimoto, 1955), many triploid hybrids have been produced by artificial hybridization. since these triploids are seedless, it could be considered that producing triploids is a useful way to promote seedless breeding in citrus efficiently. tachikawa et al. (1961) conducted one of the earliest programmed triploid breeding projects. first, they produced tetraploid materials (4x=36) by colchicine treatment and then triploid (2x=18) hybrids were obtained from these tetraploid and diploid cross combinations. as they showed, since triploids arise from tetraploid and diploid crossing, tetraploid plants are important for triploid breeding. therefore, various tetraploid accessions were obtained by certain methods. oiyama et al. (1980) selected spontaneous autotetraploids from nucellar seedlings of polyembryonic cultivars. they revealed the leaf morphological characteristics of tetraploids: thick and broad leaves and reduced number of stomata per area. kawase et al. (2005) obtained meiwa kumquat (fortunella crassifolia swingle) autotetraploid from 500 seedlings. an autotetraploid also arose as a bud sport (yamao et al., 1993). colchicine treatment is useful for the production of autotetraploids in many higher plants. in citrus in particular, various tetraploids were produced by this treatment. colchicine treatment of seeds was effective in polyembryonic cultivars (yahata et al., 2004). however, this treatment of monoembryonic seeds is a problem because the tetraploids obtained by this treatment are not true-to-type. oiyama and okudai (1986) resolved this problem through a combination of colchicine treatment of isolated small buds and their micrografting. they successfully produced autotetraploids from three monoembryonic cultivars. moreover, in another eight autotetraploids, monoembryonic citrus was produced using the same method (kaneyoshi et al., 2008). it has been revealed that although tetraploids arise from both diploid × tetraploid and tetraploid × diploid crosses, the latter combination is more effective (cameron and burnett, 1978; kaneyoshi et al., 2008) and an unbalanced ploidy ratio between embryo and endosperm is considered to cause this phenomenon (esen and soost, 1973). thus, artificially produced monoembryonic autotetraploids were important as seed parents for triploid breeding. on the other hand, triploid hybrids sometimes appeared from diploid-diploid crosses (esen and soost, 1971; oiyama and okudai, 1983; yasuda et al., 2010); the appearance of triploids is due to the unreduced gametophyte of one parent (esen and soost, 1971). biotechnological methods such as protoplast fusion have contributed to the progress of citrus triploid breeding. since somatic hybrids are tetraploids in general (ohgawara et al., 1985), they are important parents for triploid breeding. somatic hybrids derived from crosses between navel orange (c. sinensis) + satsuma mandarin (c. unshiu), grapefruit (c. paradisi), yuzu (c. junos) and ‘murcott’ (artificial hybrid) were registered as parental lines (kobayashi et al., 1995). triploid hybrids could be produced directly by means of protoplast fusion. somatic hybrids produced from diploid and haploid fusion became triploids (kobayashi et al., 1997). the haploid parents were obtained by diploid × triploid cross (oiyama and kobayashi, 1993). table 2 shows the triploid cultivars released in japan. among the four cultivars, ‘puchimaru’ (yoshida et al., 2003) (fig. 5), ‘white love’, tokushima 3x no. 1’ (tokufig. 5 triploid seedless kumquat ‘puchimaru’ and diploid seedy meiwa kumquat and oval kumquat. p= puchimaru, m= meiwa kumquat, and o= oval kumquat. fig. 4 self-incompatible seedless citrus ‘ariake’. 69 naga et al., 2005), and ‘yellow bell’ (kaneyoshi et al., 2014) were derived from diploid × tetraploid cross, protoplast fusion between diploid and haploid, tetraploid × diploid cross, and diploid × diploid cross, respectively. 4. conclusions seedless breeding of citrus has progressed rapidly in japan over the last few decades. the production of new seedless cultivars is increasing in contrast to the decrease in production of seedy conventional cultivars such as ‘kawano natsudaidai’ (c. natusdaidai) and ‘miyauchi iyokan’ (c. iyo). various kinds of cross combinations using cultivars or parental lines with sterility have been conducted actively and further polyploid breeding is being carried out to produce new triploid plants. in addition, methods to shorten the long juvenile period of citrus have developed in japan (okudai et al., 1980; 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h-j., nakagawa s., 1970 cyto-histological studies on the male sterility of satsuma orange (citrus unshiu marc.). j. japan. soc. hort. sci., 58: 239-245. yasuda k., yahata m., komatsu h., kurogi y., kunitake h., 2010 triploid and aneuploidy hybrids from diploid-diploid intergeneric crosses between citrus cultivar ‘kiyomi’ tangor and meiwa kumquat (fortunella crassifolia swinglw) for seedless breeding of kumquats. j. japan. soc. hort. sci., 79: 16-22. yoshida t., nesumi h., yoshioka t., ieki h., ito y., nakano m., ueno i., yamada y., murase s., takishita f., 2003 new kumquat cultivar ‘puchimaru’. bull. natl. inst. fruit tree sci., 2: 9-16. yoshida t., nesumi h., yoshioka t., ito y., ueno i., yamada y., 2005 a ‘kankitsu chukanbohon nou 5 gou’ (‘citrus parental line norin no. 5’) is useful for breeding seedless and early maturing cultivars. bull. natl. inst. fruit tree sci., 4: 47-52. yoshida t., nesumi h., yoshioka t., ito y., yano m., nakano m., ueno i., yamada y., ogawa k., murase s., takishita f., hidaka t., kawai s., 2005 72 b ‘kankitsu chukanbohon nou 6 gou’ (‘citrus parental line norin no. 6’) is useful for breeding seedless and functional component-rich cultivars. bull. natl. inst. fruit tree sci., 4: 53-59. yoshida t., nesumi h., yoshioka t., nakano m., ito y., murase s., takishita f., 2005 c new citrus cultivar ‘harehime’. bull. natl. inst. fruit tree sci., 4: 3745. impaginato 3 1. introduction drought stress can affect plant growth, development and yield. it has been estimated that up to 45% of world agricultural lands are subjected to drought (bot et al., 2000). water deficit leads to the perturbation of most of the physiological and biochemical processes and consequently reduces plant growth and yield (boutraa, 2010). abscission of reproductive organs like flower buds and flowers is a major yieldlimiting factor in vegetable crops (wien et al., 1989). the abscission of floral organs during stresses has b e e n a s s o c i a t e d w i t h c h a n g e s i n p h y s i o l o g i c a l processes (aloni et al., 1996). it has been reported that, in tomato, the abscission of flowers and flower buds and the reduction in photosynthesis were higher in susceptible cultivars compared to tolerant cultivars (bhatt et al., 2009). in soybean, flower retention and fruit set are highly sensitive to environmental stresses (kokubun et al., 2001). etsushi et al. (2009) reported that soil plant analytical development (spad) method readings significantly correlated with chlorophyll content, rubisco content, photosynthetic rate, and fv/fm ratio. a major portion of soluble protein (50%) in leaves is occupied by rubisco, a prime enzyme for carbon fixation in photosynthesis (noggle and fritz, 1986). daniel and triboi (2002) showed that heat stress decreased the duration of soluble protein accumulation in terms of days after anthesis but not in terms of thermal time. drought decreased leaf n, whereas heat stress did not influence it and, however, the total soluble protein content was decreased during drought, heat, and a combination of drought and heat. heat and drought stress induced suppression of photosynthesis by mainly decreasing the proportion of soluble protein to total leaf n, adversely affecting the rubisco protein and activity (xu and zhou, 2006). bhatt et a l . ( 2 0 0 9 ) r e p o r t e d t h e r e w a s a c o n s i d e r a b l e decrease in sps activity in flowers under water deficit condition; a relatively higher decrease was observed in the susceptible genotypes. sps activity was shown to decrease during leaf desiccation (foyer et al., 1998), or to remain constant (zrenner and stitt, 1991). however, yang et al. (2002) and niedzwiedzsiegen et al. (2004) observed an increase in sps activity in rice and wheat leaves, respectively, under adv. hort. sci., 2016 30(1): 3-11 doi: 10.13128/ahs-18696 impact of drought on flowering, yield and quality parameters in diverse genotypes of tomato (solanum lycopersicum l.) r. sivakumar (*), s. srividhya department of crop physiology, tamil nadu agricultural university, coimbatore 641 003, tamil nadu, india. key words: ascorbic acid, drought, flower abscission, lycopene, soluble protein, sps, yield. abstract: the effect of drought stress on flowering, yield and quality of tomato (solanum lycopersicum) genotypes was investigated under field conditions in rainout shelter. the drought condition was imposed on the first day after transplanting based on field capacity of soil. experimentation was undertaken with ten genotypes adopting factorial randomized block design with three replications and two treatments viz., 1.0 iw/cpe and 0.5 iw/cpe field capacity. as the stress increased from 100% field capacity to 50% field capacity, reductions in chlorophyll index, soluble protein content, days to flower initiation, sucrose phosphate synthase (sps) activity, fruit volume, fruit diameter, yield and increased flower abscission percentage were noted. significant increases in tss and lycopene were observed under drought. the genotypes le 118, le 57 and le 114 showed significantly less reduction in soluble protein content; sps activity and fruit yield during drought were considered as drought tolerant. genotypes le 1 and le 125, which gave the lowest soluble protein content, sps activity and ultimately poor yield, were considered as drought susceptible. (*) corresponding author: sivatnau5@gmail.com received for publication 26 january 2015 accepted for publication 6 november 2015 copyright: © 2016 author(s). this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. adv. hort. sci., 2016 30(1): 3-11 4 mild drought. tomato (solanum lycopersicum) is one of the most popular and widely grown vegetables in the world. considering the potentiality of this crop, there is plenty of scope for its improvement, especially under the drought situation. water is a scarce resource for irrigation. although the concept of drought tolerance has been viewed differently by molecular biologists, biochemists, physiologists and agronomists, the major concern is to enhance the biomass and yield under limited water input, which is a characteristic feature of rainfed agriculture. there are several physiological and biochemical traits contributing to the drought tolerance of horticultural crops. however, a large number of tomato genotypes have not been screened for drought tolerance or exploited for their cultivation under drought. to breed drought tolerant genotypes, it is necessary to identify physiological traits of plants which contribute to drought tolerance. chlorophyll pigments and soluble protein content in leaves which have a greater correlation to crop yield show reduced levels under drought. the reproductive stage of the crop is highly sensitive to any abiotic stress. hence, reduction in crop yield under drought is mainly attributed to the effect on flowering characters and a decrease in sps enzyme activity. therefore, the present investigation was carried out to study the physiological traits such as spad value, soluble protein content, sps activity, flowering characters, and yield to facilitate the screening and selection of tomato genotypes for drought tolerance. 2. materials and methods plant materials and cultivation the study was undertaken to determine the effect of drought on flowering and yield of tomato genotypes in the field experiment at rainout shelter of crop physiology department, tamil nadu agricultural university, coimbatore, tamil nadu during 2012-13. the experiment was conducted with 10 tomato genotypes (le 1, le 27, le 57, le 114, le 118, le 125, co 3, pkm 1, tnau thco 3 and coth 2) and two treatments (1.0 and 0.5 iw/cpe (irrigation water/ cumulative pan evaporation) with three replications following a factorial randomized block design. seeds of selected genotypes were sown in trays filled with vermicompost for nursery use. twenty-five day old seedlings were transplanted in the rainout shelter; plot size 1.35 x 1.5 m. drought was imposed on the first day after transplanting to all genotypes in both iw/cpe ratio treatments. furrow irrigation was applied when the cumulative pan evaporation reading reached 50 mm (1.0 iw/cpe ratio) and 75 mm (0.50 iw/cpe ratio). crop was supplied with fym (25 t/ha rate), npk fertilizers (75:100:50 kg/ha), borax 10 kg and zinc sulphate 50 kg/ha as basal dose and 75 kg n/ha on the 30th day after planting during earthing up. other cultivation operations including weed control (applied pendimethalin 1.0 kg a.i./ha as preemergent herbicide followed by one hand-weeding at 30 days after planting) and plant protection measures were carried out as per the recommended package of practices of tamil nadu agricultural university, coimbatore. physiological parameters spad readings were recorded using chlorophyll meter (spad 502) designed by the soil plant analytical development (spad) section, minolta, japan. data were recorded as described by peng et al. (1996). soluble protein content of the leaf was estimated as per the method of lowry et al. (1951) and expressed as mg g-1 fw. sucrose phosphate synthase (sps) activity was determined as described by pavlinova et al. (2002). sucrose was measured using anthrone reagent as modified by ashwell (1957). absorbance was measured at 630 nm and the activity was expressed in mg sucrose mg-1 protein h-1. flowering parameters days to flowering (the number of days from seedling emergence to opening of the first flower) were recorded for each plant in the three replications and the average was taken. abscission study was conducted on single flower basis. flower number of tagged plants and dropped flowers per plant were counted every three days. these records were used to calculate the flower abscission and expressed in terms of percentage. yield parameters average fruit weight was calculated by adding the weight of five fruits from each plant at second harvest and dividing it by the total number of fruits and expressed in g fruit-1. the fruit volume was estimated by water displacement method. individual fruits were immersed in 1 l of water; amount of water displaced was measured and volume was worked out per fruit and expressed as cc. polar diameter was measured from stalk end to blossom end of fruit by using vernier calipers and the average of five fruits sivakuma and srividhya drought impact on flowering, yield and quality in different tomato genotypes 5 was worked out and expressed in cm. the fruit weight per plant was recorded in control and stressed plants for each picking and fruit yield (kg per plant) was calculated as fresh weight of fruits in all the pickings. quality parameters juice extracted from cut fruit was used to determine tss with the help of a hand refractometer (0 to 32°brix) at room temperature and the value was noted in °brix. ripened fruit samples were analyzed for ascorbic acid content, using 2,6-dichlorophenol indophenol dye titrimetrically as per sadasivam and manickam (1996). lycopene content of fruit was extracted using petroleum ether and od of the extract was measured at 503 nm in uv-vis-spectrophotometer using petroleum ether as a blank (ranganna, 1986). lycopene content of the sample was calculated using the following formula and expressed in mg 100 g-1. 3.1206 x od of sample x volume made up x dilution lycopene = x 100 weight of sample x 1000 statistical analysis data from the various parameters were analyzed statistically as per the procedure of gomez and gomez (1984). 3. results decreased spad value under drought control (1.0 iw/cpe) plants showed a higher mean chlorophyll index value (47.57) than treated (0.5 iw/cpe) plants (44.04). among the genotypes, co 3 and pkm 1 recorded significantly higher chlorophyll index values of 50.1 and 46.1, and 49.5 and 46.7 at 1.0 iw/cpe and 0.5 iw/cpe conditions, respectively. during drought, the genotypes le 114 (45.8), le 57 (45.7), coth 2 (45.5) and le 118 (45.0) were found to be on par with each other. decreased soluble protein due to drought control (1.0 iw/cpe ratio) plants showed a higher mean soluble protein content (13.26) than the drought (0.5 iw/cpe ratio) imposed plants (8.98). among the genotypes, coth 2 (15.63) and thco 3 (15.18) registered the highest soluble protein content under 1.0 iw/cpe ratio level and le 57 (11.99) and le 118 (11.74) under drought conditions (0.5 iw/cpe). the lowest soluble protein content was found for le 125 (8.16) and le 1 (8.68) (table 1). altered flowering characters from drought our data on number of days to flower initiation revealed that genotypes, treatments and interactions attained statistical significance (table 2). in the case of treatments, the plants under drought initiated flowers earlier (26) than control plants (30). at 0.5 table 2 effect of drought on flowering characters of tomato genotypes during flowering stage genotypes days to flower initiation (dat) flower abscission (%) 1.0 iw/cpe 0.5 iw/cpe mean 1.0 iw/cpe 0.5 iw/cpe mean le 1 29 25 27 10.90 20.10 15.50 le 27 30 27 29 13.20 15.80 14.50 le 57 30 25 28 12.60 15.10 13.80 le 114 29 26 28 11.90 15.40 13.70 le 118 32 28 30 10.90 13.90 12.40 le 125 31 25 28 11.90 17.10 14.50 co 3 26 23 25 10.20 15.90 13.10 pkm 1 28 24 26 9.90 16.40 13.20 thco 3 32 28 30 10.90 17.40 14.20 coth 2 31 27 29 11.20 18.10 14.70 mean 30 26 28 11.40 16.50 13.90 g t g x t g t g x t sed 0:18 0:08 0:26 0.08 0.03 0.11 cd (0.05) 0:37 0:17 0:52 0.16 0.07 0.22 table 1 effect of drought on chlorophyll index spad value and soluble protein content of tomato genotypes at 60 dat genotypes spad value soluble protein (mg g-1) 1.0 iw/cpe 0.5 iw/cpe mean 1.0 iw/cpe 0.5 iw/cpe mean le 1 42.00 38.40 40.20 10.85 6.51 8.68 le 27 48.20 44.40 46.30 13.98 10.72 12.35 le 57 47.60 45.70 46.70 15.03 11.99 13.51 le 114 48.20 45.80 47.00 13.43 10.19 11.81 le 118 48.40 45.00 46.70 14.58 11.74 13.16 le 125 45.00 39.50 42.30 11.07 5.24 8.16 co 3 50.10 46.10 48.10 11.55 8.69 10.12 pkm 1 49.50 46.70 48.10 11.33 7.69 9.51 thco 3 48.40 43.30 45.90 15.18 8.46 11.82 coth 2 48.30 45.50 46.90 15.63 8.58 12.11 mean 47.57 44.04 45.82 13.26 8.98 11.12 g t g x t g t g x t sed 1.15 0.38 1.62 0.137 0.061 0.194 cd (0.05) 2.28 0.76 ns 0.278 0.124 0.393 adv. hort. sci., 2016 30(1): 3-11 6 iw/cpe ratio level, le 118, thco 3 (28), le 27 and coth 2 (27) registered a delay in flowering compared to other genotypes. the variety co 3 showed its supremacy for earlier flowering, both under control (26) and drought (23) conditions, compared to the other genotypes; pkm 1 (28 and 24) ranked next. the differences due to genotypes, treatments and interactions attained significance for flower abscission (table 2). among the treatments, the plants imposed with 1.0 iw/cpe ratio recorded lesser mean percentage of flower abscission (11.4) than 0.5 iw/cpe ratio plants (16.5). among the genotypes, le 1 recorded greater mean percentage of flower abscission (15.5) than the other genotypes considered. interestingly, among the control plants, le 27 and le 57 recorded higher flower abscission (13.2, 12.6) while, under drought, le 1 and coth 2 recorded higher flower abscission of 20.1 and 18.1, respectively. at 0.5 iw/cpe ratio level, le 118 showed its supremacy with lower abscission of 13.9 followed by le 57 (15.1), le 114 (15.4) and le 27 (15.8). sps is the plant enzyme thought to play a major role in sucrose biosynthesis. it is considered to play a major role in the re-synthesis of sucrose (wardlaw and willenbrink, 1994) and sustain the assimilatory carbon flux from source to developing sink (isopp et al., 2000). among the genotypes, le 57 recorded significantly higher enzyme activity of 2.75 under control followed by the genotypes le 27 (2.61), le 118 (2.53) and le 114 (2.46). at 0.5 iw/cpe ratio level, the highest activity of 1.97 was registered by le 118 followed by le 57 (1.90) while the lowest was recorded by le 1 (0.57) and le 125 (0.69) (fig. 1). the data on yield components such as average fruit weight, fruit volume, fruit diameter, fruit number and fruit yield attained statistical significance except fruit diameter. comparing the treatments, control (1.0 iw/cpe ratio) plants recorded higher fruit weight (20.25) than under drought condition (13.75) (table 3). relating the genotypes, coth 2 recorded higher fruit weight of 45.40 which was on par with thco 3 (44.45) at 1.0 iw/cpe ratio level while the lowest was registered by le 125 (9.06) followed by le 57 (9.48). at 0.5 iw/cpe ratio level, relatively higher fruit weight was recorded by coth 2 (29.87) followed by thco 3 (28.78), co 3 (19.93), fig. 1 effect of drought on sps activity of tomato genotypes at 60 dat. genotypes average fruit weight (g) fruit volume (cc) fruit diameter (cm) 1.0 iw/cpe 0.5 iw/cpe mean 1.0 iw/cpe 0.5 iw/cpe mean 1.0 iw/cpe 0.5 iw/cpe mean le 1 11.18 5.72 8.45 12.020 6.48 9.25 3.41 2.43 2.920 le 27 10.68 7.23 8.96 11.52 7.95 9.74 3.34 3.05 3.20 le 57 9.48 7.89 8.69 10.09 8.62 9.36 3.11 3.48 3.30 le 114 9.55 7.49 8.52 10.31 8.25 9.28 3.53 3.25 3.39 le 118 10.01 7.95 8.98 10.92 8.61 9.77 4.12 3.76 3.94 le 125 9.06 3.89 6.48 9.65 4.64 7.15 3.30 2.72 3.01 co 3 26.38 19.93 23.16 28.39 22.69 25.54 5.40 4.84 5.12 pkm 1 26.31 18.75 22.53 28.34 22.49 25.42 5.51 4.71 5.11 thco 3 44.45 28.78 36.62 48.02 29.64 98.33 6.57 5.59 6.08 coth 2 45.40 29.87 37.64 48.95 30.69 39.82 6.31 5.73 6.02 mean 20.25 13.75 17.00 21.82 15.01 18.41 4.46 3.96 4.21 g t g x t g t g x t g t g x t sed 0.582 0.260 0.823 0.594 0.266 0.841 0.053 0.024 0.075 cd (0.05) 1.178 0.527 1.667 1.203 0.538 1.702 0.108 0.048 0.152 table 3 effect of water deficit on the yield parameters of tomato genotypes sivakuma and srividhya drought impact on flowering, yield and quality in different tomato genotypes 7 pkm 1 (18.75), le 118 (7.95) and le 57 (7.89). the data on fruit volume recorded similar trend of fruit weight. regarding the treatments, the plants imposed with 1.0 iw/cpe ratio recorded the fruit volume of 21.82 than 0.5 iw/cpe ratio (15.01) (table 3). under control, higher volume of fruit was recorded in the genotype coth 2 (48.95) which was on par with thco 3 (48.02). under 0.5 iw/cpe ratio level, the lowest fruit volume was recorded by le 125 (4.64) followed by le 1 (6.48). other than hybrids and varieties, le 57 showed higher fruit volume of 8.62 followed by le 118 (8.61), le 114 (8.25) and le 27 (7.95) at 0.5 iw/cpe ratio level. with regard to fruit diameter, thco 3 (6.08) and coth 2 (6.02) recorded higher average diameter of fruits which was on par with each other. for treatments, plants imposed with 1.0 iw/cpe ratio recorded higher fruit diameter (4.46) than 0.5 iw/cpe ratio (3.96) (table 3). among the genotypes, coth 2 registered higher fruit diameter of 5.73 followed by thco 3 (5.59), co 3 (4.84) and pkm 1 (4.71) at 0.5 iw/cpe ratio level. other than hybrids and varieties, le 118 showed higher fruit diameter of 3.76 followed by le 57 (3.48), le 114 (3.25) and le 27 (3.05) at 0.5 iw/cpe ratio condition while the lowest was recorded by le 1 (2.43) and le 125 (2.72). comparing the irrigation treatments, plants that received 1.0 iw/cpe ratio recorded higher fruit yield than 0.5 iw/cpe ratio (fig. 2). among the genotypes, le 57 recorded significantly superior fruit yield of 16.64 followed by coth 2 (15.89), le 118 (15.02), thco 3 (14.67) and le 27 (14.42) with 1.0 iw/cpe ratio level. but, at 0.5 iw/cpe ratio condition, le 57 documented higher fruit yield of 11.12 followed by le 118 (10.14), le 114 (8.54) and le 27 (8.13) while the lowest yield of 2.22 was recorded by le 125 followed by le 1 (2.57). quality parameters were altered under drought the data on tss content of the fruits revealed that the genotypes, treatments and interactions attained statistical significance (table 4). among the treatments, plants imposed with 0.5 iw/cpe ratio recorded higher brix value (3.01) than 1.0 iw/cpe ratio (2.89). among the genotypes, thco 3 recorded higher average brix value of 4.00 than the rest of the genotypes. at 0.5 iw/cpe ratio condition, the highest tss value was recorded by thco 3 (4.1) followed by coth 2 (3.9), pkm 1 (3.6) and co 3 (3.4) while the lowest was registered by le 125 (2.2). plants imposed with 0.5 iw/cpe ratio recorded fig. 2 effect of drought on the yield of tomato genotypes. table 4 effect of water deficit on fruit quality of tomato genotypes genotypes tss (° brix) lycopene (mg 100 g-1) vitamin c (mg 100 g-1) 1.0 iw/cpe 0.5 iw/cpe mean 1.0 iw/cpe 0.5 iw/cpe mean 1.0 iw/cpe 0.5 iw/cpe mean le 1 2.50 2.70 2.60 2.21 2.39 2.30 14.45 14.42 14.44 le 27 2.50 2.60 2.55 2.52 2.73 2.63 14.76 14.96 14.86 le 57 2.40 2.60 2.50 2.46 2.68 2.57 14.95 15.30 15.13 le 114 2.40 2.50 2.45 2.82 2.88 2.85 13.97 14.05 14.01 le 118 2.40 2.50 2.45 2.85 2.95 2.90 14.36 14.36 14.36 le 125 2.20 2.20 2.20 2.13 2.67 2.40 13.46 13.43 13.45 co 3 3.30 3.40 3.35 4.54 4.84 4.69 24.05 24.17 24.11 pkm 1 3.50 3.60 3.55 3.78 4.05 3.92 23.06 23.21 23.14 thco 3 3.90 4.10 4.00 3.35 3.53 3.44 15.42 15.64 15.53 coth 2 3.80 3.90 3.85 3.54 3.55 3.55 16.19 16.30 16.25 mean 2.89 3.01 2.95 3.02 3.23 3.12 16.47 16.58 16.53 g t g x t g t g x t g t g x t sed 0.03 0.01 0.04 0.048 0.022 0.068 0.147 0.066 0.208 cd (0.05) 0.05 0.02 0.07 0.097 0.044 0.138 0.297 ns ns adv. hort. sci., 2016 30(1): 3-11 8 higher lycopene content (3.23) than 1.0 iw/cpe ratio (3.02). with respect to the genotypes, co 3 recorded significantly higher average lycopene content (4.69). at 0.5 iw/cpe ratio level, lowest lycopene content was recorded by le 1 (2.39) and le 125 (2.67). the data on vitamin c content indicated that a narrow increment under drought compared to control (table 4). 0.5 iw/cpe ratio recorded higher ascorbic acid (16.58) than 1.0 iw/cpe ratio (16.47). among the genotypes, co 3 recorded higher average vitamin content of 24.11 followed by pkm 1 (23.14). at 0.5 iw/cpe ratio level, the higher value was registered by co 3 (24.17) followed by pkm 1 (23.21), coth 2 (16.30) and thco 3 (15.64) while the lowest values were recorded by the genotypes le 125 (13.43) and le 114 (14.05). interestingly, only genotypes attained significant difference not the treatments or interactions in the case of ascorbic acid. 4. discussion and conclusions in the present study, spad value, an index for total chlorophyll content in plants, showed a reduction under drought stress. hawkins et al. (2009) reported that spad values can be used to evaluate the response of plant species to drought and heat stresses in the field. the adverse effect of drought on greenness of the leaf in the current investigation could be observed in the susceptible genotypes le 125 and tnau thco 3 which depicted the highest reduction of spad values at the time of reproductive development stage. on the contrary, the tolerant genotype le 57 showed only a very low reduction in spad value. hence, the intensity of greenness in terms of spad values of the plant influenced the photosynthetic rate and thereby plant efficiency for increased biomass production. ma et al. (1995) also reported a highly significant correlation of spad readings with photosynthetic rate in soybean. the ability of the genotypes le 57 and le 114 to maintain high spad values under field conditions in response to water deficit has been revealed. therefore, these genotypes were able to endure drought injury better than the sensitive lines. the soluble protein content of the leaf, a measurement of rubp carboxylase activity, was considered an index for photosynthetic efficiency. rubisco enzyme makes up nearly 80% of the soluble proteins in leaves of many plants (joseph et al., 1981). diethelm and shibles (1989) opinioned that, the rubisco content per unit leaf area was positively correlated with that of soluble protein content of the leaf. several studies have reported that drought stress in tomato (bartholomew et al., 1991), arabidopsis (williams et al., 1994), and rice (vu et al., 1999) leads to a rapid decrease in the abundance of rubisco small subunit (rbcs) transcripts, which may indicate the decreased synthesis of soluble protein. the present study also confirms the above findings with a 32.3% reduction of soluble protein content under drought compared to control. drought stress induces degradation of soluble proteins and this effect could be revealed through a reduction in leaf soluble protein content of various genotypes. in the present study, the reduction was, however, low in le 118 and high in coth 2 and thco 3 under drought stress. maintenance by the genotypes of soluble protein content could be attributed to higher rubisco activity, leading to more carbon fixation and ultimately to higher photosynthetic efficiency under drought, which is one of the important traits for drought tolerance. reproduction is the crucial stage to be affected by any abiotic stress in any crop. an increase in the frequency of water stress days during flower development affects plant reproduction with immediate and long-term effects (srivastava et al., 2012). drought stress, in general, induces early flowering and in the present study as well flower initiation occurred three days earlier than the control. this early flowering under drought might be due to rapid phenological development in order to complete the life cycle under an unfavorable environmental condition. differences due to genotypes, treatments and interactions attained significance for flower abscission (table 2). among the treatments, the plants imposed with 1.0 iw/cpe ratio recorded a lower mean percentage of flower abscission than plants under 0.5 iw/cpe. among the genotypes, le 1 recorded the highest percentage of flower abscission. interestingly, under control conditions, genotypes le 27 and le 57 gave high flower abscission, while le 1 and coth 2 recorded higher flower abscission under drought. an earlier finding by bhatt et al. (2009) in tomato strongly supports the results of the present study. a lower rate of flower abscission in the tolerant genotypes might be due to the maintenance of photosynthesis and efficient translocation of photosynthates to the reproductive parts under drought. the reduction in photosynthesis during stress may decrease the availability of assimilates to the develsivakuma and srividhya drought impact on flowering, yield and quality in different tomato genotypes 9 oping floral organs and leads to the abscission of flowers and flower buds in susceptible cultivars. however, some workers are of the opinion that the abortion of reproductive organs is not solely due to a poor assimilate supply but also due to other factors such as assimilate utilization (ruiz and guardiola, 1994; aloni et al., 1996). in the present study, there was a reduction in sps activity under drought conditions compared to control. the highest percent reduction (74.3) was observed in the genotype le 1, however the lowest reduction was noted for le 118. as observed also by huber and huber (1996), there was a significant elevation in sps activity in response to water stress. in contrast to this finding, the present study revealed a decreased activity of sps under drought conditions. bhatt et al. (2009) observed a considerable reduction in sps activity in susceptible cultivars during stress. the present study corroborates these findings. the reduced photosynthesis during water stress may also lead to a reduction in the capacity for both starch and sucrose synthesis and cause a decline in the sps activity (vassey and sharkey, 1989). our results reveal that drought stress caused the reduction in fruit weight up to 30% under field conditions. among the genotypes, le 57, le 118, and le 114 had a lower reduction in fruit weight. all the genotypes exhibited a similar trend in fruit volume and fruit diameter in response to drought stress, which also caused remarkable changes in fruit number and an overall reduction up to 31% was observed compared to control. the reduction in fruit weight, in response to drought stress, had a direct influence on fruit yield of the various genotypes of tomato. drought stress resulted in an overall yield loss of tomato fruits up to 55%. the greatest yield loss (70 to 80%) was exhibited by le 1 and le 125. the varieties and hybrids showed a reduction of fruit yield from 40.5 to 50.4% compared to control. significantly less reduction (35 to 40%) was exhibited by le 118, le 57, le 114, and le 27 showing their somewhat tolerant nature toward drought stress (fig. 2). therefore, it can be clearly stated that water deficit, as a result of soil drying, caused a major adverse effect on yield and yield components even in tolerant genotypes. the present study confirms previous findings by farooq et al. (2009) and manjunatha et al. (2004). doorenbos and kassam (1979) indicated that the highest demand for water supply in tomato plants occurs at the flowering phase. water deficit during this stage would have reduced the number of flowers produced and, as suggested by mahendran and bandara (2000), limitation of water at flowering stage not only reduces flower formation but also increases flower shedding. purseglove et al. (1981) stated that, although the cultivar has a dominant influence over quality determinant properties, the environment in which it grows also has a significant role in the quality characters. fruit quality, mainly total soluble solids, vitamin c, and acid contents have been reported to change under moisture stress (kozlowski, 1972). however, in the present study, a slight enhancement in ascorbic acid content was noticed in all the genotypes in response to drought stress. furthermore, tss, lycopene and citric acid content of the fruit also increased slightly. our work corroborates earlier findings by ali et al. (1980) in tomato. also nahar et al. (2011) explained that the fruit quality improvement under water deficit conditions in tomato might be due to the synthesis of ascorbic, citric, and malic acid. in the present study, le 118, le 57, and le 27 showed their primacy with the highest ascorbic acid content, as well as higher tss and lycopene content. this finding is strongly supported by tambussi et al. (2000), who also reported that the increase in ascorbic acid might be an effective strategy to protect membranes from oxidative damage in water stressed condition. from perusal of the results obtained for spad value, soluble protein, sps activity, fruit characters, lycopene, ascorbic acid, tss and yield, it can be inferred that genotypes le 114, le 57, le 118, and le 27 performed better under drought conditions and can be categorized as drought tolerant genotypes compared to genotypes le 1 and le 125, drought susceptible ones. however, further studies are required to confirm the results by molecular evidence. the tolerant genotypes could be utilized for further breeding programmes to evolve new tomato genotypes for better drought tolerance with higher yield. acknowledgements the authors wish to express their sincere thanks to the professor and head, department of vegetable science, for providing seed materials for this research and all the faculty members of the department of crop physiology, tamil nadu agricultural university, coimbatore for providing valuable input for the research work. adv. hort. sci., 2016 30(1): 3-11 10 references ali a.k., delbert w.h., william o.p., 1980 evaluating leaf water potential, stomatal resistance and canopy surface temperature of tomatoes as indices for irrigation timing. acta horticulturae, 100: 181-192. aloni b., karni l., zaidman z., schaffer a.a., 1996 changes of carbohydrates in pepper (capsicum annuum l.) owers in relation to their abscission under different shading regimes. ann. bot., 78: 163-168. ashwell g., 1957 colorimetric analysis of sugars, pp. 73105. in: colowick s.p., and n.o. kaplan (eds.) methods in enzymology, iii. academic press, new york, usa. bartholomew d.m., bartley g.e., scolnik p.a., 1991 abscisic-acid control of rbcs and cab transcript ion in tomato leaves. plant physiol., 96: 291-296. bhatt r.m., rao n.k.s., upreti k.k., shobha h.s., 2009 floral abscission and changes in sucrose phosphate synthase and 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environ., 14(9): 939-946. impaginato 199 adv. hort. sci., 2017 31(3): 199-203 doi: 10.13128/ahs-20585 aggressiveness of four fusarium head blight species on wheat cultivars n. sakr department of agriculture, atomic energy commission of syria, p.o. box 6091, damascus, syria. key words: disease development, diseased-head severity, fusarium species, soft dough stage, syrian wheat cultivars. abstract: aggressiveness of four fusarium head blight species (f. culmorum, f. solani, f. verticillioides and f. equiesti) was studied on six syrian wheat cultivars under controlled conditions. two aggressiveness criteria: diseased-head severity (dhs, fusarium infection) and disease development (dd, fusarium spread) were visually estimated as percentage of heads showing fusarium symptoms in wheat cultivars at the soft dough stage. results showed significant differences among fungal isolates and wheat cultivars for the two tested criteria. the mean values of dhs evaluations ranged from 33.27 to 45.49% among fungal isolates, and from 29.62 to 42.22% among tested cultivars. the mean dd rating varied from 25.58 to 35.43% among fungal isolates, and from 25.33 to 34.01% among tested cultivars. results in the current research highlighted that the level of resistance in syrian cultivars to fusarium species is characterized with low to moderate dhs and dd evaluations (%). also, the results were comparable with those previously obtained using the same fungal isolates and wheat cultivars in vitro. the current study confirmed the suitability of in vitro method to be used as fast and reliable test to analyze aggressiveness in fusarium species. 1. introduction fusarium head blight is one of the most destructive global diseases of wheat. in infected plants, it leads to kill the developing seed (prematurely bleached spikes) within moist conditions and moderate temperatures prevail during flowering. since it was identified in 1884, severe epidemic outbreaks caused quantitative losses in yield of up to 50-75% (parry et al., 1995; mcmullen et al., 2012). it also reduces grain quality due to contamination of harvest with large amount of mycotoxins that cause toxicities to human and livestock (maresca, 2013). at least seventeen fusarium species with several habitats and types of mycotoxins produced have been associated with fusarium species (parry et al., 1995). fusarium graminearum is the main causal agent of this disease and has been subdivided into at least 11 cryptic species (o’donnell et al., 2004). other species can cause fusarium disease on a lesser scale such as f. avenaceum, f. culmorum, f. solani, f. equiseti, f. verticillioides and f. poae (*) corresponding author: ascientific@aec.org.sy citation: sakr n., 2017 aggressiveness of four fusarium head blight species on wheat cultivars. adv. hort. sci., 31(3): 199-203. copyright: © 2017 sakr n. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 24 april 2017 accepted for publication 6 june 2017 ahs advances in horticultural science adv. hort. sci., 2017 31(3): 199-203 200 (xu et al., 2008). understanding the interaction between wheat plants and fusarium populations requires more detailed knowledge about the variation of aggressiveness (wu et al., 2005). van der plank (1968) defined aggressiveness as a quantitative ability of an isolate to cause disease on a susceptible host plant in a non-race-specific pathosystem. aggressiveness is an important factor determining the potential ability of fusarium isolates to cause fusarium epidemics. variability of quantitative component of pathogenicity in f. graminearum has been the subject of several studies (parry et al., 1995; leonard and bushnell, 2003; wu et al., 2005; mcmullen et al., 2012). however, other fusarium species have attracted less pathogenic analyses (xu et al., 2008; bakri et al., 2012; sakr, 2017). fusarium resistance in wheat plants is conferred by quantitative trait loci (qtl) detected on all chromosomes (loffler et al., 2009). it is necessary to combine type i (resistance to initial infection) and type ii (resistance to spreading) to get fusarium resistant wheat plants (loffler et al., 2009). in syria, about 1.7 million hectares were sown to wheat, with an annual production of 3.9 million tons in 2011. host-pathogen interactions were evaluated for several local wheat cultivars inoculated with fungal isolates of different species associated with fusarium species, and differential reactions on cultivars were detected (alazem, 2007; talas et al., 2011; bakri et al., 2012). recently, sakr (2017) analyzed aggressiveness of four fusarium species in vitro, and significant differences were detected between pathogen isolates and wheat genotype. in order to underline pathogenic variation for fusarium species collected from ghab plain, one of the principal syrian wheat production areas, the objectives of the current study were to (1) evaluate aggressiveness of four isolates [f2 (f. culmorum), f27 (f. verticillioides), f35 (f. solani), and f43 (f. equiesti)] on six wheat syrian cultivars under controlled conditions, and (2) compare results previously obtained by sakr (2017) in vitro with the current data from floret inoculation under controlled conditions. 2. materials and methods fungal isolates and inoculum production the fungal isolates of four fusarium species [f. culmorum (f2), f. verticillioides (f27), f. solani (f35), and f. equiesti (f43)] were collected in 2015 from naturally wheat spikes exhibiting fusarium symptoms from different locations of ghab plain in 2015. isolates were identified morphologically according to nelson et al., (1983). the cultures were maintained in sterile distilled water at 4°c and freezing at -16°c until needed. for inoculum preparation, four to six agar plugs out of the stored isolates were put over the surface of pda in 9-cm petri dishes and incubated for 10 days, at 22°c in the dark to allow mycelial growth and sporulation. ten ml of sterile distilled water were added to each dish, and the resulting spore suspensions were adjusted to 5 × 104 spores/ml for inoculation following a count in a hemacytometer (bakri et al., 2012). wheat cultivars and growth chamber conditions in the current study, aggressiveness for the four fusarium head blight isolates was measured on six wheat cultivars previously analyzed in vitro (‘cham1’, ‘cham7’, ‘acsad65’, ‘cham4’, ‘cham6’ and ‘douma4’, most cultivated in different syrian areas) under controlled conditions. wheat seeds were surface-sterilized with 5% sodium hypochlorite solution for 8 min and then washed six times in sterile distilled water (purahong et al., 2012). they were sown into plastic pots (15-cm) filled with 2 kg of sterilized soil (ten seeds per pot), and arranged in a complete randomized design with three replicates. three plots per replicate were left non-inoculated as control treatment. pots were placed in a growth chamber operated at 20°c during day and night with an 16-h photoperiod. following emergence, plants were thinned to three per pot and nitrogen fertilizer was applied twice at two dates: emergence and tillering. aggressiveness tests at 10-14 days after heading, spore suspensions of the four fusarium head blight isolates or sterile distilled water (control) were sprayed one time into flowering spikes. six flowering spikes were randomly selected within each replicate of the six cultivars. after the inoculum dried for 30 min, inoculated spikes were then kept covered for 48 h using polythene bags to ensure 100% rh. head blight symptoms were evaluated as percentage of spikes showing fusarium symptoms after 7, 14, and 21 days, when plants were at the soft dough stage. fusarium disease severity was visually estimated in situ for each inoculated spike using the xue’s et al., (2004) scale. this scale includes nine levels of incidence expressed in percentage of bleached spike area on a head: 0 (no visible fusarium symptoms) to sakr aggressiveness of four fusarium head blight species on wheat cultivars 201 9 (severely diseased, spike dead). each head was assessed separately in all experiments. the values of diseased-head severity as percentage of infected spikes measured 21 days after inoculation (dai) were considered, for each cultivar, a parameter to determine initial infection. the values of disease development calculated by the means of each evaluation; 7, 14 and 21 dai over the estimation time were considered, for each cultivar, a parameter to determine pathogen spreading. statistical analyses statistical analyses of aggressiveness data were performed using statview, 4.57® abacus concepts, berkley, canada. before statistical analysis, the percentages were transformed using the arcsines function. a complete randomized design with two factors (fusarium isolate and wheat genotype) and 3 replications was used for aggressiveness analysis. fisher’s lsd test was used to compare the means at p <0.05. 3. results and discussion understanding the interaction between fusarium head blight species and wheat plants requires knowledge of the variation of quantitative component of pathogenicity (wu et al., 2005). with this in mind, aggressiveness variability for four local fusarium species was analyzed by using a floret inoculation in a growth chamber on six wheat cultivars most cultivated in different syrian areas. differences in aggressiveness of four fusarium species (f. culmorum, f. verticillioides, f. solani, and f. equiesti) are indicated when isolates vary in the amount of damage that they cause in wheat plants. the results demonstrated that none of the six tested cultivars was immune from disease. however, typical fusarium symptoms induced by the four isolates (f2, f27, f35 and f43) were clear and easy to score in the inoculated spikes, while no symptoms were present in the control (fig. 1). the mean values of diseasedhead severity (dhs) ranged from 33.27% to 45.49% as compared with 0% for the control treatment (table 1). there were significant differences among four isolates (f isolates=4.376; probability=0.0084). the mean values of disease development (dd) varied from 25.58% to 35.43% (table 1). significant differe n c e s a m o n g f o u r i s o l a t e s ( f i s o l a t e s = 4 . 2 5 7 ; probability=0.0096) were detected. results in the current study showed that there was no interaction between fusarium isolates and host plant for the two parameters. this indicates to non-race-specific interaction described for this pathosystem (loffler et al., 2009). high diseased-head severity and disease development values represent high aggressiveness (parry et al., 1995; wu et al., 2005; xu et al., 2008). results shown in table 1 indicate that the isolate f35 of f. solani was the most aggressive with a mean value of dhs of 45.49% and dd of 35.43%; followed by other tested isolates. these results are in accordance with previous analysis on the aggressiveness of theses isolates in vitro; sakr (2017) observed that the isolate f35 was the most aggressive one, followed by other analyzed isolates. the current study confirmed the suitability of in vitro modified petri-dish method to be used as fast and reliable test to analyze aggressiveness in fusarium species. results in the current study are comparable with those found by alazem (2007) and bakri et al., (2012) for f. culmorum, f. solani, f. verticillioides and f. equiesti in which significant differences were detected for aggressiveness among fungal isolates in each fusarium head blight species in a growth chamber. the mean value of dhs and dd rating for six fig. 1 fusarium head blight symptoms on spike of syrian wheat cultivar cham4 inoculated with isolate f35 (fusarium solani) compare with control (water). adv. hort. sci., 2017 31(3): 199-203 202 wheat cultivars (table 1) reflects the ability of the same isolate of the pathogen (f2, f27, f35 and f43) t o d i s t i n g u i s h d i f f e r e n t l e v e l s o f r e s i s t a n c e a s observed for the same pathosystem (alazem, 2007; talas et al., 2011). also, the resistance of a given wheat cultivar is not related to a certain fusarium species (table 1). significant differences were underlined for dhs (f cultivars=2.907; probability=0.0226) and dd (f cultivars=1.870; probability=0.1171) criteria among wheat cultivars (table 1). the mean values of dhs evaluations ranged from 29.62 to 42.22% among tested cultivars. the mean dd rating varied f r o m 2 5 . 3 3 t o 3 4 . 0 1 % a m o n g t e s t e d c u l t i v a r s . quantitative resistant wheat cultivars are identified by low dhs and dd values of the fungus compared with the susceptible one (parry et al., 1995). results in the current research highlighted that the level of resistance in syrian cultivars to fusarium species is characterized with low to moderate dhs and dd evaluations (%). these results are in accordance with previous analysis on the comportment of local wheat cultivars in which differential reactions on cultivars were detected (alazem, 2007; bakri et al., 2012; talas et al., 2011). however, fusarium resistance scores ranged one fold and half between resistant and susceptible cultivars for the two tested parameters (table 1). thus our observation suggests that in resist a n t w h e a t c u l t i v a r s , t h e d e v e l o p m e n t o f t h e pathogen was slowed, and may be due to resistance mechanisms expressed by accumulation of qtl in host cultivars (alazem, 2007; talas et al., 2011). results in the current study showed that the level of quantitative resistance in the six wheat cultivars made it possible to detect significant differences between isolates of four fusarium species. these results are in accordance with our previous analysis on the behavior of theses cultivars in vitro (sakr, 2017). the variability of resistance for the syrian cultivars is interesting and promising for ecological framing/breeding and also for improving resistance of wheat cultivars. for f. graminearum, purahong et al., (2012) validated the modified petri-dish method (used by sakr, 2017) by highly significant correlation with the data from floret inoculation in adult plants in a growth chamber. results indicted that the petri-dish aggressiveness test conducted on other fusarium species (f. culmorum, f. verticillioides, f. solani, and f. equiesti) is repeatable and stable with the six wheat cultivars in a growth chamber (table 1). it will be necessary to analyze the pathogenic variation in a large number of fusarium isolates on several wheat cultivars under controlled and field conditions to screen fusarium resistance in syrian wheat cultivars. acknowledgements the author would like to thank director general of aecs, and the head of the agriculture department fusarium head blight incidence scores were evaluated as percentage of spikes showing fusarium species symptoms using the xue’s et al., (2004) scale. f tests (p<0.05), ns= not significant. table 1 diseased-head severity and disease development scores in % among isolates of four fusarium head blight species measured on six syrian wheat cultivars isolate ‘cham1’ cham7' acsad65' cham4' cham6' douma4' mean diseased-head severity scores (%) f2 25.92 25.92 29.62 37.03 44.44 44.44 35.24 f27 29.62 29.62 37.03 33.33 37.04 36.66 33.27 f35 33.33 33.33 40.74 58.88 47.77 47.77 45.49 f43 29.62 29.62 44.44 40.00 37.04 40 37.40 mean 29.62 29.62 37.96 42.31 41.57 42.22 f isolates=4.376; probability=0.0084 f cultivars=2.907; probability=0.0226 f interactions=0.601 ns; probability=0.8594 diseased development scores (%) f2 23.56 24.69 21.16 30.86 40.4 29.62 28.38 f27 24.69 23.56 28.49 25.64 24.69 26.45 25.58 f35 30.30 31.74 29.10 45.58 32.09 43.77 35.43 f43 22.79 25.64 37.03 33.95 26.45 29.10 29.16 mean 25.33 26.41 28.94 34.01 30.91 32.23 f isolates=4.257; probability=0.0096 f cultivars=1.870; probability=0.1171 f interactions=1.199 ns; probability=0.3051 sakr aggressiveness of four fusarium head blight species on wheat cultivars 203 for their support. references a l a z e m m . , 2 0 0 7 e v a l u a t i n g g e n e t i c v a r i a t i o n o f fusarium head blight by molecular markers. master, faculty of agriculture, university of damascus, syria, pp. 72. bakri y., jawhar m., arabi m.i.e., 2012 correlative analysis of fusarium species pathogenicity and in vitro xylanase activity. j. plant biol. res., 1(2): 86-92. leonard k.j., bushnell w.r., 2003 fusarium head blight of wheat and barley. vol. i. the american phytopathological society, minnesota, usa, pp. 530. loffler m., schon c.c., miedaner t., 2009 revealing the genetic architecture of fhb resistance in hexaploid wheat (triticum aestivum l.) by qtl meta-analysis. mol. breed., 23(3): 473-488. maresca m., 2013 from the gut to the brain: journey and pathophysiological effects of the food-associated trichothecene mycotoxin deoxynivalenol. toxins, 5(4): 784-820. mcmullen m., bergstrom g., de wolf e., dill-macky r., hershman d., shaner g., van sanford d., 2012 a unified effort to fight an enemy of wheat and barley: fusarium head blight. plant dis. 96(12): 17121728. nelson p.e., toussoun t.a., marasas w.f.o., 1983 f u s a r i u m s p e c i e s : a n i l l u s t r a t e d m a n u a l f o r identification. the pennsylvania state univ. press, university park, usa, pp. 226. o’donnell k., ward t.j., geiser d.m., kistler h.c., aoki t., 2004 genealogical concordance between the mating type locus and seven other nuclear genes supports formal recognition of nine phylogenetically distinct species within the fusarium graminearum clade. fungal genet. biol., 41(6): 600-623. parry d.w., jekinson p., mcleod l., 1995 fusarium ear blight (scab) in small grain cereals-a review. plant pathol., 44(2): 207-238. purahong w., alkadri d., nipoti p., pisi a., lemmens m., prodi a., 2012 validation of a modified petri-dish test to quantify aggressiveness of fusarium graminearum in durum wheat. eur. j. plant pathol., 132(3): 381-391. sakr n., 2017 in vitro assessment of fusarium head blight spp. on wheat cultivars. arch. phytopathol. plant protect., 50(5-6): 254-261. talas f., longin f., miedaner t., 2011 sources of resistance to fusarium head blight within syrian durum wheat landraces. plant breed., 130(3): 398-400. van der plank j.e., 1968 disease resistance in plants. vol. i. academic press, new york and london, pp. 206. wu a.b., li h.p., zhao c.s., liao y.c., 2005 comparative pathogenicity of fusarium graminearum isolates from china revealed by wheat coleoptile and floret inoculations. mycopathologia, 160(1): 75-83. xu x.m., parry d.w., nicholson p., thomsett m.a., simpson d., edwards s.g., cooke b., mdoohan f.m., monaghan s., moretti a., tocco g., mule g., hornok l., béki e., tantnell j., ritieni a., 2008 w i t h i n f i e l d v a r i a b i l i t y o f f u s a r i u m h e a d b l i g h t pathogens and their associated mycotoxins. eur. j. plant pathol., 120(1): 21-34. xue a.g., armstrong k.c., voldeng h.d., fedak g., babcock c., 2004 comparative aggressiveness of isolates of fusarium species causing head blight on wheat in canada. can. j. plant pathol., 26: 81-88. impaginato 25 1. introduction the increasing demand for basil resulted in cropping area extension by 66% since 2001 in italy (www.istat.it), with the frequent use of local ecotypes (zecchinelli, 1999; tesi and lenzi, 2002). basil (ocimum basilicum l.) is a high marketable value vegetable, which is consumed both as a fresh aromatic ingredient and combined with pasta in a cooked dish (pesto). as today’s consumer choices are oriented towards high quality produce, not only from a sensorial point of view but also in terms of nutritional properties, a particular emphasis is given to this product. in this direction, soilless growing could represent an effective crop management in order to enhance product quality attributes (sgherri et al., 2010), which mainly depend on variety (tesi et al., 1991) but they are also affected by crop system (tesi et al., 1997). plant growing in pots sown with several seeds per pot, to be sold when the plants set reaches a scheduled size, is one of the current farm strategies and interesting market perspectives mainly arise from the winter crop cycle. in this season, light intensity is sufficient for carrying out efficient crop cycles (beaman et al., 2009), but it is necessary ensuring the adequate minimum temperature, which is also positively correlated with basil flavour (chang et al., 2007). moreover, the nutritive solution supplied to plants plays a crucial role as it significantly affects yield (bekhradi et al., 2015) and plant features, such as stem height and dry weight (adler et al., 1989; bione et al., 2014); in this respect, basil is considered a moderately tolerant species (herrera, 2005). the plants density is also of primary importance for the adv. hort. sci., 2017 31(1): 25-30 doi: 10.13128/ahs-20722 effects of nutritive solution electrical conductivity and plant density on growth, yield and quality of sweet basil grown in gullies by subirrigation morano g. 1, amalfitano c. 1, sellitto m. 2, cuciniello a. 1, maiello r. 1, caruso g. 1 (*) 1 dipartimento di scienze agrarie, università degli studi di napoli federico ii, via università, 100, 80055 portici (na), italy. 2 microspore s.p.a., 86035 larino, cb, italy. key words: leaves quality, nutrient uptake, ocimum basilicum l., plants number per pot, production, soilless. abstract: the increasing demand for basil in the last decade has arisen from consumer tendency towards high nourishing produce. soilless growing of this crop is a current farm strategy and the quality targets are affected by nutritive solution as well as by plants density per pot. research was carried out with the aim of assessing plant growth, yield and leaves quality of basil (ocimum basilicum l., cv. gecom ft) grown in pots (peat-lapil) and fed by subirrigation inside plastic gullies, under a heated greenhouse. comparisons were made of four electrical conductivities (ec: 2.2, 2.5, 2.8, 3.1 ms·cm-1) in factorial combination with four plant densities (9, 12, 15, 18 plants per pot) and a split plot design was arranged with three replicates. the 2.8 ms.cm-1 ec resulted in the best yield, growth indexes and biometrical parameters values. water absorption was highest under the 2.8 ms.cm-1 ec, whereas the highest nutrient consumptions as well as the best quality indicators and chemical composition corresponded to the 2.8 to 3.1 ms.cm-1 ec range. the 12 plants per pot density gave the best results, in terms of yield, growth indexes and biometrical parameters, also showing the highest plant water and nutrient uptakes. the leaves quality attributes and chemical composition always displayed decreasing trends as a function of the plant density increase, the highest values corresponding to 9 and 12 plants per pot; only the nitrates concentration showed an opposite trend compared to the other nutrients. in conclusion, the 2.8 ms.cm-1 nutritive solution and the 12 plants per pot density resulted in the best yield and leaves quality. further enhancement of both experimental factors level even caused the reduction of water and nutrient efficiency use. (*) corresponding author: gcaruso@unina.it received for publication 11 september 2016 accepted for publication 23 december 2016 copyright: © 2017 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2017 31(1): 25-30 26 produce quality (bohme and pinker, 2014), as the individual space available has a major impact on the balance among the different plant parts. due to literature shortage on the above mentioned topics, research was carried out on soilless pot-grown basil in naples (southern italy), with the aim of evaluating the effects of nutritive solution electrical conductivity and pot plants density on crop growth, yield and leaves quality. 2. materials and methods r e s e a r c h w a s c a r r i e d o u t o n b a s i l ( o c i m u m basilicum l. cv. gecom ft) at the experimental site of naples university federico ii in portici (naples, southe r n i t a l y , 4 0 ° 4 9 ’ n , 1 4 ° 2 0 ’ e , 6 3 m a . s . l . ) , i n a mediterranean or csa climate (peel et al., 2007), during winter season in 2007 and 2008. the crops were soilless grown in pots, placed in gullies and fed by sub-irrigation, under plastic (ir-pe) tunnels equipped with an air heating system set to 16°c. comparisons were made of four electrical conductivities (ec: 2.2, 2.5, 2.8, 3.1 ms·cm-1) in factorial combination with four plant densities (9, 12, 15, 18 plants per pot) and a split plot design was arranged with three replications; each treatment included 30 pots. the hydroponic equipment consisted of: a) 48 rigid pvc gullies (each 12 cm wide and deep, 300 cm long) supported by plastic elements at 70 cm above ground level, according to 1% slope; b) 12 plastic tanks holding 220 l; c) 12 submerged pumps of 90 watt unit power; d) 24 delivery and return overhead lines. the sowing was performed on 15 january in pots (ø = 10 cm) filled with peat and lapil (1:1 in volume), placed in the gullies through a pierced white pe film and spaced 10 cm along and between the rows (18 pots per m2). they were fed by nutritive solutions (table 1), with 1-6 interventions per day of 5 minutes each, which were never adjusted but they were completely changed at the end of each weekly cycle. besides, during the crop, an insecticide application against aphids was practiced. the crop cycles ended when each plants set reached the scheduled size for pot commercialization, i.e. the plants had four fully expanded leaves couples, and at that time the following determinations were made on plant samples obtained by 8 pots per plot: fresh and dry weight of whole plants and of leaves; leaf area; stems thickness and height. moreover, water consumption was calculated assessing the volume of nutritive solution in each tank at the beginning and at the end of each weekly cycle. concurrently, nutritive solution samples were collected in order to assess nutrient consumption through laboratory analyses of nitrogen, phosphorus, potassium, calcium, magnesium and iron, using the same methods as described below for leaf cation and anion determinations. at the crop cycles end, leaves samples were also collected from 8 pots per plot, in order to perform laboratory analyses. in this respect, two hundred grams of leaves per plot were homogenized in a 1.0 l waring blender (waring laboratory, torrington, ct, usa) and aliquots of this raw homogenate were used for the analyses of cations. the raw homogenate was centrifuged at 10,000 x g for 30 min at 6°c in an 5810r eppendorf refrigerated centrifuge (eppendorf hq, hamburg, germany). the resulting supernatant w a s p a s s e d t h r o u g h a 0 . 4 5 µ m a c r o d i s c f i l t e r (gelman sciences, mi, usa). samples of this filtered leaves extract were used for assessing anion, sugar and ascorbic acid contents. the laboratory determinations were performed as follows: the dry residue was assessed in an oven at 70°c with a vacuum; the soluble solids content or ssc (in °brix) was measured at 20°c with a bellingham & stanley, model rfm 81 digital refractometer on the supernatant obtained from raw homogenate centrifugation; anions, sugars and ascorbic acid were determined by high performance liquid chromatography (hplc) as previously described (caruso et al., 2011); table 1 chemical composition of the soilless nutrient solutions for all treatments ph was adjusted to 6.0 and nh4/no3 ratio was 1/9. nutritive solution ec (ms·cm-1) macronutrients (mmol·l-1) micronutrients (m mol·l-1) n p k ca mg s cl fe cu mn zn b mo 2.2 12.3 1.6 5.3 3.8 2.2 1.9 1 35 1 15 5 35 1 2.5 13.8 1.7 5.9 4.3 2.5 2.5 1 35 1 15 5 35 1 2.8 15.7 2.0 6.7 4.8 2.8 2.8 1 35 1 15 5 35 1 3.1 17.5 2.2 7.6 5.4 3.0 3.3 1 35 1 15 5 35 1 morano et al. effects of nutritive solution electrical conductivity on sweet basil grown in subirrigated gullies 27 titratable acidity of the leaves homogenate was determined as previously described (caruso et al., 2014) and it was expressed as grams of anhydrous citric acid per 100 g of leaf fresh weight; c a t i o n s ( c a , m g , k ) c o n t e n t i n t h e l e a v e s homogenate was determined by atomic adsorption spectrophotometry as previously described (caruso et al., 2011). data statistical processing was performed by analysis of variance using the spss software version 21, referring to 0.05 probability level, and duncan multiple test was used for mean separation. 3. results and discussion from the data statistical processing, the year of research resulted to have no significant effect either as a main effect or as an interaction with the two experimental factors, therefore in the following tables the mean values of the experimental data of the years 2007 and 2008 are reported. as for yield results relevant to the comparison among the nutritive solution electrical conductivities (table 2), the 2.8 ms.cm-1 ec resulted in the highest basil yield, both as whole plants and of leaves, and in the shortest crop cycle; the lowest nutrient solution strength (2.2 ms.cm-1) showed the worst performances. among the plant densities, the 12 plants per pot treatment resulted in the highest yields and both the 9 and 12 plants per pot led to the shortest crop cycle (table 2). in terms of growth indexes and biometrical parameters (table 3), the 2.8 ms.cm-1 ec also produced the highest values of plant dry matter, leaf area and stem thickness, though the latter was not statistically different from that obtained with the highest ec level (3.1 ms.cm-1); conversely, nutritive solution dilution caused the internodes extension and, indeed, the two lowest electrical conductivities enhanced plant height. with regard to plant density (table 3), the 12 plants per pot treatment resulted in the highest dry weight and lai, whereas the lowest density produced the thickest stems; the 15 and 18 plants per pot densities proved excessive and, in particular, the highest one caused the most unbalanced growth of plants. in fact, the plants grown under the 18 plants per pot treatment showed thinner stems and smaller leaves compared to the other experimental treatments, and they were also taller than the most spaced ones. in contrast with our findings, in previous research (tesi et al., 1995) the 1.6 ms·cm-1 ec showed the best effect on basil yield. moreover, other authors reported that doubling the nutrient availability did not affect basil yield (raimondi et al., 2006), but it led to the increase of leaf dry matter percentage and lai (chen et al., 2004). in our research, the depressing effects of salt stress caused by the 3.1 ms.cm-1 ec on plant vegetative growth and in particular on leaf area corresponds to the rapid plant adaptation to water deficit (munns, 2002). moreover, in our investigation, the density increase presumably caused the light conditions worsening within the canopy and, accordingly, the reduction of plant photosynthetic efficiency. chang et al. (2007) also reported that basil plant weight is adversely correlated with canopy shading. consistently, tesi et al. (1995) recorded the plant weight increase per soil unit area up to a critical density value, over which a decrease occurred; however, they also found that a doubled density, compared to our best treatment of 12 plants per pot provided with the best results using 10 cm diameter pots. moreover, raimondi et al. (2006) found that plant density increase from 66 to 100 plants per m2 results in total yield increase. further, in studies on canopy dynamics simulation (van oosteron et al., 2001), leaf table 2 basil yield results table 3 basil growth and biometrical parameters nutritive solution ec (ms·cm-1) yield crop cycle duration (days) whole plants (g·m-2) leaves (g·m-2) leaves/ plant (%) 2.2 579.1 d 418.7 d 72.3 c 64.0 a 2.5 733.9 c 538.7 c 73.4 b 62.7 b 2.8 958.9 a 713.4 a 74.4 a 61.3 c 3.1 845.5 b 629.9 b 74.5 a 61.0 c no. plants per pot 9 761.9 b 570.7 b 74.9 a 61.2 c 12 931.1 a 695.5 a 74.7 a 61.4 c 15 776.3 b 569.8 b 73.4 a 62.6 b 18 648.5 c 465.6 c 71.8 c 63.9 a nutritive solution ec (ms·cm-1) plant dry matter (g·m-2) lai (m2·m-2) leaf area (cm2·pt-1) plant height (cm) stem thickness (mm) 2.2 45.0 d 0.66 c 28.5 c 17.2 a 2.76 b 2.5 64.8 c 0.92 b 39.3 b 17.0 a 2.90 b 2.8 90.0 a 1.11 a 47.1 a 16.7 b 3.11 a 3.1 82.8 b 0.98 b 41.6 b 16.6 b 3.16 a no. plants per pot 9 73.6 b 0.86 c 51.9 a 16.8 b 3.33 a 12 88.2 a 1.06 a 47.6 b 16.9 ab 3.12 b 15 67.9 b 0.93 b 33.1 c 16.9 ab 2.85 c 18 52.7 c 0.83 c 24.3 d 17.1 a 2.60 d adv. hort. sci., 2017 31(1): 25-30 28 area index showed increasing trend with the plant density enhancement. the highest water and nutrient absorptions were recorded in the last crops week, when plant leaf area reached the highest expansion and the greenhouse temperature showed the highest value of 26.4°c (as an average of the two research years). as reported in table 4, plant water consumption showed a similar trend to the yield one, with the highest values corresponding to 2.8 ms.cm-1 ec, whereas the highest nutrients consumption was assessed under the 2.83.1 ms.cm-1 ec range; the most diluted nutritive solution always resulted in the lowest absorption rates. as for the comparison among the plant densities (table 4), the highest daily values of both water and nutrient absorption occurred in the 12 plants per pot treatment, which also resulted in the best yield (table 2); the highest plant density (18 plants per pot) always showed the lowest consumptions. compared to our research findings, a similar plant response to water deficit was recorded in previous investigations (savvas et al., 2007), where the increase of the nutrient solution strength caused the reduction of plant water absorption. the latter represents a salinity adaptation mechanism, consisting of leaf area and stomata decrease which in turn contributes to reducing transpiration and increasing water use efficiency (chartzoulakis and klapaki, 2000). the quality indicators were significantly affected by the nutritive solution strength (table 5), as their trends were always increasing with the electrical conductivity raise from 2.2 to 2.8 ms.cm-1, whereas no further increases were recorded in the last 0.3 ms.cm-1 rise. the quality parameters showed decreasing trends as a function of the plant density increase (table 5), with the 9 and 12 plants per pot treatments generally attaining the highest values and the 18 plants per pot treatment displaying the worst performances. in previous investigation (adams and ho, 1989), an increase in sugar content and titratable acidity was reported as a consequence of salinity increase or water deficit. moreover, raimondi et al. (2006) found that nutrient solution ec interacted with the cultivars in modifying leaf antioxidant content: i.e. napoletano leaves showed an ascorbate increase with the ec enhancement, whereas genovese displayed opposite trend. the same authors also recorded that the plant density increase from 66 to 100 plants per m2 did not table 4 basil water and nutrient absorptions nutritive solution ec (ms·cm-1) maximum daily absorptions water (l·m-2) nitrogen (g·m-2) phosphorus (g·m-2) potassium (g·m-2) calcium (g·m-2) magnesium (g·m-2) iron (mg·m-2) 2.2 1.6 d 0.35 c 0.11 c 0.42 c 0.29 c 0.11 c 5.76 c 2.5 2.0 c 0.51 b 0.15 b 0.61 b 0.42 b 0.16 b 7.20 b 2.8 2.6 a 0.76 a 0.21 a 0.92 a 0.64 a 0.23 a 9.00 a 3.1 2.4 b 0.78 a 0.22 a 0.93 a 0.65 a 0.24 a 8.46 a no. plants per pot 9 2.2 b 0.62 b 0.19 b 0.74 b 0.53 b 0.19 b 7.56 b 12 2.6 a 0.73 a 0.21 a 0.90 a 0.58 a 0.23 a 9.00 a 15 2.1 b 0.58 b 0.17 c 0.70 b 0.49 c 0.17 c 7.38 b 18 1.7 c 0.47 c 0.13 d 0.54 c 0.40 d 0.15 d 5.94 c table 5 basil leaves quality indicators nutritive solution ec (ms·cm-1) dry residue (%) soluble solids (°brix) titratable acidity (g · 100 g-1 d.w.) glucose (g · 100 g-1 d.w.) fructose (g · 100 g-1 d.w.) sucrose (g · 100 g-1 d.w.) ascorbic acid (mg·100 g-1 d.w.) 2.2 9.5 c 3.2 c 0.76 c 1.80 c 2.25 c 0.40 c 508.4 c 2.5 9.7 bc 3.4 bc 0.84 b 2.12 b 2.52 b 0.52 b 585.7 b 2.8 10.0 ab 3.6 ab 0.96 a 2.34 a 2.83 a 0.63 a 703.8 a 3.1 10.0 a 3.7 a 1.02 a 2.47 a 2.98 a 0.67 a 744.6 a no. plants per pot 9 10.0 a 3.6 a 0.98 a 2.30 a 2.86 a 0.64 a 708 a 12 10.0 a 3.6 a 0.96 a 2.24 a 2.78 a 0.62 a 689.5 a 15 9.8 ab 3.4 ab 0.88 b 2.14 ab 2.54 b 0.52 b 617.3 b 18 9.6 b 3.3 b 0.78 c 2.02 b 2.40 b 0.45 c 527.4 c morano et al. effects of nutritive solution electrical conductivity on sweet basil grown in subirrigated gullies 29 affect fresh produce quality, but it just lowered the soluble solids content. as reported in table 6, mineral nutrient concentrations were significantly affected by the nutritive solution strength, as their trends were always increasing with the electrical conductivity raise from 2.2 to 2.8 ms.cm-1, whereas no further increases were recorded in the last 0.3 ms.cm-1 rise. with regard to plant density (table 6), the 9 and 12 plants per pot treatments always resulted in the highest nutrient accumulation in the leaves, except for the nitrates which showed an opposite trend, increasing from the lowest to the highest density. the decreasing trend of the leaves mineral ion concentrations as a function of the pot plant density increase resulted in relation with the plant nutrient absorptions (table 4). notably, the increase of mineral cations concentration in the plant tissues is caused by salt ion accumulation in the rizhosphere (sonneveld, 2002) as a c o n s e q u e n c e o f t h e p l a n t a c t i v e e x c l u s i o n i n response to salt occurrence in the external solution (bethke and drew, 1992). moreover, the nitrate concentration increase in response to nutritive solution strength raise recorded in our research is consistent with the reports of previous investigations (tesi et al., 1997; raimondi et al., 2006). as for plant density, the increasing nitrate accumulation corresponding to the enhancement of the plants number per pot was presumably caused by the gradual light conditions worsening. contrastingly, in previous research (tesi et al., 1995) a decreasing trend of nitrate accumulation as a function of plant density increase was reported. interestingly, in our research the nitrate concentration was always very low and, accordingly, basil leaves consumption not exceeding 563 g per day complies with the acceptable daily intake for nitrate (222 mg·d-1 for 60 kg adult) (authority efs, 2008). 4. conclusions from research carried out in southern italy on soilless pot-grown basil, it can be inferred that the 2.8 ms.cm-1 nutritive solution resulted in the best product yield and quality, whereas a further increase to 3.1 ms.cm-1 caused the reduction of the water and nutrient efficiency use. moreover, the 12 plants per p o t d e n s i t y s h o w e d t h e o p t i m a l c o m p r o m i s e between the individual plants and the pot plants set performances, providing with the highest production and leaves quality. indeed, density intensification to 15 and further to 18 plants per pot caused the reduced efficiency use of water and nutrients and accordingly the plant growth worsening, as well as the crop cycle extension up to 2.7 days. acknowledgements the authors wish to thank mr. rosario nocerino and mr. luigi sannino for their valuable assistance with the greenhouse equipments and farming practices. references adams p., ho l.c., 1989 effects of constant and fluctuating salinity on the yield, quality and calcium status of tomatoes. j. hortic. sci., 64: 725-732. adler p.r., simon j.e., wilcox g.e., 1989 nitrogen form alters basil growth and essential oil content and composition. hortscience, 24: 789-790. authority efs, 2008 nitrate in vegetables: scientific opinion of the panel on contaminants in the food chain. the efsa journal, 689: 1-79. beaman a.r., gladon r.j., schrader j.a., 2009 sweet basil requires an irradiance of 500 µmol·m-2·s-1 for greatest edible biomass production. hortscience, 44: 64-67. bekhradi f., delshad m., marin a., luna m.c., garritable 6 basil leaves chemical composition nutritive solution ec (ms·cm-1) nitrates (g·kg-1 d.w.) phosphates (g·kg-1 d.w.) sulphates (g·kg-1 d.w.) calcium (g·kg-1 d.w.) magnesium (g·kg-1 d.w.) potassium (g·kg-1 d.w.) 2.2 3.2 c 3.6 c 1.5 c 5.9 c 3.3 c 45.3 c 2.5 3.7 b 4.1 b 1.8 b 6.4 b 3.8 b 48.8 b 2.8 4.4 a 5.0 a 2.2 a 7.1 a 4.3 a 53.4 a 3.1 4.8 a 5.3 a 2.3 a 7.3 a 4.4 a 55.0 a no. plants per pot 9 3.4 d 4.8 a 2.1 a 7.1 a 4.2 a 54.2 a 12 3.8 c 4.7 ab 2.1 a 7.0 ab 4.1 ab 52.4 ab 15 4.2 b 4.4 bc 1.9 ab 6.5 bc 3.8 bc 49.7 bc 18 4.7 a 4.1 c 1.7 b 6.1 c 3.6 c 46.2 c adv. hort. sci., 2017 31(1): 25-30 30 do y., kashi a., babalar m., gil m.i., 2015 effects of salt stress on physiological 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with a proliferation of diverse food attribute labels, such as organic, local, humane, fair trade, etc. (berlin et al., 2009). in particular, locally products, such as fruits and vegetables, not offered by conventional grocery stores, are looking for niche markets, in country and urban areas. studies indicate that many consumers associate the local foods with higher perceived quality as well as increased freshness and naturalness of the products (denver and jensen, 2014). the appeal for local varieties, represented by ancient genotypes, is due to the major environmental hardiness (negri, 2005) able to tolerate extreme climate events such as an increase in temperatures and frequency or intensity of precipitation. this peculiarity makes ancient genotypes suitable for sustainable cultivations as support for farmers and consumers addressed to choose healthy products. fruits and vegetables are well recognized to be important source of vitamins, minerals, fibers and many hundreds of compounds with potential antioxidant activity which, acting against cellular oxidation reactions, may have beneficial effects on human health (halliwell, 1996). the antioxidant activity of fruits is strongly affected by the species and, within species, by the variety which is the main factor in determining fruit nutritional quality (scalzo et al., 2005). among other preand post-harvest factors influencing the antioxidant properties, such as cultivation techniques, ripening season, shelf-life and processing, the environmental conditions have an important role. in particular, it has been found that light intensity, temperature together with water availability are related to the antioxidant activity in different fruit species (lee and kader, 2000). the fruit nutritional quality of many commercially important varieties has been characterized, but still scanty is knowledge on the antioxidant properties of ancient genotypes that can show high quality performances, in terms of flavor and nutritional properties (donno et al., 2012; jakobek et al., 2013). recently, a trend to discover and to reintroduce into marketplace genotypes native of a restricted geographical adv. hort. sci., 2017 31(2): 77-84 doi: 10.13128/ahs-21086 chemical characterization and sensory analysis by blind and visually impaired people of local peach varieties s. bartolini 1 (*), r. viti 2, e. ducci 1 1 istituto scienza della vita, scuola superiore sant’anna, piazza martiri della libertà, 33, 56127 pisa, italy. 2 dipartimento di scienze agrarie, alimentari e agro-ambientali, università di pisa, via del borghetto, 80, 56124, pisa, italy. key words: ancient varieties, physicochemical traits, prunus persica (l.) batsch, total antioxidant capacity, total phenols. abstract: the interest in locally produced foods by the reintroduction of old varieties is due to their environmental hardiness and suitability for low-input agricultural systems. these managements can often produce fruits with imperfections, preventing the consumer acceptance. we have settled a new sensory evaluation going beyond the appearances, involving blind and visually impaired people to provide a quality evaluation of fruits linked to intrinsic rather than exterior characteristics. the research was conducted over two consecutive harvest seasons on three peach old local varieties (‘alberta’, ‘mora di dolfo’ and ‘regina di weinberger’ called in loco ‘regina di bember’) grown in central italy. on fruits, physicochemical traits (fruit weight, peel and flesh color, flesh firmness, ph, total soluble solids, titratable acidity), antioxidant content (total antioxidant capacity and total phenols) and sensory analysis were assessed. the three local peach varieties showed interesting fruit attributes in both studied growth-ripening seasons. the white-fleshed ‘mora di dolfo’, characterized by the highest antioxidant contents, was particularly appreciated by panellists for its aroma. the new sensory analysis, providing an evaluation based on judgment of intrinsic characteristics of peaches, emerges as a valid tool to assess the interest and appreciation of fruits for a conscious consumer’s choice. (*) corresponding author: susanna.bartolini@santannapisa.it received for publication 13 december 2016 accepted for publication 11 may 2017 copyright: © 2017 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2017 31(2): 77-84 78 area took place (krawitzkyab et al., 2014). the local varieties, mainly managed by low-input agricultural systems, often produce fruits with defects, preventing the consumer acceptance (donno et al., 2012). it has been showed that the appearance of fresh fruits is the primary criterion in making purchasing decisions (kays, 1991). to overcome this approach we have settled a new sensory evaluation going beyond t h e a p p e a r a n c e s , i n v o l v i n g b l i n d a n d v i s u a l l y impaired people (bartolini et al., 2015 a), who having a higher sense of taste and smell (luers et al., 2014), can provide a quality assessment of fruits linked to intrinsic rather than exterior characteristics. the aim of this research was to determine some fruit quality traits of three peach local varieties origin a t e d f r o m l u c c a p r o v i n c e ( c e n t r a l i t a l y ) . physicochemical and antioxidant parameters were studied, and the innovative sensory analysis involving people with visual disability was introduced. 2. materials and methods plant material and cultivation site the research was conducted over two consecutive harvesting seasons (2014-15) on full bearing peach trees of old local varieties: ‘alberta’, ‘mora di dolfo’ and ‘regina di bember’. this latter variety is an example of synonymy; its name was modified by local citizen from the original ‘regina di weinberger’ (bartolini et al., 2015 a), a californian variety widespread at the end of fifties which nowadays has lost importance at national level. the orchards were located in a traditional area at high agricultural vocation (tuscany, lucca province, lat. 44.02 n, long. 10.27 e) and trees were grown in similar soil conditions, using low input farming practices. the main climatic parameters, minimum-maximum temperatures and rainfall data were provided by the regional agro-meteorological service of florence (sir) (http://www.sir.toscana.it). at physiological maturity stage, samples of peaches were collected and analyzed to determine the main physicochemical, antioxidant and sensory traits. physicochemical analysis from each fruit (n = 30 per variety), measurements of weight, peel and flesh color, flesh firmness (ff), total soluble solids (tss), ph and titratable acidity (ta) were determined. fruit weight was expressed according to the following size categories (della strada et al., 1984): very small (≤90 g), small (91-125 g), medium (126-160 g), large (161-195 g), very large (>195 g). peel and flesh color was evaluated using color charts according to upov code (international union for the protection of new varieties of plants, geneva) for peach. the red cover color area of peel (cc) was visually evaluated and expressed as percentage. firmness was assessed with a manual penetrometer on two peeled opposite areas at the equatorial region of peach, using an 11-mm-wide plunger (model 53200sp tr, tr-turoni & c. inc forlì, italy). tss were measured using a refractometer (model 53015c tr, tr-turoni & c. inc forlì, italy) and expressed in °brix at room temperature. the fruit ph was recorded with the help of an electronic ph meter, and ta was determined on fruit juice by titrating a known volume of juice with 0.1n sodium hydroxide (naoh) to ph 8.1. ta was expressed as milliequivalents of malic acid per 100 grams of fresh weight (meq malic ac. 100 g fw-1). total antioxidant capacity (tac) and total phenol (tp) analysis t o t a l a n t i o x i d a n t c a p a c i t y ( t a c ) a n d t o t a l phenols (tp) analyses were carried out on the same fruits that had been previously subjected to the physicochemical determinations. fresh fruit samples (flesh with peel) of 3 g (in triplicate) were immediately frozen and stored at -20°c until extraction. the samples were homogenized using an ultra-turrax blender at 4°c to avoid oxidation. the extraction was performed in 80% ethanol for 1 h in a shaker in the dark and subsequently centrifuged at 4°c for 10 min at 2600 g. the supernatant was used for tac (total antioxidant capacity) and tp (total phenol) analysis. tac was evaluated using the improved trolox equivalent antioxidant capacity (teac) method (arts et al., 2004). the teac value was calculated in relation to the reactivity of trolox, a water-soluble vitamin e analogue that was used as an antioxidant standard. in the assay, 40 μl of the diluted samples, controls or blanks added to 1,9690 μl abts•+ solution, resulted in a 20-80% inhibition of the absorbance. the decrease in absorbance at 734 nm was recorded 6 min after an initial mixing, and plotted against a dose-response curve calculated for trolox (0-30 μm). antioxidant activity was expressed as micromoles of trolox equivalents per gram of fresh weight (μmol te g fw −1). trolox was purchased from sigma chemical co. (st. louis, mo). tp content was determined according to the improved folin-ciocalteu (f-c) method (waterhouse, 2001). the assay provides a rapid indication of the bartolini et al. chemical characterization and sensory analysis of local peach varieties 79 antioxidant status of the studied material and is valuable for different food samples. the standard compound for the calibration curve was gallic acid (ga, sigma chemical co, st. louis, mo). total phenol content was calculated as milligrams of ga equivalent (gae) per gram of fresh fruit weight (mg gae g fw−1). the absorbance of the blue colored solutions was read at 765 nm after incubation for 2 h at room temperature. sensory evaluation the sensory evaluation was carried out on fruits of the varieties ‘alberta’ and ‘mora di dolfo’ as they have the same harvest time (end of july) but different characteristics, such as taste, ground peel (yellow and light-green) and flesh color (yellow and white), respectively. the test was performed without visual inspection, in collaboration with blind and visually impaired persons of the ‘blind and visually impaired people italian association’ of lucca (italy). this is an original experiment on stone-fruits realized for ‘going beyond the appearances’ that, to our knowledge, is the only one after an experience carried out in sicily ( h t t p : / / w w w . c o n s o r z i o p a r s i f a l . i t ) . t a k i n g i n t o account the disability of the assessors-consumers, a tentative of protocol was established using an acceptability test, as a pertinent option for specific applications (varela and ares, 2012). in both years, the same 10 blind and visually impaired persons, aged between 26 and 65, performed the evaluations on the selected varieties. in order to make the evaluation phase easier, prior the acceptability test a short training stage (5 hours) was provided outlining methodology and procedure of the sensory method. a room equipped with individual sites was used. to each panellist, room-tempered and washed halfunpeeled anonymous peaches were served in randomized order, and samples were evaluated in one session. the panellist were invited to rinse the mouth with mineral water and eat saltless bread between samples, in order to avoid tiring effects. the fruit attributes evaluated were the following: shape, size, texture, aroma, sweetness, acid taste, juiciness (shinya et al., 2014). moreover, participants were asked to assess the fruit global appreciation. a continuous non-structured 9-point hedonic scale was utilized for evaluation: 1= dislike extremely; 2= dislike very much; 3= dislike moderately; 4= dislike slightly; 5= neither like nor dislike; 6= like slightly; 7= like moderately; 8= like very much; 9= like extremely (porretta, 2000). the blind judges were requested to indicate the score for each attribute and they were supported by sighted persons helping them to fill the data form. judges were not informed about the characteristic of fruits such as peel and flesh color. statistical analysis physicochemical data of each variety were compared using a student’s t-test analysis with two treatments (2014 and 2015 harvest seasons); for each season, 30 repetitions/parameter were used, excluding ta and ph, where three composite samples containing ten fruit per sample were considered. a two-way anova analysis were performed to test the effects of year and variety on the main physicochemical and antioxidant parameters. differences on the content of antioxidants and phenolics between the three varieties were investigated with the analysis of variance (anova), using tukey’s post hoc test at p≤ 0.05. a correlation analysis of antioxidant capacity versus total phenol content was calculated and correlation coefficient was reported in terms of goodness of fit. for the sensory evaluation, taking judges as factors, results were compared through the student’s t-test analysis between harvest seasons. spearman’s correlations were performed to estimate relationships among sensory and physicochemical traits of fruits. data are reported as means ± standard errors of the means (sem), and the analysis were all performed using the statistical package graphpad prism 5 (graphpad software, inc.). 3. results and discussion weather conditions during the fruit growth, from may to july, the considered harvest seasons were characterized by different weather conditions, in terms of temperatures and rainfall events (table 1). in particular, at the final stages of fruit ripening the strongest differences were observed: the amount of precipitation was consistently higher in 2014 when, only on july, more than 300 mm of rainfall occurred. this condition was unusual for lucca area in comparison to table 1 monthly minimum and maximum temperatures (°c) and cumulative rainfall (mm) from may to july, over a 2-year period (2014-2015) month 2014 2015 t. min. t. max. average rainfall t. min. t. max. average rainfall may 11.8 23.7 17.8 39.8 13.0 24.5 18.8 34.4 june 16.0 29.4 22.7 48.0 16.8 30.2 23.5 29.8 july 17.4 28.7 23.2 314.6 20.7 33.9 27.3 7.4 average 15.1 27.3 21.2 16.8 29.5 23.2 total rainfall 40.4 71.6 adv. hort. sci., 2017 31(2): 77-84 80 average values of the last 10 years, ranging from 8.6 to 60.2 mm (sir toscana). the cumulative rainfall of the early summer seasons (may-july) was about 400 and 72 mm in 2014 and 2015, respectively. the second year was particularly drought and warm by mean temperatures of 4-5°c more than 2014. physicochemical and antioxidant fruit parameters harvest time and mean values of the main physicochemical traits of the peach varieties, recorded over a 2-year period, are presented in table 2. under the e n v i r o n m e n t o f l u c c a a r e a , t h e h a r v e s t t i m e occurred between the first (‘regina di bember’) and third decade of july (‘alberta’ and ‘mora di dolfo’). considering the physical traits of fruit, all varieties showed attractiveness: ‘alberta’ was characterized by fruit of medium size with yellow ground peel (1050% red cover color) and deep yellow flesh; ‘mora di dolfo’ had a large fruit size, light-green peel (3050% red cover color) and white flesh; ‘regina di bember’ had a medium-large fruit size, yellow ground peel, (50-90% extensive red cover color) and deep yellow flesh (fig. 1). within each variety, values of chemical parameters showed variations between years, particularly for soluble sugars (tss), titratable acidity (ta) and ph. ‘regina di bember’ had the greatest differences in relation to the weather conditions occurred during the ripening period of the considered years. tss ranged from about 9 to 12 °brix, and ta changed between 8 and 6 meq malic ac. 100 g fw-1 in the wettest (2014) and driest (2015) year, respectively. as a consequence, the tss/ta ratio varied greatly with the lowest values in the wettest year. a similar trend was recorded in ‘alberta’ where ta changed while tss did not differ as much. no changes in chemical parameters were observed in ‘mora di dolfo’, performing well under the rainy climatic conditions of 2014. in both years, fruits exhibited similar values in tss (11.8-13.9 °brix), ta (9.6-9.8 meq malic ac. 100 g fw-1) and, as a consequence, in their ratio. the anova results comparing harvest year and variety effect showed significant interactions ‘year x variety’ for ta and tss/ta (table 3). during the latter stages of fruit development, the influence of weather *, **, ***: significant at p≤0.05, 0.01, 0.001, respectively. ns= not significant. table 2 harvest time, peel and flesh color, red cover color (cc, %), and main physicochemical parameters recorded on three local peach varieties, over a 2-year period: fruit size, flesh firmness (ff, kg/0.5 cm2), total soluble sugars (tss, °brix), ph, titratable acidity (ta, meq malic ac. 100 g fw-1), sugars/acids ratio (tss/ta) harvest time ‘alberta’ ‘mora di dolfo’ ‘regina di bember’ end july end july early july peel color yellow, 10-50% cc light green, 30-50% cc yellow, 50-90% cc flesh color deep yellow white deep yellow fruit size medium large medium-large 2014 2015 2014 2015 2014 2015 ff 1.2±0.3 1.7±0.1 3.9±0.5 5.0±0.4 2.8±0.5 3.3±0.4 tss 10.7±0.4 11.6±0.2 11.8±0.9 13.9±0.4 9.2±0.4 12.2±0.7 * ph 3.80±0.1 4.15±0.1* 3.71±0.0 3.75±0.0 3.81±0.0 3.76±0.0 ta 10.2±0.1 6.1±0.1 * 9.6±0.1 9.8±0.1 8.3±0.1 6.2±0.1 * tss/ta 1 1.9 * 1.2 1.4 1.1 2.0 * fig. 1 the peach old local varieties ‘alberta’, ‘mora di dolfo’ and ‘regina di bember’ (originally ‘regina di weinberger’). table 3 two-way anova results. variables: tss (°brix), ta (meq malic ac. 100 g fw-1), tss/ta, tac (μmol te g fw-1), and tp (mg gae g fw-1) main effects tss ta tss/ta tac tp p year 0.0001 *** 0.0015 ** < 0.0001 *** 0.6980 ns 0.2792 ns variety 0.0015 ** 0.0001 *** <0.0001 *** <0.0001 *** <0.0001 *** interaction year x variety 0.5636 ns 0.0389 * 0.0016 ** 0.0113 * 0.2500 ns mean±sem. within each variety, asterisk indicates significant differences between years by student t-test (p≤0.05). bartolini et al. chemical characterization and sensory analysis of local peach varieties 81 conditions on pomological and quality properties has been proved: among fruit quality parameters, soluble solids content can vary substantially from year to year, and rainfall and summer sunshine have been identified as the main affecting factors (choi et al., 2003). in peach, extensive rain may reduce the sweetness, while severe water stress or regulated irrigation deficit techniques caused a soluble sugar increased, as a recognized physiological response to stress (kwon et al., 2008). as regards tac (fig. 2a), related to several bioactive compounds such as phenolics, flavonoids, anthocyanins and vitamin c (gil et al., 2002), the considered local varieties showed higher values than those reported in literature for commercial varieties of which, generally, only the flesh was analysed (scalzo et al., 2005). by the contrary, our investigation assessed samples constituted by flesh with peel, fraction of fruit with a key role in determining the antioxidant properties of the whole fruit (remorini et al., 2008; zhao et al., 2015). in both years, significant differences were observed: ‘mora di dolfo’ was characterized by the highest tac levels, on an average of about 14 μmol te g fw–1; ‘alberta’ and ‘regina di bember’ had similar average values (8.5-9.5 μmol te g fw–1). the tp content (fig. 2b), likewise tac results, was the highest in ‘mora di dolfo’ (about 2 gae mg fw–1), while the other varieties showed values from 0.7 to 0.9 gae mg fw–1. the relevant antioxidant content of the white-fleshed variety ‘mora di dolfo’ is in agreement with works reporting the tendency of white-fleshed cultivars to have significantly higher antioxidant content than yellow-fleshed peaches and nectarines (cantìn et al., 2009). a significant relationship between tac and tp content was found (fig. 3), confirming phenolic compounds as important contributors to the antioxidant activity of peach fruits (karav and eksi, 2012). the similar tac and tp values observed between years suggest that different rainfall amounts, occurred during the last growth-ripening period of fruit in 2014 (wet year) and 2015 (dry year), did not impact on the antioxidant power of varieties. these interesting results are not in agreement with studies carried out on several fruit species, where a variability in antioxidant levels in relation to climatic conditions has been found. in particular, the water availability markedly determined changes in fruit nutritional properties, and a linear significant inverse relationship between water status and antioxidant content was reported by several authors (leccese et al., 2010; laribi et al., 2013; bartolini et al., 2015 b). also in peach (cv. suncrest) the irrigation stress induced a fig. 3 linear regression between total antioxidant capacity (tac) and total phenols (tp) of the studied varieties (2year period). fig. 2 (a) total antioxidant capacity (tac) and (b) total phenols (tp) in peach fruits of local varieties recorded over a 2year period. values (means ± sem) are expressed for tac as μmol of trolox equivalent per g of fresh weight (μmol te g fw -1), and for tp as mg of gallic acid equivalent per g of fresh weight (mg gae g fw-1). different letters indicate significant differences at p≤0.01. adv. hort. sci., 2017 31(2): 77-84 82 higher biosynthesis of phytochemical compounds (tavarini et al., 2011). analysis of variance (table 3) showed that variation in tac and tp between varieties was much greater than between harvest years, indicating that genotype plays a more important role than growing season in influencing antioxidant content in peach, as found in other fruit species (howard et al., 2003; leccese et al., 2012). the significant interaction ‘variety x year’ for tac revealed that environmental growing conditions may impact the capacity of genotype to synthesise antioxidants which are influenced by biotic and abiotic factors (biesiada and tomczak, 2012). although investigation on the behavior of these varieties has to be continued, the high antioxidant capacity of peaches can be considered as a part of cultivar value (dalla valle et al., 2007). this peculiarity would be crucial for breeding strategies selecting genotypes with enhanced antioxidant levels which m a y p r o v i d e h e a l t h b e n e f i t s t o c o n s u m e r s . moreover, the availability of peach hardy genotypes will be a key factor for future, in view of the climate variability responsible of extreme event increases, such as drought or intense rainfall. sensory evaluation the sensory evaluation, carried out by blind and visually impaired panellists over a 2-year period, was conducted on varieties ‘alberta’ and ‘mora di dolfo’, characterized by yellow and white flesh color, respectively. considering that color and exterior feature of fruits are nowadays important commercial sensorial traits to attract consumers (kays, 1991), the judgment carried out by sightless assessors allowed to attain a real intrinsic quality evaluation of fruits. ‘going beyond the appearances’ revealed its importance particularly under the wet growing season of 2014; in fact, this condition, favoring the presence of defects on peach peel, could have prevented the consumer’s acceptance. on the basis of panellist’s appreciation, expressed as average degree of linking (1-9), the morphological parameters of fruits (shape and size) were similarly evaluated in both 2014 and 2015 (data not shown), while the organoleptic descriptors (acid taste, aroma, texture, juiciness, sweetness) were differently assessed depending on the harvest season (table 4). concerning the global appreciation, the lowest score was attributed in the year (2014) characterized by exceptional rainy events over the ripening period. these conditions could have negatively influenced components linked to the sensory profile, in agreement with authors who reported that high levels of water before harvest can reduce organoleptic quality and consumer liking degree (navarro et al., 2010). in fact, blind and visually impaired panellists expressed the best agreement for peaches sampled in 2015 when summer drought conditions occurred. most of descriptors showed higher scores and, particularly in ‘mora di dolfo’, the global appreciation was associated with an increase of aroma and juiciness, as observed in other peach varieties (di miceli et al., 2010). moreover, this variety could seem less susceptible to unfavorable climatic conditions since positively judged also in 2014 (score 6.2), although chemical attributes (tss, ta) were similar to ‘alberta’ that, instead, had a lower score (5.2). it has been established that a different sensory perception of peaches could be linked not only to basic organic components (sugars, organic acids, fibers, micro and macro elements) but in great part to the volatile compounds, which define the flavor impact (bononi et al., 2012). the correlation coefficient (table 5) between global appreciation and some sensory and physicochemical traits of peaches showed that panel’s acceptability was highly and positively correlated with aroma, sweetness and tss. moreover, these attributes were significantly correlated each other scores were based on a nine-point hedonic scale: 1= extremely dislike; 5= neither like nor dislike; and 9= extremely likewithin years asterisk indicates significant differences between varieties by student t-test (p≤0.05). table 4 mean scores (acid taste, aroma, texture, juiciness, sweetness and global appreciation) recorded for peach varieties ‘alberta’ and ‘mora di dolfo’, over a 2-year period variety/year ac. taste aroma texture juiciness sweetness glob. appr. 2014 ‘alberta’ 4.0±0.5 5.4±0.7 5.8±0.4 5.8±0.6 5.4±0.7 5.2±0.6 ‘mora di dolfo’ 4.8±1.1 6.2±0.8 5.4±0.9 6.8±0.5 6.2±0.8 6.2±1.0 2015 ‘alberta’ 2.8±0.5 5.8±0.6 5.0±0.5 7.0±0.4 5.7±0.7 6.0±0.4 ‘mora di dolfo’ 3.3±0.6 7.7±0.2 * 5.3±0.5 8.0±0.1* 7.3±0.5* 7.3±0.3 * table 5 coefficients of correlation between scores of global appreciation and some sensory and physicochemical traits of peach fruits attributes r spearman acid taste 0.1 aroma 0.99* juiciness 0.8 sweetness 0.99* texture -0.4 tss 0.99* tss/ta 0.4 significant coefficients are denoted by an asterisk at p≤0.05. bartolini et al. chemical characterization and sensory analysis of local peach varieties 83 (table 6), confirming tss as one of the most important quality indicators in determining acceptance judgments for peaches, due to its influence on the perceived sweetness (crisosto et al., 2006). on the contrary, texture appeared inversely related to global appreciation. the comparison between sensory and the main chemical attributes showed significant relationships: sweetness and aroma and total soluble sugars content were directly related each other. 4. conclusions the three local peach varieties showed interesting quality traits as well as a very high antioxidant content in both studied growth-ripening seasons. the capacity to maintain the bioactive components of fruits also under unfavorable weather events confirms the importance of old genotypes to be more stable in comparison to modern ones (donno et al., 2012). the white-fleshed ‘mora di dolfo’, characterized by the highest tac and tp content, was particularly appreciated by panellists for its excellent eating quality denoted by aroma, sweetness and well balanced sugar/acid ratio. although fruits of this variety are perishable, like the most white-fleshed peach varieties, it could represent a valid hardiness genotype for farmers who are focused to establish local markets where consumers are obviously willing to pay a premium price for niche products. this variety, as source of appreciable attributes, could be useful for specific breeding programs to develop new peach cultivars combining quality traits, hedonic and enhanced nutritional value, actually of high relevance for the consumer. the new sensory analysis, carried out by blind and visually impaired assessors, provided a quality evaluation linked to intrinsic rather than exterior characteristics of peaches that could prevent consumers if defects in appearance are present. this type of test could represent a valid tool to assess the interest and appreciation of fruits for a conscious consumer’s choice, going beyond the appearances, for a reintroduction of interesting ancient peach varieties characterized by unconventional fruit quality traits. acknowledgements research partially supported by ‘comune di capannori (lucca)’. thanks to ‘unione italiana ciechi e ipovedenti di lucca’ for kindly participation to the panel test. references a r t s m . j . t . j . , d a l l i n g a j . s . , v o s s h . p . , h a e n e n g.r.m.m., bast a, 2004 a new approach to assess the total antioxidant capacity using the teac assay. food chem., 88: 567-570. bartolini s., leccese a., viti r., 2015 b quality and antioxidant properties of 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waterhouse a.l., 2001 determination of total phenolics. in: wroslad re (eds) current protocols in food analytical chemistry. john wiley & sons inc., new york, i1.1.1 -i1.1.8 zhao xx., zhang w., yin x., su m., sun g., li x., chen k., 2015 phenolic composition and antioxidant properties of different peach [prunus persica (l.) batsch] cultivars in china. int. j. mol. sci., 16: 5762-5778. impaginato 11 1. introduction the polyamines as natural compounds are present ubiquitously in almost all living organisms. the m a i n p o l y a m i n e s i n s i g n i f i c a n t a m o u n t s a r e putrescine, spermidine, and spermine which are crucial for the growth and development of plant and fruit as well as stress responses (valero and serrano, 2010). they are known as anti-senescent agents that decrease the rate of fruit softening and senescence by suppression of ethylene production (kramer et al., 1991). reduced values of polyamines have been attributed with enhanced ethylene production and vice versa (walden et al., 1997). this mechanism is correlated to a competition between polyamine and ethylene for the common precursor s-adenosyl methionine (sam) (pandey et al., 2000). the use of polyamines has been claimed to decrease ethylene synthesis in a wide range of plants by decreasing acc synthase (acs) and acc oxidase (aco) enzymes activities (ke and romani, 1988; kakkar and rai, 1993; lee et al., 1997; martinez-romero et al., 2001; bregoli et al., 2002; perez-vicente et al., 2002; serrano et al., 2003; petkou et al., 2004; malik and singh, 2005; de dios et al., 2006; khan et al., 2007). in several investigations putrescine applied exogenously have been reported to increase storage life and quality attributes of mango (razzaq et al., 2014), pear (franco-mora et al., 2005), apricot (martinezr o m e r o e t a l . , 2 0 0 2 ) , s t r a w b e r r y ( z o k a e e khosroshahi et al., 2007), plum (abu-kpawoh et al., 2002; pérez-vicente et al., 2002), grapes (harindra champa et al., 2015; mirdehghan and rahimi, 2016), pomegranate (mirdehghan et al., 2007; barman et al., 2011) and litchi (jiang and chen, 1995). therefore, the aim of this study was to investigate the role of preharvest putrescine treatment on maintaining postharvest quality of pear fruit cv. spadona. 2. materials and methods the experiments were conducted on pear trees (p. communis cv. spadona) in the center of horticultural research of the university of tehran, karaj, iran. eighteen 16-year-old trees (250 cm height) were selected in terms of uniformity in size and fruit load then sprayed with putrescine at 0.5, 1 and 2 mm (3.5 adv. hort. sci., 2017 31(1): 11-17 doi: 10.13128/ahs-20720 effect of pre-harvest putrescine treatment on quality and postharvest life of pear cv. spadona m.s. hosseini (*), z. fakhar, m. babalar, m.a. askari department of horticulture science, college of agriculture and natural resources, university of tehran, 31587 karaj, iran. key words: cold storage, color, fruits, quality assessment. abstract: the study was conducted to determine the effect of pre-harvest foliar spraying with putrescine (at 0.5, 1 and 2 mm) on quality and postharvest life of pyrus communis cv. spadona during cold storage. fruit quality assessment such as weight loss, firmness, total soluble solids (tss), titratable acidity (ta), flavor index, skin color (l*, hue angle), vitamin c total phenol (tp), and total antioxidant activity (taa) were made at harvest and after 3, 6, 9, 12, 15, 18 and 21 weeks of storage at 0±1°c, 80-85% relative humidity. weight loss, fruit softening, tss and ph increased during storage but the rate of changes was significantly lower in fruit treated with putrescine at 1 and 2 mm. putrescine application maintained higher levels of ta, vitamin c, tp, taa, l*, hue angle and reduced decay incidence compared to control. furthermore, higher doses of putrescine were effective in terms of prolonging the storage and marketability of fruits more than 127142 days. in conclusion, pre-harvest application of putrescine could be an effective means for extending the postharvest life of pear cv. spadona. (*) corresponding author: m.hosseini79@yahoo.com received for publication 28 august 2016 accepted for publication 12 december 2016 copyright: © 2017 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2017 31(1): 11-17 12 l per tree) at different stages of fruit development in may, june, and july. six trees sprayed with water (3.5 l per tree) were used as control. fruits were harvested manually and transported to the postharvest laboratory, and selected for absence of visual symptoms of disease or blemishes, then stored (5 fruits per basket) at 0±1°c, 80-85% rh for 21 weeks. quality attributes were measured in five fruits of each replicate at harvest and after 3, 6, 9, 12, 15, 18 and 21 weeks of cold storage. fruit quality assessments fruit color changes were calculated at two opposite sides of fruit with a minolta chroma meter cr400 (osaka, japan). the values of l*(0 black; 100 white), a* (green to red), b* (blue to yellow) and hue angle (h°=180 + tan-1 b*/a*, if a* < 0) were recorded (fernando et al., 2007; pek et al., 2010). the percentage of weight loss was recorded by using following equation: % weight loss = (a-b)/b × 100 in which a was the initial fruit weight and b was the final fruit weight. fruit firmness was determined using a penetrometer ft327 (gffeci, italy) fitted with an 8 mm tip on the equatorial position of fruit. the results were expressed in newton (n). total soluble solids (tss) in the extracted juice of each treatment were measured by a refractometer (atago n1, japan) at 20°c and the result was recorded as percentage. five ml of diluted juice titrated a g a i n s t 0 . 1 n n a o h t o p h 8 . 2 t o a s s e s s t a . phenolphthalein was used as an indicator. the ta was expressed as malic acid percentage (saini et al., 2001). the ph of fruit juice was calculated using a mtt65 (japan) ph meter calibrated by ph 4 and 7 buffer solutions. flavor index was estimated by dividing tss with the corresponding ta value. vitamin c was measured using the procedures of tian et al. (2002). total phenol (tp) content and total antioxidant activity (taa) tp and taa were assessed according to koushesh saba et al. (2012). decay incidence determination fruit decay was determined based on the procedure of khademi and ershadi (2013). scales from 1 to 5 were given to individual treatment group whereas: 1= normal (without decay), 2= (up to 5 % decay), 3= (5-20 % decay), 4= (20-50% decay) and 5= (more than 50% of fruits skin was decayed). statistical analysis this experiment was conducted in a randomized experimental design with three levels of putrescine (0.5, 1 and 2 mm), using plants sprayed with water as control in three replications and two trees in each experimental unit. to estimate storability of pear fruit cv. spadona, a factorial design in completely randomized were carried out and the experimental data analyzed using sas statistical software package 9.4 for windows and mean comparisons were conducted using duncan’s multiple range tests. 3. results and discussion color a high rate of color changes was observed in control fruits and 0.5 mm putrescine treated fruits, whereas, they exhibited lower l* and hue angel than others during storage (fig. 1 a and b). therefore, the conversion rate of green to yellow and degradation fig. 1 the effect of putrescine at different concentrations on l* (a) and hue angle (b) of pear cv. spadona along the storage. values are the mean ± se. hosseini et al. putrescine application for improving storage life of pear 13 of chlorophyll were shown slower in putrescine treated fruits by 1 and 2 mm. the effect of putrescine in retarding skin color changes throughout the storage by decreasing senescence rate has also been reported in table grape (harindra champa et al., 2015), and pomegranate (barman et al., 2011). weight loss and firmness the weight loss increased in all fruit samples during the 21 weeks cold storage. as shown in figure 2 a, putrescine at 1 and 2 mm reduced the weight loss value than control at the end of storage. however, fruit treated with 2 mm putrescine showed inferior weight loss which started at the third sampling date (6th week), while it was not seen in those treated with 1 mm before the fifth sampling date (12th week). reduction of weight loss in putrescine treated fruits can be ascribed to conjugation of polyamines to the cell membrane phospholipids and consequently stabilization as well as consolidation of both cell integrity and permeability (barman et al., 2011; mirdehghan and rahimi, 2016). irrespective of treatments, fruit firmness decreased with the advancement of storage but putrescine treatment at 1 and 2 mm maintained highest fruit firmness compared to control (fig. 2b). it is suggested that polyamines maintain fruit firmness by their cross-linkage to the pectin substances carboxyl groups in the cell wall and lead to strengthening of cell wall and consequently decreasing cell wall degrading enzymes activities of pectin methyl esterase (pme), pectin esterase (pe) and polygalactouronase (pg) (valero et al., 2002). the role of putrescine in reducing weight loss and maintaining fruit firmness has been reported for peach (zokaee khosroshahi and esna-ashari, 2008) and pear (franco-mora et al., 2005). tss, ta, ph and flavor index the contents of tss (in the first 12 weeks of storage), ph and flavor index increased in all treated and untreated fruits while ta showed reverse trend along the storage. however, the lowest tss, ph and flavor index were observed in treated fruits by 1 and 2 mm (fig. 3 a, b, c and d). the role of putrescine on maintaining tss, ta and ph in treated fruits would be attributed to the reduction of respiration rate (valero et al., 2002), ethylene synthesis (barman et al., 2011) and subsequently retarding the ripening process. similar results have been reported in peach (zokaee khosroshahi and esna-ashari, 2008) and apricot (enas et al., 2010). vitamin c vitamin c significantly declined as the storage advanced. however, this trend was slower in 1 and 2 putrescine treated fruits (fig. 4). this effect can be associated with the property of putrescine on reducing or delaying the activity of ascorbate oxidase and consequently maintaining vitamin c (ascorbic acid) content (ishaq et al., 2009). similar results have been reported in mango (razzaq et al., 2014) and apricot (davarynejad et al., 2013). total phenol (tp) and total antioxidant activity (taa) measurement irrespective of treatments, total phenolic content and total antioxidant activity decreased at the end of storage; while these decreases were significantly higher at 1 and 2 mm putrescine treated fruits (fig. 5 a and b). in spite of taa, the tp changes were not constant during storage, it reached the highest value at the 9th week in fruits treated with 1 and 2 mm with the maximal values of 28 and 31 mg of gae/100 g of fw at the 9th week respectively, then followed by reducing tp during the rest of storage period. fig. 2 the effect of putrescine at different concentrations on weight loss (a) and firmness (b) of pear cv. spadona along the storage. values are the mean ± se. adv. hort. sci., 2017 31(1): 11-17 14 fig. 3 the effect of putrescine at different concentrations on tss (a), ta (b), ph (c) and flavor index (d) of pear cv. spadona along the storage. values are the mean ± se. fig. 4 the effect of putrescine at different concentrations on vitamin c of pear cv. spadona along the storage. values are the mean ± se. fig. 5 the effect of putrescine at different concentrations (0.5, 1 and 2 mm) on total phenol content (a) and total antioxidant activity (b) of pear cv. spadona along the storage. values are the mean ± se. hosseini et al. putrescine application for improving storage life of pear 15 the changes in the level of tp content may be associated to the breakdown of cell structure and subsequently senescence (ghasemnezhad et al., 2010). the role of putrescine treatment to maintain tp could be ascribed to the delay of senescence process (arora et al., 2002; razzaq et al., 2014). as shown in figure 5, the value of taa decreased along with a decrease of tp during storage. it may be ascribed to a direct correlation among tp content and taa (razzaq et al., 2014). however, putrescine treatment at 1 and 2 mm maintained taa compared to control during storage. similar results demonstrated a positive correlation among tp and taa in mango (palafox-carlos et al., 2012) and apricot (ghasemnezhad et al., 2010). decay incidence the lowest rate of fruit decay percentage was o b s e r v e d i n f r u i t s t r e a t e d w i t h 1 a n d 2 m m putrescine contrary to control at the end of storage (fig. 6). it is suggested that polyamines have all requirements of an alternative approach for management of postharvest decay (romanazzi et al., 2012). storage life the application of putrescine at higher doses (1 and 2 mm) extended storage life of pear fruits, and consequently they were suitable to be exposed in the market more than 127-142 days after the beginning of storage in comparison to control (109 days) (fig. 7). 4. conclusions t h e p r e h a r v e s t a p p l i c a 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technol., 1(3): 278-287. zokaee khosroshahi m.r., esna-ashari m., ershadi a., 2007 effect of exogenous putrescine on post-harvest life of strawberry (fragaria ananassa) fruit, cultivar selva. sci. hortic., 114(1): 27-32. impaginato 13 adv. hort. sci., 2018 32(1): 13-17 doi: 10.13128/ahs-21241 storage conditions of soft x-ray irradiated pollen for producing seedless watermelons m. akutsu1, 2 (*) 1 national agricultural research center for hokkaido region, hokkaido, japan. 2 department of electrical engineering and computer science, tokai university, kumamoto, japan. key words: fruit quality, n2 storage, seedless watermelon, soft x-ray, storage temperature, vacuum storage. abstract: a safe and efficient method for preserving viable soft x-ray-irradiated pollen for the production of seedless watermelon (citrullus lanatus l.) from diploid plants was tested by packing the pollen under vacuum, o2, co2 or n2 gas at 25°c, 4°c or -25°c. pollen germination rates decreased most rapidly with storage at 25°c and slowest with storage at -25°c. oxygen as a storage gas was not good for storage of pollen, but pollen stored in n2 or co2 gave good germination. pollen stored at -25°c for 90 days germinated, but pollen stored at 4°c for 90 days did not, and n2 storage tended to result in higher fruit set than vacuum storage. fruit set was significantly affected by pollen storage conditions, with n2 storage being more effective than vacuum storage at 4°c. storage at -25°c produced little difference in fruit set between vacuum and n2 storage. thus, temperature was the major factor for maintaining viable and effective pollen, and the use of n2 gas was an effective adjunct. fruit quality was not significantly affected by storage parameters in this experiment. 1. introduction generally, seedless watermelons are produced from triploid plants that are the product of crosses between tetraploid and diploid plants (terada and masuda, 1943; kihara and nishiyama, 1947; kihara, 1951). diploid seedless watermelons have also been produced by pollination with soft x-ray irradiated pollen (sugiyama and morishita, 2000; sugiyama et al., 2002 a). using irradiated pollen is advantageous because seedless watermelons can be produced with ordinary cultivation methods due to the use of diploid plants. however, mass production of seedless watermelon seed by this method will require the production, irradiation, preservation and storage of a lot of pollen. pollen viability after freezing and low temperature storage at low relative humidity has been reported (*) corresponding author: akutsu@tsc.u-tokai.ac.jp citation: akutsu m., 2018 storage conditions of soft xray irradiated pollen for producing seedless watermelons. adv. hort. sci., 32(1): 13-17 copyright: © 2018 akutsu m. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 12 september 2017 accepted for publication 8 november 2017 ahs advances in horticultural science adv. hort. sci., 2018 32(1): 13-17 14 in many plant species (holman and brubaker, 1926; mcguire, 1952; king, 1961; fatmer and barnett, 1974; nath and anderson, 1975; anderson et al., 1978). miyaji and shirazawa (1977) reported that the best storage conditions for watermelon pollen are 5°c at 20 to 40% humidity, which extended the viability of pollen to 60 days on germination medium, but did not investigate fruit set. watermelon pollen had fruit setting ability for 13 days when stored at 5°c under dry conditions (silica gel) (araki et al., 1987). pollen stored under temperature and humidity control, tend to have rapid germination rate decreases (miyaji and shirazawa, 1977; araki et al., 1987; sugiyama et al., 1998). watermelon pollen storage in organic solvents has also been investigated (kodani and omura, 1981; shimizu, 1983; sugiyama et al., 2002 b). among the organic solvents, ethyl acetate and ethyl ether were the most suitable for watermelon pollen storage (shimizu, 1983). pollen stored in ethyl acetate at -20°c had a germination rate of over 4 0 % f o r o n e m o n t h o n g e r m i n a t i o n m e d i u m (morishita et al., 2000), and fruit set was observed up to 79 days of storage in ethyl acetate at -20°c (sugiyama et al., 2002 b). these methods are effective for watermelon pollen, but are not suitable for long-term storage because of a decrease in vigor. also, it is very difficult for farmers to recover and use pollen that has been stored in organic solvents. n2, co2 and/or o2 gas have been used as a general storage medium in the food industry, but there are few reports using gas for pollen storage. in this study, we describe effective new pollen storage method using atmospheric gases. 2. materials and methods storage conditions for watermelon pollen pollen from watermelon cultivar green seeded was collected in october 2005 by cutting the anthers from the flowers and shaking the contents through a stai nl ess steel filter into a stainless steel cup. collected pollen was packed in paraffin paper and irradiated with 600 gy soft x-ray (soft x-ray unit om60r, ohmic ltd.) at 14.5 gy·min-1. irradiated pollen aliquots of about 1 g each were packed with a vacuum packaging machine (‘tospack’ v-380g, tosei, shizuoka, japan), in air or under vacuum storage at 25°c, 4°c or -25°c for storage over 35 days. a second similar storage test was initiated in may 2007 to investigate effect of atmospheric gases on storage. irradiated pollen aliquots of about 3 g each were packed with a vacuum packaging machine under n2, o2, co2, air or vacuum for storage at 25°c for 1-6 days. viability of the stored pollen was judged on the basis of germination on an artificial medium consisting of 14% sucrose, 0.1% boric acid, and 1.5% agar (wako, ltd, osaka, japan). germination percentages were determined after 3 hour incubation at 25°c by counting random samples of 100 pollen or more for each storage condition. the first test was replicated 8 times, and the second test was replicated 5 times. tukey’s multiplerange test (p<0.05) on statistical software (statcel, oms-publishing, saitama, japan) was used to test differences between treatment means. fruit set ability of stored pollen, and fruits quality using stored pollen experiments were performed at the hokkaido research center, sapporo, japan. during cultivation, greenhouse temperatures were recorded (‘ondotori’, t&d corporation, nagano, japan), and monthly mean temperatures were determined. the average temperatures during cultivation in the greenhouse were 2 5 ° c i n j u l y , 3 0 . 4 ° c i n a u g u s t , a n d 2 5 . 1 ° c i n september. watermelon cultivar fujihikari tr seeds were sown in pots on june 15, 2006 in a greenhouse. seedlings with 5 to 6 leaves were transplanted 50 cm apart in a bed (2.3 x 35 m) in a greenhouse on july 10, 2006. the bed was covered with black and gray polyethylene mulch and fertilized with 9n-9p-9k (in kg·ha-1) before transplanting. plants were topped at the five leaf stage and three lateral vines were allowed to grow. male flowers were pollinated with a paint brash at about the 15th node of lateral branches. all treatments were arranged in a randomized complete-block design with four single plant replications. four fruits were harvested for each treatment. values were compared by tukey’s multiple-range test (p<0.05). source of pollen watermelon cultivar green seeded seeds were sown as a source of pollen for the storage experiments in february 2006, before ‘fujihikari tr’ was sown. male flowers of ‘green seeded’ were harvested in the morning to obtain pollen that would be stored for 14, 28 and 90 days, from april through july. collected pollen was irradiated with 600 gy soft x-ray and packed with a vacuum packaging machine either under n2 gas or vacuum storage at 4 or -25°c for 14, 28 or 90 days. soft x-ray irradiated pollen processed on the morning of the experiment was akutsu pollen preservation for watermelons 15 used as a non-storage control. the stored pollen was used to pollinate female flowers of ‘fujihikari tr’. female flowers of ‘fujihikari tr’ were bagged with cellophane before anthesis. prepared irradiated pollen was applied with a brush. after pollination, female flowers were covered again with cellophane bags to prevent contact with insect-borne pollen for about 3 days. pollination occurred during the period of august 2-6. one or two female flowers on each of the 3 lateral shoots were pollinated from each storage treatment, and fruit set was confirmed about 7 days later. the fruit set rate for each plant was calculated. fruits were selectively thinned, leaving one fruit per plant to mature for each treatment. mature fruits were harvested after about 42 days. after harvesting, fruit weight, shape, rind thickness, flesh color, sugar content (brix) and number of empty seeds exceeding 6 mm in length were recorded for each fruit. fruit shape index is expressed as the ratio of length from peduncle to blossom end, to equatorial diameter. mature harvested watermelons were cut in half, and flesh color was measured with a colorimeter [a*= hue relates to red (+60) green (-60) color axes, nihondenshokukogyo, tokyo, japan]. the germination ability of stored pollen was assayed as in experiment 1 before pollination (the period of august 2-6). fruit set and pollen germination rates were compared by tukey’s multiple-range test (p<0.05). 3. results and discussion storage condition for watermelon pollen pollen germination rates decreased rapidly at 25°c to nearly zero after 7 days (fig. 1). when stored in atmospheric air at 4°c, the germination rate was significantly lower than with other treatments (except at 25°c) after 21 days. after 35 days of storage under vacuum at 4°c, pollen viability was significantly lower than either treatment at -25°c (p<0.05). thus, high temperature is not suitable for storage of pollen, and packing pollen under vacuum at 4°c prolonged viability. the viability of watermelon pollen stored under vacuum or o2 at 25°c decreased during storage to levels that make o2 and vacuum conditions unacceptable as atmospheric media for pollen storage (table 1). pollen stored for 7 days under n2 and co2 showed good germination, indicating that storage of soft x-ray irradiated watermelon pollen under n2 or co2 would enhance its viability. fruit set and quality produced from stored pollen the ability of stored polled to germinate, and to produce agronomically acceptable fruit are two distinct qualities, both of which are necessary for the adoption of soft x-ray irradiated pollen on a commercial scale. there were observable differences in fruit set between pollen stored at 4°c and -25°c (table 2). lower temperatures resulted in good germination rates, and pollen which had been stored at -25°c had a longer shelf-life than at 4°c. pollen stored at 4°c for table 1 the differences of germination ability for cultured periods and treatments at 25°c treatments periods (days) 1 2 3 4 5 n2 25.1 a z 21.2 a 16.5 a 13.8 a 7.9 a o2 11.2 b 4.7 b 0.0 d 0.0 c 0.0 b co2 19.4 ab 17.6 ab 16.3 a 15.1 a 6.9 a vacuum 16.9 ab 10.9 b 2.0 c 1.7 b 0.0 b air 20.3 ab 19.5 ab 9.3 b 3.3 b 0.2 b (z) means followed by the same letter are not significantly different at the 5% level by tukey's multiple-range test. table 2 relationship between storage condition and fruit set treatment storage pollination fruiting fruit set (%) temperature (°c) period (days) control 0 9 8 88.9 nitrogen 4 14 17 17 100.0 nitrogen 4 28 18 7 38.9 nitrogen 4 90 16 0 0.0 nitrogen -25 14 11 10 90.9 nitrogen -25 28 16 14 87.5 nitrogen -25 90 16 15 93.8 vacuum 4 14 17 11 64.7 vacuum 4 28 7 0 0.0 vacuum 4 90 13 0 0.0 vacuum -25 14 15 15 100.0 vacuum -25 28 15 14 93.3 vacuum -25 90 15 13 86.7 fig. 1 germination of pollen after storage at different temperature and treatments. bars indicate ± standard errors (n=8). adv. hort. sci., 2018 32(1): 13-17 16 90 days did not set fruit whether or not it was stored under n2 or vacuum. although germination rates under n2 and vacuum storage were almost the same, fruit set with pollen stored at 4°c for 14 or 28 days was more effective under n2 than under vacuum. pollen stored at 4°c for 28 days under n2 had the ability to set fruit, but pollen stored at 4°c under vacuum did not. snope and ellison (1963) also reported that germination from freeze-dried pollen that had been stored under n2 was got good. these results indicate that pollen vigor is maintained more effectively under n2 than under vacuum. the percentage of germinating pollen stored at -25°c for 28 days was 24.8% and there was fruit set following hand pollination. however, the percentage of germinated pollen stored at 4°c for 28 days was 22.8%, but no fruit was set with this treatment. n2 gas was an effective atmospheric medium for storage at 4°c in both tests. there were many reports that low temperature provides a good environment for stored pollen (araki et al., 1987; sugiyama et al., 1998; morishita et al., 2000). low temperature provides the major benefit, with storage in n2 gas as an adjunct for pollen storage. inert gases may be effective for the maintenance of pollen vitality because they may suppress metabolism of the pollen by displacing oxygen. the additive effect of n2 with low temperature suggests that low metabolic rates are the key factor in maintaining pollen viability over time. there was no significant difference in weight or shape between control fruit and fruit produced by pollen under any of the storage conditions (table 3). rind thickness and flesh color were also the same, except for fruits produced with pollen stored at -25°c for 90 or 14 days, respectively. although the brix of fruit from pollen stored at -25°c for 14 days was the lowest, there was no consistent relationship between storage conditions and brix. sugiyama and morishita (2000) has observed that the number of empty seeds differs widely in individual fruits. in this experiment there were significant differences in the number of empty seeds, but there was no apparent relationship between empty seed numbers and storage conditions. thus, there is no consistent relationship between pollen storage conditions and fruit quality. further statistical analysis of the relationships between treatment, temperature and periods of pollen storage, was done for the survival of pollen, but there were no statistically significant differences. organic solvents are useful as a pollen storage method (kodani and omura, 1981). however, organic solvents carry the risk of environmental pollution and are difficult to use outside the laboratory. storage of pollen at low temperatures and under n2 eliminates the need for organic solvents, thus providing an ecofriendly storage solution which should be applicable for pollen export or import. furthermore, pollen stored under n2 at 25°c for 5 days had germination ability (table 1) and then the temperature in the greenhouse was around 25-30°c, so it might be also no problem to bring the pollen from freezer to the greenhouse during pollination. table 3 fruit quality of seedless watermelon pollination with irradiated pollen in several conditions treatment storage fruit weight (kg) flesh shape (z) thickness of rind (mm) flesh color brix (%) no. of empty seeds temperature (°c) period (days) control 0 5.5±1.1(y) 1.14 13.9±0.0 a (x) 21.4±1.1 a 10.3 a 96.5±12.1 b nitrogen 4 14 4.6±0.6 1.11 13.2±1.0 a 20.4±1.7 a 9.9 a 102.5±86.3 b nitrogen 4 28 4.9±0.5 1.12 13.5±0.2 a 23.9±2.8 a 9.7 b 104.5±17.6 b nitrogen -25 14 5.1±1.0 1.11 12.2±1.0 a 18.4±3.6 b 9.6 bc 150.0±88.3 a nitrogen -25 28 5.5±0.9 1.10 13.5±0.6 a 23.9±1.5 a 9.9 a 104.8±25.2 b nitrogen -25 90 5.2±0.5 1.14 11.9±0.4 b 22.5±1.0 a 9.7 b 222.8±71.7 a vacuum 4 14 4.9±0.3 1.13 12.2±1.4 a 24.0±3.9 a 9.7 b 103.0±44.9 b vacuum -25 14 5.4±0.5 1.10 12.7±1.9 a 20.6±2.5 a 10.2 a 134.8±21.1a vacuum -25 28 5.1±1.5 1.15 12.3±1.6 a 21.0±1.9 a 10.1 a 158.5±81.0a vacuum -25 90 5.7±0.4 1.17 12.5±0.6 a 22.5±0.6 a 10.0 a 127.0±24.5b (z) flesh shape is expressed as the ratio of height to width. (y) mean ± se. (x) value within columns with the same letter are not significantly different (5% level). means followed by the same letter are not significantly different at the 5% level by tukey's multiple-range test. akutsu pollen preservation for watermelons 17 plant breeders may also be able to overcome differences in crop or varietal flowering times by storing pollen with this method. references anderson j.o., nath j., harner e.j., 1978 effects of freeze-preservation on some enzyme. ii. freezing and freeze-drying stresses. cryobiology, 15: 469-477. araki m., hashizume t., hagihara t., 1987 effects of stored watermelon pollen on fruit set. j. jpn. soc. hort. sci., 56(2): 344-345. fatmer r.e. jr., barnett p.e., 1974 low-temperature storage of black walnut pollen. cryobiolgy, 11: 366367. holman r.m., brubaker f., 1926 on the longevity of pollen. univ. cal. publ. bot., 13: 179-204. kihara h., 1951 triploid watermelon. proc. amer. soc. hort. sci., 58: 217-230. kihara h., nishiyama i., 1947 an application of sterility of autotriploids to the breeding of seedless watermelons. seiken ziho, 3: 93-103. in japanese with english summary. king j.r., 1961 the freez-drying of pollens. econ. bot., 15(1): 91-98. kodani t., omura s., 1981 on the collection and storage of pollens of watermelon by the utilization organic solvents. bulletin of tottori agri. exp. stn., 2: 35-46. in japanese with english summary. mcguire dc., 1952 storage of tomato pollen. proc. amer. soc. hort. sci., 60: 419-424. miyaji r., shirazawa m., 1977 influence of temperature and the humidity for melon’s and watermelon’s pollens on their storage. bulletin of the kagoshima agric. exp. stn., 5: 203-206. morishita m., hori t., sakata y., sugiyama m., 2000 storage of pollen irradiated with soft x-ray for producing seedless watermelon. j. jpn. soc. hort. sci., 69(2): 346. nath j., anderson j.o., 1975 effect of freezing and freeze-drying on the viability and storage of lilium longiflorum l. and zea mays l. pollen. cryobiology, 12: 81-88. shimizu t., 1983 studies on the artificial pollination of watermelon. 1. 0n the storage of pollens of watermelon by the utilization of organic solvents. bulletin of the tottori agric. exp. stn., 4: 9-17. snope j.a., ellison j.h., 1963 storage of asparagus pollen under various conditions of temperature, humidity and pressure. amer. soc. hort. sci., 83: 447-452. sugiyama k., morishita m., 2000 production of seedless watermelon using soft-x-irradiated pollen. scientia horticulturae, 84: 255-264. sugiyama k., morishita m., nishino e., 2002 a seedless watermelons produced via soft-x-irradiated pollen. hortscience, 37: 292-295. sugiyama k., morishita m., sakata y., 1998 storage of soft-x-irradiated pollen and effects of stored pollen on fruit quality of seedless watermelon. j. jpn. soc. hort. sci., 67(2): 283. sugiyama m., sakata y., kitadani e., morishita m., sugiyama k., 2002 b pollen storage for production of seedless watermelon (citrullus lanatus) using soft-xirradiated pollen. acta horticulturae, 588: 269-272. terada j., masuda k., 1943 parthenocarpy of triploid watermelon. agric. hort., 18: 15-16. impaginato 183 adv. hort. sci., 2017 31(3): 183-189 doi: 10.13128/ahs-20575 field evaluation of three biopesticides for control of the raspberry cane midge, resseliella theobaldi (barnes) in bulgaria m. mohamedova department of entomology, agricultural university plovdiv, 12 mendeleev str., 4000 plovdiv, bulgaria. key words: azadirachtin, bacillus subtilis, biocontrol, efficacy, resseliella theobaldi, spinosad. abstract: the raspberry cane midge, resseliella theobaldi is a key pest on red raspberry, rubus idaeus. the larvae of the insect severely attack the raspberry canes, resulting in premature death of the plant canes. in the last decade, organic production of raspberry fruits has significantly increased in bulgaria. at the same time there are few products of botanical or microbiological origin that might be used for control of this pest. in present study the effect of neemazal® t/c (azadirachtin a), sineis 480 sc® (spinosad), and bacillus subtilis on r. theobaldi was evaluated. the experiments were conducted in two raspberry fields at different altitude. in the field at lower altitude (196 m), the raspberry cane midge has developed four generation per year, while in the field at higher altitude (960 m) three generations of the pest have been completed. lowest number of larvae in raspberry canes was observed after application of neemazal® t/c, and b. subtilis in both raspberry fields. both products demonstrated highest efficacy at 7th day after treatment, when the number of larvae per splits was 67.1-82.5% for neemazal® t/c, and 75.1-81.2% for b. subtilis lower compared with the control at the two experimental sites. 1. introduction among the small fruit crops grown in bulgaria, the red raspberry (rubus idaeus l.) is the most valuable. during the last five years, the total area of raspberry plantation has increased by 43% and the yield has reached 3620 kg-1 ha. at the same time approximately 54% of raspberry production is organic. in 2015 and 2016, about 75% of the total raspberry yield was exported mainly to western european countries, but also to several markets in asia (agrostatistics, 2015). the most serious pest of raspberry is the raspberry cane midge, resseliella theobaldi (barnes) (diptera: cecidomyiidae), which causes premature death of the plant canes. in bulgaria, the insect was first reported by stoyanov in 1960. the author examined the life cycle of r. theobaldi (*) corresponding author: m.mohamedova@au-plovdiv.bg citation: mohamedova m., 2017 field evalutation of three biopesticides for control of the raspberry cane midge, resseliella theobaldi (barnes) in bulgaria. adv. hort. sci., 31(3): 183-189. copyright: © 2017 mohamedova m. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 20 april 2017 accepted for publication 15 may 2017 ahs advances in horticultural science adv. hort. sci., 2017 31(3): 183-189 184 under different conditions and reported the development of three-five overlapping generations a year. the larvae of the insect attack the plant primocanes, both of those fruiting in june-july as well as those fruiting in august-september (stoyanov, 1963). the larvae feed under the bark of canes and clearly define dark brown spots appearing on the green surface of the canes. pitcher (1952) stated that damage to raspberry plants caused by r. theobaldi was usually associated with fungal pathogens such as botrytis cinerea, fusarium avenaceum and didymella applanta. the fungi cause necrosis of vascular cylinder through the larval feeding sites (williamson, 1987). the complex of damage involved the raspberry cane midge and mycoses is known as “midge blight” (pitcher and webb, 1952). as a result of usually present of midge blight, there is no established relationship between population level of r. theobaldi and d e g r e e o f p l a n t d a m a g e c a u s e d b y t h e i n s e c t (williamson and hargreaves, 1979). according to shternshis et al. (2002), the effect of the control treatments against the pest should be assessed by estimating the severity of midge blight including fungal lesions. to date, the biological control of raspberry cane midge is still poorly investigated. there are few reports concerning alternatives to chemical control for r. theobaldi. sex-pheromone-based strategies are promising techniques to control of many economically important pests. pheromone traps for raspberry cane midge were used for the first time in the uk in 2005 (milenković et al., 2006). cross and hall (2006) and hall et al. (2009) identified 2-acetoxy-5-undecanone as a major component of r. theobaldi female sex pheromone. over the past ten years, the sex pheromones have been tested for monitoring the male emergence (cross et al., 2008, tanasković and milenković, 2010, sipos et al., 2012). therefore, little information concerning the application of biopesticides for control of r. theobaldi in raspberry organic production is currently available. for instance, use of products based on entomopathogenic bacteria, bacillus thuringiensis (bt) and streptomyces avermitilis against raspberry midge blight have been reported (shternshis et al., 2002). further, there are no publications on the biological control of this raspberry pathogen complex. the objective of this study was to evaluate the possibility to control raspberry cane midge using two commercial biopesticides and one noncommercial bacterial strain under field conditions. in particular, i attempted to assess the role of used products in reducing the number of r. theobaldi larvae in raspberry canes. 2. materials and methods biopesticides and bacterium cultivation c o m m e r c i a l f o r m u l a t i o n s n e e m a z a l ® t / c (azadirachtin a, trifolio-m, germany), and sineis 480 sc® (spinosad, dowagrosciences, bulgaria), and a bacterium bacillus subtilis were used against midge in raspberry fields. the strain of b. subtilis was grown in the dark for 48 h at 24°c on tryptic soy broth agar (tsba). for inoculum production a loop of the bacteria was transferred into 100 ml of tsb and allowed to multiply for 48 h on a rotary shaker (160 rpm) at the same temperature. bacterial suspensions were centrifuged at 4000 rpm for 20 min, and the bacterial pellet was resuspended in sterile¼ strength ringer’s solution (merck). the bacterial suspension was adjusted to a final concentration of 108 cfu ml-1 by dilution with ringer’s solution. the bacterial strain identification was determined by fame analysis, following by biolog analysis. experimental design the trials were conducted in 2016 in two commerc i a l r a s p b e r r y p l a n t a t i o n s i n t h e r e g i o n s o f bogdanovo (196 m) and samokov (960 m). the first location (bogdanovo) has a flat topography, with small hills. the soil type is leptosols (bulgarian soil taxonomy), and the landscape is dominated by agricultural land use. according to the climatic data (national institute of meteorology and hydrology, bas), the average air temperature from april through october 2016 was 22.4°c. the average rainfall for the investigation period was 284 mm. the geographical coordinates of the raspberry field in bogdanovo are 42°36’ n, 26°00’ e. the second location (samokov) is a valley between two mountains rila and verila. the soil type is fluvisols, and the landscape is dominated by arable land. the average air temperature from april through october 2016 was 15.4°c. for the same period, the average rainfall was 531 mm. the geographical coordinates of the raspberry field in samokov are 42°21’ n, 23°34’ e. the cultivar heritage (usa) was grown in both three years old fields. plants were located in spacing of 50 cm within rows and 2.0 m between rows. experimental plots were 10 m2, each plot containing approximately 100-120 raspberry canes. the size of mohamedova field evaluation of three biopesticides for control of the raspberry cane midge in bulgaria 185 the buffer zone between the plots was 4 m. the treatments were arranged in a completely randomized block design with four replications. treatment and application methods the pheronorm® standard large delta traps (andermatt biocontrol ag, switzerland) were used to determine the population dynamic of the cane midge. the traps containing 10 µl cane midge sex pheromone lure per trap were mounted on bamboo sticks at height of 60 cm the 10th of april. three traps were used in bogdanovo (11 ha), and two in samokov (7,5 ha). two applications were made in samokov, one against the first generation (on the 17th may), and one against the second generation (on the 14th july). three applications were made in bogdanovo on 9th may, 8th july and 13th august against the first, second and third generations of r. theobaldi, respectively. the timing of each treatment was chosen according to the number of males caught by the traps. the treatments were made during the period of midge oviposition and larvae hatching. t h e t e s t s u s p e n s i o n s w e r e : n e e m a z a l ® t / c (0.2%), sineis 480 sc® (0,025%), and b. subtilis (20 ml). the suspensions were applied at a volume application rate of 0.1 l m-2, using a hand held sprayer. the plants in the control were treated with equal quantity (0.1 l m-2) of water. data collection and analysis the observations were made on the 3rd, 6th, and 12th day after treatments. twenty canes per replicate, 20 canes were examined under stereomicroscope and the number of larvae in the natural splits was counted. obtained data were subjected to one-way analysis of variance (anova) and the treatment means were compared with the control plants, according to the duncan’s test (p<0.05). 3. results in 2016, the raspberry midge cane flight pattern demonstrated four generations in bogadonovo (196 m) and three generations in samokov (960 m) (fig. 1). in both commercial fields, the flight of midges started in the second half of april, a week later in samokov (25.04) compared with bogdanovo (18.04). at lower altitude, the first, the second, and the third generations of the pest showed three pronounced peaks of its flight dynamic. in the higher altitude, there were two peaks of midge cane flight the first one between 19th and 25th of may, and the second one between 18th and 21th of july, for the first and second generations respectively (fig. 1). in general, the population density of the males was higher in bogdanovo than at samokov. the highest number of males was recorded during the intensive flight of the first generation of the midge in both bogdanovo (824) and samokov (683) sites (fig. 1). later in the season, the number of the males attracted by the traps in bogdanovo was 623, 698 and 193 for the second, third and fourth generation, respectively. in samokov, the number of the males was 528 and 267 for the second and third generation, respectively. the flight of the fourth generation of r. t h e o b a l d i c o n t i n u e d u n t i l 1 2 t h o f o c t o b e r i n bogdanovo, while the flight of the third generation of the midge in samokov was completed on 27th of september (fig. 1). the results obtained from this observation allowed finding the most appropriate date for treatments. the evaluated bioinsecticides demonstrated biological activity against the raspberry midge cane applied during the period of pest oviposition and larvae hatching. except for the treatment against the second generation of r. theobaldi in samokov, in both raspberry fields, bogdanovo and samokov, the highest efficiency was found for the insecticide neemazal® t/c (tables 1 and 2). this biopesticide also demonstrate the rapid initial effect against the penetration of larvae into raspberry canes. the number of midge larvae per split in raspberry canes treated with neemazal® t/c was significantly lower than the control at observations at 3rd, 7th and 12th day after applying the insecticide in bogdanovo (table 1, p<0.05). there was no significant difference between the efficacy demonstrated by neemazal® t/c and b. subtilis. after the treatment against the first generation the number of midge larvae per split varied fig. 1 trap catches of males of resseliella theobaldi in the fields of bogdanovo and samokov during the spring, summer and autumn of 2016. adv. hort. sci., 2017 31(3): 183-189 186 between 1.08 and 1.54 for neemazal® t/c, and b e t w e e n 1 . 5 5 a n d 2 . 0 7 f o r b . s u b t i l i s . s i m i l a r results were observed after the second and third treatments. the insecticide sineis 480 sc® demons t r a t e d t h e l o w e s t e f f i c a c y c o m p a r e d w i t h neemazal® t/c and b. subtilis (table 1, p<0.05). at the observation at 3rd day after treatments against the first and third generation of raspberry midge in bogdanovo, the number of larvae in plots treated with sineis 480 sc® was not significantly different from the number of larvae in control plots. in the second generation there was a statistical difference between the variant with the bioinsecticide and the control variant. after the treatments at 7th and 12th day, the insecticide showed better effect, and the number of larvae into the canes was significantly lower than the control but still higher compared with neemazal® t/c and b. subtilis (table 1, p<0.05). in the experiments conducted in samokov sineis 480 sc® demonstrated higher efficacy against raspberry cane midge and the number of larvae per split was statistically different than the control at all three observations (table 2, p<0.05). in this field, b. subtilis was more effective than neemazal® t/c and the number of larvae per split varied between 2.86 and 2.91 after first treatment. after the same treatment, the number of larvae into the canes treated with neemazal® t/c varied between 3.15 and 2.94. after the treatment against the second generation of r. theobaldi, b. subtilis showed rapid initial effect than neemazal® t/c. there was significant difference in number of larvae at 3rd day 2.63 and 4.86 for b. subtilis and neemazal® t/c, respectively (table 2, p<0.05). table 1 efficacy of three biopesticides using against raspberry midge cane in bogdanovo means within each column followed by the same letter are not significantly different, duncan’s test (p<0.05). treatments products/active ingredients number of midge larvae/split (day after treatment) 3rd (±sd) 7th (±sd) 12th (±sd) first generation neemazal® t/c (azadirachtin a) 1.54 (±0.18) a 1.12 (±0.07) a 1.08 (±0.37) a sineis 480 sc® (spinosad) 4.25 (±0.45) b 2.93 (±1.04) b 3.41 (±1.35) b b. subtilis 2.07 (±0.67) a 1.35 (±0.11) a 1.55 (±0.64) a control 5.62 (±1.02) b 6.28 (±1.22) c 6.78 (±0.56) c second generation neemazal® t/c (azadirachtin a) 2.48 (±1.11) a 1.74 (±0.64) a 1.62 (±0.19) a sineis 480 sc® (spinosad) 5.77 (±1.32) b 3.82 (±1.04) b 3.87 (±0.94) b b. subtilis 3.19 (±0.44) a 2.17 (±0.05) a 2.28 (±0.14) a control 8.44 (±1.12) c 8.92 (±1.65) c 9.41 (±0.27) c third generation neemazal® t/c (azadirachtin a) 1.24 (±0.72) a 0.92 (±0.08) a 0.98 (±0.34) a sineis 480 sc® (spinosad) 3.48 (±1.13) b 2.14 (±0.22) a 2.21 (±0.75) a b. subtilis 1.37 (±0.18) a 1.22 (±0.31) a 1.43 (±0.86) a control 4.05 (±0.93) b 4.89 (±1.16) b 5.12 (±0.99) b table 2 efficacy of three biopesticides using against raspberry midge cane in samokov means within each column followed by the same letter are not significantly different, duncan’s test (p<0.05). treatments products/active ingredients number of midge larvae/split (day after treatment) 3rd (±sd) 7th (±sd) 12th (±sd) first generation neemazal® t/c (azadirachtin a) 3.15 (±1.11) a 2.13 (±0.82) a 2.94 (±0.54) a sineis 480 sc® (spinosad) 6.05 (±1.32) b 4.74 (±1.08) b 5.38 (±0.67) b b. subtilis 2.92 (±0.97) a 2.39 (±0.17) a 2.86 (±0.91) a control 10.21 (±1.24) c 12.17 (±1.98) c 14.71 (±1.15) c second generation neemazal® t/c (azadirachtin a) 4.86 (±0.46) b 3.77 (±0.21) b 3.58 (±1.06) a sineis 480 sc® (spinosad) 5.14 (±1.11) b 3.98 (±0.87) b 3.43 (±0.35) a b. subtilis 2.63 (±1.07) a 2.05 (±0.57) a 2.73 (±0.97) a control 8.73 (±0.48) c 10.84 (±1.54) c 13.22 (±1.18) b mohamedova field evaluation of three biopesticides for control of the raspberry cane midge in bulgaria 187 4. discussion and conclusions r. theobaldi was described by theobald in 1920 (barnes, 1926). since then, almost a century later, it has become a pest of economic importance of raspberry crop throughout europe. the midge cane is widely distributed in bulgaria, greece, rumania, italy, f r a n c e , i r e l a n d , u k , s w e d e n , c z e c h r e p u b l i c , slovakia, hungary and poland. in these countries the insect has been introduced mainly with infested planting materials and somewhere with infested soil. when establishing a new raspberry plantation, it is critical to choose the cultivar that is well adapted to local soil and climatic conditions and it is less susceptible to infestation by the raspberry cane midge, as well. normally, infested raspberry plants have demonstrated the symptoms of dark brown, clearly defined spots in the canes 3 to 5 weeks after laying the eggs (stoyanov, 1963). for this reason, it is important to determine the most appropriate timing for control of r. theobaldi. the treatments have to be done before the larval feeding sites become visible. organic production of raspberry in many countries, including bulgaria, is a challenge because of lack of products for plant protection, which have nonchemical origin. moreover, in bulgaria there are no officially registered biopesticides or even chemical insecticides, that can be specifically used for control of the midge cane. meanwhile, neemazal® t/c has been registered for control of tomato borer, tuta absoluta, meyr. considering this need, the present study met its objective the results showing the possibility of biological control of the r. theobaldi. the tested biologically based preparations demonstrated their efficacy against the larvae of midge cane. the pest had four generations in the plantation at lower altitude (bogdanovo) and three generation in the plantation at higher altitude (samokov). further, the data from the pheromone traps showed that the first generation had the highest population density in both raspberry plantations. the forth and the third generations in both sites showed the lowest population density. according to this information, the time and the number of treatments were determined. among the tested biopesticides, neemazal® t/c and b. subtilis demonstrated the highest efficacy, causing up to 82.5% (the former) and 81.2% (the later) reduction of number of midge larvae in the splits compared to the control. sineis 480 sc® demonstrated slower initial effect than neemazal® t/c and b. subtilis, but comparatively high efficacy, causing up to 63.26% reduction in number of larvae in the splits compared with the control. in fact, the present evaluation is the second attempt to apply only environmentally safe products for control of r. theobaldi. the first one was made by shternshis et al. (2002). the authors tested the preparations based on bt (bacticidae®), and s. avermitilis (phytoverm®) for control of the raspberry midge blight and reported significant reduction of disease complex severity compared with the control variants. s. avermitilis is the base of spinosad, the active ingredient of sineis 480 sc®. spinosad as an active ingredient of the product audienz®, was tested by barrofio et al. (2011) against the raspberry midge cane. the result from this experiment is ambivalent, showing comparatively high efficacy against the midge larvae, but not significantly differ compared to the control. in this study spinosad showed to be less effective to the raspberry cane midge compared with both, azadirachtin a and b. subtilis. the result is interesting, because the spinosad penetrates translaminarly in plant tissues and this suggests higher efficacy compared with the other tested products. deleva and harizanova (2014) stated the rapid initial effect of spinosad against the larvae of tomato borer, t. absoluta. the authors reported 73.33% larval mortality in tomato leaves at 3rd day after treatment. neem-based products have been evaluated for their efficacy against the different pest on berry plants. kim (2014) reported insufficient activity of azadirachtin against the rednecked cane borer, agrilus ruficollis f. on blackberries in usa. contrary, aguilera et al. (2009) commented the high efficacy of neem against the raspberry weevil, aegorhinus superciliosus g. in chile. the authors reported significant embryogenesis inhibition after applying the neem. the lowest number of larvae in raspberry canes observed in this evaluation is probably due to affect of azidarachtin on both larvae and adult of raspberry cane midge. the results obtained after application of, b. subtilis indicate that the bacterium might be considered as an effective agent for control of r. theobaldi. b. subtilis was originally isolated from the soil and has been tested as a biocontrol agent of root-knot nemat o d e , m e l o i d o g y n e a r e n a r i a o n t o m a t o (mohamedova and samaliev, 2011). the bacterium is able to colonize successfully the rhizosphere of the plants and affect different pathogens in this zone. this suggests that b. subtilis could influence the pupae of the raspberry cane midge in the soil. i have not observed any phytotoxicity or negative adv. hort. sci., 2017 31(3): 183-189 188 influence of the three biopesticides on raspberry plants and beneficial insects. in several raspberry canes collected from the plantation in samokov was observed parasitized 4th instar midge larvae of the third generation (fig. 2). the midge larvae probably were infested by the parasitic larvae of aprostectus genus. therefore the results of the present evaluation show the possibility of the tested biopesticides to control of the raspberry cane midge. these products are a good alternative to chemical insecticide and might be successfully integrated in the control strategies of r. theobaldi in both, conventional and organic raspberry production. further research should focus on screening the pesticides and bioagents, which could be able to control the disease complex “midge blight”, causing very often the dead of raspberry plants. acknowledgements the present study was partially supported by association of raspberry growers in bulgaria. the author would like to thank to s. velikova for her kind help and the growers who allowed access to their plantations. i am grateful to prof. svetoslav bobev for his comments concerning the “midge blight” symptoms and his advice and experience during the process of writhing of this manuscript. references agrostatistics, 2015 fruit production. ministry of agriculture and food, sofia, bull. no., 289. aguilera p.a., zampezzi v.m., araneda d.x., klein k.c., rebolledo r.r., 2009 azadirachtin effectivity in embryogenesis inhibition of aegorhinus superciliosus (guérin) (coleoptera: curculionidae). idesia, 27(1): 47-55. barnes h.f., 1926 the gall midges of blackberries and raspberries. j. pomol. hort. sci., 5: 137-140. baroffio c.a., mittaz c., bedard f., 2011 flight monitoring and efficacy trials against ressliella theobaldi in switzerland. iobc/wprs bulletin, 70: 45-49. c r o s s j . v . , b a r o f f i o c . , g r a s s i a . , h a l l d . r . , łabanowska b., milencović s., nilsson t., shternshis m., tornéus c., trandem n., vétek g., 2008 monitoring raspberry cane midge resseliella theobaldi w i y h s e x p h e r o m o n e t r a p s : r e s u l t s f r o m 2 0 0 6 . iobc/wprs bulletin, 39: 11-17. cross j.v., hall d.r., 2006 sex pheromone of raspberry cane midge. iobc/wprs bulletin, 29(9): 105-109. deleva e., harizanova v., 2014 efficacy evaluation of insecticides on larvae of the tomato borer tuta absoluta, meyrick (lepidoptera: gelechiidae) under laboratory conditions. agriculture & food, 2: 158-164. hall d.r., farman d.i., cross j.v., pope t.w., ando t., yamamoto m., 2009 (s)-2-acetoxy-5-undecanone, female sex pheromone of the raspberry cane midge resseliella theobaldi (barnes). j. chem. ecol., 35: 230242. kim s.-h., 2014 control of agrilus ruficollis (coleoptera: buprestidae) with insecticides and identifying visual attractants for use in a monitoring trap. phd thesis, university of arkanzas, usa. milencović s., tanasković s., sretenović d., 2006 monitoring flight of raspberry midge resseliella theobaldi (diptera: cecidomyiidae) by the pheromone trap. vii counseling on plant protection. zlatibor, 27 november 2 december 2006. book of abstracts, pp. 117-118 (in serbian). mohamedova m., samaliev h., 2011 effect of the rhizobacterium, bacillus subtilis on meloidogyne arenaria at different temperature. agric. sci. technol., 3(4): fig. 2 parasitized and nonparasitized 4th instar larvae of resseliella theobaldi in splits of raspberry canes collected from samokov raspberry plantation (september, 2016) mohamedova field evaluation of three biopesticides for control of the raspberry cane midge in bulgaria 189 378-383. pitcher r.s., 1952 observations on the raspberry cane midge (thomassiniana theobaldi barnes). i. biology. j. hortic. sci., 27: 71-94. pitcher r.s., webb p.c., 1952 observations on the raspberry cane midge thomassiniana theobaldi barne. ii. “midge blight”, a fungal invasion of the raspberry cane following injury by t. theobaldi. j. hortic. sci., 27: 95100. shternshis m.v., beljaev a.a., shpatova t.v., bokova j.v., duzhak a.b., 2002 field testing of bacticidae®, phytoverm® and chitinase for control of the raspberry midge blight in siberia. biocontrol, 47: 697-706. sipos k., madár s., markó, m., pénzes b., 2012 the possibility of automation of sex pheromone trapping: tested on resseliella theobaldi (barnes) (diptera, cecidomyiidae). afr. j. agric. res, 7(5): 1410-1413. stoyanov d., 1960 protection against the pests and diseases of raspberry in due time. ovostarstvo, 7: 24-29 (in bulgarian). stoyanov d., 1963 investigations on the raspberry cane midge thomassiniana thebaldi barnes in bulgaria. izvestija na instituta za zastita na rastenijata, gara kostinbrod, 4: 41-46 (in bulgarian). tanasković s., milencović s., 2010 monitoring of flight phenology of raspberry cane midge resseliella theobaldi barnes (diptera: cecidomyiidae) by the pheromone traps in western serbia. acta entomol. serbica, 15(1): 81-90. williamson b., 1987 effect of fenitrothion and benomyl sprays on raspberry cane midge (resseliella theobaldi) a n d m i d g e b l i g h t , w i t h p a r t i c u l a r r e f e r e n c e t o leptospheria coniothyrium in the disease complex. j. hortic. sci., 62: 171-175. williamson b., hargreaves a.j., 1979 a technique for scoring midge blight of red raspberry a disease complex caused by resseliella theobaldi and associated fungi. ann. appl. biol., 91(3): 297-301. 163 1. introduction cultivated olive (olea europaea subsp. europaea var. europaea) (green, 2002) ranks 21st among agricultural species worldwide and first among woody fruit species in terms of surface area (9.5 million ha), producing more than 3 000 000 t of virgin olive oil (fao, 2010) and more than 2 200 000 t of table olives per year (iooc, 2010). virgin olive oil, which makes up slightly more than 2% of total vegetal oil production, accounts for a return in the agricultural sector of more than 22 billion dollars, while the figure for palm oil (30% of vegetal oil production) amounts to a little more than 31 billion dollars (fao, 2010). olive cultivation worldwide has grown over the past 20 years by more than 20% in terms of surface area with increases noted on all continents. there has also been a doubling in production, which is more than proportional to the increase in surface area (fao, 2010). these increases have been due to improvements in cultivation technique in traditional plantings as well as entry into production of new, more rational plantings. the species is in general very efficient in exploiting environmental resources and can produce as much as 2 t ha-1 of healthy oil food. little more than 50 years have passed since olive first expanded beyond its traditional cultivation areas, and the species has shown to be flexible and adaptable in various agronomic, climatic and environmental situations, able to add value to extensive areas and to face the climate changes currently taking place. 2. taxonomy cultivated olive is an evergreen bush that can be, with suitable intervention, grown as a tree and it is considered typical of mediterranean flora. the taxonomic position of the species olea europaea l. has been reviewed (green, 2002), also in light of the data that has emerged from new technologies of molecular identification (besnard et al., 2002). the olea genus is divided into three subgenera and in the subgenus olea, sez. olea, the species olea europaea is grouped as a “complex” with potentially infertile forms, compatible for grafting, and characterized by the presence of flavonoid glucosides in plant tissues and fruit. the olea europaea complex is further divided into six subspecies: cerasiformis (island of madeira), cuspidata (from the south to northeast of africa and from southwest asia to the arid zones of the yunnan and sichuan in china), europaea (mediterranean basin as far as mesopotamia), guanchica (canary islands), laperrinei (hoggar high plain in the south of algeria as far as al jebel marra in the western sudan), and maroccana (southern slope of the atlas mountains in morocco). these subspecies can be considered “geographic entities” with notable molecular differences (for example cerasiformis and maroccana are polyploid) (besnard et al., 2007 a) but their morphologic characteristics are so similar that in certain cases descripoleiculture in progress e. marone*, p. fiorino** * dipartimento di scienze degli alimenti, università degli studi di teramo, piazzale aldo moro, 45, 64100 teramo, italy. ** dipartimento di scienze delle produzioni agroalimentari e dell’ambiente, sezione colture arboree, università degli studi di firenze, viale delle idee, 30, 50019 sesto fiorentino (fi), italy. kew words: aging, branching, chilling requirement, cold resistance, flower buds, planting density, triglycerides. abstract: the present work evaluates the limits and possibilities of development with regard to the most recent olivegrowing techniques in light of up-to-date knowledge of species characteristics. after a brief introduction regarding the productive capacity of olive, the new taxonomic position of the cultivated species and a reorganization of the genus olea is presented in the first part of the work. examination follows of the assumed stages of domestication, spread (from the bronze age until decline in the 6-10th centuries a.d.) and then globalization of the species from the 19th century until the present. the second part addresses the spread of olive to the different continents, environmental limitations to its cultivation and the growth model that distinguishes it from most of the other cultivated woody species. the various problems that arise when olive is cultivated outside its areal of origin are considered, from induction processes to effective chilling requirements, as well as the effects of climatic environment on plant growth and product quality following shifts in areal. the paper concludes with a brief analysis of open questions relative to new models of cultivation. adv. hort. sci., 2012 26(3-4): 163-175 received for publication 28 january 2013 accepted for publication 15 february 2013 164 tions can overlap. this fact justifies past imprecision in classifications. many entities which in the past were considered to be separate species, including progenitors of cultivated olive such as o. crysophylla and o. ferruginea (simmonds, 1976), have been placed within the subspecies o. europaea cuspidata. based on this classification, the areal of the olea europaea complex occupies three continents: starting from south africa it crosses the central part of the continent and the horn of africa, from egypt and the red sea it reaches the mediterranean and toward the west into the islands of macaronesia (island of madeira and the canary islands), while in the east it passes through palestine, syria, mesopotamia and western and eastern zones of the himalaya chain as far as southwestern china. 3. origin and domestication of the species due to a scarcity of fossil evidence, it is difficult to determine the geologic period during which this complex taxa became defined and began to evolve. it is believed that both a floristic element of african paleotropical origin and the long evolutionary history of this group spanned a large part of the tertiary (besnard et al., 2009). it seems that the genus olea had its origins in the oligocene and that numerous taxa diversified thanks to catastrophic climatic and tectonic events which were characteristic during the tertiary (from the great glacial period of the oligocene to the raising of mountains in eastern africa and desertification of the sahara). a study on the phylogenesis of the olea genus, conducted by besnard and coworkers (besnard et al., 2007 b) using plastidial dna and nuclear ribosomal dna as biological clocks, positions the principal phylogenetic nodes for determining articulations of the genus from the oligocene (c. 59.2 mya) until the pliocene (c. 4.4 mya), by which time the main taxa of the genus had clearly separated. the desertification of continental africa seems to be correlated to subsequent differentiation of taxa adapted to arid conditions. the new dry environments may have favored establishment of foliar morphotypes in olea. recurrent segregation and hybridization events could very well have been caused by geographic barriers (e.g. the sahara) and land bridges. probably until the end of the last ice age (15 000-12 000 b.c.) the distribution of olive was prevalently in the area of africa where desertification led to isolation of saharan populations (subsp. laperrinei). only later were the southern coasts of the western mediterranean colonized through the spread of glacial refugia. thus, spontaneous olive in the western mediterranean would be of african origin, redistributed as far as the coasts of spain, france and the tyrrhenian portion of italy after the last glaciations (besnard et al., 2001). the subspecies o. europaea europaea is divided into two botanical varieties: europaea, which corresponds to the old denomination olea sativa (weston) and includes the cultivars having seedlings called olivasters; and silvestris (mill.) which corresponds to the old presumed species olea oleaster (hoffman and link), spontaneous olive or oleasters. the wild oleasters forms represent the original postglacial mediterranean population. in the area between ancient palestine (zohary, 1994; zohary and spiegel roy, 1975) and the caucasus, the earliest plants adapted to the needs of proto-cultivators (around the 5th millennium b.c.) were individuated, utilized and multiplied. considering the ease of agamic reproduction and longevity of the plant, it is possible that few generations of crosses divide contemporary cultivars from these not-sodistant progenitors. as colonizers advanced westward, carrying with them plant material and expanding cultivation, they individuated and utilized autochthonous material, progressively diversifying the genetic base of their cultivars and at the same time enriching the local forms. generally speaking, significant molecular differences exist between the oleasters of eastern and western zones of the mediterranean (breton et al., 2006 a) and it is possible to identify separate data groups with a separation line that passes through the adriatic sea and the libyan desert (breton et al., 2006 b). from the cited work, the cultivated genotypes seem rather dispersed which can point to a mixing among the various markers, suggesting repeated attempts at domestication and subsequent crossing among cultivars of different origins. evidence of these events are illustrated by hannachi and coworkers (hannachi et al., 2010): comparing plastidial dna of traditional tunisian cultivars and wild tunisian forms, they demonstrated that seven of the 15 cultivars currently under cultivation in that country are of oriental origin, while the others are linked to wild material of maghrebian origin. even if the center of domestication for olive was in the eastern mediterranean, pre-domestication episodes seem to have occurred more or less at the same time, starting from the 5th millennium b.c., in various areas around the basin, for example on the island of crete and southern spain. in these areas “stratifications” have been found with cultivated olive seeds overlaying oleaster seeds, as well as the contrary with “imported” seeds from cultivated forms supplanted by autochthonous forms (terral et al., 2004). initially olive was used for different purposes compared to its modern use: it was employed for shade, to produce forage and firewood, and to make tools such as poles and staffs; it took thousands of years of cohabitation to arrive at use of the fruit and oil. when cultivation advanced beyond the phase of simply collecting spontaneous fruits, the proto-farmers decided it was more practical, in a preliminary form of cultivation, to group together the best examples from spontaneous forms through agamic propagation by part of stump. this multiplication technique remained in use until the 1970s in many traditional olive-growing countries of the southern mediterranean. 165 4. the spread of cultivation in the beginning, direct use of the fruits must have been limited since the drupes are very bitter, even when extremely ripe, due to the presence of elevated quantities of oleuropein and other phenolic glucosides, and the technology required to remove the bitterness and for brining was too complex for the means available at the time. however, the oil could be extracted through milling using implements similar to those used for grains, making it available for medicinal use, as fuel, for illumination (both sacred and not) and as an unguent. the use of olive oil in the diet came later in this plant’s history, around the first half of the 2nd millennium b.c., when use of this product spread via sea routes first from the eastern mediterranean and aegean sea toward greece, then thanks to the phoenicians along the coast of africa toward the west as far as (and beyond) the pillars of hercules. it is probable that with this progression, attempts at domestication by the inhabitants of the various zones brought advantages. it should be no surprise that ample deposits of knossos oil have been found, which testify to the presence of active olive oil commerce in the minoan period (second half of the 2nd millennium b.c.), nor is transportation of oil from spain toward carthage a surprise as cultivation on the iberian peninsula probably dates back farther than evidence and documentation demonstrate. at the beginning of the 1st millennium b.c. olive cultivation and oil use had reached the various coasts of the mediterranean and the type of “domestication” of the plant is revealed through differentiation: the greeks, as far back as the oldest writings, distinguished the oleaster as κότινος for wood usage from ελαία for oil production. according to pliny, the romans didn’t know olive cultivation until the time of tarquinio prisco (6th century b.c.) but in the 1st century a.d. oil from the italian peninsula was exported to the provinces of the empire. in rome, oil was principally used for external treatment of the body. as the saying went, “wine on the inside, oil on the outside” (intus vini fori olei)1 between the 2nd and 4th centuries the spread of this plant reached its greatest development in the entire mediterranean region. oil was distributed free of charge to the plebs of rome as a food source and sent to the legions in germany. the trade surrounding oil was so important that remains from the jars and amphorae in the port of rome of the time left a hill which is today one of the recognized neighborhoods of the city (testaccio). 5. globalization of cultivation with the fall of the western roman empire in 476 ad and loss of control of the sea routes, trade and use of olive in the western mediterranean declined rapidly. evidence 1 plinio, storia naturale, xiv, 150. of introduction of oil from northern africa in the 6th century can be found (brugnoli and varanini, 2005) but from the 7th century sea transport of oil toward rome ceased, at least in an organized way, to exist. at the same time a period of instability in north africa began, marking a generalized abandonment of olive cultivation in the region; it did not pick up again until after the arab conquest around the 10th century. in europe, olive oil acquired new importance through the christian religion, and for which substitutes were not possible (e.g. for illumination of altars, anointing of the sick, use of plant oils during lent or other periods of abstinence from foods of animal origin). for this reason, cultivation was undertaken in some areas considered marginal in terms of climate for the cultivation of olive (northcentral italy until the alpine valleys) as a way to at least guarantee the needs for churches and monasteries. olive growing zones along the coasts receded with the fall of commerce and only the oldest cultivation areas remained important: palestine, syria, and the island of crete provided oil for venice and constantinople while andalusia furnished muslim areas. the olive oil trade was revived in the 11th and 12th centuries thanks to merchants from genoa and venice. they supplied monasteries, cities in italy, france and constantinople with this precious product needed not only as a food source but also for religious and liturgical purposes, illumination, and soap and wool production. an intense freeze in 1009 killed off the remains of ancient olive growing on the italian peninsula along the entire adriatic and in particular in puglia. subsequently this region experienced a notable increase in olive cultivation with new plantings, at the expense of grain cultivation, thanks to the development of trade by the venetians. in this period apulian markets were open to traffic from venice, genoa and byzantium, thus progressively establishing this area as an important zone for olive cultivation (iorio, 2005). even today puglia accounts for the most extensive and productive portion of italian olive production, with more than 20 million trees of 300-500 years of age. after about 1300, olive oil (for illumination and soap and wool production) became one of the most important products necessary for industrial development in northern italian cities. as it was not possible to rely exclusively on commercial trade, it became necessary for the economies of many zones to provide incentives for cultivation, also in areas with extreme soil and environmental conditions. the 15th century represents a turning point not only in the history of olive but also for humanity. in 1453 constantinople fell, the last remnant of a civilization born with the cultivation of olive, while in 1492 two important events occurred: the fall of granada, the last vestige of muslim dominion in spain, and the landing of colombo on san salvador. the interest of europe shifted westward and with it went the olive, a colonizing plant par excellence. olive was introduced in spanish colonies, first in cuba around 1520 and then in california. the original plant material left the port of seville as seeds or seedlings and so 166 genetically the material can be considered as andalusian in origin. the spanish colonists carried olive with them as they travelled southward from california along the pacific, introducing it in peru and from there crossing to argentina. an olive plant considered to be more than 400 years old exists in the province of la rioja in argentina, which would place its establishment at the time of the first european settlements in that region when the capital, ciudad de todos los santos de le nueva rioja, was founded (1591) (fig. 1). during its trip from california to argentina, the genetic material of olive underwent further selection with identification of cultivars such as ‘mission’ (california) and ‘arauco’ (argentina), the 400-year-old tree belonging to this latter. with the spread of colonialism and the need to transfer plant productions able to satisfy industrial and dietary requirements in the colonies, also olive began to be valued, utilized and cultivated in various areas considered to be suitable. in 1661 dutch merchants carried olive to south africa where spontaneous forms of olea europaea cuspidata were already present. between the end of the 18th and beginning of the 19th centuries cultivated olive, together with cuspidata, landed in southern australia where the two subspecies found a favorable environment, giving rise to a vast phenomenon of spontaneity (breton et al., 2008). the first documented introduction dates back to 1800 when olive officially arrived in sydney (spennemann, 1999). from then until before the second world war, olive from various provenances was introduced (from spain, italy, greece and later israel). starting in 1956 there was interest in olive cultivation in china as well, where the plant was known but its cultivation was difficult due to intense summer rainfall. currently, the cultivation area of olive is spread in both hemispheres between 45° and 30° of latitude with extension toward the warm-temperate zones of our planet. the agronomic success of this species is based on two fundamental characteristics that distinguish olive from all other cultivated fruit species: its particular growth model and flower formation make it easy to manage and the reliability of its production have been determinant for its cultivation since neolithic times. it is adaptable to highly variable climatic conditions and its localities of origin for domestication are characterized by different conditions. 6. environmental limitations the limits for cultivation of olive are attributable to environmental factors and their annual cycles. beyond the degree of latitude (45°) for cultivation for this species, plants are potentially exposed to damage from cold temperatures that can compromise production or even the life of the plant, depending on intensity and timing of the event. cultivated olive is only moderately tolerant of low temperatures and even if it is suitably prepared for winter cold (acclimatization), it can resist only a few degrees below zero due to a protection mechanism (supercooling) that keeps water in cells in the liquid state down to several degrees below the freezing point (table 1). the most resistant tissues are the bud meristems (in ‘ascolana tenera’ the recorded lethal temperature is -19.3°c) (fiorino and mancuso, 2000), so that vegetation severely damaged by fig. 1 old picture (dated around 1950) of the oldest ‘arauco’ plant in argentina. table 1 lethal temperature (°c) as evaluated by differential thermal analysis for various organs in two acclimatized olive cultivars having different resistance to cold: ‘ascolana tenera’, very resistant; ‘coratina’, poorly resistant part of plant lethal temperature (°c) ‘ascolana tenera’ ‘coratina’ leaves -14.5 ± 0.2 -11.8 ± 0.2 shoots -18.6 ± 0.6 -12.6 ± 0.4 buds -19.3 ± 0.6 -13.5 ± 0.4 roots -9.1 ± 0.3 -8.6 ± 0.3 from fiorino and mancuso, 2000. 167 intense winter freeze can copiously resprout in the following spring from latent and adventitious buds, in particular on the stump. differences exist among the cultivars: comparing 21 prevalently italian cultivars for their resistance to cold, employing three different evaluation methods in leaves and sprouts, the authors (azzarello et al., 2009) were able to divide the material into three groups in relation to lethal temperature. with regard to phylogenesis, and considering knowledge of other subspecies such as olea europaea subsp. cuspidata which is poorly resistant to cold stress, it is possible that the varieties cultivated today have reached their maximum result possible through genetic pressure applied by humans in terms of advancement toward colder regions. furthermore, due to its tropical origin, the species is characterized by a relatively high critical temperature (1012°c) (mancuso, 2000), thus pushing cultivation toward northern limits would result in a growing season that is too short. the highest latitude areas with olive cultivation are the alpine lakes in italy, istria (slovenia, croatia), and the olive-growing area of odessa (crimea, ukraine). the factors which limit expansion toward the equator are less evident. olive is a species that can support high temperature: the lethal temperature for leaf tissues varies from 46 to 50°c depending on the cultivar and can reach 52°c in sprouts (mancuso and azzarello, 2002). the greatest limiting factor seems to be the need by flower buds for a period of low temperature in order to pass from an “inductive” phase to that of tissue differentiation and development of the inflorescence. this rest phase is generally divided into two periods with very different thermal needs. in the first phase low temperatures (6-9°c) are needed to remove inhibition for subsequent growth, and in the second warmer temperatures (above 8.5°c) are required to accelerate evolution of the phenomenon. the currently used reference model for olive (de melo-abreu et al., 2004) is based on that of richardson to study periods of rest in peach (richardson et al., 1974). this model considers the period from 1 october to 31 january useful for overcoming chilling need, with 7.3°c being the optimal temperature. temperatures from 0 to 18.5°c can also be considered acceptable with a weighted effect of their distance from the optimal temperature, similar to the richardson model. the authors terminate calculation of the chilling units on 1 february, as they consider that for their study area (cordoba and mas bové reus terragona in spain, elvas and santarém in portugal) by that date the needs of olive for cold should already be satisfied. the authors underline that temperature is not the only factor toward which olive is sensitive and thus the data needed for this calculation should be verified every time there is a change in cultivation zone or variety. interest in the effect of low temperatures for normal development of flower buds is relatively recent: only in the 1950s was it proposed that the amount of flowering is in some way linked to the duration of low winter temperatures (hartmann, 1953; hartmann and porlingis, 1957). in the beginning researchers (hackett and hartmann, 1964) believed that the role of low temperature was more incisive and able, together with other environmental factors, to influence flowering processes from the first phases of induction, but later it was demonstrated (rallo and martin, 1991) that its role is limited to development subsequent to the induction phase. there are however ample areas of research that have only barely been considered. for example, in 1975 it was noted in a study (hartmann and whisler, 1975) that: 1) by applying a suitable period of cold, flowering can be stimulated in any time of the year (this somehow confirms the theory of “aging” of apical meristems that when mature produce buds able to directly develop flowers) (fiorino and marone, 2010); 2) there are ample differences between cultivars in regard to their response to various quantities (constant or variable) of low temperatures (varietal differences represent the weak point of all experimentation in this field); and 3) thermal thresholds of response seem extremely various among cultivars themselves as evidenced by the behavior of local cv. mission which can, by flowering continuously throughout the cool summer typical of the california coast, lead progressively to a modest number of inflorescences. in 1983 a work was published (denney and mceachern, 1983) aimed at improving understanding of responses of this species to various environments. the results were obtained by elaborating temperature data (october-may) from 15 olive-growing stations to determine the effect of the “cold”, measured as the capacity of the olive plant to flower in the subsequent spring, when two conditions have been met: 1) active growth has concluded; 2) daily temperature trend does not exceed an average of 12.5°c. the greatest interest with regard to this study concerns the concept of “effective chilling” which indicates, for regular flowering to occur, the number of days when the average temperature must not drop above 12.5°c. in order to satisfy the thermal needs of olive, 70-80 days of “effective chilling” are necessary, thus setting geographic limits for the species. a comparative analysis of the thermal conditions of some geographical zones at the warmer limits for cultivation (ico, peru and caborca, mexico) and where olive growing is undergoing development (gran chaco, argentina) (ayerza and sibbett, 2001) points out that often in “new”, “warm” olive-growing areas the number of days for wintering are fewer than those considered necessary (70-80 days). for example, at ico (peru) the possibility for wintering does not exist as average daily temperatures are never below 12.5°c, although there are cultivars that produce well enough in the area to allow commercial plantings. the area is characterized by a lack of rainfall despite frequently cloudy skies, making it possible to control growth through drastic reductions in irrigation water which oblige the plant to reduce or interrupt vegetative growth, despite year round temperatures that are relatively high. the authors of the study admit that the reasons for this 168 behavior are not clear and hypothesize that in the autumnwinter period, as the sky is almost constantly overcast, the thermal lows needed for the buds are attained, although perhaps they wouldn’t be in sunny areas. sensitivity to different thermal thresholds or chilling needs to bring about flowering is appearing in new plantings, in areas considered to be homogeneous with consistent seasonality, but where there are specific zones with cold periods of different duration and intensity. for instance, this can be the case in the mediterranean basin where notable differences are recorded for florence (central italy), seville (southern spain) and cairo (egypt). the millennia-long history of olive has made it possible to identify local cultivars perfectly suited to specific environments, thus creating varietal standards able to adapt themselves to the thermal trends of that particular area. in recent olive-growing, cultivars famous in a specific territory have often simply been used elsewhere, although this has led to unsuccessful attempts generally due to a lack of adaptability to the relative high winter temperatures of the new environment. examples of this are the negative results obtained from the transfer of ‘frantoio’ (cultivar from tuscany) to southern coasts of the mediterranean or warm areas of the planet. this sensitivity of the plant to specific thermal requirements for flowering confirms the fundamental role of temperature in all aspects of the life of olive, from the progression of phenological phases to the biochemistry of the oil. there are notable differences among mediterranean germplasm with regard to the effective chilling requirements of autochthonous material which do not necessarily depend on where the material was selected. thus, two varieties selected in similar environments and at approximately the same latitude, for example ‘arbequina’ and ‘frantoio’, have demonstrated opposing behaviors when cultivated in warm areas: the former flowers copiously and early, while the latter rarely flowers much and often not at all. in olive, the effect of low temperature is more complex than in temperate species which are often referred to: for some varieties the “chill” that results from diurnal temperature variation is sufficient to permit normal flowering (e.g. in ‘arbequina’) and the thermal threshold of chilling must be rather elevated since nocturnal temperatures of less than +2 to -1°c markedly diminish flowering under controlled conditions (malik and bradford, 2009). 7. current trends over the course of the millennia of expansion (4th-2nd millennia b.c.) and cultivation (1st millennium b.c. 2nd millennium a.d.) the techniques for growing olive remained essentially unchanged, just as the needs of agriculture to have long-living, hardy, easily managed plants able to produce oil for nutrition, illumination, industries (textile and mechanical) and other purposes did not change over time. it was only in the 19th century that olive-growing began to gain advantage from technological development, in particular with regard to oil extraction, while agronomic techniques remained anchored in acquisitions and requirements from the past. research pertaining to olive-growing was only blandly affected by the innovative spirit that invested agriculture and in the first half of the 20th century knowledge based on millennia-old, empirical observations persisted or models and concepts were adapted that pertained to other cultivated woody species, for example fruit-bearing species from temperate zones. at the turn from the 19th to 20th centuries, in europe olive oil was considered a strategic food but it was only in the middle of the 20th century that the true importance of this product for human nutrition was understood in terms of its nutritional, organoleptic and functional profile. with economic and geographic expansion of olive cultivation in the second half of the last century came awareness of the peculiarity, potentiality and lack of scientific knowledge about this ancient species. olive possesses all the fundamental requisites to become a modern crop: good productivity (more than 2 t ha-1 per year of oil), very early entry into production for plantings that are well positioned in terms of water and nutritional availability (on average the third year after planting), good adaptability to diversified environments and availability of light, soil and other resources, good availability of plant material that can be obtained using new techniques (mist-propagation and micropropagation), and great flexibility of plant material for adaptation to various breeding systems, all depending on the various destinations of the product (for oil or the table) and in relation to the needs of the market. 8. architecture of the plant olive owes its agronomic success to the longevity of plantings, simplicity of its constitution and reconstruction of the canopy, and predisposition for flowering. in nature, the plant grows as a bush with a stump rich in vegetative meristems able to produce suckers that can form trunk-like structures able to survive for hundreds of years; it can grow to have canopy heights and diameters of 10-12 m. the olive model is not greatly described in literature. each vegetative meristem on the stump (or positioned plantlet) can give rise to a sprout with acrotonous, orthotropous attitude and continuous growth that will form the vegetative axis. early on, this sprout is poorly lignified, slender in relation to its height and tends to curve if not supported. at its point of curvature (the new tip of the branch), another vegetative meristem grows and repeats the cycle, while the distal portion of the original branch continues his growth in a lateral direction. with lignification and subsequent radial growth, the insertion angle of the two consecutive, opposing sections attenuates and the structure takes on the form of a single trunk resulting from fusion of the segments (fiorino et al., 2012). the originally-vegetative apical meristems, over time and with growth, mature, change in function, lose their orthotro169 pous characteristics, become plagiotropous and take on reproductive functions (“aging”) (fiorino and marone, 2010) with subsequent formation of fruit-producing vegetation. this particular type of growth and shift to production makes olive a very flexible plant as it can be grown either with a central axis and short branchlets arranged at various heights or with varying vase-like forms, obtained via subsequent vegetative axes, having or not a central trunk. having these characteristics, the parts of the canopy destined to support the vegetation are semi-permanent structures that periodically need to regenerate the growth-agingflowering cycle typical of olive branches. this growing habitus simplifies pruning principles and operations. 9. induction and differentiation growth and lengthening of the branch in subsequent years is almost completely delegated to apical meristem activity, creating a continuous linear structure (branchlimb) with persistent (three years), opposite leaves and lateral flowering that allows further apical growth. the branch-limb complex is made up of a succession of nodes generated by an evolving meristem (vegetative vs. reproductive) that will determine bud functions. the growth-aging process continues until a progressive weakening leads to the loss of the apical meristem or until itself transforms into a flower (fig. 2). two types of buds coexist at each node. the main, most evident ones that can remain on the plant for no more than two vegetative cycles, and at least two accessory buds positioned above the main one (fig. 3). these accessory buds are poorly visible and often covered after their formation by cortical tissues (and therefore also called hidden buds) of the growing branch to the point of not even being considered (lavee, 2007), and are destined to become latent and able to burst many years after their formation. the evolutionary phases of a flower meristem are not distinguishable to the naked eye before the burst of the buds (barone and di marco, 2003; andreini et al., 2008) and sometimes, even under morpho-anatomical analysis, some early manifestations of differentiation are ambiguous and difficult to interpret (troncoso, 1966). histological and histochemical indications point out that: a) there are histochemical differences in the formation and development of lateral buds in different genotypes (e.g. ‘leccino’ and ‘puntino’); b) there are differences in zeatin levels in buds of the same cultivar (e.g. ‘leccino’) taken from plants with different fruit loads and these hormone level differences start early, from the month of july (andreini et al., 2008); c) there are differences in development (timing and forming structures) among homologous buds removed from rising branches or identified in peripheral parts of the canopy and these also start early, from the month of july (fabbri and alerci, 1999). it was believed (lavee, 2007) that in growing sprouts the principal buds of originally mixed function could shift toward flower formation only when a specific sequence of events occurred, which are successive (preinductionconfirmation), temporally separated and controlled by endogenous and exogenous factors. more recent research has indicated the primary role of apical growth in the formation of fruit-bearing vegetation and in the evolution that is determined by growth. in order to reach adequate levels of aging, in relation to the cultivar, buds with defined function flowers for the principal buds, vegetative for the accessory buds form along the fig. 2 terminal bud transformed in floral grape in cv. koroneiki (photo e. marone). fig. 3 node showing the two main buds producing floral grapes and in upper position the two accessory buds (photo e. marone). 170 lengthening sprouts (fiorino and marone, 2010; marone et al., 2013). in this way, and possible due to an adequate intensification of cultivation, the amount of annual growth of mature branch-limbs increases leading directly to an increase in the number of flower clusters and thus fruit load on the branch. the fruits, due to their weight, pull the vegetation downward forming fruit-bearing cascades that are particularly suitable for mechanical harvesting by horizontal shakers (fiorino et al., 2010). 10. adaptability due to olive’s adaptability to xeric environments, currently expansion of cultivation takes place with success mainly in warm-temperate dry zones below 30-35° latitude in both hemispheres: these areas, where land is available, are characterized by short winters, early-spring temperature rise and hot, sunny summers, all factors which affect the quantity of growth, progression of phenological phases and oil characteristics as particularly influenced by temperature, according to results presented in the literature. in a study carried out at one location (montepaldi, tuscany, 43° 40’ n lat., 11° 09’ e long., 266 m a.s.l.) to evaluate the behavior of cultivars selected over time from areas having different climatic conditions (mancuso et al., 2002), analysis of the relationship between variations in annual climatic conditions and development of phenological phases revealed the following roles played by temperature: a) in the determination and evolution of the various phases together with different behavior of cultivars coming from different areas. cultivars ‘coratina’ and ‘carolea’, selected from southern italy where spring temperatures are warmer, have more accelerated bud burst and phenological phases up until fruit set compared to cultivars (‘moraiolo’ and ‘leccino’) selected in the cool, test area; and b) in the appearance of phenological phases in relation to average increase in temperature and duration of insulation. from a data pool pertaining to five cultivars (the four previously mentioned plus ‘picholine languedoc’) it was found that an increase of 0.5°c leads to an anticipation of flower bud burst by more than three days, with notable effects from the time of pit hardening. in addition, an increase in average insulation seems to increase the speed with which these phenological phases progress. the influence of spring temperatures on the date of flowering in different cultivars was confirmed by de melo-abreu and coworkers (de melo-abreu et al., 2004). more recently, orlandi et al. (2012) found that pollen release was generally determined by meteorological factors in the period before flowering, with effects on both pollen amounts and timing of flowering (early or delayed). the role of temperature in oil composition is more extensive. independently in each cultivar, temperature trend during fruit growth and maturation influences the ratio among fatty acids of the triglycerides in the oil (fiorino and ottanelli, 2003; marone et al., 2003) with a reduction of the percentage normally expected for oleic acid in years characterized by prolonged periods of elevated temperature. with increased average seasonal temperatures (expressed as thermal sums, gdh, with 10°c threshold) (software for the calculation of gdh with variable thresholds was elaborated by marone, 2003), the percentage of oleic acid content drops and is substituted by increases in palmitic and linoleic acid percentages. employing a collection of italian germplasm, with plants grown in the same environment (mirto, calabria) and with adequate agronomic techniques, and through analysis of the variations between levels and ratios of the three principal fatty acids (palmitic, oleic, linoleic) and the thermal sums values of gdh for the relative years, it was possible to measure the existing regression between thermal sums and levels of oleic acid percentages in the oils obtained from the cultivated accessions (fig. 4) (lombardo et al., 2008). each cultivar constantly adapts its triglycerides composition, modifying equilibriums and increasing palmitic or linoleic acid (lombardo et al., 2008). the graph shown in figure 5 illustrates the division of a population of cultivars into three clusters separating samples with different characteristics. the first, the most numerous, groups the cultivars that compensate for the reduction in percentage of oleic acid with linoleic acid and subordinately palmitic acid (cv. canino, nera di gonnos, tonda di cagliari and moraiolo). the second group reacts in the opposite manner (cv. raja sabina and moraiolo t. corsini), while only 5% of the tested population is composed of stable triglycerides (cv. nocellara messinese). the intensity of the response to temperature changes can be such that the oil from some cultivars fig. 4 regression between the % content of oleic acid in the tags of different cultivars and gdd (population 188 cultivars in total with at least two analytical data in the five years; p≤ 0.01 and r=0.473). on the left, the coolest year (2005), on the right, 2003 (the warmest, practically superposed with 2001) (from: lombardo et al., 2008). 171 approaches or exceeds the limits established by law in some countries with regard to triglyceride composition. working in the province of rome, italy (41° 53’ n lat., 12° 14’ e long., 30 m a.s.l.) on ‘arbequina’ and ‘arbosana’ plants grown as hedges under superintensive cultivation, the acidic composition of the oil was well balanced and characterized by elevated amounts of oleic acid and low or very low levels of linoleic acid in both cultivars (marone et al., 2009). however, when the same varieties were grown in warm, dry valleys in the northwest of argentina, with a prolonged growing period and high temperatures during the growing season, the acidic composition was modified: there was a drop in oleic acid and a rapid rise in level of linoleic acid (rondanini et al., 2011). due to this sensitivity to high temperatures during fruit growth and maturation, the need to review the limits for some parameters in the oil, as prescribed by international laws intended to verify the genuineness of the product, becomes necessary with the extension of olive growing in warm areas or the marketing of local products that had previously been destined for self-consumption. unadulterated productions can, for example, present not only variations in the ratios among triglycerides (often the amount of linoleic acid exceeds the allowed limit of 1%), but also alterations with regard to the phytosterols, with increases in particular in campesterol levels beyond established limits and which could indicate an addition of seed oils to the olive oil. there have been few observations of the result of transfer of cultivars from warm to cooler zones. in this case, an opposite phenomenon has been noted with an increase in oleic acid percentage (fiorino and marone, unpublished data); it is possible that special characteristics of some productions from cool olive cultivation zones derives precisely from the specific interactions between some cultivars and the various climatic zones. 11. open questions new plantings require the transformation of current cultivation techniques, which are still tied to millenniaold traditions. this shift calls for not only radical cultural changes, but also notable investment for establishment of new or modification of existing plantings as well as for machinery. the objective of new plantings is to produce oil, with a lengthening of the production chain from the fruit to the finished product extra virgin olive oil which remains unique among other vegetal oils on the market for the presence of complex antioxidants (i.e. polyphenols) that derive directly from this plant’s origins. for researchers and technicians who direct their efforts toward globalization of cultivation, the weak point remains the modest amount of basic knowledge available in order to respond to questions that arise regarding varietal adaptability to different environments, regularity of production and canopy management. in particular, with regard to the spread of new intensive or superintensive olive-growing models (terms linked to the elevated number of plants per hectare), there are a series of questions: what is the productive lifespan of orchards? what are the characteristics of the product? what is the varietal platform? the reduced productive lifespan of orchards represents the greatest difficulty to overcome for the spread of this model in countries where olive-growing is deeply rooted in culture and history. today, plantings last only a few years: about 10 for peach, 15 for apple and pear, perhaps 20 or a bit more for olive, with plants having performed their economic duty in this period of time. as for the characteristics of the product, research has pointed out that it depends more on cultivar/environment interaction than on the growth system. initial data seem to confirm that, in any case, the chemical and sensory characteristics of the oil do not change in intensive or superintensive plantings (marone et al., 2009). the varietal platform suitable for superintensive plantings is made up of few cultivars typical of specific areas (‘arbequina’ and ‘arbosana’, spain; ‘koroneiki’, greece). growth and productivity characteristics are known for the new planting models (rallo, 2006; tous et al., 2003) as well as the characteristics of the product (marone et al., 2009). novelties, such as tosca 07® and chiquitita® (redacción olint, 2007; rallo et al., 2008), have been more recently introduced in new plantings and still need to be better defined agronomically. the longevity and vitality of olive, and the marginality of its cultivation and production have penalized genetic improvement of this plant: it was undertaken unconsciously in the past but has been more targeted in the last century. the ideotypes must combine different characteristics (fiorino, 1999). in addition to growth habit, fructification, fig. 5 fuzzy clustering in a ternary plot (fuzziness f= 2.25), utilizing the absolute value’s differences for the two compared years between 69 cultivars for saturated, monounsaturated and polyunsaturated fas (from: lombardo et al., 2008). 172 and resistance to biotic and abiotic stress, they have to respond to precise composite characteristics for oil which indicate the nutritional value for humans and are by now used internationally for marketing of the product. despite the existence of a broad genetic base, with a total of more than 1200 cultivars in germplasm collections (bartolini, 2008), there is little reliable information available about genetic determination of traits and their heredity (bellini et al., 2003) and this lack makes it difficult to choose the genetic material to use for crossing programs. a recent review (rugini et al., 2011) about the major needs for genetic improvement of olive, indicated systems and aim to achieve different goals in this species, but at present it often ends up being based simply on the phenotypic behavior of parental lines. also variability in the triglyceride and unsaponifiable fraction in descendents from crosses with the same parents was wide (tables 2-4) and sometimes with percentages of some fatty acids outside normal values (fiorino, 2001). the table 2 reports the values for fatty acids, polyphenols and tocopherols in oils obtained from eight genotypes of open-pollinated ‘arbequina’ grown in the same environment. the lineage is characterized by great variability in acidic composition, above all for linoleic acid contents. it has only been in the past ten years that indications regarding the technological characteristics of the various germplasm collections are present in terms of the qualitative and organoleptic characteristics of the oil (rotundo and marone, 2002). furthermore, characterization of the oils produced by single cultivars in specific environments has begun in this past decade as well (cimato et al., 2004; di vaio, 2012). new plantings call for control of canopy dimensions, and as most varieties selected over the millennia are very vigorous, new input has come from selection of clonal rootstocks able to influence growth and vigor in olive. by using rootstocks from specific varieties (‘tosca 07® and ‘leccino dwarf’) vigor and architecture have been modified in plants of ‘cerasuola’ highly vigorous and ‘biancotable 2 oil characteristics of genotypes obtained from open-pollinated ‘arbequina’ (ghiza, egypt) genotypes fatty acids (%) 16 52 56 61 67 68 94 105 c 16:0 10.7 14.2 14.2 16.2 19.3 14.9 15.7 18.6 c 16:1 1.0 0.6 1.2 2.2 3.4 2.0 2.1 3.4 c 18:0 2.5 2.1 2.3 2.2 1.8 1.7 2.5 1.7 c 18:1 75.3 66.7 50.7 60.6 42.0 45. 9 61.1 58.5 c 18:2 8.5 14.2 29.3 16.6 31.7 33.3 16.6 15.7 c 18:3 0.7 0.9 1.3 1.2 0.9 1.2 1.0 1.0 tocopherols (mg/kg) 416 168 219 366 286 347 91 420 polyphenols (mg/kg) 34 165 67 51 77 91 50 101 from fiorino, 2001. table 3 values for the most important fatty acids in oil from crosses of ‘arbequina’ fatty acids (%) genotypes 127* 129* b28** c 16:0 13.7 11.7 19.6 c 16:1 0.9 0.8 2.4 c 18:0 2.1 2.3 2.1 c 18:1 66.1 76.6 50.2 c 18:2 14.7 6.3 23.3 c 18:3 1.0 0.8 1.2 * no. 127 and no. 129 = ‘arbequina’ x ‘aggezi shami’. ** b28 = ‘arbequina’ x ‘picholine languedoc’. from fiorino, 2001. table 4 oil characteristics of genotypes obtained from controlled crosses of ‘manzanilla’ x ‘picholine languedoc’ genotypes fatty acids (%) a2 a5 a4 a14 a18 a19 c 16:0 12.0 10.9 13.7 16.6 9.9 16.1 c 16:1 0.9 1.2 1.9 1.9 0.9 2.1 c 18:0 2.5 2.1 2.3 1.9 2.1 2.1 c 18:1 68.6 75.0 64.9 63.9 72.0 58.0 c 18:2 13.7 8.8 15.1 13.7 12.8 19.5 c 18:3 0.8 0.7 0.8 0.9 0.9 1.3 tocopherols (mg/kg) 173 185 215 177 214 318 polyphenols (mg/kg) 169 286 140 305 from fiorino, 2001. 173 lilla’ low vigorous (caruso et al., 2012). these results are very promising, even if they must be verified in the field and in different environments. 12. conclusions as with other woody fruit trees (e.g. citrus spp.) and prevalently herbaceous species (e.g. corn, soy, sugarcane, manioc), olive is rapidly expanding its territory into new areas, which in turn permits the latent capacities of this species to be expressed to an equal or greater extent compared to that possible in the species’ areas of domestication and millennia of cultivation. olive seems able to gain added value in some temperate-warm areas of the planet where otherwise unutilized desert zones could offer ample space. in these areas it would be possible to control growth and fructification through the water stress inherent to the climate as well as temporal space during the annual growth cycle to satisfy the modest chilling requirements of some cultivars. the current geographical limit toward the equator (30° lat.) does not seem insurmountable and, in particular conditions, the margins for maneuvering with regard to enlarging the areal still seem quite wide. the limits of this development are related to a lack of some background knowledge and uncertainties in interpretation of research data, especially with regard to control mechanisms for flowering, flower formation (inductive phase), and flower development (chilling requirements). olive oil has special characteristics that make it not only a food and condiment, but also a product able to protect the human organism from dysfunctions and pathologies, thanks to the presence of a number of components, and thus it plays an important role in a balanced diet. olive has a particular capacity 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in the second test, vocs emission for selected masses were monitored under increasing temperatures over time: at room temperature not heated oils (nh), 60, 90, 120, 150, and 180°c, respectively. the spectra were acquired using a proton transfer reaction time of flight mass spectrometer (ptr-tof-ms). the total vocs emission increased at 180°c, determined both by the rise of the amount of compounds present in the nh samples and by the formation of new masses generated by oxidative chemical reaction from triglycerides and fatty acids. from the set of results it is evident that a good control of the temperatures can be useful in reducing the quantities of masses potentially harmful to health in human food. 1. introduction a common worldwide operation extensively used in food preparation is cooking by immersion in hot oil, as for fried and deep fried operations. types of oils used for fried and deep frying operations vary with different cultures and are widely used both domestically and commercially. when frying and deep frying are used, fats and oils are carried at elevated tem(*) corresponding author: emarone@unite.it citation: sabbatini l., taiti c., redwan m., azzarello e., marone e., mancuso s., 2018 monitoring in real time the changes in vocs emission in sunflower and extra virgin olive oil upon heating by ptr-tof-ms. adv. hort. sci., 32(1): 149-153 copyright: © 2018 sabbatini l., taiti c., redwan m., azzarello e., marone e., mancuso s. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 2 december 2017 accepted for publication 23 march 2018 ahs advances in horticultural science short note adv. hort. sci., 2018 32(1): 149-153 150 peratures often over 180°c, which in presence of atmospheric oxygen (between 160 and 240°c) produce many flavor compounds (alkanes, fatty acids, aldehydes, ketones, polycyclic aromatic hydrocarbons), pleasant or unpleasant, and change their flavor stability. the flavor compounds released during the heating process are affected by some parameters such as cooking temperature, preparation time, chemical composition of the oils, physical and physicochemical constants and the presence of additives and contaminants (fullana et al., 2004). the volatile compounds and sensory characteristics from different edible oils (brewer et al., 1999; katragadda et al., 2010) were altered after intense heating and the main alterations were in the emission of volatile compounds linked to the oxidation process, compounds which are negatively related with the sensory attributes. in particular, the oxidation process in oils m a i n l y l ea d t o t h e f o rm a t i o n o f a l kyl ra d i c a l s , alkylperoxyl radicals and decomposition of hydroperoxide and cause the decay of both their nutritional and sensory quality (velasco and dobarganes, 2002; silvagni et al., 2012). moreover, there are some human health risks both linked to oil oxidations process and to many of the originated compounds released by the heating process (vaclavik et al., 2013). the ptr-tof-ms tool represents a simple, and high-throughput strategy, applicable without any laborious sample preparations and treatments, particularly suited for analysis in real time of dynamic flavor release (both in vitro and in vivo) from diverse food matrices along the food-to-fork production chain (costa et al., 2016). the aims of this study are (1) to test the ptr-tofms as a sampling system for the detection of the released compounds from not heated olive and sunflower oils and to compare with heated oils at their smoking point (180°c); (2) to monitor and evaluate in real-time the vocs emitted from different oils, upon the heating from 25°c to 180°c, to understand the dynamic of oxidation phenomena. 2. materials and methods vegetable oil material two different types of commercial oils were used for this experiment, extra virgin olive oil (evoo) and a high oleic sunflower oil (sfo). they have different heating temperatures, this latter (sfo) considered more resistant to thermal increases (smith et al., 2007). both oils were acquired at the local supermarket in florence city (italy). the analysis of volatile organic compounds (vocs) emitted from both oils virgin has been obtained through a ptr-tof-ms (ionicon analytik gmbh, innsbruck, austria), which is an instrument with high resolution mass and sensibility (5-10 ppt) and makes analysis possible without any pretreatment of samples. h3o + was used as a reagent ion for the proton transfer reaction. sample preparation and analysis first experiment. for this experiment, 100 ml of each type of oil was placed inside a 250 ml pyrex glass jar, hermetically sealed; the jar’s lid was equipped with two holes that permitted the insertion of teflon tubes, which were respectively connected to a zero-air generator (peak scientific instruments, usa) and to the ptr-tof-ms system. it was essential to avoid the entry of foreign substances inside the jar, and that was obtained by means of a sealing paste placed around the lid and the two entry holes. oil samples were analyzed at two points: room temperature (25°c) and at 180°c. second experiment. the same steps of the first experiment was followed, in addition each oil sample was heated by using a round electric heating plate and analyzed in real time to verify the vocs emitted from both types of oil at different temperatures: room temperature (25°c), 60, 90, 120, 150, 180°c. once reached each of the five thermal steps, the temperature was kept constant for 5 minutes and then passed to the subsequent thermal step. the evaluation of the aromatic profile of each oil examined was carried out in real time in order to monitor the behavior of the various volatile substances during the heating process. for each sample the analysis lasted for a period of about 50 minutes, recording a spectrum per second for a total of 60 spectra per minute. to determine the achievement of the selected temperatures, a trial was performed in parallel; in details, a thermometer was placed into the jar contained 100 ml of different kinds of oil and the times required to reach each temperature, for both samples, were measured as well. the whole analyses were carried out inside a conditioned room, with an internal temperature of 25°c in order not to condition the chemical reactions, as they are strongly sensitive to variations in temperature and humidity. the value of the white (control) represented by an empty sealant jar was recorded before starting with samples analysis, and the volatile profile of the white (control) has always been subtracted from the final value of sabbatini et al. vocs emission in sunflower and extra virgin olive oil upon heating by ptr-tof-ms 151 each sample. the vocs in the headspace were observed by direct injection into the ptr-tof-ms and separation of single ions happened accordingly to their mass to charge (m/z) ratio. all the instrumental parameters were settled in the following way as in taiti et al. (2017): drift tube ionization condition at 600 v and a continuous pressure of 2.20±0.02 mbar; while instrument internal calibration was based on: m/z = 29.997 (no+); m/z = 59.049 (c3h7o +) and m/z = 21.022 (h3o +) and was performed off-line. the experiment data was acquired through the tofdaq software (tofwerk ag, switzerland) and all spectra were analyzed and acquired using a dead time of 20 ns for the poisson c o r r e c t i o n a n d p e a k e x t r a c t i o n f o l l o w i n g t h e methodology described in cappellin et al. (2011), using a modified gaussian peak shape. 3. results the signal intensity (ncps) generally vary among different oils. in figure 1 the signal intensity (ncps) of protonated m/z detected at 25°c and 180°c in sunflower oil (sfo) and extra virgin olive oil (evoo) are clearly indicated. as a general consideration, the intensity of the signals is always higher in evoo than in the sfo at the same temperature. at room temperature (25°c) the sfo spectrum is smoother, characterized by only 16 signals, being a very refined oil compared to evoo. the evoo presents instead 33 signals at room temperature when not heated, which are characteristics of the extra virgin olive oil in general, even if among them there are some masses that could generate off-flavor (m/z = 47.049, m/z = 61.028, m/z = 89.060) (marone et al., 2017; taiti and marone, 2017). at temperatures of 180°c, 43 m/z were measured in evoo and 39 m/z in sfo, with the formation of 23 new masses generated as a result of the high temperature in sfo and 11 new m/z in evoo, of which only 7 common to both matrices. to better verify the intensity of the signals at temperatures below 180°c, some masses of particular relevance have been selected in sfo and evoo, found in the most recent literature (klein et al., 2016). in particular, in the sfo emerge the masses m/z = 57, m/z = 101, and m/z = 113 (fig. 2); these masses are also reported in klein et al. (2016) as particularly relevant emissions by sfo. for olive oil emerge the masses m/z = 57, m/z = 101, m/z = 115, m/z = 143, also reported in klein et al. (2016). moreover, as its peculiar trend, it should be noted the mass m/z = 45 (fig. 2), that sharply increases in evoo starting from 150°c. the emission assessment of the selected masses by ptr-tof-ms allows to directly examine in real time the trend of their quantities in sfo and evoo in relation to the heating temperatures, avoiding alterations due to foods present together with the cooking oil, as generally found in the current scientific literature (ontanón et al., 2013; klein et al., 2016). the increasing temperature determines the increase of the emission of the total vocs produced at each temperature point for both sfo and evoo. the intensity of vocs emission by the refined sfo, always appears lower than that of the evoo. in both oils the increases are progressive, and related to the increasing temperature, in agreement with katragadda et al. (2010). both in sfo and evoo the mass m/z = 57, still absent at 90°c, present at 120°c, showed a dramatic increase at 150°c according to katragadda et al. (2010), and subsequently restart a trend proportional to the increase in temperatures. for each matrix all the other selected masses showed a common trend; fig. 1 signal intensity (ncps) of vocs compounds in sunflower oil (sfo) and extra virgin olive oil (evoo) at 25°c and 180°c. bars represent standard deviations (n = 3). adv. hort. sci., 2018 32(1): 149-153 152 while they were absent, or present in small quantities, up to a temperature of 90°c, they have a relatively modest rise to 120°c, and showed a dramatic increase from 150°c, according to nunes et al. (2013). the mass m/z = 57, always absent in the unheated oils, would derive from the dehydration of glycerol subjected to high temperatures (klein et al., 2016), and this would be a phenomenon common to all vegetable oils: the aldehydes that may be present in virgin olive oils as a result of a natural enzymatic o x i d a t i o n o f p o l y u n s a t u r a t e d f a t t y a c i d s (lypoxigenase cascade, lox) increase due to the temperature through a chemical oxidative reaction of the same polyunsaturated fatty acids (muik et al., 2005). 4. discussion and conclusions the ptr-tof-ms tool represents a simple, and high-throughput strategy, applicable without any laborious sample preparations and treatments, for the real-time detection of oxidative changes occurred during the heating process of both vegetable oils. there are numerous studies that examine the quality of the oil, as well as its composition and characteristics during the heating or frying process; nevertheless, a fewer number of studies examine the composition of the fumes generated during this process. in this work, it is pointed out that evoo emissions are always higher than those of the sfo; the effect of the heating causes an increase in the emission of the masses present at the origin and the generation of new compounds, deriving from transformations of triglycerides and above all of the polyunsaturated fatty acids (moreno et al., 1999). in particular, the trend of emissions in relation to the temperature has been highlighted for some selected masses, and it is clear that change due to chemical oxidation occur between 120 and 150°c, temperatures above which the production of volatiles increases enormously for all the examined masses. since the increase in oil temperature proportionally increases the emission of vocs, including some compounds that could be considered as pollutants, it is clear that an accurate control of the temperatures of oils used for frying food is important for the industry and for the home cooking. many vocs depend on the temperature, emissions at temperatures below the smoke point will be actually reduced. some compounds, such as mass m/z = 57, start to form consistently already at relatively low temperatures (150°c). it should be noted that in the case of evoo, masses of naturally occurring compounds are also very high, as they are derived from an oxidative process linked to enzymatic reactions of the polyunsaturated fatty acids (lipoxygenase pathway, lox) (campestre et al., 2017). the increase determined by high temperatures is no longer enzymatic but chemical oxidative. further studies are needed to deepen the knowledge on the effect of heating temperature in the formation of compounds potentially harmful to human health, in order to reduce the effect of pollution not only at commercial but also at the domestic level, for the daily use of fried oils. references brewer m.s., vega j.d., perkins e.g., 1999 volatile compounds and sensory characteristics of frying fats. j. food lipids, 6(1): 47-61. cappellin l., biasioli f., granitto p.b., schuhfried e., soukoulis c., costa f., tillman m.d., gasperi f., 2011 on data analysis in ptr-tof-ms: from raw specfig. 2 trend of signal intensity (ncps) of different protonated m/z in sunflower oil (sfo) and extra virgin olive oil (evoo) at different temperatures (25, 60, 90, 120, 150, and 180°c) upon heating sabbatini et al. vocs emission in sunflower and extra virgin olive oil upon heating by ptr-tof-ms 153 tra to data mining. sens. actuators b chem., 155: 183-190. campestre c., angelini g., gasbarri c., angerosa f., 2017 review. the compounds responsible for the sens o r y p r o f i l e i n m o n o v a r i e t a l v i r g i n o l i v e o i l s . molecules, 22: 1833. costa c., taiti c., strano m.c., morone g., antonucci f., mancuso s., claps s., pallottino f., sepe l., bazihizina n., menesatti p., 2016 multivariate approaches to electronic nose and ptr-tof-ms technologies in agro-food products, pp. 73-82. in: rodrígez mendéz m.l. (ed.) electronic noses and tongues in food science. elsevier inc., academic press, oxford, uk, pp. 309. fullana a., carbonell-barrachina a.a., sidhu s., 2004 comparison of volatile aldehydes present in the cooking fumes of extra virgin olive, olive, and canola oils. j. agric. food chem., 52(16): 5207-5214. katragadda h.r., fullana a., sidhu s., carbonell-barrachina a.a., 2010 emissions of volatile aldehydes from heated cooking oils. food chemistry, 120: 59-65. klein f., platt s.m., farren n.j., detournay a., bruns e.a., bozzetti c., daellenbach k.r., kilic d., kumar n.k., pieber s.m., slowik j.g., temime-roussel b., marchand n., hamilton j.f., baltensperger u., prévôt a.s.h., el haddad i., 2016 characterization of gas-phase organics using proton transfer reaction time-of-flight mass spectrometry: cooking emissions. environ. sci. technol., 50: 1243-1250. marone e., masi e., taiti c., pandolfi c., bazihizina n., azzarello e., fiorino p., mancuso s., 2017 sensory, spectrometric (ptr-tof-ms) and chemometric analyses to distinguish extra virgin from virgin olive oils. j. food sci. technol., 54(6): 1568-1376. moreno m.m., olivares d.m., lopez f.a., adelantado j.g., reig f.b., 1999 analytical evaluation of polyunsaturated fatty acids degradation during thermal oxidation of edible oils by fourier transform infrared spectroscopy. talanta, 50(2): 269-275. muik b., lendl b., molina-díaz a., 2005 direct monitoring of lipid oxidation in edible oils by fourier transform raman spectroscopy. chemistry physics lipids, 134(2): 173-182. nunes c.a., rios de souza v., corrêa s.c., de cássia da costa e silva m., carvalho bastos s., marques pinheiro a.c., 2013 heating on the volatile composition and sensory aspects of extra-virgin olive. oil ciênc. agrotec., lavras, 37(6): 566-572. ontanón i., culleré l., zapata j., villanueva b., ferreira v., escudero a., 2013 application of a new sampling device for determination of volatile compounds released during heating olive and sunflower oil: sensory evaluation of those identified compounds. eur. food res. technol., 236: 1031-1040. silvagni a., franco l., bagno a., rastrelli f., 2012 thermo-induced lipid oxidation of a culinary oil: the effect of materials used in common food processing on the evolution of oxidised species. food chemistry, 133(3): 754-759. smith s.a., king r.e., min d.b., 2007 oxidative and thermal stabilities of genetically modified high oleic sunflower oil. food chemistry, 102: 1208-1213. taiti c., marone e., 2017 evoo or not evoo? a new precise and simple analytical tool to discriminate virgin olive oils. adv. hort. sci., 31(4): 329-337. taiti c., marone e., lanza m., azzarello e., masi e., pandolfi c., giordani e., mancuso s., 2017 nashi or williams pear fruits? use of volatile organic compounds, physicochemical parameters, and sensory evaluation to understand the consumer’s preference. european food res. techn., 243(11): 1917-1931. vaclavik l., belkova b., reblova z., riddellova k., hajslova j., 2013 rapid monitoring of heat-accelerated reactions in vegetable oils using direct analysis in real time ionization coupled with high resolution mass spectrometry. food chem., 138(4): 2312-2320. velasco j., dobarganes c., 2002 oxidative stability of v i r g i n o l i v e o i l . e u r o p e a n j . l i p i d s c i . t e c h n . , 104(9-10): 661-676. impaginato 39 1. introduction berberis microphylla g. forst. is a patagonian native shrub commonly named “calafate”, with a large distribution from neuquén (37° sl) to tierra del fuego (54° 8´ sl) (orsi, 1984). this species has a growing economic potential due to the production of fruits as a non-timber forest product (tacón clavaín, 2004). in fact, its dark blue berries are consumed fresh, as jams and preserves, and are used for the production of soft drinks and ice cream. moreover, the fruits have a high content of carbohydrates, phenols and antioxidants (arena and curvetto, 2008; arena et al., 2011, 2012, 2013 b). the genetic and morphological analysis of spontaneous accessions in natural populations of b. microphylla grown on tierra del fuego (giordani et al., 2016), as well as the changes in form and leaf anatomy due to weather conditions (radice and arena, 2015) were recently studied. the study of flower anatomy related to blooming is an important step for programs of genetic resource conservation and improvement, complementing basic studies of floral biology (de castro nunes et al., 2012; wetzstein et al., 2014). flower structure and floral biology was well described by arena et al. (2011), like the phenological stages (arena et al., 2013 a) and flower bud differentiation (arena and radice, 2014). more recently, a comprehensive study of pollen grain was published (radice and arena, 2016 a). nevertheless, pollination was not clear until now. fertilization of patagonian berberis has been classified as cross-pollination by orsi (1984). however, hegi (1958) and romeo et al. (2005) referred the berberis species as autogamous due to the absence of visiting insects together with extreme climatic cond i t i o n s p r e v a l e n t i n m o s t a r e a s o f p a t a g o n i a . however, during flowering, the activity of different syrphidae was observed (radice et al., 2016). on the other hand, floral movements have been appointed as mechanisms to facilitate self-pollination (darwin, 1862). stamen movement has been documented in a few plant families, among them berberidaceae (lechowski and bialczyk, 1992). however, results of controlled treatments of selfand cross-pollination compared with those of open-pollination performed during three different periods in b. microphylla do adv. hort. sci., 2017 31(1): 39-44 doi: 10.13128/ahs-20724 flower anatomy related to blooming development of berberis microphylla g. forst (berberidaceae) s. radice, m. arena department of plant physiology, facultad de agronomía y ciencias agroalimentarias um-conicet, machado 914, lab. 501, b1708eoh, morón. prov. de buenos aires, argentina. key words: anthesis, barberry, nectar, patagonia, pollen grain, stigma. abstract: berberis microphylla g. forst. is a patagonian native shrub commonly named “calafate”, which has a growing economic potential due to its dark blue berries that are consumed fresh, as jams and preserves, and are used for the production of soft drinks and ice cream. moreover, the fruits have a high content of carbohydrates, phenols and antioxidants. the objective of this work was to show the changes observed in the flower from the emergence in relation to the floral phases and the importance that they have on pollination and fertilization. during the anthesis, the nectar is excreted inside and outside of the petal through the epidermis of the secretory tissue. the epidermis of the stigma is papillae with cells of greater length in the periphery of this structure simulating an additional ring. secretory tissue is also present on the area of the fusion carpel. during anthesis, the epidermis glands of the stigma showed active secretion and these conditions favor pollen grain germination. germinated pollen grains were observed after 12 hours of pollination and ten days later the pollen tube reached the ovule area. pollen tube grew surrounded the ovules and probably some of them already accomplished the fertilization. (*) corresponding author: siradice@yahoo.com received for publication 19 november 2016 accepted for publication 15 february 2017 copyright: © 2017 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2017 31(1): 39-44 40 not support this hypothesis (radice and arena, 2016 b). thus, self-pollination resulted only in pollen germination on the stigmas but the pollen tubes were not able to reach the ovules. according to these antecedents, anatomical studies are necessary for a better understanding of the physiological processes during floral biology and pollination that interact with syrphidae activity. the objective of this work was to show the changes observed in the flower from its emergence in relation to the floral phases and the importance that they have on pollination and fertilization. 2. materials and methods plant material flowers of berberis microphylla g. forst. (n = 20) on growth stages ranging from 53 to 68 on the bbch scale proposed for b. buxifolia (arena et al., 2013 a) were collected on plants grown near ushuaia city, tierra del fuego (54° 48’ sl, 68° 19’ wl and 30 m asl), and were fixed in faa (formaldehyde, 100 ml; ethyl alcohol, 500 ml; acetic acid, 50 ml; distilled water, 350 ml). light microscopy button flowers were dehydrated in an ethanol series and embedded in spurr’s resin. thin sections (75-90 nm thick) were stained with uranyl acetate and lead citrate. fluorescent microscopy flowers fixed in faa were washed with distilled water and softened with naoh (8n) as described by martin (1959). then, they were stained with aniline blue to study pollen tube growth. squash material was observed by a leica microscope (dm 2500) with dapi filter. scanning electron microscopy (sem) button flowers fixed in faa were dehydrated in an ethanol series and critical point drying technique was employed. samples were sputter coated with 20 nm gold and observed with a philips xl 30 sem. ovules and seeds relation the number of ovules on button flowers (n= 130) and the number of seeds on formed fruits (n=100) were counted. 3. results pistil of b. microphylla is similar to a bottle (fig. 1a). flowers before anthesis (growth stage 54) showed anthers with microspores at an advanced stage of development and underdeveloped ovules (figs. 1a-d). in effect, microsporangia contain tapetal cells metabolized, i.e. a thick portion was deposited on the wall of the microspore and inside it is possible to observe vegetative and generative cells that are surrounded by a thin delicate wall (fig. 1d). on the other hand, ovules are rudimentary with an active cellular proliferation on the nucellus and integuments (fig. 1c). the epidermis of the stigma is covered by secretory cells (figs. 2 a, c, e) and the short style is also recovered by glands (fig. 2b). petals have two thick nectaries on the basal position (fig. 3a). both petal epidermises are also formed by glandular cells (figs. 3b, d, e, f). on a later flower phase (growth stage 59), the epidermis and nectariferous cells of the nectar have dense cytoplasm and conspicuous nucleus (figs. 3c). nectar is abundant in the intercellular spaces (fig. 3c). flowers on the anthesis phase (growth stage 60) showed nectariferous cells with a gradual diminution of staining density (fig. 3g). total production of nectar per flower is poor; it is secreted through the gland cells that are present on the epidermis of the nectaries (figs. 3c, g) and two epiderfig. 1 bottom flower of b. microphylla g. forst (light micrograph). a, longitudinal section of a flower on stage 54. b, anther with mature pollen grains; c, rudimentary ovule with nucellus (n) and integuments (i) in development; d, detail of pollen grain with exine (e), vacuole (v) and generative cell (gc) contained into the vegetative cell; n= nucleus. bars: a = 200 µm; b, d = 10 µm; c = 50 µm. radice and arena flower anatomy related to blooming development of berberi microphylla g. forst 41 mal layers of petals (figs. 3e,f). nectar is exuded through the cuticle with rupture of its outer layer. greater presence of vacuoles is observed in both nectar tissues (fig. 3g). flowers on growth stage 59 showed a monocarpellary pistil with a clavate shaped stigma (fig. 4a). epidermis of the stigma is papillae (figs. 4b, c, e) with cells of greater length in the periphery of this structure simulating an additional ring (fig. 4e). these cells secrete a sticky substance that keeps always the stigma hydrated during the anthesis phase (fig. 2f). secretory tissue is also present on the area of the fusion carpel (figs. 4b, d). on the other hand, stigmas on growth stage 59 are receptive, i.e. they can promote pollen germination while stigma on less developed growth phases is not receptive (fig. 2e). flowers on anthesis phase (growth stage 60) fig. 2 sem micrograph of the pistil of b. microphylla g. forst. a, stigma of a flower on stage 59; b, detail of box in the picture a, periphery of the upper end of the ovary with glands; c, stigma front view shown in a; d, stigma front view shown of a flower on stage 60; e, detail of box in the picture d, long hairs peripheral stigma with pollen grains attached; f, detail of circle in the picture c, epidermal cells of stigma; g, detail of circle in the picture d, active secretions glands; h, mixture of pollen grains wrapped in stigmatic mucilage, arrow points to a grain of foreign pollen. bars: a-e =100 µm; f-h= 50 µm. fig. 3 nectary of b. microphylla g. forst. a-d sem micrograph. bc, e-g light micrograph. a, nectary view at the base of petal; b, section of a petal and a nectariferous area; c, detail of tissue of nectariferous area of a flower on stage 59; intercellular space with nectar (arrows); d, detail of circle in the picture a, epidermis with glands, arrows show secretory glands. e, detail of circle in the picture b, petal outer epidermis; f, detail of circle in the picture b, petal inner epidermis; g, detail of tissue of nectariferous area of a flower on stage 60, arrow indicates an epidermal cell in active secretion. bars: a-b= 100 µm; c= 10 µm; d= 50 µm; e-g= 10 µm. fig. 4 light micrograph of the pistil of b. microphylla g. forst. a, longitudinal section of a flower on stage 59.b, view of stigma and the upper end of the ovary; c, glandular cells of the flat part of stigma; d, detail of glandular cells of fusion area of the carpel; e, detail of hairs surrounding the stigma. bars: a= 100 µm; b= 200 µm; c-e= 50 µm. adv. hort. sci., 2017 31(1): 39-44 42 showed mature pollen grain with a well-formed external wall and the cytoplasm of the vegetative cell rich of starch (fig. 5e). in this phase anthers are dehiscent. on the contrary, ovules are externally coated by the integuments and attached to the ovary by the funiculus on basal placentation (fig. 5d). internally, the ovules present some delay in comparison to the pollen grain development. pollinated flowers show ovules with the embryo sac with egg cell, synergists, antipodes and polar nuclei cells developed (figs. 5 a-c); nevertheless, no pollinated pistils show ovules with megaspore mother cells without development. pollen is deposited on the stigma mainly between the secretory cells that surround this structure (figs. 2d, g). germinated pollen grains were observed after 12 hours from pollination (fig. 6b). after 24 hours pollen tube crosses the stigma (fig. 6a) and ten days later the pollen tube reached the ovule area. pollen tube grew surrounded the ovules and probably some of them accomplished the fertilization (figs. 7a, b). subsequently seed growth is observed (fig. 7c) but this process is not given in all the ovules. in effect, on a selected natural population the ovules and seeds were counted, and an average of 8.95 (ranged from 6.9 to 10.0) and 5.28 (ranged from 3.3 to 7.2), respectively, were registered, i.e. 40.97% of all ovules produced aborted. 4. discussion and conclusions flowering plants are associated with a broad spectrum of animal pollinators, among these bees constitute an important but not exclusive one (dötterl and vereecken, 2010). in effect, it has already been demonstrated that b. microphylla is not self-fertile so it depends on insect pollination (radice and arena, 2016 b). tierra del fuego (argentina) offers an extreme climatic situation where the bees cannot prosper; accordingly calafate flowers are visited by different syrphids (radice et al., 2016). pollination by insects, including flies, is commonly a mutualistic interaction, in which both the plant and the insect benefit; thus, anatomical organization and pollination strategies developed on the flowers must be adapted to the environmental conditions. calafate shrubs bloom with abundant yellow flowers that produce aromatic nectar (radice et al., 2016), that exudates inside the flower as well as the outside through the petals mainly in their insertion fig. 5 details of the ovary and pollen grains of a flower on anthesis stage of b. g. forst. a-c, embryo sac with egg cell (a, arrow), synergids (b, arrow) and antipodes (c, arrow) and polar nuclei (c, arrows head); d, sem micrograph of ovules on the basal insertion of ovary; e, mature pollen grains with cytoplasm rich in starch grains. bars: a-c= 200 µm; d= 100 µm; e= 10 µm. fig. 6 fluorescent light micrograph of pollen tube germinated on pistils of b. microphylla g. forst. a, view of stigma with pollen grain germinated and pollen tubes inserted in the ovary; b, pollen tubes on the first stage of growth; c, pollen tube penetrating the ovule. bars: a-c= 100 µm. radice and arena flower anatomy related to blooming development of berberi microphylla g. forst 43 area. this particularity is very important because the fluorescent emission of nectar attracts pollinating insects. b. microphylla have perigonial nectaries type “1a” according to the topographic classification of fahn (1982). unlike what was found on the nectar tissue of berberis corymbosa by bernardello et al. (2000), no stomata were found on this species either on histological sections or through sem observations. in effect, nectar is exuded through the epidermis as cited by bernardello (2007). berberis produce small amounts of nectar per flower; effectively it was registered less than 1 µl on b. corymbosa (bernardello et al., 2000) and 1.57 µl on b. microphylla (radice al., 2016). nectar concentration was considered as intermediate for b. buxifolia (31.2±14.8%) (chalcoff et al., 2006). this result is in coincidence with radice et al. (2016) who measured 36.28 °brix in nectar of the berberis population studied. stigma epidermis is covered by hairs that secrete a stigmatic fluid to promote pollen germination. surface hairs on the stigma can be seen in others species like papaver rhoeas, or lupinus luteus (fahn, 1982). once overcome the stigma, the pollen tubes grow through the carpel wall. in effect the margin of the carpel is covered by glands that nourish the germinated pollen. the berberidaceae are generally considered to have originated in some part of the ranalian complex (chapman, 1936), i.e., it is belonging to the group of the oldest dicotyledons which is confirmed by its structure devoid of carpelar style (fahn, 1982). there are three important elements to attract the pollinating insect on b. microphylla such as color, scent and nectar. it has long been known that bees utilize not only visual but also olfactory flower cues f o r f i n d i n g s u i t a b l e h o s t p l a n t s ( d ö t t e r l a n d vereecken, 2010). this pollination strategies present in calafate could be useful in other growth areas of the species. on the other hand, young flies in the presence of generic floral scent respond more strongly to a uniformly yellow cue than to any other uniform color cue (green, white, black, blue, red) except for ultraviolet (brodie et al., 2015). nectar is the most commonly sought reward by flower-visiting flies (woodcock et al., 2014) because carbohydrates contained in the nectar provide short-term energy supply. so the abundant number of yellow flowers plus the fluorescent emitted cue by nectar must constitute a strong attraction for syrphids. acknowledgements authors acknowledge to the prefectura naval argentina, the technical assistance of isabel farías. this research was supported by grants pip 0314 subsidized by conicet. fig. 7 fertility of b. microphylla g. forst. a-b, time of discharge of sperm nuclei (arrows) into the egg cell (ec) and polar nuclei (pn), c, growing seed. bars: a-b= 200 µm; c= 100 µm. adv. hort. sci., 2017 31(1): 39-44 44 references arena m.e., curvetto n., 2008 berberis buxifolia fruiting: kinetic growth behavior and evolution of chemical properties during the fruiting period and different growing seasons. sci. hortic., 118(2): 120-127. arena m.e., giordani e., radice s., 2011 flowering, fruiting and leaf and seed variability in berberis buxifolia, a patagonian native fruit species, pp. 117-136. in: m a r i n l . , a n d d . k o v a è ( e d s . ) n a t i v e s p e c i e s : identification, conservation and restoration. nova science publishers, new york, usa, pp. 176. a r e n a m . e . , g i o r d a n i e . , r a d i c e s . , 2 0 1 3 a phenological growth and development stages of the native patagonian fruit species berberis buxifolia lam. j. food, agric. environ., 11(3-4): 1323-1327. arena m.e., postemsky p., curvetto n.r., 2012 accumulation patterns of phenolic compounds during fruit growth and ripening of berberis buxifolia, a native patagonian species. new zeal. j. bot., 50(1): 15-28. arena m.e., radice s., 2014 shoot growth and development of berberis buxifolia lam. in tierra del fuego (patagonia). sci. hortic., 165: 5-12. arena m.e., zuleta a., dyner l., constenla d., ceci m., curvetto n.r., 2013 b berberis buxifolia fruit growth and ripening: evolution in carbohydrate and organic acid contents. sci. hortic., 158: 52-58. bernardello g., 2007 a systematic survey of floral nectarines, pp. 19-128. in: nicolson s.w., m. nepi, and e. pacini (eds.) nectaries and nectar. springer, dordrecht, the netherlands, pp. 396. bernardello g., galetto l., anderson g.j., 2000 floral nectary structure and nectar chemical composition of some species from robinson crusoe island (chile). can. j. bot., 78(7): 862-871. brodie b.s., smith m.a., lawrence j., gries g., 2015 effects of floral scent, color and pollen on foraging decisions and oocyte development of common green bottle flies. plosone, 10(12): 1-15. chalcoff v.r., aizen m.a., galetto l., 2006 nectar concentration and composition of 26 species from the temperate forest of south america. ann bot., 97(3): 413-421. c h a p m a n m . , 1 9 3 6 c a r p e l a n a t o m y o f t h e berberidaceae. amer j. bot., 23: 340-348. darwin c., 1862 on the various contrivances by which british and foreign orchids are fertilised by insects, and on the good effects of intercrossing. john murray, london, uk. de castro nunes r., oliveira bustamante f., techio v.h., mittelman a., 2012 morphology and pollen viability of lolium multiflorum lam. ciên. agrotec., 36(2): 180-188. dötterl s., vereecken n.j., 2010 the chemical ecology and evolution of bee-flower interactions: a review and perspectives. the present review is one in the special series of reviews on animal-plant interactions. can. j. zool., 88(7): 668-697. fahn a., 1982 plant anatomy. nature, pergamon press, usa, pp. 544. giordani e., müller m., gambineri f., paffetti d., arena m., radice s., 2016 genetic and morphological analysis of berberis microphylla g. forst. accessions in southern tierra del fuego. plant biosyst., pp. 1-14. hegi c., 1958 illustrierte flora. von mittel-europa, 4(1): 1-11. lechowski z., bialczyk j., 1992 effect of external calcium on the control of stamen movement in berberis vulgaris l. biol. plant., 34: 121-130. martin f.w., 1959 staining and observing pollen tubes in the style by means of fluorescence. stain technol., 34: 125-128. orsi m.c., 1984 berberidaceae, pp. 325-348. in: correa m.n. (ed). flora patagónica sección 4ª. inta, buenos aires, argentina. radice s., arena m., 2015 environmental effect on the leaf morphology and anatomy of berberis microphylla g. forst. int. j. plant biol., 6(5677): 1-7. radice s., arena m.e., 2016 a characterization and evaluation of berberis microphylla g. forst pollen grains. adv. hort. sci., 30(1): 31-37. radice s., arena m.e., 2016 b effect of different pollination treatments of berberis microphylla g. forst, a patagonian barberry. proceedings of ii international workshop on floral biology and s-incompatibility in fruit species, murcia, spain, may, pp. 23-26. radice s., arena m.e., suárez f.j., landi l.i., caló j.f., 2016 pollination strategies of berberis microphylla g. forst, a patagonian barberry. proceedings of ii international workshop on floral biology and sincompatibility in fruit species, murcia, spain, may, pp. 23-26. r o m e o r . a . , s á n c h e z a . c . , n o v a r a l . , 2 0 0 5 berberidaceae. aportes botanicos de salta-serie flora, 7(9): 1-10. tacón clavaín a., 2004 manual de productos forestales no madereros. cipma, valdivia, chile, pp. 22. wetzstein h.y., porter j.a., janick j., ferreira j.f.s., 2014 flower morphology and floral sequence in artemisia annua (asteraceae). amer. j. bot., 101(5): 875-885. woodcock t.s., larson b.m., kevan p.g., inouye d.w., lunau k., 2014 flies and flowers ii: floral attractants and rewards. j. pollinat ecol., 12(8): 63-94. impaginato 275 adv. hort. sci., 2017 31(4): 275-280 doi: 10.13128/ahs-20694 ‘superior seedless’ grapevine grafted on three rootstocks grown on calcareous soil under diluted brackish water irrigation. ii. expression of antioxidant genes i.m. qrunfleh 1 (*), s. abu-romman 2, t.g. ammari 3 1 department of plant production and protection, faculty of agricultural technology, al-balqa applied university, al-salt 19117, jordan. 2 department of biotechnology, faculty of agricultural technology, albalqa applied university, al-salt 19117, jordan. 3 department of water resources and environmental management, faculty of agricultural technology, al-balqa applied university, al-salt 19117, jordan. key words: 41b, p1103, r110, ros, salinity. abstract: grapevine rootstocks that can absorb brackish water and maintain satisfactory growth of the grapevine scion might be a feasible management practice in areas suffering scarce water resources. the objective of this study was to evaluate the expression of antioxidant genes in ‘superior seedless’ leaves grafted on r110 (vitis berlandieri x v. rupestris), 41b (v. berlandieri x v. vinifera) and p1103 (v. berlandieri x v. rupestris) in response to diluted brackish water irrigation at three levels: 1.5, 3.0 and 5.0 ds m-1 in addition to the 0.8 ds m-1 control. results revealed that after salinity exposure for two weeks, the transcript levels of apx, mn-sod and mdar increased in ‘superior seedless’ leaves grafted on the different rootstocks. however, their expression levels in response to salinity were noticeably higher in plants grafted on p1103 and r110 compared to 41b. the expression of cat gene showed obvious enhanced level in plants grafted on p1103 in response to salt exposure. meanwhile, the expression of cat gene in ‘superior seedless’ scion grafted on 41b or r110 showed almost unchanged level in control and stressed conditions. down-regulation of cuzn-sod was recorded in leaves of ‘superior seedless’ grafted on p1103. slight up-regulation of this gene in response to saline condition was recorded when scion was grafted on 41b or r110. the expression of gpx was enhanced in scion grafted on p1103 and 41b. on the other hand, scion grafted on r110 showed decreased expression of gpx in response to salt treatment. grapevine rootstocks that have v. rupestris and v. berlandieri in their parentage are good candidates for salinity tolerance. (*) corresponding author: iqrunf@bau.edu.jo citation: qrunfleh i.m., abu-romman s., ammari t.g., 2017 ‘superior seedless’ grapevine grafted on three rootstocks grown on calcareous soil under diluted brackish water irrigation. ii. expression of antioxidant genes adv. hort. sci., 31(4): 275-280 copyright: © 2017 qrunfleh i.m., abu-romman s., ammari t.g. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 28 may 2017 accepted for publication 29 september 2017 ahs advances in horticultural science adv. hort. sci., 2017 31(4): 275-280 276 1. introduction grapes (vitis sp.) are considered as one of the world’s major commercially grown fruit crops. in jordan, grapes are ranked in second place after olives regarding the total area planted. the total area planted with grapes is 3806 ha (fao, 2014). more than half of that area is under irrigation and jordan is now ranked as the world’s second water-poorest country. irrigation with low quality water during the whole growing season of the crops, even the tolerant ones, does not always produce high yield. mixing low quality water; such as dam brackish water, with conventional quality irrigation water in ratios to keep the salinity of the irrigation water below the threshold of the target crop might be an acceptable management practice and was used by many researchers (abdel gawad and ghaibeh, 2001). alternating conventional quality water with brackish water is another management practi ce. its appl i cati on woul d be easi er because it does not need containers for mixing two different sources of irrigation water. the conventional quality irrigation water can be used during the sensitive stages of plant growth and the brackish water during the non-sensitive or less sensitive stages. considerable yields were obtained using saline irrigation water (4-12 ds m-1) in crops that had been previously defined as moderately sensitive to salt stress (bustan et al., 2004). furthermore, in some crops (e.g., tomato) the reduction in the fresh yield was compensated by an increase in fruit dry weight and other quality parameters (mizrahi et al., 1988). bustan et al. (2005) reported that the combination of fresh (1.2 ds m-1) and brackish (7 ds m-1) irrigation water increased the yield level of melon to that of fresh water plants whereas it brought about the improvement of fruit quality typical to brackish water plants, thus providing an attractive approach to optimize late-summer melon production. plants subjected to saline environment use different mechanisms to overcome such abiotic stress. to avoid a disorder of ion homeostasis under saline conditions, plant cells have to maintain a low na+ concentration and keep a high h+ concentration in the cytosol where enzymes for metabolism are located (zörb et al., 2005). usually, plants use different ways to maintain a l o w c y t o s o l i c s o d i u m c o n c e n t r a t i o n , i n c l u d i n g restricting na+ influx, maintaining active na+ efflux, and compartmentalizing na+ into the vacuole, etc. (rubio et al., 1995). these mechanisms involve a number of na+/h+ antiporter proteins that are locali z e d i n p l a n t p l a s m a a n d v a c u o l a r m e m b r a n e s (vasekina et al., 2005). they catalyze the exchange of na+ for h+ across membranes and the energy needed is generated by the h+-adenosine triphosphatase and h+-pyrophosphatase (niu et al., 1995; shi and zhu, 2002). plants exposed to salt stress showed enhanced formation of reactive oxygen species (ros) such as superoxide anion, hydrogen peroxide and singlet oxygen (mittler, 2002). the harmful effects of these molecules are referred to as oxidative stress (halliwell, 2006). plants have evolved an enzymatic antioxidant system to reduce the ros levels. this enzymatic system includes superoxide dismutase, catalase, ascorbate peroxidase and glutathione peroxidase (apel and hirt, 2004). the development of salt-tolerant crops is a practical solution to sustain agricultural productivity. because of the complexity of the trait, traditional crop-breeding programs aimed at improvi n g t o l e r a n c e t o s a l i n i t y h a v e l i m i t e d s u c c e s s . therefore, understanding the cellular and molecular bases of salinity-tolerance mechanisms is essential for marker-assisted selection and genetic engineering of salt tolerance in economic crops. understanding the responses of plants to the major environmental stressor salinity is an important topic for the biotechnological application of functional mechanisms of stress adaptation. plant engineering strategies for cellular and metabolic reprogramming to increase the efficiency of plant adaptive processes may either focus on (1) conferring stress tolerance by directly reprogramming ion transport processes and primary metabolism or (2) by modulating signaling and regulatory pathways of the adaptive mechanisms. the second approach seems to be more perspective because it is likely that signaling and regulatory factors orchestrate as key signaling components the transcriptional and translational control of group (1) adaptive mechanisms (diédhiou et al., 2008; popova et al., 2008). m o s t k n o w l e d g e o n m o l e c u l a r m e c h a n i s m s involved in plant salt responses and adaptation has been derived from analyses of the glycophytic models arabidopsis thaliana and rice. such knowledge is lacking in vitis species, therefore detecting expression changes of antioxidant defense genes is the objective of this study. since the understanding of a plants response to a stress requires an evaluation of stress induced changes in gene expression, the expression of major defense genes (superoxide disqrunfleh et al. ‘superior seedless’ grown under diluted brackish water irrigation. ii. expression of antioxidant genes 277 mutase, ascorbate peroxidase, catalase, glutathione peroxidase, monodehydroascorbate reductase) in ‘superior seedless’ grafted on different rootstocks has been examined by rt-pcr. 2. materials and methods plant material three grape rootstocks were evaluated in this study: r110 (vitis berlandieri x v. rupestris), 41b (v. berlandieri x v. vinifera) and p1103 (v. berlandieri x v. rupestris). the rootstocks were purchased from les pépiniéristes du comtat, sarrians, france. after being imported, ‘superior seedless’ bud cultivar was grafted on the rootstocks in a local nursery; al-bushra nurseries, may, 2013. grafted plant materials were planted in polyethylene bags filled with peatmoss. the one year old grafted grapevine rootstocks were grown for several months to allow for the formation of a well developed root system before applying treatments. fertilizers and fungicides were applied as necessary. soil and water the soil was brought from the southern jordan valley. soil was crushed and sieved through 1 cm sieve and plastic pots (the working volume of the pots was 44 l) were filled with 50 kg each in order to roughly have a bulk density of 1.14 g cm-3. the bulk density is within the typical range of bulk densities of agricultural soils. if bulk density was higher, infiltration would be very slower and hydraulic conductivity would be slower as well. low hydraulic conductivity would exacerbate the osmotic effect and create anoxic conditions. the pots were placed in a controlled greenhouse. grafted grapevines were transplanted in february and the growth was unified based on the number of buds and root length. the root system was cut back to 15 cm in length and the vegetative system was cut back to eight buds. the water was brought from al-karameh dam located in the jordan valley and stored in a galvanized tank. three levels of irrigation water salinity; in terms of electrical conductivity (ec), were applied: 1.5, 3.0 and 5.0 ds m-1 in addition to the 0.8 ds m-1 control. these three levels of diluted brackish water were used to find out the salt level that would result in a tolerable “adverse” effect to design alternate irrigation that would contribute to water saving. these concentrations were selected based upon the threshold ec of grapes (i.e. ec<2 ds m-1). the treatments were prepared by mixing the dam water with tap water. a portable conductivity meter (model cond 3210, wtw, germany) was used to measure the ec and to obtain the determined salinity levels. the twelve treatments were arranged in a randomized complete block design with three replicates. the grafted grapevines started to break the dormancy period during spring. composite fertilizer (20:20:20), urea and ammonium sulphate were also applied to the grapevines and growth was again unified before applying the assigned treatments. irrigation with the assigned treatments started in may. all pots received the same amount of water whenever irrigation was applied. each pot received a total amount of irrigation water equal to 446 mm. irrigation was scheduled according to evaporation readings from free water surface (in mm) taken every 48 hours and corrected using proper grapevine crop coefficient of 0.30 (according to food and agriculture organization). dna and rna extraction leaves were sampled starting from february until november, 2014 (every two weeks) and frozen in liquid nitrogen for analyzing dna and rna using kits (intron, korea). total rna was extracted from frozen leaf samples with the iqeasy™ plus plant rna extraction mini kit (intron biotechnology, korea) according to user’s manual. rna concentration and purity were estimated based on absorbance at 260 and 280 nm. two microgram of rna was used to reverse transcribe the first strand cdna using power cdna synthesis system (intron biotechnology, korea) according to the manufacturer’s protocols with oligo (dt)15 as a primer in a reaction volume of 20 µl. the first strand cdnas generated for all the samples were used for semi-quantitative rt-pcr to monitor the transcript levels. the gene-specific primers for rt-pcr were designed based on the basis of the sequences published in genbank using the software p r i m e r b l a s t ( h t t p : / / w w w . n c b i . n l m . n i h . g o v / tools/primer-blast/index.cgi?link_loc=blasthome). the ef-1α gene was selected as a reference gene. the primer sequences are listed in table (1). the pcr reaction was performed using intron i-maxtm ii system (intron, korea). the same thermal profile was used for all pcr reactions; pcr was initiated with enzyme activation at 95°c for 2 min followed by 32 cycles of 40s at 95°c, 40s at 56 °c and 1 min at 72°c. different amplification cycles were tried and the product of the 32 cycles was selected to be presented. all rt-pcr products were loaded in ethidium bromide-stained 1.5 % (w/v) agarose gel. adv. hort. sci., 2017 31(4): 275-280 278 3. results and discussion as an abiotic stress, salinity induces oxidative damage in plant cells through the increased generation of reactive oxygen species (ros) in different cell compartments (mittler, 2002). the activation of antioxidant defense system reduces the level of ros and minimizes the impact of oxidative stress and its associated damage. plant cells possess antioxidant enzymes which have the ability to detoxify toxic ros (apel and hirt, 2004). rootstock choice should be taken with careful consideration since the scion is dependent on the rootstock (creasy and creasy, 2009). novel rootstocks are frequently used to confer resistance to environmental adversities in horticultural crops (albacete et al., 2015). a promising approach to improve salt tolerance of horticultural species is the use of grafting on salt-tolerant rootstocks (colla et al., 2010; giuffrida et al., 2014; simpson et al., 2015; zrig et al., 2016). grapevine rootstock selection is a key factor and could be considered an important strategy to mitigate salinity stress. grape rootstocks do influence the scion cultivar in many growth and physiological aspects (gu, 2003). however, some contradictions can be found in the literature in terms of the salt tolerance of grapevine rootstocks implying that various factors are involved, which eventually determine grapevine response to salt stress. for example, southey and jooste (1991) found that american hybrids performed poorly in response to salinity when used as rootstocks for the cultivar ‘colombard’. in addition, cavagnaro et al. (2006) concluded that argentinean cultivars performed better than european cultivars in an in vitro salinity evaluation study. regarding differences in ranking rootstocks, dardeniz et al. (2006) indicated that 41b was the most salt resistant rootstock, followed by 140ru and p1103, and the least resistant was 5 bb. on the other hand, walker et al. (2002) showed that the highest salt resistance was obtained when p1003 was used as a rootstock. to determine whether nacl-induced stress and rootstock type were able to regulate the expression levels of genes responsible for antioxidant defense response, a semi-quantitative rt-pcr assay was performed for the analysis of six major antioxidant enzyme genes (apx, cat, cuzn-sod, mn-sod, gpx, and mdar) (fig. 1). table 1 primer pairs used in gene expression analysis gene genebank id primer pairs (5'→3') amplicon size (bp) ascorbate peroxidase eu280159 f: gacaatgaagcacccagaggag 542 (apx) r: aatgggcttcagcatagtcagc cuzn-superoxide dismutase af056622 f: ctgctccatctcgtgtctttct 452 (cuzn-sod) r: atccacaattgttgcttcagcc mn-superoxide dismutase np_001268135 f: agaaaatcgctagggttagggc 538 (mn-sod) r: tacccagcaatggaaccaagtt catalase af236127 f: aggcccagttcttcttgaggat 860 (cat) r: aggcaagcatctcattctcagc glutathione peroxidase xm_002272900 f: atgtcgaagcaaatacagcagg 472 (gpx) r: tgagaggggaagttgttgggta monodehydroascorbate reductase np_001267971 f: tcatgtttgtgttggaagcgga 868 (mdar) r: ggacagatcaaaggcacgagag elongation factor 1α xp_002277159 f: attgtggtcattggccatgttg 566 (ef-1α) r: ccttcgaaaccagagatgggaa fig. 1 semi-quantitative rt-pcr expression analysis of major antioxidant enzyme genes in ‘superior seedless’ grafted on different rootstocks and exposed to salinity for two weeks. the ef-1α gene was used as the internal control for normalization of loading. qrunfleh et al. ‘superior seedless’ grown under diluted brackish water irrigation. ii. expression of antioxidant genes 279 plants utilize antioxidant enzymes, such as: superoxide dismutase (sod), catalase (cat), glutathione peroxidase (gpx), ascorbate peroxidase (apx) and m o n o d e h y d r o a s c o r b a t e r e d u c t a s e ( m d a r ) , a s defense mechanisms against salinity and do have the ability to detoxify toxic ros (apel and hirt, 2004). their expression has been studied in many crops as previously mentioned in the introduction part. however, such knowledge is lacking in vitis species, s p e c i f i c a l l y i n v i t i s b e r l a n d i e r i a n d r u p e s t r i s . meanwhile, their expression is extensively studied in both vinifera rootstocks and cultivars (carvalho et al., 2015). after salinity exposure for two weeks, the trans c r i p t l e v e l s o f a p x , m n s o d a n d m d a r w e r e increased in leaves of ‘superior seedless’ grafted on the different rootstocks. however, their expression levels in response to salinity were noticeably higher in plants grafted on p1103 and r110 compared to 41b. the expression of cat gene showed obvious e n h a n c e d l e v e l i n p l a n t s g r a f t e d o n p 1 1 0 3 i n response to salt exposure. however, the expression of cat gene in ‘superior seedless’ scion grafted on 41b or r110 showed almost unchanged level in control and stressed conditions. down-regulation of cuzn-sod was recorded in leaves of ‘superior seedless’ grafted on p1103, while, slight up-regulation of this gene in response to saline condition was recorded when scion was grafted on 41b or r110. in response to salinity stress, the expression of gpx was enhanced in scion grafted on p1103 and 41b. on the other hand, scion grafted on r 1 1 0 s h o w e d d e c r e a s e d e x p r e s s i o n o f g p x i n response to salt treatment. the expression of these genes was evaluated every two weeks, and only the results after the first two weeks of the stress treatment were presented since no differences in expression were noticed at the other time points between different treatments. this indicates early differential responses to salt stress at the level of antioxidant defense genes. such responses were previously reported in other plant species (ellouzi et al., 2014; ranjit et al., 2016). 4. conclusions r o o t s t o c k c h o i c e i s c r i t i c a l i n d e t e r m i n i n g grapevine performance and productivity under saline conditions. expression levels of apx, mn-sod and mdar were noticeably higher in plants grafted on p1103 and r110 compared to 41b. the expression of cat and gpx genes showed obvious enhanced level in plants grafted only on p1103 in response to salt exposure. cuzn-sod was down-regulated in leaves of ‘superior seedless’ grafted on p1103 and up-regulate d w h e n s c i o n w a s g r a f t e d o n 4 1 b o r r 1 1 0 . rootstocks with enhanced expression levels of antioxidant defense genes, such as superoxide dismutase (sod) and catalase (cat) can possibly eliminate or reduce detrimental effects of ros. thus, grapevine rootstocks that possess this defense line are more preferable to be utilized for soils subjected to salinity or grapevines irrigated with diluted brackish water. moreover, additional studies are needed to investigate the salt-stress responses of enzymatic and non-enzymatic antioxidant components in grapevine grafted on contrasting rootstocks. acknowledgements t h i s r e s e a r c h w a s f u n d e d b y t h e s c 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(lc50 = 0.004%), cananga odorata (lam.) hook f. and thoms (lc50 = 0.050%), murraya koenigii (l.) spreng. (lc50 = 0.113%), and euodia hortensis forma hortensis (lc50 = 0.114%). the essential oil of m. koenigii (ri=52%) and c. citratus (ri=52%) at 5% (v/v) concentration were found to have a higher repellent toxicity against the spiralling whiteflies. the chemical composition of the selected essential oils was also determined using gc-ms. the trend in the chemical constituent of essential oils revealed that the phenolic and alcoholic compounds were the major groups of contributors to the tested activities. thus, these data suggested that essential oils from the selected medicinal plants found in the south pacific (fiji) have the potential to be employed in the pesticidal activities. (*) corresponding author: chand_rv@usp.ac.fj received for publication 10 august 2016 accepted for publication 18 october 2016 copyright: © 2016 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2016 30(3): 165-174 166 diverse use of essential oil can represent a good alternative due to its novel, safe and eco-friendly substitute for its effective insecticidal properties (li et al., 2014; palanisami et al., 2014). several essential oils from medicinal plants have been screened for the repellence and toxicity against grain storage insects, fleas, ticks and lice (leal and uchida, 1998; gandhi et al., 2010; olivero-verbel et al., 2010; caballero-gallardo et al., 2011; cheng et al., 2012; seo et al., 2012; vera et al., 2014). however, very little information on the fumigant and repellent toxicity of essential oils from medicinal plants found in the south pacific (fiji) was available against the spiralling whiteflies. the aim of this study was to assess if essential oils of determined medicinal plants could serve as bio-pesticides for the control of the whitefly pest. the chemical profile of selected essential oils was also studied in order to provide justification for the presence of active compounds in the tested activities. 2. materials and methods essential oils extraction and analysis the plant materials from cananga odorata (lam.) hook f. and thoms (makosoi flowers), cymbopogon citratus (dc.) stapf. (lemongrass leaves), murraya koenigii (l.) spreng. (curry leaves), ocimum tenuiflorum l. (tulsi leaves) and eudioa hortensis forma hortensis (uci leaves) were collected from fiji islands in april to november, 2015. the selected plant materials were verified with the voucher specimens placed at university of the south pacific herbarium and koronivia research station, suva, fiji islands. the plant materials from the medicinal plants were hydro-distilled using clevenger apparatus for 5-7 hours. a meniscus layer (essential oils) was formed in the collecting tube which was then collected in a vial. the samples were dried over anhydrous sodium sulphate (na2so4) and stored at 4°c. t h e a n a l y s i s o f e s s e n t i a l o i l s u s i n g g a s chromatography equipped with mass spectrometry (agilent technologies 6890) was performed using an hp-5ms non polar fused silica capillary column (0.25 mm, 30 m, 0.25 μm film thickness; model number: 19091s-433) with the following conditions: the oven temperature was programmed from 50°c to 325°c over 5 min, at equilibration time of 0.50 min. the transfer source and quadrupole temperatures were 150°c, 200°c, 230°c and 250°c respectively, operating at 71 ev ionization energy. for the front inlet the mode used was split with an initial temperature of 250°c at 42.5 kpa at a split ratio of 50:1 and split flow of 43.8 ml/min. helium was used as a carrier gas at a constant linear velocity of 35 cm/sec, flow rate of 0.9 ml/min; the injected sample volume was 1.0 μl which was diluted in hexane (1000 μl). the analysis w a s c a r r i e d a t t h e s o u t h e r n c r o s s u n i v e r s i t y , australia. the constituents of essential oils were identified based on mass spectra comparison of retention indices (ri) with standard compounds. for the reference purpose, the database search was done using essoils and adams library. for the purpose of semi-quantification, the normalized peak areas of reported compounds were used without any correction factors for establishing abundance. retention indices (ri) and abundance were calculated using the mean values of 3 injections (el bouzidi et al., 2011). breeding of spiralling whiteflies the adult spiralling whiteflies were brought from a nearby farm (rewa province) without any insecticidal exposure. the collected spiralling whiteflies were brought to the green house where they were introduced to the cassava plants [manihot esculenta (crantz)] in order for them to grow and multiply. the plants were maintained in the greenhouse for appropriately 6-7 months without any pesticide contact before carrying out the actual experiment. the adult spiralling whiteflies were collected in petri dish using a small paintbrush. the conditions that were set in the laboratory were similar to the environment that they were found, that is, under the condition of 28±2°c, 75±5% rh and light regime of 14:10 h (l:d). the spiralling whiteflies (aleurodicus dispersus russell) bred in the greenhouse were brought into the laboratory when required to carry out the fumigant and repellent test. fumigant toxicity assessment t h e l e a v e s o f t h e c a s s a v a p o t p l a n t s w e r e enclosed with a clear pocket plastic bag (16 cm in length) with 50 whiteflies in each bag irrespective of their sex. the treatments [0.25%, 0.5% and 5% (v/v)] were introduced into each plastic bag using a filter paper (~2 cm in diameter) based on the randomisation. the filter paper discs (~2 cm in diameter) were impregnated on the side of the plastic bag. the control filter disc had tween 20 (5%) (purchased from sigma-aldrich, australia) mixed with the distilled water. the mortality count results after 3, 6, 9, 12 chand et al. bioactivity of essential oils from medicinal plants in fiji 167 and 24 hours were calculated. repellent toxicity assessment a t-shaped olfactometer set was constructed in order to test the repellency on the adult spiralling whiteflies. the setup consisted of a long glass tube (diameter of 50 cm). the external light source was placed between site 1 and site 2. site 1 had the control leaf disc (2 cm in diameter) dipped in tween 20 (5%) solution, while site 2 had the leaf disc with selected concentration of the essential oil. the essential oil concentration for all the five plants tested were 0.25%, 0.5% and 5% (v/v). the test was performed on 50 adult whiteflies with 4 replicates for each concentration. after 6-8 hours the number of whiteflies were counted using a hand lens for each site (chamber). the repellency index (ri %) was calculated using the formula (abdellaoui et al., 2009): ri % = (c-t/ c+t) x 100 where [c= whitefly counts on the control side of the olfactometer] and [t = whitefly counts on the treatment side of the olfactometer] . if the repellency index calculated (ri %) is positive, it means that the whiteflies were repelled with the tested concentration of essential oils and vice versa if the repellency index (ri %) calculated is negative. statistical analysis fumigant test assessment. a factorial anova (5x4x5 split plot design) using tukey’s hsd test was performed. prior to performing anova (significant at p=0.05), the percentage mortalities were transformed by the arcsine of the square root. the total mortalities were converted to percentage mortality. the lethal concentration (lc50) values for the mortality after 24 hours were assessed using probit in xlstat software (version 2015.1) (kabir et al., 2007; postelnicu, 2011). the morality was corrected using abbott’s formula for those that exceed 10% by natural mortality (abbott, 1925). repellent test assessment. to evaluate the statistical difference at 5% level of significance between each essential oil with its respective control, an independent sample t-test was performed. the probit analysis in xlstat software (version 2015.1) was also used to calculate the ec50 for the repelling effect of each essenti al oil (padhy and panigrahi, 2016, olufayo and alade, 2012). 3. result and discussion chemical analysis of the essential oils a total of 88 compounds were detected in the selected essential oils from the medicinal plants, accounting for 92.76-97.88% of total composition as summarised in table 1 and 2. the main chemical compounds identified in the essential oil of c. odorata were trans, trans-farnesol (29.71%), benzyl benzoate (21.69%), linalool (16.65%) and trans, trans-farnesyl acetate (6.93%). while for m. koenigii the major compounds identified were sabinene (43.80%), βcaryophyllene (16.52%), terpinen-4-ol (7.20%) and αpinene (5.67%). in case of e. hortensis, menthofuran (55.17%) and evodone (25.91%) were the main comp o u n d s . t h e e s s e n t i a l o i l f r o m o . t e n u i f l o r u m revealed the presence of eugenol (58.20%), germacrene d (11.68%) and cis-β-ocimene (10.79%) as the major compounds. the major compounds identified i n t h e c . c i t r a t u s e s s e n t i a l o i l w e r e c i t r o n e l l a l (45.09%), citronellol (19.11%), geraniol (13.57%) and elemol (6.15%). table 1 composition of essential oils (%) from c. odorata (makasoi), m. koenigii (curry leaves), e. hortenis (uci), o. tenuiflorum (tulsi) and c. citratus (lemon grass) chemical compounds ocimum tenuiflorum (%) cymbopogon citratus (%) cananga odorata (%) euodia hortensis forma hortensis (%) murraya koenigii (%) α-thujene 0.61 0.31# 1.79 linalool 0.21 0.27 16.65 0.10# myrcene 0.38 0.11 0.37 1.84 sabinene 0.43 0.58# 43.80 iso-pulegol 1:17 α-pinene 0.32 5.67 limonene 4.64 1-octen-3-ol 0.19# citronellal 45.09 0.20 iso iso-pulegol 0.46# β-pinene 1.55 α-terpinene 0.23# 2.64 decanal 0.14# methyl benzoate 1.64 menthofuran 55.17 p-cymene 0:23 0.67 citronellol 19:11 0.13# cis-β-ocimene 10.79 0.11 neral 0:55 ethyl benzoate 0.14 limonene-10-ol 0.60 trans-β-ocimene 0.43# 0.39 geraniol 13:57 0.74 terpinen-4-ol 1:01 0.15 7.20 evodone 25.97 β-phellandrene 0.69 ϒ-terpinene 0:37 4.82 geranial 0.74 methyl salicylate 3.15 α-copaene 1.98# 0.79 citronellic acid 0.37# to be continued adv. hort. sci., 2016 30(3): 165-174 168 variability in the essential oils the results obtained also showed variability in terms of the quality, quantity and composition of essential oils in all the selected plants when compared to the available literature, that is, o. tenuiflorum (pino et al., 1998; naquvi et al., 2012), c. citratus (negrelle and gomes, 2007; olivero-verbel et al., 2010; matasyoh et al., 2011; tyagi et al., 2014), c. odorata (katague and kirch, 1963; gaydou et al., 1986; murbach teles andrade et al., 2013), e. hortensis (brophy et al., 1985) and m. koenigii (raina et al., 2002; chowdhury et al., 2008) (table 1). the variability in the composition of essential oil is mainly due to the genetic variations, climatic, ecological locations, soil composition, plant organs, age and vegetative cycle stages of the plant (pietschmann et al., 1998; masotti et al., 2003; stewart, 2005; tchoumbougnang et al., 2005; angioni et al., 2006; koba et al., 2007; nascimento et al., 2008; katoch et al., 2013; erbil et al., 2015; ríos, 2016). fumigant toxicity of selected essential oils among the five tested essential oils (fig. 1 a-c), o. tenuiflorum essential oil showed the most robust fumigant effect against the spiralling whiteflies with lc50 value of 0.003% followed by the essential oils of c. citratus (lc50 = 0.004%), c. odorata (lc50 = 0.050%), m. koenigii (lc50 = 0.113%), and e. hortensis (lc50 = 0.114%) (table 3). statistically, the fumigant activity of o. tenuiflorum and c. citratus essential oils at 0.5% and 5% (v/v) concentrations were significantly higher than the other species (p=0.00). the significant threshold was set at p<0.05. the mortality count of the spiralling whiteflies were also higher at 5% (v/v) concentration for c. odorata, e. hortensis and m. koenigii essential oils as compared to 0.25% and 0.5% (v/v) concentrations. generally the increasing conc e n t r a t i o n s o f t h e t e s t e d e s s e n t i a l o i l s l e d t o increased mortality of whiteflies. the robust effect of o. tenuiflorum essential oil chemical compounds (z) ocimum tenuiflorum (%) cymbopogon citratus (%) cananga odorata (%) euodia hortensis forma hortensis (%) murraya koenigii (%) methyl chavicol 0.45# β-cubebene 0.26 allo-ocimene 0.17# citronellyl acetate 1.05# limonene-10-yl acetate 0.60 geranyl acetate 0.44 trans-anethole 0.27# α-(2) gurjunene 0.59# trans-sabinene hydrate 0.59 α-cubebene 0.18# β-elemene 0.59# δ-elemene 0.24# βcaryophyllene 0.54 isoterpinolene 0.95# eugenol 58.20 1.38 0.33# germacrene d 11.68 0.79# 2.74 0.27# 0.14 trans-α-bergamotene 0.18# trans-p-menth-2-en-1-ol 0.47# δ-cadinene 1.44# 0.88 methyl eugenol 1.77 trans-β-farnesene 0.20# β-bourbonene 0.93 elemol 6.15 β-caryophyllene 4.31 0.49 16.52 β-funebrene 0.23# α-terpineol 0.28 4-α-hydroxyl germacral (10), 5-diene 1.15# humulene 0.33# 0.29# cis-piperitol 0.12# β-copaene 0.35 ϒ-eudesmol 0.72# β-selinene 0.31# 0.40# trans-piperitol 0.17# δ-cardinol 0.27# α-germacrene 0.35# 0.18# ar-curcumene 0.60 ϒ-muurolene 0.40# α-cardinol 3.70 βelemene 1.50 trans, trans-farnesol 29.71 cis, trans-farnesol 0.46# bicyclogermacrene 0.41# trans, trans-farnesal 0.43# benzyl benzoate 0.21# 21.69 β-curcumene 0.56# α-cardinene 0.55# ϒ-cardinene 0.22# trans, trans-farnesyl acetate 6.93 δ-cardinene 0.46# α-selinene 0.78# benzyl salicyate 2.21 caryophyllene oxide 0.24 0.75# epi-1-cubenol 0.13# trans-nerolidol 0.24# α-cadinol 0.87# intermedeol 0.27# ϒ-curcumene 3.79# (z) compounds listed in order of elution from a hp-5ms non polar fused silica capillary column. # indicate that the compounds were detected for the first time as compared to the literature. chemical groups cananga odorata (%) murraya koenigii (%) euodia hortensis forma hortensis (%) ocimum tenuiflorum (%) cymbopogon citratus (%) monoterpenes 1.32 65.51 60.18 13.64 ester 35.76 0.60 1.70 alcohol and phenol 50.85 9.08 0.83 60.61 45.88 sesquiterpenes 4.13 20:27 9.17 22.61 3.41 aldehyde 0:43 0.20 46.52 ketones 25.97 acid 0.37 miscellaneous 0.27 total (%) 92.76 94.86 96.95 96.86 97.88 table 1 (continued) table 2 the major chemical groups present in the essential oils of c. odorata, m. koenigii, e. hortensis, o. tenuiflorum, and c. citratus chand et al. bioactivity of essential oils from medicinal plants in fiji 169 could be attributed to the chemical constituents present in the oil. in this study, o. tenuiflorum essential oil had 60.61% of alcoholic and phenolic compounds as compared to c. citratus (45.88%), c. odorata (50.85%), e. hortensis (0.83%) and m. koenigii (9.08%). according to isman (2000), eugenol compounds were found to be 7-9 times more toxic than terpenes and terpinene-4-ol. this confirms that o. tenuiflorum essential oils from the present study showed a strong fumigant effect due to eugenol (58.20%) compound. in previous studies, eugenols were also reported as a major cause of toxicity against the adult beetle (callosobruchus maculatus) (ajayi et al., 2014), bean weevil (acanthoscelides obrectus) (regnault-roger and hamraoui, 1995), yellow fever mosquito (aedes aegypti) (sosan et al., 2001) and rice weevil (sitophilus oryzae) (lee et al., 2003). cymbopogon citratus essential oil also showed strong fumigant effect (fig. 1 a-c). such effect can be attributed to the major chemical compounds, alcohols and phenols (45.88%), especially citronellol (19.11%) and geraniol (13.57%). these major chemical compounds have showed toxicity and repellent effects on different pests (fradin and day, 2002; ansari et al., 2005; choochote et al., 2007; paluch et al., 2009; sakulku et al., 2009; maia and moore, 2011). similarly, the interaction of different chemical compounds could have played a major role in the repression of the fumigant effect. according to chang et al. (2009), when linalool compound from the basil oil (ocimum family) was mixed with cuelure compounds, the level of toxicity on the tested insect (melon fly, bactrocera cucurbitae) decreased. the above scenario could explain why c. odorata essential oil had the second highest percentage of alcohol and phenol compounds (50.85%) while it was not able to produce a greater fumigant effect as compared to c. citratus (45.88%) essential oil. fig. 1 fumigant effect (mean±se) of selected essential oils on the spiralling whiteflies over different time intervals using different solution concentrations: (a) 0.25%; (b) 0.5%; (c) 5% (v/v). the alphabetical letters represent the respective essential oils and the asterisks indicate results statistically different from the control at p<0.05 (*), p<0.01 (**), p<0.001 (***) using tukey’s test. essential oils time (hours) equation r2 lc50 /ec50 (%) χ 2 statistic p-value df cananga odorata 24 y = 4.998 + 4.086x 0.750 0.050 118.149 <0.0001 1 murraya koenigii 24 y = 3.408+ 3.933x 0.316 0.113 76.080 <0.0001 1 euodia hortensis forma hortensis 24 y = 3.349+3.887x 0.586 0.114 78.574 <0.0001 1 cymbopogon citratus 24 y = 8.725+3.764x 0.902 0.004 279.950 <0.0001 1 ocimum tenuiflorum 24 y = 12.286+5.020x 0.651 0.003 253.512 <0.0001 1 table 3 dose-effect analysis of the fumigant properties of essential oils on the spiralling whiteflies after 24 hours at 0.25%, 0.5% and 5% (v/v) concentrations the χ² probability ≤0.0001, indicated that the significant difference was brought by the log (concentration) variable and the repellency. each test represents the mean of four replicates of 50 whiteflies. adv. hort. sci., 2016 30(3): 165-174 170 repellent toxicity of selected essential oils the table 4 revealed that none of the essential oils showed a very strong repelling effect on the s p i r a l l i n g w h i t e f l i e s . i n o r d e r , b a s e d o n t h e repellency index (ri %) of selected essential oils at the highest concentration [5%( v/v)], we found c. citratus (52%), m. koenigii (52%), o. tenuiflorum (12%), e. hortensis (10%) and c. odorata (9%). a direct relationship was seen between the repellent effect and the concentration (table 4). statistically, it was found that only c. citratus had a strong significant difference (r2 =0.611, p=0.00) at tested concentrations. the ec50 values in ascending order of the repellent effect of selected essential oils were 3.05% (c. odorata), 2.73% (o. tenuiflorum), 0.96% (e. hortensis), 0.43% (c. citratus) and 0.41% (m. koenigii). the chemical analysis in this study revealed the presence of α-pinene (5.67%), β-pinene (1.55%) and myrcene (1.84%) only in the essential oil of m. koenigii. the other active compounds that might have contributed towards the repellent effect can be terpinene-4-ol (7.20%) and eugenol (0.33%). in previous report, these compounds were found to repel yellow fever mosquito (aedes aegypti) (coats et al., 1 9 9 1 ; d e b b o u n e t a l . , 2 0 1 4 ) , b e a n w e e v i l (callosobruchus chinensis) (haidri et al., 2014) and two-spotted spider mites (lee et al., 1997). the repellent activity of c. citratus and m. koenigii essential oils at the highest concentration [5% (v/v)] were similar. in agreement with nerio et al. (2010), essential oil from c. citratus family were found to have promising repellent properties. the active compounds from previous studies such as α-pinene, limonene, citronellol, citronellal, camphor and thymol have shown higher repellent activity against ticks (amblyomma americanum) and yellow fever mosquito (aedes aegypti) (nerio et al., 2010; debboun et al., 2014). this study also reported the presence of citronellal (45.09%), citronellol (19.11%) and geraniol (13.57%) that may have caused the repellent effect (table 1). interestingly, the essential oil activity of o. tenuif l o r u m s h o w e d a w e a k r e p e l l e n c y a g a i n s t t h e spiralling whiteflies, despite the fact that the mode of action of essential oils against the spiralling whiteflies in both fumigant and repellent test are known to be similar. in fact, the mode of action of essential oils against the spiralling whiteflies was via neurotoxicity and respiratory toxicity (tanada and kaya, 1993; isman and machial, 2006; satar et al., 2008; li et al., 2014; tehri and singh, 2015). the weak repellent activity of o. tenuiflorum essential oil could be due the eugenol content (58.20%) which could have a t t r a c t e d t h e s p i r a l l i n g w h i t e f l i e s r a t h e r t h a n repelling. in previous study, eugenol caused attractancy to the japanese beetle (popillia japonica) (isman and machial, 2006). the other chemical compounds from literature that were found to attract the insects were cinnamyl alcohol, 4-methoxy-cinnamaldehyde, cinnamaldehyde, geranylacetone and α-terpineol (hammack, 1996; petroski and hammack, 1998). the overall trend of repellent effect of selected essential oils on the spiralling whiteflies can be ranked as c. citratus and m. koenigii followed by e. hortensis, o. tenuiflorum and c. odorata. 4. conclusions all the five essential oils from medicinal plants essential oils conc (v/v) (%) ri (%) equation r2 ec50 (%) χ 2 statistic p-value df cananga odorata 0.25 -29 y = -0.140+0.290x 0.0795 3.046 5.93 0.015 1 0.05 -17 5 9 murraya koenigii 0.25 -13 y = 0.260+0.663x 0.3232 0.406 38.214 < 0.0001 1 0.05 8 5 52 euodia hortensis forma hortensis 0.25 -10 y=0.003+0.188x 0.028 0.964 3.277 0.070 1 0.05 -3 5 10 cymbopogon citratus 0.25 -9 y = -0.285+0.953x 0.6111 0.434 27.474 < 0.0001 1 0.05 3 5 52 ocimum tenuiflorum 0.25 -18 y = -0.163+0.374x 0.1582 2.728 13.928 0.000 1 0.05 -11 5 12 table 4 summary of repellent effect (6-8 hours) on the adult whiteflies at different concentrations (using probit analysis) the χ² probability ≤0.0001, indicated that the significant difference was brought by the log (concentration) variable and the repellency. each test represents the mean of four replicates of 50 whiteflies. chand et al. bioactivity of essential oils from medicinal plants in fiji 171 tested against the spiralling whiteflies showed fumigant and repellent effects. the strongest fumigant effect was shown by o. tenuiflorum essential oils, while for repellent test none of the essential oils showed strong effect. however the m. koenigii and c. citratus showed higher repellency when compared with other tested essential oils. in addition, the results presented in this study are the first given information on the chemical composition of essential oils from the south pacific on the selected plant species. so far only e. hortensis essential oil composition data from fiji is reported (brophy et al., 1985). the selected essential oils from medicinal plants showed potential for the development of possible natural form of controlling the whitefly but needs to be further evaluated to enhance their activity and safety to the humans. acknowledgements a warm thanks to mr ashley dowell and the team f r o m s o u t h e r n c r o s s u n i v e r s i t y , q u e e n s l a n d , australia for assisting in the identification of compounds in the selected essential oils. a special thanks to the chief scientist dr rajeswara rao, dr. karuna shanker and the team from 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(gomez-campo, 2003). among these, the subsp sativa, also called eruca sativa, has been spreaded in different part of the world as cultivated rocket and is the most consumed and economically relevant. this species is diploid with eleven chromosomes (2n = 22) (padulosi and pignone, 1996) with annual life cycle flowering at begin of spring and ending with the prod u c t i o n o f s e e d s i n l a t e s p r i n g / e a r l y s u m m e r . nowadays it is cultivated in all continents in both marginal areas and/or fertile soils. plants of this species are characterized by a height of about 15.0 cm, flowers with calyx caduceus, sepals only two cucullate and corolla cream or whitish (gomezcampo, 2003). d i p l o t a x i s g e n u s i n c l u d e s a b o u t 3 3 s p e c i e s (www.theplantlist.org) with great variability related to morphological traits and chromosome number (table 1). the genus includes both annual and perennial plants with leaves of different shape, thickness, adv. hort. sci., 2017 31(2): 107-113 doi: 10.13128/ahs-21087 mini review rocket salad: crop description, bioactive compounds and breeding perspectives p. tripodi (*), g. francese, g. mennella consiglio per la ricerca in agricoltura e l’analisi dell’economia agraria, centro di ricerca orticoltura e florovivaismo (crea-of), 84098 pontecangnano-faiano (sa), italy. key words: diplotaxis tenuifolia, eruca sativa, flavonols, genetic improvement, glucosinolates, phytochemicals, rocket salad. abstract: rocket salad is a plant member of the brassicaceae family whose name encloses species of the eruca and diplotaxis genera characterized by leaves with peculiar pungent taste and strong flavour. it has been originated in the mediterranean area and nowadays is worldwide cultivated and consumed as food condiment and in ready-to-use mixed salad packages. several other uses are recognized in cosmetics and medicine. this crop represents a valuable source of health benefits due to the presence of a range of health-promoting phytochemicals including carotenoids, vitamin c, fiber, polyphenols, and glucosinolates. these compounds are potentially linked in the prevention of certain diseases and types of cancer. glucosinolates, represent the major class of compounds in rocket, and their hydrolysis products are responsible of the typical pungent aromas and flavours. despite the continuous increase of the global consumption during the recent years, few efforts have been carried out in genetic improvement programs aimed to constitute new varieties due to biological and reproductive barriers. in the present article is provided a brief overview of the principal species of rocket salad used in dietary and discussed the qualitative properties as well as the potentiality and constraints for breeding. (*) corresponding author: pasquale.tripodi@crea.gov.it received for publication 7 march 2017 accepted for publication 21 june 2017 copyright: © 2017 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2017 31(2): 107-113 108 indentation, and flower colour (white, yellow and purple). the most common species cultivated across all continents are diplotaxis tenuifolia and diplotaxis muralis. both are perennial being cultivated in the winter and producing new sprouts in the spring. this aspect, combined to the dehiscence of the siliqua and the large number of viable seeds, helps to spread these species as weeds. diplotaxis tenuifolia is the most used for human consumption; plants are characterized by average height of 80 cm, a deep tap root, fleshy leaves, and oblong, lobed with pointed apexes. main differences occurred between eruca and diplotaxis genus in terms of plant architecture, leaf morphology, chromosomal number and phytochemical compound contents. eruca species, being annuals, tends to have a higher growth rate, increased size of leaves and early flowering. these characteristics result in a high production of biomass, which make the system of cultivation different respect to the diplotaxis spp. and requiring a lower seeding density and a lower number of harvests. another trait discriminating the two genera is the larger seed dimension of the eruca species (1.5 mm in length) with respect to diplotaxis spp. (about 0.7 mm in length) (padulosi and pignone, 1996). a wider germination temperature range and greater speed of germination is also observed within the eruca species, which is probably due to their annual nature, requiring greater energy of the plant to produce viable seeds (hall et al., 2015). the consumption of rocket salad dates back since ancient time and included food and non food uses such as oil, deodorant, cosmetic and medical purposes (hall et al., 2012). aphrodisiac properties and medical uses related to anti inflammatory and depurative effects (padulosi and pignone, 1996) were emphasized by ancient poets during greek and roman times. nowadays leaves are eaten fresh in salads or as topping of many dishes (e.g. pizza) or cooked in soups. several recipes provide the preparation of pureed, sauces and pesto. other cosmetic uses concern the production of creams and lotions for body. rocket salad is worldwide cultivated and commercialized in many countries as mix salad packages. in europe, the needing of prepared products ready to use as well as the major attention given to a well balanced and assorted diet, composed of a variety of health-promoting compounds, has facilitated its consumption. in central and northern markets, over half of the rocket comes from italy and spain, which, table 1 list of diplotaxis species recognized and their chromosome number * na = not available. species chromosome number d. acris (forsk.) boiss. var. acris 11 var. duveyrieriana (coss.) coss. na * d. antoniensis rustan na d. assurgens (del.) thell. 9 d. berthautii br.-bl. and maire 9 d. brachycarpa godr. 9 d. brevisiliqua (coss.) mart.-laborde 8 d. catholica (l.) dc. var. catholica 9 var. rivulorum (br.-bl. and maire) maire na d. erucoides (l.) dc. subsp. erucoides 7 subsp. longisiliqua (coss.) gómez-campo 7 d. glauca (j.a. schmidt) o.e. schulz 13 d. gorgadensis rustan subsp. gorgadensis na subsp. brochmanii rustan na d. gracilis (webb) o.e. schulz 13 d. griffitthii (hook.f. and thomps.) boiss. na d. harra (forsk.) boiss. subsp. crassifolia (rafin.) maire 13 subsp. harra 13 subsp. lagascana (dc.) o. bolòs and vigo 13 subsp. confusa mart.-lab. 13 d. hirta (chev.) rustan and borgen 13 d. ibicensis (pau) gómez-campo 8 d. ilorcitana (sennen) aedo et al. 8 d. kohlaanensis a. miller and j. nyberg na d. muralis (l.) dc. subsp. ceratophylla (batt.) mart.-laborde na subsp. muralis 21 d. nepalensis hara na d. ollivierii maire na d. pitardiana maire na d. scaposa dc. 9 d. siettiana maire 8 d. siifolia kunze subsp. bipinnatifida (coss.) mart.-laborde na subsp. siifolia 10 subsp. vicentina (samp.) mart.-laborde 10 d. simplex (viv.) sprengel 11 d. sundingii rustan 13 d. tenuifolia (l.) dc. subsp. cretacea (kotov) sobr. vesp. 11 subsp. tenuifolia 11 d. tenuisiliqua subsp. rupestris (j. ball) mart.-laborde na subsp. tenuisiliqua 9 d. varia rustan na d. villosa boulos and jallad na d. viminea (l.) dc. var. viminea and var. integrifo10 d. virgata (cav. dc.) subsp. sahariensis coss. na subsp. virgata 9 subsp. rivulorum (br.-b1. and maire) mart.na subsp. australis mart.-lab. na d. vogelii (webb) o.e. schulz na tripodi et al. qualitative properties and breeding perspectives in rocket salad 109 thanks to their geographical position and mild climati c c o n d i t i o n s , r e p r e s e n t t h e m a i n p r o d u c e r s . diplotaxis is much more cultivated, fitting better to the needs of the farmers and being better suited to commercial utilization, thanks to the possibility to perform several harvests per cycle with yield increasing after the first harvest (hall et al., 2015). arugula is recommended in diets, having a very low-calorie vegetables (25 calories per 100 grams of fresh leaves) and being a very good source of vitamins and minerals (table 2). furthermore, it contains a r a n g e o f v i t a l c o m p o u n d s , w i t h i m p o r t a n t nutraceutical and anticancer properties, which are discussed in the next paragraph. 2. bioactive compounds many studies associate a highly significant reduction in the risk of cancer as well as a tumorogenesis inhibition and hepatoprotective effects with increasing consumption of cruciferae (lynn et al., 2006; juge et al., 2007; lamy et al., 2008; alqasoumi et al., 2009). rocket contains a range of health-promoting phytochemicals including carotenoids, vitamin c, fiber, polyphenols and glucosinolates (bennett et al., 2006; heimler et al., 2007). glucosinolates (glss) represent the major class of compounds in rocket and their contents in this crop h a v e b e e n w e l l d o c u m e n t e d i n t h e l i t e r a t u r e (d’antuono et al., 2008; pasini et al., 2012). when g l u c o s i n o l a t e s a r e e x p o s e d t o m y r o s i n a s e ( e c 3.2.1.147, thioglucoside glucohydrolase) during tissue damage, glucose and an unstable intermediate are formed. this intermediate degrades to produce a sulfate ion, and a variety of products including isothiocyanates, nitriles and, to a lesser extent, thiocyanates, epithionitriles and oxazolidines. the relative proportion of these hydrolysis products depends on the plant species studied, on the glucosinolate itself (as side chain substitution), and reaction conditions like ph, metal ions or epithiospecifier protein (bennett et al., 2007). both eruca and diplotaxis species contain similar profiles of glss within the leaf tissue, the most prominent of which are glucosativin (4-mercaptobutyl-gls), glucoerucin [4-(methylthio)butyl-gls] and g l u c o r a p h a n i n [ 4 ( m e t h y l s u l f i n y l ) b u t y l g l s ] . glucosativin and glucoerucin breakdown products are thought to contribute most to pungency and flavour in rocket (pasini et al., 2012). numerous other glss have also been identified within rocket tissue, for example diglucothiobeinin [4-(b-d-glucopyranosyldisulfanyl) butyl-gls] (kim et al., 2007), 4hydroxyglucobrassicin (4-hydroxy-3-indolymethylgls) (cataldi et al., 2007) and 4-methoxyglucobrassicin (4-methoxy-3-indolymethyl-gls) (kim and ishii, 2006). phenolics are the most abundant antioxidants in the human diet. considerable evidence indicates that some of the protective effects of phenols in fruits and vegetables may be due to flavonoids (clifford and brown, 2006). rocket species also contain large concentrations of polyglycosylated flavonol compounds, which are known to infer numerous beneficial health effects in humans and other animals. particularly of note are their effects on the gastrointestinal tract and in cardiovascular health (bjorkman et al., 2011; traka and mithen, 2011). several studies in rocket have identified and quantified polyglycosylated flavonols, which belong to three core aglycones: table 2 rocket salad nutritional values for 100 g of fresh leaves (usda nutrient database *) *https://ndb.nal.usda.gov/ndb/foods/show/3569?manu=&fgcd= &ds= (z) dietary folate equivalents. (y) retinol activity equivalents. nutrient unit value energy kcal 25 water g 91.71 carbohydrate g 3.65 protein g 2.58 sugars g 2.05 fiber g 1.6 lipid g 0.66 vitamins vitamin c mg 15 thiamin (vitamin b 1 ) mg 0.044 riboflavin (vitamin b 2 ) mg 0.086 niacin (vitamin b 3 ) mg 0.305 pyridoxine (vitamin b 6 ) mg 0.073 folate (vitamin b 9 ), dfe (z) µg 97 vitamin a, rae (y) µg 119 vitamin a iu 2373 vitamin e mg 0.43 vitamin k µg 108.6 minerals calcium, ca mg 160 iron, fe mg 1.46 magnesium, mg mg 47 phosphorus, p mg 52 potassium, k mg 369 sodium, na mg 27 zinc, zn mg 0.47 adv. hort. sci., 2017 31(2): 107-113 110 isorhamnetin, kaempferol and quercetin (bennett et al., 2006). pasini et al. (2012) studied the glucosinolate and phenolic profiles of 37 rocket salad accessions (32 eruca sativa and 5 diplotaxis tenuifolia) obtained by liquid chromatography-mass spectrometry. the authors isolated eleven desulpho-glucosinolates (dsglss) and the glucosinolate profiles did not differ between the two species. total ds-gls content, expressed as sinigrin equivalents (se) revealed a certain variability, ranging from 0.76 to 2.46 mg g-1 dry weight (dw) but, again, the quantitative analysis did not discriminate eruca from diplotaxis. moreover, the polyphenol evaluation by hplc-dad-ms allowed the identification of two different classes of compounds in the two rocket salad species. qualitative differences were observed between the polyphenol profiles at specific level: quercetin derivatives were the main phenolics of diplotaxis, whereas kaempferol derivatives characterised eruca samples. the contents of total flavonoids determined as rutin equivalents (re) ranged from 4.68 to 31.39 mg g-1 dw. k a e m p f e r o l 3 , 4 ’ d i g l u c o s i d e ( 7 1 . 4 8 2 . 2 % ) a n d isorhamnetin-3,4’-di-glucoside (7.8-18.4%) were always isolated as first and second more abundant phenolic compounds in eruca samples. no marker phenolic compounds were isolated in diplotaxis samples. durazzo et al. (2013) reported significant differences in the quality of conventional and integrated cultivation practices on the nutritional properties and benefits of wild rocket [diplotaxis tenuifolia (l.) dc.], while no influence on biological activity was evidenced. the authors also determined the cytotoxicity and antiproliferative activity of rocket polyphenol extract on human colon carcinoma (caco-2) cells, evidencing a significant accumulation of cells in g1 phase and a consequent reduction in the s and g2 + m phases in response to the treatment. regarding antioxidant properties, they found frap (ferric reducing antioxidant power) values ranged from 4.44±0.11 mmol kg-1 fresh weight (fw) to 9.92±0.46 mmol kg -1 fw for conventional rocket and from 4.13±0.17 mmol kg-1 fw to 11.02±0.45 mmol kg-1 fw for integrated rocket. villatoro-pulido et al. (2013) analysing four e. sativa accessions reported the total content of glucosinolates ranged from 6.12 to 12.33 mg g−1 of dw. glucoraphanin represented up to 52% of the total glucosinolates in leaves of one accession. accessions showed differences in the hydrolysis of glucoraphanin to the isothiocyanate sulforaphane. no correlation between these compounds was observed, which insisted differences in the myrosinase activity within accessions. the same authors highlighted that rocket leaves had variable phenolic profiles represented by quercetin-3-glucoside, rutin, myricetin, quercetin and ferulic and p-coumaric acids. a high variability was observed for the total carotenoids ranged from 16.2 to 275 μg g-1 with lutein as the main carotenoid. moreover, they found glucose was the predominant sugar, representing >70% of the total soluble carbohydrates. bell and collaborators (2015) used liquid chromatography mass spectrometry (lc-ms) to obtain glucosinolate and flavonol content for 35 rocket accessions and commercial varieties. they identified 13 glucosinolates and 11 flavonol compounds; semiquantitative methods were used to estimate concentrations of both groups of compounds. minor glucosin o l a t e c o m p o s i t i o n w a s f o u n d t o b e d i f f e r e n t between accessions; concentrations varied significantly. according to pasini et al. (2012) they confirmed flavonols differentiation between genera, with diplotaxis accumulating quercetin glucosides and eruca accumulating kaempferol glucosides. the authors detected several compounds in each genus that have only previously been reported in the other. recently, we investigated the qualitative and quantitative profiles of glucosinolates and polyphenols, highlighting flavonoid glycoside compounds (flavonols), in 39 accessions of wild and cultivated rocket (taranto et al., 2016). seven ds-glss were detected in rocket leaves belonging to two chemical classes: five aliphatic compounds (glucoerucin, glucoraphanin, progoitrin, glucoalyssin, and glucosativin) and two structurally related compounds containing one intermolecular disulfide linkage, 4-(β-d-glucopyranosyldisulfanyl)butyl-gls and dimeric 4-mercaptobutyl-gls. the species studied significantly differed for gls content: total average concentrations being 29.61 and 19.41 mg g-1 dw for e. sativa (21 accessions) and d. tenuifolia (16 accessions), respectively. total gls content ranged from 2.10 to 40.96 mg g-1 dw and from 11.61 to 26.96 mg g-1 dw, for eruca and diplotaxis accessions, respectively. additional accessions of d. muralis and erucastrum spp. were evaluated exhibiting an average gls content of 17.39 and 3.63 mg g-1 dw, respectively. fifteen flavonol compounds were tentatively identified in the thirty-nine accessions studied. diplotaxis accessions were characterized by nine diftripodi et al. qualitative properties and breeding perspectives in rocket salad 111 ferent flavonols mainly represented by quercetin derivatives, total average content being 7.17 mg g-1 dw with a range from 4.91 to 8.57 mg g−1 dw. the most abundant flavonol compound in diplotaxis was quercetin 3,4’-diglucoside-3’-(6-sinapoylglucoside). as regards eruca accessions, the more abundant flavonoid group was represented by kaempferol derivatives, in agreement with a previous report (martínez-sánchez et al., 2007). the 21 eruca sativa accessions showed a flavonol total average concentration of 8.13 mg g−1 dw, the lowest and the highest content being 0.82 and 10.16 mg g−1 dw, respectively. the most abundant flavonol was kaempferol 3,4’diglucoside. according to previous research, isorhamnetin 3,4’-diglucoside was the only compound common to diplotaxis and eruca accessions studied (martínezsánchez et al., 2008; pasini et al., 2012; bell et al., 2 0 1 5 ) . h o w e v e r , s o m e e x c e p t i o n s h a v e b e e n observed. specific compounds mainly detected in eruca such as kaempferol 3-glucoside and kaempferol 3-diglucoside-7-glucoside have been reported also in diplotaxis commercial varieties (bell et al., 2015). o t h e r c o m p o u n d s s p e c i f i c f o r d i p l o t a x i s ( i . e . , quercetin 3,4’-diglucoside-3’-(6-caffeoylglucoside) and quercetin 3,4’-diglucoside-3’-(6-sinapoylglucoside) have been also identified in eruca (bell et al., 2015). these inconsistencies could be related to the genetic material used. overall the results of the analysis of glucosinolates and flavonols evidenced how the eruca sativa gene pool contains potential candidates to use in breeding programs for quality. 3. potentiality and perspectives for breeding despite the global consumption of rocket salad has increased in the recent years, little efforts have been spent by both private and public breeding programs aimed to constitute new varieties. the importance in phytochemicals has been above discussed and novel knowledge as source of resistances have b e e n r e c e n t l y d e s c r i b e d ( p a n e e t a l . , 2 0 1 7 ) . n o w a d a y s , c o n s t r a i n t s a r e m a i n l y c a u s e d b y pathogens, nitrate accumulation, early flowering and physiological disorders due to intensive culture system. accessions of eruca sativa are reported to be late-bolting (kenigsbuch et al., 2014) and to accumulate less nitrate than diplotaxis tenuifolia (cavaiuolo and ferrante, 2014), being good candidates for the improvement with respect to the latter. however, several limitations for the transfer of these useful traits are linked to the failure of intergeneric crosses between eruca and diplotaxis due to post-zygotic barriers (tripodi unpublished) resulting in the absence of a cost effective hybridization system available for rocket. moreover, interspecific crosses among diplotaxis species are difficult due to their different chromosomes number (table 1). e r u c a s a t i v a x b r a s s i c a r a p a a n d d i p l o t a x i s tenuifolia x brassica rapa hybrids are instead possible using embryo rescue (agnihotri et al., 1990; jeong et al., 2009) and somatic hybridization (zhang et al., 2008) techniques, making the two rocket salad species a good source to use for the improvement of brassica rapa. the possibility of intercross has been applied in the development of cytoplasmic male sterile (cms) eruca sativa plants transferring a male sterile cytoplasm from brassica oleracea or brassica napus (merete et al., 2012). two approaches have been used: one requiring the application of embryo rescue after the first cross hybridization, subsequent chromosome doubling and backcrossing of the resulting hybrid to eruca sativa, another, using protoplast fusion from cytoplasmic male sterile brassica, subsequent regeneration of allogenic cells and crossing of the regenerated plant with pollen from eruca s a t i v a . t h e s a m e a p p r o a c h h a s b e e n u s e d b y hosemans and leviell (2012) by transferring cytoplasmic male sterility from raphanus sativus to diplotaxis tenuifolia. raphanus sativus has been also used to transfer cms in eruca sativa (nothangel et al., 2016). despite these achievements, breeding activities are still carried out by means of traditional selection schemes such as mass selection or single seed descent. new possibilities may be obtained by tilling (mccallum et al., 2000) in order to select mutants for gene of interest or genome wide association approaches (gwas) (huang and han, 2014) for the dissection of the genetic basis of complex traits and the development of markers for breeding assiste d s e l e c t i o n . m u t a g e n e s i s m e d i a t e d b y e t h y l methanesulfonate (ems) is already reported with success in diplotaxis tenuifolia (kenigsbuch et al., 2014), resulting in a mutant showing late flowering a n d d e l a y e d p o s t h a r v e s t s e n e s c e n c e . t h e s e a p p r o a c h e s s u c c e s s f u l i n b r a s s i c a s p e c i e s (stephenson et al., 2010; xu et al., 2016) may be also applied in rocket salad for a better exploitation of the genetic potentiality of this crop, and furthermore, to address the challenges of the modern agriculture that demands major security and quality of foods. adv. hort. sci., 2017 31(2): 107-113 112 acknowledgements the authors wish to acknowledge the project: “innovazione e potenziamento della filiera sementiera della rucola per la iv gamma (fiser)” funded through the f.e.a.s.r. european funding program (p.s.r. campania 207/2013, measure 124). references agnihotri a., gupta v., lakshmikumaran m.s., shivanna k.r., prakash s., jagannathan v., 1990 production of eruca brassica hybrids by embryo rescue. plant breeding, 104: 281-289. alqasoumi s., al-sohaibani m., al-howiriny t., alyahya m., rafatullah s., 2009 rocket “eruca sativa”: a salad herb with potential gastric anti-ulcer activity. world j. gastroenterol., 15: 1958-1965. bell l., oruna concha m.j., wagstaff c., 2015 identification and quantification of glucosinolate and flavonol compounds in rocket salad (eruca sativa, eruca v e s i c a r i a a n d d i p l o t a x i s t e n u i f o l i a ) b y l c − m s : highlighting the potential for improving nutritional value of rocket crops. food chem., 172: 852-861. bennett r.n., carvalho r., mellon f.a., eagles j., rosa e.a.s., 2007 identification and quantification of glucosinolates in sprouts derived from seeds of wild eruca sativa l. 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haro-bailón a., font r., del río-celestino m., 2013 an approach to the phytochemical profiling of rocket [eruca sativa (mill.) thell]. j. sci. food agric., 93(15): 3809-3819. xu l., hu k., zhang z., guan c., chen s., hua w., jiana l., jing w., bin y., jinxiong s., chaozhi m., jinxing t., tingdong f., 2016 genome-wide association study reveals the genetic architecture of flowering time in rapeseed (brassica napus l.). dna res., 23: 43-52. zhang c., yang z., gui x., liu y., mao x., xia g., lin l., 2008 somatic hybridization between brassica napus and eruca sativa mill. chin j. biotechnol., 24: 793-802. impaginato 239 1. introduction a variety of horticultural products is grown in afghanistan with the potential to be (re)developed into valuable export commodities and brands. the primary engine of the afghan economy is its agriculture industry, which engages approximately 80% of the working population. fruit trees, such as peach, plum, apricot or almond and grape are therefore of significant economic importance to the country, with importing or local exchange of plant material for propagation or commercial growing of these fruits likely. the absence of certain viruses is an essential pre-requisite for virus-free certification of plant material in general in many parts of the world (golino and savino, 2005; barba et al., 2015). in afghanistan, there is a tremendous increase in fruit tree nursery production and regulation of phytosanitary certification of plant propagation material is therefore of major importance. in 2005 the ministry of agriculture, irrigation and livestock (mail) stated in the agriculture master plan that the rejuvenation of the horticulture sector would be a government priority. thriving in the 1970s, this sector has become incapable of competing in the international market. three interconnected problems have been identified for the industry: 1) the existing orchards are run-down and production is adv. hort. sci., 2016 30(4): 239-248 doi: 10.13128/ahs-20350 the phytosanitary status of the national collection of fruits and nuts of afghanistan and the private mother stock nurseries: a virus survey s. rehman 1, j. ahmad 1, c. lanzoni 2, c. rubies autonell 2, c. ratti 2 (*) 1 plant biotecnology laboratory, seed and planting material certification directorate, ministry of agriculture, irrigation and livestock, kabul, afghanistan. 2 dipartimento di scienze agrarie, università di bologna, via g. fanin, 40, 40127 bologna, italy. key words: germplasm, plant pathology, virus disease. abstract: the horticultural industry is a vital component of the agriculture sector of afghanistan, the primary engine of the country’s recovering economy which engages approximately 80% of the working population. this sector was thriving in the 1970s, but is today incapable of competing in the international market. to recover and develop the horticulture of the country, the european community (ec) supports the phdp (perennial horticulture development project), to provide true to type/ecotype and healthy planting materials, and the plant biotechnology laboratory, to ensure the health status of local germplasm. this laboratory started screening the health status of the afghan germplasm national collections in order to ensure the multiplication of not only the best-selected varieties or ecotype, but also to avoid production and distribution of virus-infected trees. inspection for symptoms and sample collection for viral diseases was carried out in all the national collection fields, including cherry, pear, peach, plum, apricot, almond, apple, grape and citrus plants, located in different areas of the country. stone fruit plants infected by apple chlorotic leaf spot virus or prunus necrotic ringspot virus have been identified in the national collection experimental farms located in different provinces of afghanistan. moreover, many grape plants included in the national collection located in herat and kandahar resulted infected by grapevine fanleaf virus, but only few imported plants by grapevine leafroll associated virus 1, grapevine leafroll associated virus 3 or grapevine virus a. finally, in jalalabad (nangarhar province) citrus plants showing vein flecking, yellowing and plant decline symptoms were found to be infected by citrus tristeza virus. some of the identified viral isolates have been characterized molecularly, amplifying a fragment corresponding to the coat protein gene from a selection of positive samples. the presence of those viruses in different accessions of the national collection is of concern for afghan horticulture. implementation of the certification schemes is therefore necessary to quarantine the production and for the employment of virus-free propagating material. (*) corresponding author: claudio.ratti@unibo.it received for publication 4 february 2016 accepted for publication 16 february 2016 copyright: © 2016 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2016 30(4): 239-248 240 dwindling in quantity and quality; 2) nursery owners lack access to quality inputs and services; and 3) the regulatory framework to develop and support a competitive horticulture industry is minimal. to support and develop the horticultural industry of the country, a nursery certification system was s t a r t e d i n 2 0 0 6 f r o m t h e c o m p l e t e c o l l e c t i o n afghanistan’s fruit and nut trees crop varieties by the perennial horticulture development project (phdp) funded by european union (eu). one of the main causes for the decrease in production is deleterious pests and diseases, especially plant viruses and viruslike diseases present in afghanistan. virus infection in plants is particularly difficult to detect as viral infection may be sectorial or latent, with symptoms masked or even absent under certain conditions. virus-free certification is currently implemented using conventional techniques for virus detection in vegetal material such as biological indexing, serological or molecular assays. biological indexing requires, in many instances, grafting in greenhouse facilities which is expensive and time consuming. serological assays and molecular techniques, particularly elisa and rt-pcr, have been tested in several laboratories and shown to be efficient for detection and quantification of virus infection in pome and stone fruits (gambino and gribaudo, 2006; faggioli et al., 2013). in a country such as afghanistan, the establishment of a laboratory where sensitive, reliable and specific techniques for routine detection of selected viruses of fruit trees can performed is therefore essential for virus-free certification. many viral diseases can affect fruit trees, resulting in latent or symptomatic infection. according to their effect on the cultivated host, presence of the most important viruses in propagation plant material is regulated at international level. in particular, plum pox virus (ppv) is the causal agent of sharka, the most damaging virus disease of stonefruits. some strains of apple chlorotic leafspot virus (aclsv) can also induce serious diseases in stone fruits. prune dwarf virus (pdv) and prunus necrotic ringspot virus (pnrsv) are of considerable economic significance in stonefruits as well, while citrus tristeza virus (ctv) is considered to be the most destructive virus of citrus crops. apple mosaic virus (apmv), apple stem grooving virus (asgv) and apple stem pitting virus (aspv) are spread worldwide and able to causes significant losses in mixed infections (hadidi et al., 2011; barba et al., 2015). arabis mosaic virus (armv), grapevine fleck virus (gfkv). grapevine fanleaf virus (gflv), grapevine virus a (gva) and “grapevine leafroll-associated viruses” (glravs) such as grapevine leafroll associated virus 1, 2 and 3 (glrav-1, glrav-2 and glrav-3) are of great economic impact in grape and their absence in propagation material is an essential requirement in the certification schemes of most countries (martelli, 2014). national collection centres were established in six agro-ecological zones in afghanistan to collect germplasm. after registration and cataloguing, the mother stock nurseries were established from the same planting material to ensure the availability of high quality and healthy planting material to nursery growers and farmers. theoretical and practical knowledge are essential to develop a complete profile for viral diseases in order to improve the quality of planting material within the national fruit tree germplasm (rizzo et al., 2012, 2015). we therefore developed an in-depth study regarding the detection and identification of viral pathogens through the use of advance and sensitive methodologies and techniques to characterize identified plant viral pathogens and to develop control strategies for the management of diseases. these efforts are aimed at improving the quality of planting material and will ensure access to quality and certified planting material for nursery growers and orchard owners for the establishment of new orchard/motherstock nurseries, as well as for the rehabilitation of existing orchards. screening agricultural products according to the protocols and regulations of global plant quarantine will also positively affect the import and export of afghan agricultural entities. following the standards of international sanitary and phytosanitary rules and regulations is the only way to compete in the international market. 2. materials and methods plant material plant material was collected from the national c o l l e c t i o n c e n t r e s ( n c c s ) a n d m o t h e r s t o c k nurseries (msns) from 2009 to 2015. in total, more than 12,000 samples were collected from different species and tested for different viruses. in particular, almond, apricot, cherry, peach and plum samples were tested for the presence of aclsv, apmv, pdv, pnrsv and ppv; apple and pear samples for aclsv, apmv, asgv and aspv; citrus samples for ctv; and grape samples for armv, gva, gfkv, gflv, glrav-1, glrav-2 and glrav-3. rehman et al. phytosanitary status of afghanistan fruits and nuts collection. a virus survay 241 about 32% of the fruit tree samples were collected from the six nccs located in kabul (apricot and plum), balkh (apricot and almond), kunduz (almond), herat (grape and plum), kandahar (grape and pomegranate) and nangarhar (citrus and pomegranate). moreover, samples were also collected from msns (26%) and from private orchards (nearly 40%) (fig. 1). each year stone fruit samples were collected duri n g t h e v e g e t a t i v e s t a g e i n s p r i n g o r s u m m e r , between march and september, according to the climatic conditions of each location. similarly, apple and pear samples were collected in may, june, july or september; citrus in march, may or november; and grape in dormant stage in january, october or december. the collecting method consisted of sampling leaves (in the case of stone and pome fruits), shoots or canes (grape) or leaves and flowers (citrus) homogeneously distributed around the canopy of the plant. all collected material was maintained under refrigeration (below 10°c) during transport from the field to the laboratory, then kept at -20°c until analyses. serological and molecular analyses all samples were ground in “universal” extraction bags (12x15 cm) using the homex 6 homogenizer (bioreba) then analysed by das-elisa using reagent kits from bioreba (reinach, switzerland) specific for each virus assayed and following the manufacturer’s instructions. samples that resulted positive by das-elisa test were subsequently analysed by rt-pcr reaction using specific primer pairs (table 1). fig. 1 location of the six national collection centres and of the 57 mother stock nurseries in afghanistan. virus primers primers sequences (5’ 3’) size of amplif. (bp) cycling (temp./time)* n° of cycles reference denat. anneal. exten. aclsv sense ttcatggaaagacaggggcaa 677 94°c/20 s 58°c/20 s 72°c/20 s 35 menzel et al., 2002 antisense aagtctacaggctatttattataagtctaa pnrsv mg1 atggtttgccgaatttgc 675 94°c/60 s 55°c/45 s 72°c/60 s 35 glasa et al., 2002 mg2 actctagatctcaagcaggtc gflv m3 atgctggatatcgtgaccctgt 118 94°c/10 s 54°c/10 s 72°c/45 s 35 gambino and gribaudo, 2006 m4 gaaggtatgcctgcttcagtgg ctv ctvf taatggacgacgaacaaaga 655 94°c/40 s 56°c/40 s 72°c/60 s 30 rehman et al., 2012 ctvr ccaagctgcctgacattagt table 1 list of primer pairs, size of amplicons, and rt-pcr conditions used to confirm viral infection in all samples resulting positive to the das-elisa test * pcr amplifications were performed at 94°c for 5 min for initial denaturation and 72°c for 5 min for final extension. adv. hort. sci., 2016 30(4): 239-248 242 total rnas were extracted from each sample using ctab rna extraction method, as previous reported (ratti et al., 2004). complementary dna (cdna) was synthesized using m-mlv reverse transcriptase (promega, usa) in a final volume of 5.0 µl. rna (1.0 µl), mixed with m-mlv 5x buffer, 0.5 µl 10 mm dntps, 1.0 µl 10nmol/ml specific reverse primer, 0.25 µl of 200 u/µl m-mlv and 2.25 µl of nucleasefree water, then incubated at 4 2°c (or 37°c for aclsv and pnrsv) for 1 h. for pcr amplification, 5.0 µl of cdna template, 5.0 µl of 5x green gotaq buffer, 1.5 µl of 25 mm mgcl2, 1.0 µl 10 nmol/ml of specific forward primer, 0.5 µl of 10 mm dntps and 0.2 µl of 5 u/µl gotaq polymerase (promega, usa) were mixed in a total volume of 25 µl. the number of cycles and cycling conditions for each primer set are given in table 1. the amplified pcr products were analysed in 1% agarose gel stained by ethidium-bromi de and visualized under uv light after el ectrophoresis. 3. results das-elisa analyses all apple and pear plants analysed (180 samples) resulted free from aclsv, apmv, asgv and aspv. symptoms such as yellowing, leaf distortion and puckering were observed in some stone fruit plants during collection of the 5521 samples that were tested by das-elisa (fig. 2). among them, 23 (0.4%) samples collected from several msns in kabul, khandahar, parwan, herat and baghlan provinces resulted positive to aclsv. in particular, there were two plants of european plum (clone 364/local name alu bokharashalili), one cross of myrobalan and japanese plum (4031/grangej zard), 17 plants of peach (five from 804/turki sorkh, five from 812/dir ras and seven from 811/turkizard), one plant of almond (159/sattarbai bakhmali) and two plants of apricot (373/shakarpara and 4037/aqa banu). two (0.03%) almond samples, both collected from the ncc in balkh province, resulted infected by apmv; in was interesting that one of them also tested positive to pnrsv. analyses against pnrsv evidenced 38 samples (0.6%), collected from nccs or msns located in balkh, herat, kabul, khandaharand paktya as infected by the virus (13 almond, five plum, nine peach, and 11 cherry plants). finally, one plum sample (cv. alu bokhara shalili), from a msn in kabul, resulted infected by pdv while none of the stone fruit samples tested resulted positive to ppv (tables 2-6 and fig. 3). among the 895 grape samples analysed, one plant from the ncc in kandahar (cv. shir ahmadi herat) resulted infected by gva and only one sample of the cv. pizzutello bianco, imported from italy and collected in the ncc in herat, tested positive to glrav-1 (it also resulted as infected by glrav-3). moreover, glrav-3 was detected in two samples (cv. pizzutello bianco and uva palieri both imported from italy), collected from the nnc in herat. regarding gflv, all the 62 samples which resulted infected by the virus (6.9%) belong to afghan cultivars, with the exception of the cv. regina dei vigneti imported from italy. all grape samples tested resulted negative to armv, gfkv and glrav-2 (table 7) (fig. 3). in addition, the plant biotechnology laboratory analyzed 5400 citrus plants and 318 of them (5.9%) resulted infected by ctv. in particular, among the positive samples, eight plants, showing vein clearing and flecking, yellowing and plant decline symptoms (fig. 4), were collected from the ncc in jalalabad and belong to six different accessions: kumquat cv. fig. 2 yellowing observed in an apricot accession during sample collection in a national collection centre. rehman et al. phytosanitary status of afghanistan fruits and nuts collection. a virus survay 243 table 3 apricot samples tested and resulting positive by das-elisa during the first seven years of activity of the plant biotechnology laboratory based in kabul virus region 2009 2010 2011 2012 2013 2014 2015 total tested positive tested positive tested positive tested positive tested positive tested positive tested positive tested positive ppv north 174 0 0 0 205 0 174 0 53 0 82 0 78 0 766 0 central 2 0 0 0 24 0 136 0 103 0 69 0 42 0 376 0 south 0 0 0 0 0 0 0 0 47 0 0 0 0 0 47 0 pdv north 174 0 0 0 205 0 174 0 53 0 82 0 78 0 766 0 central 2 0 0 0 24 0 136 0 103 0 69 0 42 0 376 0 south 0 0 0 0 0 0 0 0 47 0 0 0 0 0 47 0 pnrsv north 174 6 (balkh) 0 0 205 5 (balkh) 174 0 53 0 82 0 78 0 766 11 central 2 1 (herat) 0 0 24 0 136 0 103 0 69 0 42 1 (herat) 376 2 south 0 0 0 0 0 0 0 0 47 0 0 0 0 0 47 0 apmv north 174 0 0 0 205 0 174 0 53 0 82 0 78 0 766 0 central 2 0 0 0 24 0 136 0 103 0 69 0 42 0 376 0 south 0 0 0 0 0 0 0 0 47 0 0 0 0 0 47 0 aclsv north 0 0 0 0 205 0 174 0 53 0 82 0 78 0 592 0 central 0 0 0 0 24 0 136 0 103 0 69 0 42 1 (herat) 374 1 south 0 0 0 0 0 0 0 0 47 0 0 0 0 0 47 0 total 176 7 0 0 229 5 310 0 203 0 151 0 120 2 1189 14 table 2 almond samples tested and resulting positive by das-elisa during the first seven years of activity of the plant biotechnology laboratory based in kabul northern regions: badakhshan, baghlan, balkh, faryab, jowzjan, kunduz, samangan, sar-e pol and takhar. central regions: badghis, bamiyan, farah, ghor, herat, kabul, kapisa, kunar, laghman, logar, nangarhar, nurestan, panjshir, parwan and wardak. southern regions: daykundi, ghazni, helmand, kandahar, khost, nimruz, orūzgān, paktia, paktika, zabul. virus region 2009 2010 2011 2012 2013 2014 2015 total tested positive tested positive tested positive tested positive tested positive tested positive tested positive tested positive ppv north 0 0 0 0 0 0 68 0 0 0 40 0 80 0 188 0 central 278 0 833 0 0 0 221 0 141 0 69 0 48 0 1590 0 south 0 0 0 0 0 0 0 0 29 0 0 0 0 0 29 0 pdv north 0 0 0 0 0 0 68 0 0 0 40 0 80 0 188 0 central 278 0 833 0 0 0 221 0 141 0 69 0 48 0 1590 0 south 0 0 0 0 0 0 0 0 29 0 0 0 0 0 29 0 pnrsv north 0 0 0 0 0 0 68 0 0 0 40 0 80 0 188 0 central 278 0 833 0 0 0 221 0 141 0 69 0 48 0 1590 0 south 0 0 0 0 0 0 0 0 29 0 0 0 0 0 29 0 apmv north 0 0 0 0 0 0 68 0 0 0 40 0 80 0 188 0 central 278 0 833 0 0 0 221 0 141 0 69 0 48 0 1590 0 south 0 0 0 0 0 0 0 0 29 0 0 0 0 0 29 0 aclsv north 0 0 0 0 0 0 68 0 0 0 40 0 80 2 (baghlan) 188 2 central 0 0 0 0 318 0 221 0 141 0 69 0 48 0 797 0 south 0 0 0 0 0 0 0 0 29 0 0 0 0 0 29 0 total 278 0 833 0 318 0 289 0 170 0 109 0 128 2 2125 2 see legend of table 2 for provinces included in northern, central and southern regions. table 4 cherry samples tested and resulting positive by das-elisa during the first seven years of activity of the plant biotechnology laboratory based in kabul see legend of table 2 for provinces included in northern, central and southern regions. virus region 2009 2010 2011 2012 2013 2014 2015 total tested positive tested positive tested positive tested positive tested positive tested positive tested positive tested positive ppv north 0 0 0 0 0 0 7 0 0 0 0 0 0 0 7 0 central 0 0 0 0 0 0 1 0 93 0 58 0 35 0 187 0 south 0 0 0 0 0 0 0 0 18 0 0 0 0 0 18 0 pdv north 0 0 0 0 0 0 7 0 0 0 0 0 0 0 7 0 central 0 0 0 0 0 0 1 0 93 0 58 0 35 0 187 0 south 0 0 0 0 0 0 0 0 18 0 0 0 0 0 18 0 pnrsv north 0 0 0 0 0 0 7 0 0 0 0 0 0 0 7 0 central 0 0 0 0 0 0 1 0 93 6 (kabul) 58 1 (kabul) 35 3 (herat) 187 10 south 0 0 0 0 0 0 0 0 18 1 (paktya) 0 0 0 0 18 1 apmv north 0 0 0 0 0 0 7 0 0 0 0 0 0 0 7 0 central 0 0 0 0 0 0 1 0 93 0 58 0 35 0 187 0 south 0 0 0 0 0 0 0 0 18 0 0 0 0 0 18 0 aclsv north 0 0 0 0 0 0 7 0 0 0 0 0 0 0 7 0 central 0 0 0 0 0 0 1 0 93 0 58 0 35 0 187 0 south 0 0 0 0 0 0 0 0 18 0 0 0 0 0 18 0 total 0 0 0 0 0 0 8 0 111 7 58 1 35 3 212 11 adv. hort. sci., 2016 30(4): 239-248 244 table 6 plum samples tested and resulting positive by das-elisa during the first seven years of activity of the plant biotechnology laboratory based in kabul see legend of table 2 for provinces included in northern, central and southern regions. virus region 2009 2010 2011 2012 2013 2014 2015 total tested positive tested positive tested positive tested positive tested positive tested positive tested positive tested positive ppv north 0 0 0 0 0 0 48 0 31 0 0 0 45 0 124 0 central 160 0 0 0 190 0 168 0 140 0 31 0 15 0 704 0 south 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 pdv north 0 0 0 0 0 0 48 0 31 0 0 0 45 0 124 0 central 160 0 0 0 190 0 168 0 140 0 31 0 15 0 704 0 south 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 pnrsv north 0 0 0 0 0 0 48 0 31 0 0 0 45 0 124 0 central 160 5 (herat) 0 0 190 0 168 0 140 0 31 0 15 0 704 5 south 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 apmv north 0 0 0 0 0 0 48 0 31 0 0 0 45 0 124 0 central 160 0 0 0 190 0 168 0 140 0 31 0 15 0 704 0 south 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 aclsv north 0 0 0 0 0 0 48 0 31 0 0 0 45 0 124 0 central 0 0 0 0 190 0 168 0 140 2 (kabul) 31 0 15 0 544 2 south 0 0 0 0 0 0 0 0 33 1 (kandahar) 0 0 0 0 33 1 total 160 5 0 0 190 0 216 0 204 3 31 0 60 0 861 8 fig. 3 distribution, among afghan provinces, of samples resulting positive to aclsv, apmv, ctv, gflv, glrav-1, glrav-3, gva, pdv or pnrsv. see legend of table 2 for provinces included in northern, central and southern regions. table 5 peach samples tested and resulting positive by das-elisa during the first seven years of activity of the plant biotechnology laboratory based in kabul virus region 2009 2010 2011 2012 2013 2014 2015 total tested positive tested positive tested positive tested positive tested positive tested positive tested positive tested positive ppv north 0 0 0 0 0 0 75 0 41 0 9 0 65 0 190 0 central 4 0 0 0 267 0 374 0 186 0 21 0 13 0 865 0 south 0 0 0 0 0 0 0 0 75 0 0 0 0 0 75 0 pdv north 0 0 0 0 0 0 75 0 41 0 9 0 65 0 190 0 central 4 0 0 0 267 0 374 0 186 0 21 0 13 0 865 0 south 0 0 0 0 0 0 0 0 75 0 0 0 0 0 75 0 pnrsv north 0 0 0 0 0 0 75 0 41 0 9 0 65 0 190 0 central 4 0 0 0 267 0 374 7 (herat) 186 1 (kabul) 21 0 13 0 865 8 south 0 0 0 0 0 0 0 0 75 1 (kandahar) 0 0 0 0 75 1 apmv north 0 0 0 0 0 0 75 0 41 0 9 0 65 0 190 0 central 4 0 0 0 267 0 374 0 186 0 21 0 13 0 865 0 south 0 0 0 0 0 0 0 0 75 0 0 0 0 0 75 0 aclsv north 0 0 0 0 0 0 75 0 41 0 9 0 65 0 190 0 central 0 0 0 0 267 0 374 0 186 17 (kabul) 21 0 13 0 861 17 south 0 0 0 0 0 0 0 0 75 0 0 0 0 0 75 0 total 4 0 0 0 267 0 449 7 302 19 30 0 78 0 1130 26 rehman et al. phytosanitary status of afghanistan fruits and nuts collection. a virus survay 245 margarita (isolates j4 and j8), orange cv. mahali (j61), rough lemon cv. mahali (j101) and mandarin g r o u p c v s . f r u t e r ( j 7 6 ) , t a n g e l o m a p o a n d clementine di nules. the other 312 plants positive to ctv were collected from private orchards located in the provinces of kunar, laghman, and nangarhar (table 8, fig. 3). rt-pcr and molecular characterization samples from stone fruit plants which resulted infected by aclsv, or pnrsv following das-elisa technique were analysed also by rt-pcr reaction. the results obtained confirmed the presence of each viral agent assayed in the samples tested. in particular, regarding aclsv isolates, a 677-long nucleotide fragment, corresponding to partial coat protein gene, was amplified from all elisa-positive samples by rt-pcr using aclsv sense and antisense primers (menzel et al., 2002). eleven of the identified isolates of aclsv were then characterized molecularly. sequence analysis revealed similarity ranging from 83.6 to 100.0% within aclsv isolates detected in afghanistan. blast analysis showed that sequences of two peach isolates, 812/dir ras, shared the highest nucleotide similarity (95.8 and 96.2%) with the g e n b a n k a c l s v i s o l a t e s a p r 3 f r o m j o r d a n (aj586631). moreover, the same afghan isolates showed nucleotide identity between 95.0 and 95.7% with isolates s4, pp23, pp63, and hl2 (jn849008, gu327991, gu328003 and gq334211, respectively) from china. sequence analysis of isolate 364/alu table 7 grape samples tested and resulting positive by das-elisa during the first seven years of activity of the plant biotechnology laboratory based in kabul virus region 2009 2010 2011 2012 2013 2014 2015 total tested positive tested positive tested positive tested positive tested positive tested positive tested positive tested positive gflv herat 0 0 0 0 156 19 117 13 271 22 0 0 0 0 544 54 kandahar 0 0 0 0 0 0 0 0 268 0 0 0 0 268 0 armv herat 0 0 0 0 156 0 117 0 271 0 0 0 0 0 544 0 kandahar 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 gva herat 83 0 0 0 156 0 117 0 271 0 0 0 0 0 627 0 kandahar 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 1 gfkv herat 83 0 0 0 156 0 117 0 271 0 0 0 0 0 627 0 kandahar 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 glrav-1 herat 83 0 0 0 156 0 117 1 271 0 0 0 0 0 627 1 kandahar 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 glrav-2 herat 83 0 0 0 156 0 117 0 271 0 0 0 0 0 627 0 kandahar 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 glrav-3 herat 83 0 0 0 156 0 117 2 271 0 0 0 0 0 627 2 kandahar 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 total 83 0 0 0 156 19 117 16 539 23 0 0 0 0 895 58 fig. 4 vein clearing and yellowing symptoms observed in citrus accessions collected from the national collection centre in jalalabad (nangarhar province). table 8 citrus samples tested and resulting positive by das-elisa during the first seven years of activity of the plant biotechnology laboratory based in kabul. virus region 2009 2010 2011 2012 2013 2014 2015 total tested positive tested positive tested positive tested positive tested positive tested positive tested positive tested positive ctv nangarhar 0 0 193 6 263 2 1276 153 1554 110 131 0 492 0 3909 271 laghman 0 0 0 0 0 0 180 9 200 4 0 0 296 3 676 16 kunar 0 0 0 0 0 0 318 21 246 9 0 0 249 1 813 31 total 0 0 193 6 263 2 1774 183 2000 123 131 0 1037 4 5398 318 adv. hort. sci., 2016 30(4): 239-248 246 bokhara shalili, 804/turki sorkh and 811/turki zard proved that they are 99.6 to 100.0% identical with each other and share high (94.3 to 94.7%) nucleotide identity with the iranian isolate t12ja (km586376). lower nucleotide similarity (86.2%) was shown by one 804/turki sorkh isolate with the isolate 274-chal (fn391009) from greece. finally, analysis of the sequence of isolates 804/turki sorkh, 811/turki zard and 812/dir ras revealed a high degree of identity (86.2 to 88.4%) with the corresponding nucleotide sequences of the isolate sk-92-pl (hq398253) from slovakia. rt-pcr reaction performed on samples infected by pnrsv, according to das-elisa results, amplified a fragment of 675 nucleotides in length when pnrsv specific primer pair mg1/mg2 was used (table 1). regarding grape samples, specific pcr products of 118 nucleotides where also observed on das-elisa positive samples, using specific primers pair m3/m4 against gflv as reported in table 1. some of the citrus samples tested positive by das-elisa were also tested using primers ctvf and ctvr allowing amplification of specific pcr products 655 nucleotides in length (table 1). moreover, six ctv isolates collected at the jalalabad station were characterized. sequence analysis revealed high similarity, ranging from 91.1 to 99.8%, within the ctv isolates detected. in accordance with the previously defined phylogenetic groups (zemzami et al., 2002), nucleotide sequences of afghan ctv isolates cluster in group 1 (j4 and j8), group 4 (j61 and j76) and group 5 (j101). in particular, j4 and j8 isolates show, respectively, 99.4 and 99.2% identity with reference i s o l a t e t 3 6 ( m 7 6 4 8 5 ) f r o m t h e u s a ( f l o r i d a ) . furthermore, in group 4, isolate j61 and j76 were more similar to ano-1 isolate (dq211658) from egypt (98.5 and 98.0% identity, respectively) than to isolate 443-4 (ay791844) from croatia (97.4 and 97.5%, respectively). finally, isolate j101, in group 5, s h o w s 9 5 . 6 % i d e n t i t y w i t h i s o l a t e s c 2 6 8 2 (ay750770) and c269-6 (ay750775) from argentina. 4. discussion and conclusions to improve afghanistan’s agricultural sector, the e c s u p p o r t s t h e p h d p ( p e r e n n i a l h o r t i c u l t u r e development project), for true to type/ecotype and h e a l t h y p l a n t i n g m a t e r i a l s , a n d t h e p l a n t biotechnology laboratory, to ensure the health status of local germplasm. the laboratory started screening the health status of the afghan germplasm national collection in order to ensure the multiplication of not only the best-selected varieties or ecotype, but also to avoid production and distribution of virus-infected fruit trees. symptom inspection and sample collection for viral diseases was carried out in all the national collection fields, including peach, plum, apricot, almond, apple, grape and citrus plants, located in different areas of the country. during the first seven years of its activity, the plant biotechnology laboratory based in kabul, played a key role by satisfying the increasing need for certified propagation plant material to establish new plantations in afghanistan. moreover, it contributed to protecting the country from undesirable introduction of pathogens due to increasing exchanges of propagation plant material among different countries and between different continents. until now, the plant biotechnology laboratory has verified the absence of ppv (the most devastating virus for stone fruit) in the afghan germplasm and detected and verified the presence of several import a n t p l a n t v i r u s e s i n s t o n e f r u i t s , c i t r u s a n d grapevines. further activity will be addressed to confirming, by rt-pcr, infection by apmv, pdv, gva, glrav-1 and glrav-3 in almond, plum and grape accessions which resulted positive by das-elisa, using specific primer pairs (parakh et al., 1995; menzel et al., 2002; goszczynski and jooste, 2003; osman et al., 2008). the plant biotechnology laboratory first reported aclsv occurrence in peach, plum, almond and apricot plants in afghanistan. according to the preliminary results of sequence analysis, due to the relative low identity with other aclsv isolates present in genbank, our mother stock nurseries appear to be infected by several afghan isolates of the virus, but further studies will be addressed to understanding if aclsv has been introduced into the country through infected propagation material. in any case, the presence of aclsv in important cultivars of peach and plum is a worrying aspect of the afghan certification program. similarly, our results identified, for the first time, p n r s v , a p m v , a n d p d v i n f e c t e d p l a n t s i n afghanistan. even if the latter two viruses were detected by das-elisa test, the presence of the viruses in accessions of the national collection field is cause for worry for afghan horticulture as all three viruses spread through infected propagating material and nursery productions (mink, 1992). moreover, pnrsv and pdv are also seedand pollen-transmitrehman et al. phytosanitary status of afghanistan fruits and nuts collection. a virus survay 247 ted and can be spread by pollinating insects (kelley and cameron, 1986; aparicio et al., 1999; glasa et al., 2002; amari et al., 2007) while seed transmission of apmv is known only in hazelnuts (cameron and thompson, 1985). a comparative sequence and phylogenetic analysis will be performed (zindović et al., 2015) to show if clustering of various isolates is associated or not with geographic and host origin. regarding the detection of viruses in grape, according to our results the presence in the ncc in kandahar of gva and glrav-1, and glrav-3 in herat seems to be restricted to only a few accessions. early detection of these viruses by the plant biotechnology laboratory avoided multiplication and therefore the spread of infected grape material in afghanistan. in contrast, the noticeable presence of gflv (nearly 7%) in local cultivars maintained at the ncc in herat suggests the presence of the virus in afghan germplasm of grape for some time. this hypothesis should be confirmed by studying the genetic variability of afghan isolates in order to investigate the relationship among their geographical origin, sequence variability, and grapevine cultivar (meng et al., 2006; vigne et al., 2009; terlizzi et al., 2015). finally, our analyses identified, for the first time, ctv infected plants in afghanistan (rehman et al., 2012). the presence of a dangerous viral disease such as ctv in many citrus trees (nearly 6% of the a s s a y e d p l a n t s ) f r o m n c c s , m s n s a n d p r i v a t e orchards represents a serious problem for the afghan citrus industry. high sequence identity with many isolates from usa, egypt, croatia, and argentina together with the large spread of the virus in afghan citrus-cultivating areas suggests the introduction of the virus into the country a long time ago. the presence of this virus seems to be, therefore, endemic; further investigations showed that infected plants did not exhibit quite as dramatic symptoms as those usually found in other citrus cultivation areas (e.g. spain, brazil, argentina etc.) where the virus is extremely dangerous and causes in some cases plants death (rochapeña et al., 1995). a specific research program is in progress to investigate if a mild strain of ctv is pres e n t i n t h e e a s t e r n r e g i o n ( n a n g a r h a r , k u n a r , laghman) environment, if tolerant/resistant sour orange rootstock clones exist, or if the environmental (climate and soil conditions) influences the infectivity of ctv. due to the economic importance of these crops, implementation of the certification schemes is therefore necessary in afghanistan in order to guarantee the production and employment of virus-free propagating material. it is well known that data regarding the presence and spread of different plant viruses are important for individual countries, their neighbours, and for the whole agricultural community where different viruses can be spread by vegetative propagation via plant material. a continuous survey is therefore necessary to protect afghanistan from the introduction and/or spread of dangerous pests. as previously experienced, most effective management programs require prompt removal of infected trees and replacement with healthy planting material from certified nurse r i e s ( h a d i d i e t a l . , 2 0 1 1 ; p a l l a s e t a l . , 2 0 1 2 ) . establishment of rigid phytosanitary controls related to the importation of propagation material, as 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the most important herbs widely used for its medicinal properties and as food ingredient. the marketing of this product highlights the problem that these plants have long and slender stems, which are easy to break off and thus making difficult their market distribution. in this work, two cultivars of basil (genovese and profumo) at the adequate development stage for sale were used. we evaluated the effect of supplemental ultraviolet (uv)-b irradiation (15 w m-2; 3 h day-1) on plant growth and market quality. both cultivars of basil plants under uv-b irradiation resulted in increased leaf size and biomass, and decreased shoot length in comparison to that of under control growth conditions. these results indicate that the application of uv-b irradiation beneficially influenced plant architecture in basil improving their greenhouse production for fresh market. 1. introduction many species in the genus ocimum (labiatae) are ranked among the most important herbs for their medicinal properties, that are associated to high content of secondary metabolites including essential oils and caffeic acid derivatives (gülҫin et al., 2007). the most significant species of the genus is sweet basil (ocimum basilicum l.), which originated from tropical areas, such as india, africa and southern asia. sweet basil is an annual herbaceous species that is usually cultivated as an aromatic plant. the minimum temperature for the growth of sweet basil has been determined to be 10.9°c (vågen et al., 2003; chang, 2004). it is typically used in italian and asian cuisines because of the pronounced scent of its leaves, which depends on genotype (chang et al., 2009), nitrogen nutrition (sifola and barbieri, 2006) and harvesting system (may et al., 2008). sweet basil is not only cultivated for the use of aroma additives in food but also for other house-hold purposes, pharmaceuticals, cosmetics and folk medicine. numerous different chemo-types exist in both wild and cultivated (*) corresponding author: thais.huarancca@agr.unipi.it citation: ciurli a., huarancca reyes t., guglielminetti l., 2017 commercial advantages on basil architecture by ultraviolet-b irradiation. adv. hort. sci., 31(3): 215-221 copyright: © 2017 ciurli a., huarancca reyes t., guglielminetti l. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 10 march 2017 accepted for publication 22 june 2017 ahs advances in horticultural science adv. hort. sci., 2017 31(3): 215-221 216 basil. for instance, sweet basil contains high levels of phenylpropanoids, e.g. eugenol and methyleugenol, a n d t e r p e n o i d s e . g . l i n a l o o l a n d 1 , 8 c i n e o l e (lachowicz et al., 1997). in recent years, the consumption of fresh basil has b e e n e x p a n d e d i n s u p e r m a r k e t s w h e r e y o u n g seedlings are directly sold in pots obtained from greenhouses. these plants are intended for family use, which after to be transplanted, are placed on balconies, vegetable gardens or gardens. the marketing of fresh basil highlights the problem that these plants have long and slender stems, which are easy to break off and thus making difficult their market distributions. therefore, the objective of this study was to facilitate the sale of fresh basil in pots resulting not only in plants more compact and resistant to movements along the supply chain, but also healthy products in the market because of the avoidance of chemicals to control the plant height (körner and van straten, 2008; nagashima et al., 2011). the interest on the effects of ultraviolet (uv) irradiation on plants has considerably increased in the last ten years due to the continuous depletion of the ozone layer (ballaré et al., 2011). the stratospheric ozone layer completely absorbs solar uv-c (200-280 nm) which is extremely active and biologically lethal. uv-b (280-320 nm) is the most susceptible to ozone layer depletion because it is efficiently absorbed although small proportion is transmitted to the earth surface. uv-a (320-400 nm) is hardly absorbed by ozone and thus passes almost unaltered through the s t r a t o s p h e r i c l a y e r r e a c h i n g t h e e a r t h s u r f a c e (houghton et al., 2001; solomon et al., 2007). despite the small proportion of uv-b in the natural daylight and higher energy than uv-a, uv-b has substantive effects on plant growth and metabolism (kolb et al., 2001). moreover, plants are differently sensitive to uv-b levels which strongly depend on the latitude, hours of direct sunlight and variation in the thickness of the ozone layer (ballaré et al., 2011). for instance, plant species grown in mediterranean and tropical environments and/or at high altitudes have developed defensive mechanisms to protect themselves against uv radiation (zheljazkov et al., 2008). many studies found that uv-b irradiation significantly affects secondary compounds such as the biosynthetic pathway of phenylpropanoids, which are antioxidant agents (korkina, 2007) that also act as protection against uv (johnson et al., 1999; ioannidis et al., 2002). previous studies reported that uv-b irradiation also increases essential oils and total phenolic compounds content in plants (kumari et al., 2009; kumari and agrawal, 2011). moreover, it has been demonstrated that the uv-b irradiation stimulates the production of volatiles organic compounds in fresh herbs which usually are depleted when plants grown under glass or plastic greenhouses conditions in the absence of natural levels of uv-b (johnson et al., 1999; ioannidis et al., 2002). in the case of plant morphology, several studies have been demonstrated the role of uv-b in controlling the growth of various plants such as lycopersicon esculentum mill. and salvia splendens l. (garner and björkman, 1996; giannini et al., 1996; del corso and lercari, 1997). additional techniques to control plant growth have been proposed: temperature manipulation, induction of mild water or mechanical stress, and the use of chemical compounds, however all these methods present many drawbacks (moe and mortensen, 1992; garner and björkman, 1996; barreiro et al. 2006; sun et al., 2008). therefore, we focused on the role of uv-b as a growth regulator of potted basil intended for fresh consumption. 2. materials and methods plant material and growth conditions two cultivars of basil were used in this study, genovese and profumo. basil seeds were sowed into peat-based growing medium in 8 cm diameter pots (10 seeds each pot). pots were covered with nonwoven fabric and incubated in a growth chamber at 23±1°c, relative humidity 60-65%, 12 h light (150 μmol m-2 s-1). after one week of incubation, the coverage was removed and half of the pots were kept at the same conditions while the other half were under supplementary uv-b irradiation (fluorescent tubes with uv-b 10%, 15 w m-2, exo terra). uv-b was applied for 3 h starting 1 h before darkness, and lamps were placed at 0.5 m above plants. basil plants were regularly irrigated. control and uv-b treated plants were sampled 11 and 22 days after the onset of uv treatment. morphological analysis leaf length and width, shoot length, fresh and dry weight of shoot and leaves were determined upon treatment completion. all measurements were the mean of three independent experiments. analysis of pigments pigments were extracted and analysed from full expanded leaves as previously described (pompeiano et al., 2013). all the analyses were conducted in tripciurli et al. commercial advantages on basil architecture by uv-b 217 licate. statistical analysis the statistical analyses of biometric and physiologic traits were subjected to an analysis of variance (anova). differences between treatments were assessed using the f-test, and the least significant difference (lsd) was calculated at p≤0.05. all computations were performed with r 2.14.2 r development core team 2012. 3. results in this study, we analyzed the plant architecture and photosynthetic pigments content of two basil cultivars, profumo and genovese, at time zero (t0) and after treatment. two different time points of treatment were analyzed: 11 and 22 days after uv-b treatment (uv) and their respective control (c) conditions. morphological analysis was determined on leaves and shoots of both basil cultivars (fig. 1, 2). length and width of cotiledonary leaves did not show differences between cultivars at t0, and also between treated and control plants during all the experimental time (fig. 1). the first true leaves, which appeared at the first time point of the experiment, showed a significant increased size from 11 to 22 days at control conditions in both cultivars (fig. 1). it was also o b s e r v e d t h a t u v t r e a t e d p l a n t s s i g n i f i c a n t l y increase, about two times, the leaf expansion in both cultivars in comparison with their respective control (fig. 1). interestingly, only 22 days uv treated seedlings showed the second leaves pair without morphological differences between the cultivars (fig. 1). in the case of shoot length evaluation, no differe n c e s w e r e o b s e r v e d b e t w e e n c u l t i v a r s a t t 0 , although ‘genovese’ showed longer shoot than ‘profumo’ at 11 and 22 days control conditions (fig. 2). moreover, the uv irradiated plants showed a considerably shorter shoots than that of control plants at both 11 and 22 days after treatment, and in all cases ‘genovese’ still longer than ‘profumo’ (fig. 2). the effect of uv irradiation on basil biomass of both cultivars was also analyzed (fig. 3). at t0, no differences were exhibited in fresh weight (fw) and dry weight (dw) leaves (only cotyledon) between cultivars, while fw and dw shoots were almost double in ‘genovese’ in comparison with ‘profumo’ (fig. 3 a-b). fig. 1 effect of uv-b radiation on the morphology of basil leaves. two cultivars of basil were use: (a, c) cv. profumo and (b, d) cv. genovese. (a, b) length and (c, d) width of basil leaves were measured at time zero (7 days old basil seedlings, t0) and after treatment. two different time points of treatment were evaluated: 11 and 22 days (d) after uv-b treatment (uv) or control (c) conditions. cotyledonary (white bars), first (grey bars) and second leaves (black bars) were measured separately. each value is the mean ± sd of three independent experiments. asterisk indicates significant differences among treatments (p≤0.05). fig. 2 effect of uv-b radiation on the length of basil shoots. two cultivars of basil were use: (a) cv. profumo and (b) cv. genovese. shoot length of basil cultivars was measured at time zero (7 days old basil seedlings, t0) and after treatment. two different time points of treatment were evaluated: 11 and 22 days (d) after uv-b treatment (uv) or control (c) conditions. each value is the mean ± sd of three independent experiments. asterisk indicates significant differences among treatments (p≤0.05). adv. hort. sci., 2017 31(3): 215-221 218 at 11 days after treatment, no differences were reported in fw and dw of shoot or leaves (cotyledon plus first pair) between cultivars (fig. 3 c-d). both cultivars treated with uv irradiation showed significant increase of fw and dw leaves in comparison with that of the control conditions, while no differences were reported in shoots (fig. 3 c-d). at 22 days after treatment the biomass in both cultivars was increased in comparison with that of 11 d, while similar pattern was maintained when it was compared the biomass before and after uv irradiation (fig. 3 ef). the photosynthetic pigments analyzed in both cultivars include chlorophyll a, chlorophyll b and carotenoids (fig. 4). all pigments were detected at t0 without differences among cultivars. then, the amount was dramatically reduced after 11 days under control conditions as well as uv irradiation, showing no statistical differences even between cultivars (fig. 4). at 22 days after treatment, the level of chlorophyll b was maintained similar to that of 11 days and no differences were registered between control and uv conditions (fig. 4). chlorophyll a and carotenoids levels at 22 days under control conditions were increased in respect to that of 11 days, and no differences were detected when compared with that of after uv treatment (fig. 4). 4. discussion and conclusions results of the present study pointed towards a positive impact of supplementary uv-b irradiation on basil architecture, resulting on the reduction of stem e l o n g a t i o n , i n c r e a s e o f l e a f l e n g t h a n d w i d t h , increase of biomass, induction of second leaf sproutfig. 3 effect of uv-b radiation on fresh and dry biomass of basil. two cultivars of basil were use: (a, c, e) cv. profumo and (b, d, f) cv. genovese. measurements were monitored at (a, b) time zero (7 days old basil seedlings) and after treatment. two different time points of treatment were evaluated: (c, d) 11 days and (e, f) 22 days after uv-b treatment (uv) or control (c) conditions. s: shoot, l: leaf, fw: fresh weight, dw: dry weight. each value is the mean ± sd of three independent experiments. asterisk indicates significant differences among treatments (p≤0.05). fig. 4 effect of uv-b radiation on photosynthetic pigments in basil leaves. two cultivars of basil were use: (a) cv. profumo and (b) cv. genovese. measurements were monitored at time zero (7 days old basil seedlings, t0) and after treatment. two different time points of treatment were evaluated: 11 and 22 days (d) after uv-b treatment (uv) or control (c) conditions. chlorophyll a (white bars), chlorophyll b (grey bars) and carotenoids (black bars) content are expressed as mg g fw-1. each value is the mean ± sd of three independent experiments ciurli et al. commercial advantages on basil architecture by uv-b 219 ing and with a similar trend of photosynthetic pigments content in comparison to that of plants under control conditions. these uv-b effects on plants are in accordance with previous studies (giannini et al., 1996; del corso and lercari, 1997). the reduction of stem elongation found in our research was also reported previously in other species such as wheat (yuan et al., 1998). this phenomenon could be related to the impact of uv-b radiation on phytohormones metabolism, such as photo-oxidation of indole-3-acetic acid which plays an important role in stem elongation and lateral shoots (ros and tevini, 1995; mark and tevini, 1996). in addition, effects of uv-b radiation on leaf thickening, leaf elongation and biomass accumulation are highly dependent on uv-b dose and source, experimental parameters and species of study (kakani et al., 2003). therefore, in this study, we used growth chamber instead of open fields and greenhouses in order to control all the environmental conditions, making this condition replicable in any situation and avoiding the influence of environmental and seasonal variations. our results showed also an increase of leaf thickening, leaf elongation and biomass after uv-b irradiation, which could be related to a morphological architecture strategy to protect plants to deleterious effects of uv-b radiation (maffei and scannerini, 2000; jansen, 2002; santos et al., 2004; chang et al., 2009). moreover, the generation of second leaves in basil plants was earlier promoted after uv-b treatment which may also involve phytohormones metabolism changes; this effect was also previously reported (barnes et al., 1988). thus, structural changes on l e a v e s i n r e s p o n s e t o t h e a p p l i e d u v b w e r e observed, although no changes on the content of chloroplast pigments was registered between uv treated and control plants. photosynthetic pigments are useful indicators of uv-b tolerance or sensitivity (kataria et al., 2014). lower pigments content was obtained at 11 days of treatment in comparison with t0, which could be due to the stress arising from the removal of the non-woven fabric that protected plants from excessive light. however, this phenomenon was recovered in a tendency to the start point t0, suggesting the acclimatization of plants, as observed similarly in previous report (teramura and sullivan, 1994; radyukina et al., 2012). therefore, our data suggest that genovese and profumo basil cultivars are tolerant to the supplementary uv-b irradiation (15 w m-2; 3 h day-1) at chlorophyll level, but producing other strategy to prevent uv-b penetration to the mesophyll cell at plant architecture level. although the impacts of uv-b radiation on plant growth and development have been widely studied (strid et al., 1994; ballaré et al., 1995; giannini et al., 1996; del corso and lercari, 1997; santos et al., 2004; zu et al., 2004; körner and van straten, 2008), the detailed mechanism of how uv-b radiation affects plant morphogenesis is still unclear. however, previous evidences found that uv-induced morphological changes are associated with the induction of the phenylpropanoids pathway resulting in accumulation of flavonoids (jansen et al., 1998). moreover, it has been demonstrated that flavonoids regulate auxin transport affecting plant architecture (jansen, 2002; robson et al., 2015). in conclusion, the results of this work have practical implications for greenhouse production of pot basil. in fact, supplementary uv-b radiation reduced plant height but increased biomass and leaf number, width and length with no significant effect on photosynthetic pigments. therefore, the application of uv lamps could replace chemical growth retardants and/or other techniques adopted for growth control in the production of potted basil for fresh market. acknowledgements we thank prof. alberto pardossi (university of pisa, italy) for helpful discussions. this research was supported by a grant from the social cooperative parvus flos. references ballaré c.l., barnes p.w., flint s.d., 1995 inhibition of hypocotyl elongation by ultraviolet-b radiation in deetiolating tomato seedlings. i. the photoreceptor. physiol. plant., 93(4): 584-592. ballaré c.l., caldwell m.m., flint s.d., robinson s.a., bornman j.f., 2011 effects of solar ultraviolet radiation on terrestrial ecosystems. patterns, mechan i s m s , a n d i n t e r a c t i o n s w i t h c l i m a t e c h a n g e . photochem. photobiol. sci., 10(2): 226-241. barnes p.w., jordan p.w., gold w.g., flint s.d., caldwell m.m., 1988 competition, morphology and canopy structure in wheat (titicum aestivum l.) and wild oat (avena fatua l.) exposed to enhanced ultraviolet-b radiation. funct. ecol., 2(3): 319-330. barreiro a.p., zucareli v., ono e.o., rodrigues j.d., 2006 growth analysis of basil plants submitted to plant growth regulators. bragantia, 65(4): 563-567. chang x., 2004 effects of light and temperature on adv. hort. sci., 2017 31(3): 215-221 220 volatile oil compounds and growth in basil (ocimum basilicum l.). ph.d. thesis, university of nottingham. chang x., alderson p.g., wright c.j., 2009 enhanced uv-b radiation alters basil (ocimum basilicum l.) growth and stimulates the synthesis of volatile oils. j. hort. sci. biotech., 1(2): 27-31. del corso g., lercari b., 1997 use of uv radiation for control of height and conditioning of tomato transplants (lycopersicon esculentum mill.). sci. hortic., 71 (1-2): 27-34. garner l.c., björkman t., 1996 mechanical conditioning for controlling excessive elongation in tomato transplants: sensitivity to dose, frequency, and timing of brushing. j. amer. soc. hort. sci., 121(5): 894-900. giannini a., pardossi a., lercari b., 1996 the use of uv radiation to control the architecture of salvia splendens plants. i. effects on plant growth, water relations and gas exchange. photochem. photobiol., 64(1): 123130. gülçin i̇., elmastaş m., aboul-enein h.y., 2007 determination of antioxidant and radical scavenging a c t i v i t y o f b a s i l ( o c i m u m b a s i l i c u m l . f a m i l y lamiaceae) assayed by different methodologies. phytother. res., 21(4): 354-361. houghton j.t., ding y., griggs d.j., noguer m., van der linden p.j., dai x., maskell k., johnson c.a., 2 0 0 1 c l i m a t e c h a n g e 2 0 0 1 . r e p o r t o f t h e intergovernmental panel on climate change (ipcc). cambridge university press, cambridge, uk. ioannidis d., bonner l., johnson c.b., 2002 uv-b is required for normal development of oil glands in ocimum basilicum l. 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b, 74(2-3): 95-100. impaginato 151 1. introduction tomato as one of the most widely produced and consumed ‘vegetable’ in the world (heuvelink, 2005) contains high levels of antioxidant active compounds such as vitamin c, polyphenlos and carotenoids (tommonaro et al., 2012). salicylic acid (sa) has been the focus of intensive research due to its role in plant defense mechanisms and response to abiotic stresses (rivas-san vicente and plasencia, 2011). besides, it is stated that sa plays a crucial role in physiological and biochemical processes during the entire lifespan of the plant (rivas-san vicente and plasencia, 2011). sa as an endogenous plant growth regulator controls a large variety of physiological processes: from regulatory signal in plants mediating defense against pathogens, to ethylene biosynthesis, action and inhibition. it is also involved in plant responses to abiotic stress conditions such as salt and osmotic stresses (khalil, 2014). exogenous application of sa also results in m a n y d i f f e r e n t c h a n g e s i n p l a n t p h y s i o l o g i c a l processes and reactions such as prevention of ethylene production (khan et al., 2003); increases in plant height, number of branches, number of leaves (saharkhiz et al., 2011) and antioxidant activity (ananieva et al., 2004). most of the researches on sa have focused on mediating local and systemic plant defense and resistance to biotic and abiotic stresses (atkinson and urwin, 2012), while studies on its effect on physiological, biochemical and quality features of fruit are limited (ali et al., 2014). since plant growth, development and the level of bioactive compounds especially antioxidant active substances depend on the cultivar, and by agronomic and environmental conditions (tommonaro et al., 2012), the aim of this work was to study the influences of sa on growth, fruit quality attributes including fruit firmness and storability, vitamin c, antioxidant activity, total phenolic, flavonoids and yield of tomato under greenhouse conditions. adv. hort. sci., 2016 30(3): 151-157 doi: 10.13128/ahs-20277 salicylic acid at different plant growth stages affects secondary metabolites and phisico-chemical parameters of greenhouse tomato j. javanmardi, n. akbari department of horticultural sciences, college of agriculture, shiraz university, shiraz, iran. key words: antioxidant activity, flavonoids, solanum lycopersicum, total phenolics, yield components. abstract: most of the researches on salicylic acid (sa) have focused on postharvest application or acquiring stress resistance, while studies on its effect on plant growth, secondary metabolites and fruit quality are limited. sa as foliar application (0, 150, 300 and 450 mg/l) at different plant growth stages on fruit yield, secondary metabolites and quality features of tomato (solanum lycopersicum l. cv. kardelen) under greenhouse conditions were evaluated. the highest fruit yield per plant (about 1.3-fold greater than control) was obtained from 300 mg/l sa when applied three weeks after fruit set. comparing to control plants, the highest fruit firmness, 10 days prolonged storability, highest total phenolics (22.6 mg gallic acid equivalent per 100 g fw); and highest antioxidant activity (65.11) were observed when 450 mg/l sa applied at fruiting stage and 3 weeks later. an increasing pattern in ascorbic acid content was observed with increasing sa concentration irrespective to application time. the same concentration effect was observed in flavonoid content when plants treated at 3 weeks after fruiting. the highest effect of flavonoids on antioxidant activity was calculated using pearson correlation (r=0.82). sa concentrations greater than 450 mg/l showed significant adverse effects on all measured traits. the effect of exogenous sa on tomato plant depends on the developmental stage and sa concentrations tested. improved fruit quality factors may happen in a certain concentration range, while over that may have negative or adverse effect. (*) corresponding author: javanm@shirazu.ac.ir received for publication 1 may 2016 accepted for publication 9 august 2016 copyright: © 2016 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2016 30(3): 151-157 152 2. materials and methods plant material and experimental design the tomato (solanum lycopersicum cv. kardelen) seeds were obtained from gento seeds® co., turkey. six weeks old seedlings having 4-5 leaves were transplanted into a 500 m2 polycarbonate greenhouse siltloam soil (greenhouse research center, college of agriculture, shiraz university, shiraz, iran) at 50 cm double rows and 150 cm between rows spacing (density of 2 plants/m2). the greenhouse conditions were set to temperature of 25±2°c and relative humidity of 60-70% during the entire study. soil was already fertilized according to the soil test results and certified lab recommendations for growing greenhouse tomato (papadopoulos, 1991). treatments consisted of sa (merck millipore corporation, germany) foliar application at four concentrations (0, 150, 300, and 450 mg/l) at different growth stages (transplant establishment, onset of flowering, fruit set, 3 weeks after fruit set) and their all possible combinations (table 1). the experiment was arranged in a completely randomized design with three replications. each sample for analyses consisted of three fruits (per plant) of four plants for each replicate per treatment. measurement methods fruits were harvested at mature red stage based on the “color classification requirement in united states standards for grades of fresh tomatoes” chart, published by usda. tomato produces fruits on clusters. fruits of two first clusters were considered as yield and expressed as kg/plant. fruit firmness was measured as penetration force on the fruit flesh (over the fruit locules) using force gauge, fg-5005 (lutron electronic enterprise co. taipei, taiwan) with a probe diameter of 8 mm. the average values obtained for each fruit was calculated and expressed as newton. fruit storability was measured as the number of days from keeping red fruits in a storage at 12±1°c and 80% relative humidity to starting fruit shrinkage or lose their shiny appearance. vitamin c quantification was performed according to the method described by the aoac (1984) and results were expressed as mg ascorbic acid per 100 grams of fruit dw. total phenolic content (tpc) was determined with the folin-ciocalteau reagent using the method of spanos and wrolstad (1990) and the results of three replicates were expressed as mg gallic acid equivalent per 100 grams of fruit dw (mg gae/100g dw). measured trait salicylic acid (mg/l) application time establishment flowering fruiting 3 weeks after fruiting establishment + flowering establishment + fruiting establishment + 3 weeks after fruiting flowering + fruiting flowering + 3 weeks after fruiting fruiting + 3 weeks after fruiting all 4 times fruit firmness (n) 150 9.97±0.47 no 10.76±0.59 mn 12.59±0.13 kl 12.20±0.21 lm 12.33±0.14 lm 12.62±0.24 kl 12.27±0.05 lm 12.67±0.36 kl 10.89±0.64 mn 13.49±0.99 j-l 13.94±0.20 i-k 300 13.96±0.35 i-k 14.69±0.68 g-j 16.14±0.82 e-g 14.51±0.39 g-i 15.07±0.24 f-j 15.56±0.12 e-h 14±0.50 h-k 15.33±0.28 f-i 14.38±0.07 g-i 16.11±0.52 e-g 16.10±0.65 e-g 450 16.19±0.83 e-g 16.28±0.87 e-g 19.92±0.78 a 16.95±0.21 c-e 16.23±0.40 e-g 18.28±0.31 bc 16.45±0.37 ef 18.86±0.12 ab 16.60±0.32 d-f 18.89±1.17 ab 18.10±0.54 b-d control 9.07±0.51 o shelf life (days at 12°c) 150 21.00±0.57 d 21.66±0.33 b-d 21.66±0.33 b-d 22.00±1.0 b-d 21.66±0.23 b-d 21.66±0.66 b-d 21.66±0.31 b-d 21.33±0.33 cd 21.66±0.41 b-d 22±0.57 b-d 22±0 b-d 300 21.66±0.33 b-d 22±0.57 b-d 22±0.51 b-d 22.33±0.33 b-d 22.00±0.54 b-d 22.33±0.33 b-d 22.33±0.13 b-d 22.33±0.34 b-d 22±0.02 b-d 22.33±0.33 b-d 22.33±0.33 b-d 450 22.33±0.66 b-d 22±0.57 b-d 22.33±0.88 b-d 24.66±1.33 a 21.83±0.44 b-d 22.33±0.33 b-d 22.66±0.88 bc 22.66±0.23 bc 23±0.57 b 23±0.57 b 22.33±0.33 b-d control 17.00±0.00 e vitamin c (mg/100 g dw) 150 13.88±0.35 pq 13.99±0.35 pq 14 39±0.42 o-q 18.28±0.26 l-o 15.42±0.81 n-q 16.92±0.62 m-p 19.12±0.51 l-n 14.74±0.39 o-q 20.37±0.92 k-m 21.03±1.24 j-l 21.87±1.90 i-l 300 23.56±0.22 h-k 24.91±1.63 h-j 27.15±1.25 e-h 28.47±1.63 b-g 25.10±0.68 h-g 25.13±0.13 h-g 26.01±0.48 f-h 27.30±0.51 d-h 27.52±1.53 d-h 27.81±1.06 c-g 29.35±2.28 b-f 450 31.11±0.79 a-e 31.62±0.89 a-c 32.47±0.12 ab 32.50±0.09 ab 31.18±0.73 a-e 31.26±0.72 a-d 31.95±0.92 ab 31.73±0.92 a-c 32.25±0.38 ab 34.34±2.07 a 32.50±0.84 ab control 11.82±0.24 q total phenolics (mg gae/100 g dw) 150 14.19±0.09 l 14.51±0.16 k 15.60±0.25 i 16.14±0.14 gh 15.12±0.10 j 15.18±0.10 j 15.71±0.05 i 14.59±0.23 k 15.70±0.20 i 15.84±0.11 hi 16.26±0.22 g 300 19.66±0.17 f 19.73±0.14 f 20.13±0.12 e 20.24±0.06 e 20.04±0.06 ef 20.12±0.065 e 20.13±0.06 e 19.99±0.12 ef 20.22±0.03 e 20.26±0.05 e 21.45±0.06 d 450 21.47±0.03 d 21.71±0.29 b-d 22.64±0.09 a 22.67±0.12 a 21.54±0.07 cd 21.51±0.09 cd 21.59±0.11 cd 21.49±0.02 cd 21.88±0.18 bc 21.73±0.19 b-d 22.01±0.06 b control 10.84±0.10 m flavonoids (mg gae/100 g dw) 150 1.05±0.08 n 1.06±0.08 n 1.06±0.01 n 1.09±0.03 kl 1.09±0.08 kl 1.07±0.03 mn 1.09±0.08 lm 1.08±0.03 lm 1.08±0.01 lm 1.08±0.09 lm 1.09±0.06 j-l 300 1.09±0.06 j-l 1.13±0.04 i-k 1.11±0.07 h-j 1.14±0.01 b-e 1.09±0.06 j-l 1.09±0.09 j-l 1.12±0.06 g-i 1.10±0.05 j-l 1.12±0.03 f-h 1.14±0.09 d-g 1.09±0.10 j-l 450 1.14±0.06 d-g 1.14±0.06 c-f 1.13±0.01 e-g 1.18±0.05 a 1.14±0.07 c-f 1.14±0.05 c-f 1.16±0.06 bc 1.14±0.01 d-g 1.15±0.07 b-d 1.16±0.08 ab 1.14±0.08 c-f control 0.99±0.07 o table 1 effect of salicylic acid foliar application at different tomato plant phonological stages on some fruit characteristics averages±sd for each measured trait with the same letters showing no significant differences using lsd test at p<0.05. control plants did not receive any sa at any time. javanmardi and akbari salicylic acid effects on greenhouse tomato 153 total antioxidant activity (taa) was measured using dpph (2,2-diphenil-1-picrylhydrazyl) (merck millipore corporation, germany) assay as described by patras et al. (2009) at the absorbance of 517 nm using micro plate reader (epoch, germany). taa was calculated according to the following equation: taa = [1(a sample at 517nm /a control at 517nm )]×100 total flavonoid content (tfc) was determined by the aluminum chloride colorimetric assay based on the formation of a complex flavonoid-aluminum, having a maximum absorbance at 510 nm (toor and savage, 2005) and results expressed as mg gallic acid equivalent per 100 gram of fruit dw (mg gae/100 g dw). statistics data were analyzed using sas 9.1 statistical software (sas institute inc., cary, nc, usa). means were compared using lsd test at p ≤ 0.05. pearson correlation statistical method was used to determine the correlation between secondary metabolites and antioxidant activity. 3. results and discussion our preliminary tests on different concentrations of sa application showed significant adverse effects on all measured traits when concentration was higher than 450 mg/l (data not shown). this could be due to the nature of sa that acts as a plant growth regulator. it is stated that the responses to sa are highly concentration dependent, so that moderate doses of sa improve features such as antioxidant status and induce stress resistance, while higher concentrations trigger a hypersensitive cell death pathway (tounekti et al., 2013). fruit yield fruits of the first two clusters were evaluated as yield. the greatest yield was obtained from 300 mg/l sa applied at three weeks after fruit set (fig. 1). this was 1.26 fold greater than control. the stimulatory effect of sa on flowering regulation which has been well known for a long time (raskin, 1992; rivas-san vicente and plasencia, 2011) would eventually affect total number of fruits (ondrašek et al., 2007) and enhance efficiency in fruit production. previously, increased yield of strawberry (aghaeifard et al., 2015) and tomato (javaheri et al., 2012) has been related to promoted cell division and cell enlargement due to sa (hayat et al., 2010) through its influe n c e o n o t h e r p l a n t h o r m o n e s s u c h a s a u x i n , cytokinin and aba balances (shakirova, 2007) and enhanced net photosynthetic rate, internal co2 concentration and water use efficiency (fariduddin et al., 2003). it is reported that in non-thermogenic plants such as tobacco, sa levels increase 5and 2-fold in their leaves at the initiation of or during transition to flowering, respectively (abreu and munné-bosch, 2009). fruit firmness firmness is an important physical parameter for postharvest storage, transportation and monitoring the fruit ripening process. sa and its derivatives are widely in use to enhance fruits postharvest life by enhancing fruit firmness during storage (wang et al., 2006). in our experiment, sa significantly affected fruit firmness. the firmness became 2.9 times higher than the control group when 450 mg/l sa was applied at fruiting stage. plants treated with 150 mg/l sa at establishment stage showed the lowest fruit firmness as equal to control plants (table 1). a 2-fold greater tomato fruit skin thickness due to 0.01m sa application comparing to control has been previously reported (javaheri et al., 2012). metabolic reactions respiration, ethylene production (aktas et al., 2012) breakdown of cell wall and activation of enzymes involved therein are key events in fruit ripening and softening (prasanna et al., 2007). these metabolic activities can be harmful to maintain fruit quality. there are reports that sa could prevent the activity of such enzymes, while affect the swelling of cells in a manner that results in firmer fruit (zhang et al., 2003; prasanna et al., 2007). sa prevents fruit softening. shafiee et al. (2010) have cited several reports indicating that rapid softening of fruits during ripening was simultaneous with rapid decrease in endogenous sa of fruits. increased firmness in climacteric fruits due to pre-harvest sa spray has been attributed to the role of sa in preventing cell wall and membrane degrading enzymes (polygalacturonase, fig. 1 effect of salicylic acid (sa) foliar application at different tomato plant phenological stages on yield (two first clusters) at greenhouse condition. transplant establishment (est), flowering (fl), fruiting (fr), three weeks after fruiting (3wfr). adv. hort. sci., 2016 30(3): 151-157 154 lipoxygenase, cellulase, pectinemethylesterase), ethylene production (zhang et al., 2003) and reduced hydrolysis of soluble starch and therefore higher firmness (tareen et al., 2012 b), while in non-climacteric fruits such as “flame seedless” grape it has been attributed to the role of sa in preventing decay (khalil, 2014). fruit storability the greatest fruit storability period was observed in 450 mg/l sa when applied at 3 weeks after fruiting (table 1). this was over 10 days more than control plants. aghdam et al. (2014) attributed longer storability and higher chilling resistance of detached tomato fruits treated with sa to increased endogenous proline content. lowered ethylene biosynthesis has been also considered to be the main cause of prolonged storability due to regulatory potential of exogenous sa on fruit ripening of green mature tomato fruits (kant et al., 2013). parallel to this, sa can activate the alternative respiration pathway in many plant tissues which results in a lower respiratory rate and delay in the climacteric peak (raskin, 1992). srivastava and dwivedi (2000) stated that the concentration of sa determines the extent to which these effects actualize. reduced the quality loss during storage due to sa were previously reported for tomato (ding et al., 2001) and sweet peppers (fung et al., 2004). vitamin c human diet consists of about 91% of ascorbic acid coming from fruits and vegetables (tareen et al., 2012 a). an increasing pattern of ascorbic acid content was observed with increasing sa concentration irrespective to application time (table 1). the greatest ascorbic acid content was obtained from 450 mg/l sa. in most cases, this increase was about 3 times than control plants. some researches indicated that the treatment of tomatoes (javaheri et al., 2012; kalarani et al., 2002) and strawberry (aghaeifard et al., 2015) with sa caused them to acquire higher levels of ascorbic acid comparing to control plants. sa can activate ascorbate peroxidase, which is the precursor to ascorbic acid in fruits and prevents vitamin c from being destroyed in cells and therefore causes t h e a c c u m u l a t i o n o f a s c o r b i c a c i d i n t h e f r u i t (wiśniewska and chełkowski, 1999). total phenolics phenolic compounds as secondary plant metabolites are synthesized by all plants and responsible for the flavor and color of fruit products (jeong et al., 2008). the highest amount of phenolic compounds was observed in plants treated with 450 mg/l sa during the fruiting stage and three weeks after the fruiting (table 1). in those treatments, a 2.9 times higher phenolic compounds content than control plants was observed. although aghdam et al. (2012) reported no significant effect of sa application on total phenolics content of mature green tomato fruits, our results were similar to reports on sweet cherry (valero et al., 2011) and grapes (ranjbaran et al., 2011; khalil, 2014), which all concluded sa application induced greater total phenolics and other secondary metabolites with antioxidant properties (ranjbaran et al., 2011). previously, the application time of sa on sweet cherry at three fruit developmental stages (pit hardening, initial color changes and onset of ripening) increased fruit weight and led to higher concentration of total phenolics and total anthocyanins, as well as higher antioxidant activity (giménez et al., 2014). antioxidant activity it is well known that the positive effect on health associated with tomato consumption is exerted by the pool of antioxidants, with noticeable synergistic effects (tommonaro et al., 2012). the highest antioxidant activity was observed in 450 mg/l sa treatment when applied on fruiting stage plus three weeks after fruiting. this was 1.84 times greater than the control, which had the least antioxidant activity. an increasing pattern in antioxidant activity was observed with increasing sa concentration, irrespective to application time (fig. 2). the same pattern was previously found in orange (huang et al., 2008 b) and pears (cao et al., 2006). increased total antioxidant activity of strawberry due to sa has been previously reported (aghaeifard et al., 2015). regular applications of salicylic acid at different stages of plant growth and fruit development can increase the antioxidant activity fig. 2 effect of salicylic acid (sa) foliar application at different tomato plant phenological stages on fruit total antioxidant activity at greenhouse condition. transplant establishment (est), flowering (fl), fruiting (fr), three weeks after fruiting (3wfr). javanmardi and akbari salicylic acid effects on greenhouse tomato 155 (shakirova, 2007). it is frequently hypothesized that sa has direct physiological effects on the activity of antioxidant enzymes which promote the synthesis of metabolites existing in fruits and vegetables, especially those with nutritional value in the product (huang et al., 2008 a). flavonoids flavonoids comprise a diverse group of natural compounds and are among the best-known natural phenols, exhibiting an array of chemical and biological pathways such as radical scavenging and antimicrobial activities. an increasing pattern in the amount of flavonoids was found by increasing sa concentration irrespective to application time. the highest amount of flavonoids was observed in 450 mg/l sa treatment when plants were treated either in three weeks after fruiting or fruiting plus three weeks after fruiting stages with about 1.18 times greater than control treatment (table 1). reports have been cited stating that exogenous sa applications boost the accumulation of flavonoids in several plant species (tounekti et al., 2013). on the other hand, research has proved that flavonoids possess antibiotic activities (al-matani et al., 2015). this can generate debate as to whether higher amounts of flavonoids can contribute to a longer shelf life against the rot of perishable fruits like tomato. when vegetables are heated for special purposes in food industries, excess heat can cause degradations in flavonoids and thus can reduce its overall content (sharma et al., 2015). correlation between secondary metabolites and antioxidant activity a pearson correlation analysis was performed to determine relationships between the individual parameters phenolic compounds, flavonoids and ascorbic a c i d w h i c h c o n t r i b u t e i n a n t i o x i d a n t a c t i v i t y . significant correlations were found for all measured traits with the highest effect of flavonoids on antioxidant activity; however, it was not a simple sum of their contribution (table 2). this has been related to synergistic effect among all antioxidants and their interactions with other constituents of the fraction (jimenez et al., 2002; lenucci et al., 2006). similar to our results, ilahy et al. (2011) found a good significant correlation between antioxidant activity and main antioxidants (vitamin c, flavonoids and total phenols). given the key role of sa in increasing ascorbic acid (dat et al., 1998), total phenolics and other secondary metabolites with antioxidant properties (ranjbaran et al., 2011), the rise in antioxidant activity can thus be explained. 4. conclusions treatments with sa could be a promising tool to improve tomato yield, fruit quality attributes and health beneficial compounds (including phenolic compounds, vitamin c and flavonoids having antioxidant activity) because of its diverse regulatory roles in plant metabolism. the effect of exogenous sa on plant depends on the plant species, developmental stage, and the sa concentrations tested. fruit setting stage and 3 weeks later are the best two important stages for sa application. a concentration of 300 mg/l sa for increased yield and 450 mg/l sa for improved fruit quality attributes are recommended. it is 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pp. 389. valero d., díaz-mula h.m., zapata p.j., castillo s., guillén f.n., martínez-romero d., serrano m.a., 2011 postharvest treatments with salicylic acid, acetylsalicylic acid or oxalic acid delayed ripening and enhanced bioactive compounds and antioxidant capacity in sweet cherry. j. agric. food chem., 59: 54835489. wang l., chen s., kong w., li s., archbold d.d., 2006 salicylic acid pretreatment alleviates chilling injury and affects the antioxidant system and heat shock proteins of peaches during cold storage. postharvest biol. technol., 41: 244-251. wiśniewska h., chełkowski j., 1999 influence of exogenic salicylic acid on fusarium seedling blight reduction in barley. acta physiologiae plantarum, 21: 6366. zhang y., chen k., zhang s., ferguson i., 2003 the role of salicylic acid in postharvest ripening of kiwifruit. postharvest biology and technology, 28: 67-74. impaginato 183 1. introduction tomatoes are widely used, and those are a rich source of fiber, phenolics, vitamins a, c, and small amounts of vitamin e and lycopene. lycopene prevents the harmful effects of free radicals and different types of cancers as well as cardiovascular disease (pila et al., 2010; orabi et al., 2015). major problem of postharvest tomato is softening and ripening during storage, distribution, and marketing because of their susceptibility to damage (batu, 2004; agamy et al., 2013). fruit firmness and color are as effective factors of tomato quality, which are used as fruit quality indicator (batu, 2004; agamy et al., 2013). tomato is climacteric so its ripening continues after harvesting and it can become overripe quickly. hence, its quality decreases and its shelf life limits (batu, 2004). pila et al. (2010) reported that owing to lack of information on appropriate postharvest treatments, packaging, temperature, etc., the fruits not only lose their quality but also encounter a substantial postharvest loss. cold storage is one of the most efficient and most practical postharvest procedures that maintains quality of products from the harvest to consumption time (bourne, 2006), and extending the storage life of fresh horticultural products, tomatoes can be stored successfully for weeks (hatami et al., 2013), but the main problem is the postharvest handling, because the tropical and subtropical products are sensitive to chilling injury (ci) (soleimani aghdam et al., 2012). hatami et al. (2013) reported that improper temperature management is the primary cause of many postharvest diseases and disorders. elhadi and jeffrey (2012) reported that mature green tomatoes are the most sensitive to low temperatures among the commercial fruit, and there is a risk to develop chilling injury if they are held below 13°c or 12.5°c (rugkong, 2009) and 12°c (galvez et al., 2010; zhang et al., 2010). sevillano et al. (2009) reported that chilling injury reduced tomato fruit quality. commonly visible ci comprise several symptoms such as surface pitting (soleimani aghdam et al., 2012) and alteration of ripening process as indicated by delayed or even total failure offruit color d e v e l o p m e n t a n d s o f t e n i n g ( r u g k o n g , 2 0 0 9 ) , adv. hort. sci., 2016 30(3): 183-192 doi: 10.13128/ahs-20281 effect of preand postharvest salicylic acid treatment on quality characteristics of tomato during cold storage e. baninaiem (*), a.m. mirzaaliandastjerdi, s. rastegar, kh. abbaszade department of horticultural science, faculty of agriculture and natural resources, university of hormozgan, bandar abbas, iran. key words: ascorbate peroxidase, decay, lycopersicon esculentum, weight loss. abstract: nowadays, there is a considerable tendency to replace dangerous chemicals with natural compounds, compatible with plant, human, and nature. this study was aimed to assess the effect of salicylic acid on quality and storage life of tomato (lycopersicon esculentum cv. baraka). the salicylic acid application was including plant foliar application three weeks before harvest at concentration 4 mm, followed by the postharvest dipping fruits in salicylic acid at different concentrations (1, 2, 4 mm), then storing at 10°c for 40 days to investigate quantitative and qualitative characteristics. the chilling injury symptoms, electrolyte leakage, decay and a* (redness) value significantly decreased and activity ascorbate peroxidase increased. ascorbic acid content, total soluble solid, titratable acidity, firmness, and l* (lightness) retained by salicylic acid treatments. the salicylic acid application had no significant influence on weight loss and b*. application of salicylic acid in all concentrations, especially a combination of treatments preharvest to concentrations 4 mm as well as postharvest 4 mm, had the highest influence on qualitative and quantitative characteristics and increased the postharvest life of the tomato fruit. (*) corresponding author: b.elham.2013@gmail.com received for publication 21 april 2016 accepted for publication 17 november 2016 copyright: © 2016 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2016 30(3): 183-192 184 increased susceptibility to alternaria rot and decay (ding et al., 2002), decrease mealy texture when ripened (alternaria and cladosporium rots are usually associated with chilling injury) (elhadi and jeffrey, 2012). salicylic acid (sa) is a phenolic compound and plant growth regulator (zavala et al., 2004) and defenses against biotic and abiotic environmental stresses. plants produce reactive oxygen species (ros) when exposed to biotic and abiotic environmental stresses conditions (agamy et al., 2013). ros includes superoxide, hydrogen peroxide and hydroxyl ions (dat et al., 2000). thus, ros cause to damage in cellular structures. there is a mixture of non-enzymatic antioxidants (carotenoids, ascorbate) and enzymatic antioxidants in plants, such as catalase (cat) and ascorbate peroxidase (apx) which inhibit harmful effects of these ross. the enzymatic action of apx reduces h2o2 using ascorbate as an electron donor (orabi et al., 2015). salicylic acid is an antioxidant defense system and regulates different physiological and biochemical processes in plants including: plant growth (khan et al., 2003), stomatal conductivity (hayat et al., 2010), photosynthesis (fariduddin et al., 2003), seed germination (babalar et al., 2007), disease resistance (janda et al., 2007), heavy metal stress, low temperature, high temperature and salinity (hayat et al., 2008). salicylic acid treatment could be used to enhance the chilling resistance of maize, cucumber and rice (kang and saltveit, 2002), pomegranate (sayyari et al., 2009) and tomato (ding et al., 2001, 2002). salicylic acid delays the ripening of banana and kiwifruit during storage (srivastava and dwivedi, 2000; zhang et al., 2003). babalar et al. (2007) reported that pre and postharvest sa treatments caused fruit quality maintenance in strawberry. fattahi et al. (2010) reported that losses in fruit quality are mostly due to its relatively high metabolic activity during storage. salicylic acid is known as a signal molecule in the induction of defense mechanisms in plants. due to the risk of inappropriate use of substance chemicals in postharvest technology, it is essential to study the application of safe postharvest treatments along with cold storage. since the time between tomato fruit harvest and consumption may take long weeks, and during this period many changes could happen that affect the postharvest behavior of fruits. therefore, the aim of this article was to appraise the effects of pre and postharvest sa application to maintain the qualitative characteristics of tomato fruits at cold storage and increase the postharvest life of the tomato fruit. 2. materials and methods plant material and salicylic acid treatment fruits of tomato (lycopersicon esculentum cv. baraka) produced in the greenhouse at university of hormozgan (iran) were used. baraka cultivar is a hybrid seed appropriates for the tropical region. fruits were harvested at mature green stage in april 2014 and transferred to laboratory where they were selected for health and size, weight, and color uniformity. fruits were divided randomly. then, they were washed and dried in the air. each treatment consisted of 60 fruits, and each treatment was composed of three replicates (20 fruit per replicate): three sa treatments were compared (4+1, 4+2, and 4+4 mm) consisting of a 4 mm sa plant foliar application three weeks before harvest followed by the postharvest fruit dipping for five minutes in sa solutions at different concentrations (1, 2, and 4 mm). fruits harvested from non-treated plants were used as control. tomatoes were stored at 10°c temperature and 8590% relative humidity (rh) for 10, 20, 30 and 40 days (hatami et al., 2013). samples were taken at every 10 days intervals during storage for quality evaluation. firmness and weight loss fruit firmness was measured using a penetrometer equipped with a 6 mm diameter flat probe exerting maximum force on fruit. units were expressed as kg cm-2 (shafiee et al., 2010). fruit weight loss was measured immediately after harvest and storage time. the results were calculated as percentage of weight loss at the start of the experiment and at different intervals during storage by this formula: %wl = [(w1-w2)/w1] × (100), where %wl= percentage weight loss, w1= initial fruit weight in (g), w2= final fruit weight in (g) (zhang et al., 2002). superficial color superficial color of tomato was measured using a minolta chronometer model cr 400 and average readings at three points against each other in the fruits were recorded. color indices inclusive (l*, a*, and b* values) were measured. superficial color of the fruit was expressed as l* (the ratio of white to black color), a* (the ratio of red to green color) and b* (the ratio of yellow to blue color) (shafiee et al., 2007). chilling injury index (scores) and electrolyte leakage chilling injury index of fruits was evaluated at 10°c after 10, 20, 30 and 40 days in cold storage. symptoms were manifested as surface pitting and baninaiem et al. effect of preand postharvest salicylic acid treatment on tomato during cold storage 185 dehydration according to the method of sayyari et al. (2009). the severity of the symptoms was assessed with scores according to the following 3 stage scale: 0 (no symptom), 1 (1-25% of damaged area), 2 (2650% of damaged area) and 3 (>51% of the damaged area). the average extent of chilling-injury damage was expressed as a chilling-injury (ci) index, which was calculated using the following formula: ci = [(value of hedonic scale) × (number of fruit with the corresponding scale number)] / (4 × total number of fruit in the sample). the rate of electrolyte leakage (el) was measured according to the method of mirdehghan et al. (2007), using 6 discs (10 mm diameter) of peel tissue, cut with a cork borer. conductivity was measured after 4 h of incubation in 25 ml of 0.4 m mannitol under constant shaking. the conductivity of the solution ( l 1 ) w a s m e a s u r e d w i t h a c o n d u c t i v i t y m e t e r (ttracon wtw 325). after readings had been taken, the vials were autoclaved at 121°c for 15 min, and then cooled to 20°c. the conductivity of tissues (l2) was measured. ion leakage was calculated as the ratio of l1 to l2. fruit decay index (scores) decay incidence of each fruit was determined by scores. according to the amount of the decay on fruit surface scales from 1 to 5 were given to the each treatment where; 1= normal (no decay on fruit surface), 2= trace (up to 5% of fruit surface were decayed), 3= slight (5-20% of fruit surface were decayed), 4= moderate (20-50% of fruit surface were decayed), and 5= severe (>50% of fruit surface were decayed). results were expressed as fungal decay index (babalar et al., 2007). vitamin c (ascorbic acid) fruit vitamin c content was measured by using titrimetric method with the titration of filtrate against 2,6-dichlorophenol indophenol and the results of vitamin c content were expressed as mg/100 g (pila et al., 2010). titratable acidity and total soluble solids five ml of extracted fruit juice was diluted to 45 ml with distilled water. then, extract fruit juice was titrated with 0.1 n sodium hydroxide to a ph of 8.1. titratable acidity (ta) was determined as percentage of citric acid by this formula: ta (%)= [(v × n × meq)/y] × 100 where v= volume of sodium hydroxide used ml, n = sodium hydroxide normality, and meq = 0.064, y = volume of bulk fruit juice ml (saltveit, 2005). total soluble solid (tss) in the extracted fruits juice was measured with a portable refractometer (model dbr95), and the results were expressed as brix. ascorbate peroxidase ascorbate peroxidase (apx) was assayed by recording the decrease in optical density due to ascorbic acid at 290 nm for 1 min in a uv-vis spectrophotometer (model unicuv2100). samples from pulp of 0.5 g fresh tissue homogenized and the homogenized samples were centrifuged at 14000 rpm for 15 min. the supernatant was used as crude enzyme extract for apx enzyme analyses. the 3 ml reaction mixture contained 50 mm potassium phosphate buffer (ph 7.0), 0.5 mm ascorbic acid, 0.1 mm edta, 0.1 mm h2o2, and 0.1 ml enzyme. the reaction started with adding of 0.1 mm hydrogen peroxide. the enzyme activity was calculated using the extinction coefficient 2.8 mm-1 cm-1 for ascorbate (nakano and asada, 1981). statistical analysis statistical analysis was performed by sas software (sas institute inc., 1990) according to a split plot in time design on the basis of completely randomized design (crd), with 3 sa concentration and 3 replicates. data were analyzed by glm and differences among means of data were compared by with least significant difference (lsd) test at a significance level of 0.05. 3. results firmness and weight loss the results showed a rapid decrease in firmness in the control set compared to the fruits treated with sa during storage, and all treated fruits were firmer than the control set (p< 0.01). maximum fruit flesh firmness (3.2 kg cm-2) was recorded in pre and postharvest sa treatments 4+4 mm and control set had the softest fruits (0.6 kg cm-2) at the end of the experiment but, the difference was not statistically significant compared to the other sa concentrations (table 1). no significant changes were observed in weight loss during storage for any treatments, with the exception of weight loss in 30 days sa-treated fruit, which was lower than the control set (table 1). color assessment the results of this investigation showed that pre and postharvest sa treatments had an effect on fruit lightness (l*) and redness (a*) value in comparison adv. hort. sci., 2016 30(3): 183-192 186 with control set (p<0.01). no significant (b*) value were observed in treated fruits, except 20th days in 4+2 mm (34.49) and 40th days in 4+4 mm (39.76) satreated fruit, which were higher level than control set (31.34). during storage, the l* value decreased. the control set had the lowest l* value (22.60), and the highest value was recorded in pre and postharvest sa treatments 4+2-4+4 mm (28.21 and 28.87, respectively) at the end of the experiment, and their difference was not statistically significant compared to the other sa concentrations. our results indicate that sa treatments delayed the loss of l* value in tomato during storage. in general, a* value increased during storage as well as ripening. in the other hand, the color development rate of tomatoes increased with the increase in maturation. the most value of color index a* with a negative value (-6.13) was recorded in the green fruits of the control set. the negative values were observed in immature fruits and treated fruits showed negative/lower a* values than control fruits. the highest value of color index a* (19.87) was recorded in completely ripened tomatoes of the control set at 40 th days. the index a* had a sharp increase control set at 10th days with a* value changing from negative (green color) to positive (red color) (table 1). storing mature-green fruits, treated with salicylic acid at 10°c increased the postharvest life up to 40 days. chilling injury and electrolyte leakage our results showed that ci increased during storage, but applying different concentrations of sa could significantly (p<0.01) affect chilling injury index in tomato fruit. salicylic acid treatments lowered the levels of chilling injury compared to that of the fruits of control set, and the highest chilling injury was observed in control set. no chilling injury symptoms were observed in tomato fruits with pre and postharvest treatments 4+4 mm (table 2). the results obtained from the present study showed that electrolyte leakage of control set (70.27%) was significantly higher than that of sa-treated fruits end of the storage period, and there was no significant difference between concentrations used for treatment (p<0.01) (table 2). fruit decay index decay increases during storage and the results of our evaluation showed that sa, at different concentrations, significantly affected fruits decay (p<0.01). fruits with sa treatments showed lower levels of decay as compared to that of the fruits of control set. highest decay was observed in our control set. in pre and postharvest treatments 4+4 mm, there were not any decay symptoms in tomato fruit after 40 days. we did not witness any significant differences between concentrations (4+1 and 4+2 mm) (table 2). means within each column with different superscript letters are significantly different (p = 0.05) for each sampling. table 1 effect of pre and postharvest treatments salicylic acid in firmness, weight loss, l*, a*, b* of tomato fruit cv. baraka and stored at 10°c for up to 40 days time storage (days) salicylic acid treatments firmness (kg cm-2) weight loss (%) color parameter l* a* b* 0 0 4.82±1.02 a 0.00±0.00 f 34.98±2.69 a -6.13±0.63 g 15.47±1.17 e 10 0 3.38±0.18 cdef 1.62±0.82 e 29.37±1.23 fg 3.83±0.35 d 15.76±1.51 e 4+1 4.11±0.07 abc 1.32±0.03 e 33.02±1.97 abc -5.73±0.17 g 13.42±0.59 e 4+2 4.18±0.03 abc 1.28±0.03 e 33.31±0.89 ab -6.03±0.82 g 11.75±0.42 e 4+4 4.29±0.021 ab 1.22±0.02 e 33.84±1.59 ab -6.86±0.36 g 16.27±2.87 e 20 0 2.88±0.78 f 2.38±0.54 cd 26.62±0.80 hi 5.86±1.58 cd 24.59±2.97 d 4+1 3.97±0.11 bcd 2.12±0.01 d 31.89±0.96 b-e -4.94±0.59 fg 24.34±6.31 d 4+2 4.08±0.08 abc 2.10±0.02 d 32.24±0.84 bcd -2.45±0.37 ef 34.49±0.08 ab 4+4 4.15±0.14 abc 2.09±0.01 d 32.46±1.00 bcd -5.73±0.45 g 24.72±7.10 d 30 0 1.70±0.67 g 3.11±0.14 b 24.41±0.86 ij 11.84±2.14 b 32.06±1.73 bc 4+1 3.64±0.07 b-f 2.69b±0.02 c 29.73±0.47 efg -1.99±0.71 ef 31.89±0.99 bc 4+2 3.75±0.07 b-e 2.66±0.03 c 30.16±0.95 d-g 2.94±0.11 d 28.14±4.10 cd 4+4 3.78±0.09 b-e 2.65±0.03 c 30.67±0.52 c-f -4.75±0.55 efg 27.53±9.01cd 40 0 0.60±0.10 h 3.92±0.62 a 22.60±1.07 j 19.87±1.85 a 31.34±3.77 bc 4+1 3.02±0.08 ef 3.67±0.02 a 27.95±0.96 gh 5.64±0.33 cd 36.85±2.79 ab 4+2 3.08±0.08 ef 3.64±0.04 a 28.21±0.16 fgh 5.54±0.38 cd 32.40±0.32 bc 4+4 3.20±0.04 def 3.63±0.02 a 28.87±0.71 fgh -1.70±0.70 e 39.76±0.70 a baninaiem et al. effect of preand postharvest salicylic acid treatment on tomato during cold storage 187 vitamin c (ascorbic acid) tomato fruits vitamin c content was decreased during storage, and it was found to be maintained with pre and postharvest treatments of sa, and this result was statistically significant (p<0.01). tomato fruits treated with sa showed comparatively higher levels of ascorbic acid than the fruits of control set (9 mg/100 g), and the highest ascorbic acid content in pre and postharvest treatments 4+4 mm (45 mg/100 g) was observed, but there was no significant difference between two concentrations of our treatment (table 2). titratable acidity (ta) and total soluble solids (tss) tomato fruits ta content was maintained with pre and postharvest treatments of sa, and it significantly resulted in firmer fruits comparing to the controls (p<0.01). titratable acidity (ta) content in treated fruits was higher than the control set (0.86%), and our results showed that ta maintained with pre and postharvest treatments of sa and we observed the highest ta content in concentrations of 4+2-4+4 mm (1.02, 1.03%, respectively) at 40th days (table 3). also, tss increased during storage. highest and lowest tss were observed in control set and treated fruits, respectively, and this difference was significant (p<0.01). sa application in this experiment had a significant effect on soluble solids. therefore, soluble solids of control fruits (4.36 °brix) were more than of treated fruits (2.70 °brix in 4+4 mm) after 40th days storage (table 3). ascorbate peroxidase (apx) the results showed apx decreased in control fruits, it increased and then decreased again in treattable 2 effect of pre and postharvest treatments of salicylic acid in chilling injury, electrolyte leakage, decay, ascorbic acid of tomato fruit cv. baraka and stored at 10°c for up to 40 days means within each column with different superscript letters are significantly different (p = 0.05) for each sampling. time storage (days) salicylic acid treatments chilling injury electrolyte leakage (%) decay index ascorbic acid (mg/100 g) 0 0 0.00± 0.00 d 40.63±10.32 d 0.00±0.00 d 66±6.00 a 0 0.00±0.00 d 56.59±4.05 c 0.00±0.00 d 32±1.05 f 10 4+1 0.00±0.00 d 26.21±1.81 gh 0.00±0.00 d 65±1.73 a 4+2 0.00±0.00 d 24.39±0.74 h 0.00±0.00 d 66±3.00 a 4+4 0.00±0.00 d 23.98±2.00 d 0.00±0.00 d 68±3.46 a 20 0 0.00±0.00 d 61.80±1.30 bc 0.00±0.00 d 18±3.00 g 4+1 0.00±0.00 d 28.27±0.90 fgh 0.00±0.00 d 49±4.58 bcd 4+2 0.00±0.00 d 25.63±0.65 gh 0.00±0.00 d 52±4.58 bc 4+4 0.00±0.00 d 23.68±0.94 h 0.00±0.00 d 55±1.73 ab 30 0 0.60±0.20 b 65.11±5.47 ab 0.46±0.31 b 12±3.00 gh 4+1 0.20±0.00 c 33.25±2.71 defg 0.20±0.00 c 44±4.58 de 4+2 0.20±0.00 c 31.81±1.70 efgh 0.20±0.00 c 48±3.00 cd 4+4 0.00±0.00 d 30.59±0.79 efgh 0.00±0.00 d 50±1.73 bcd 40 0 1.06±0.23 a 70.27±0.92 a 1.07±0.23 a 9±3.00 h 4+1 0.33±0.12 c 37.53±1.21 de 0.40±0.20 b 39±3.00 e 4+2 0.27±0.12 c 36.65±2.95 de 0.33±0.23 bc 41±3.46 e 4+4 0.00±0.00 d 36.51±2.65 def 0.00±d 45±3.00 de means within each column with different superscript letters are significantly different (p=0.05) for each sampling. time storage (days) sa treatments ta (%) tss (°brix) apx (mg/g fw) 0 0 0.94±0.19 abcd 3.40±0.69 c 37.14±1.24 e 0 0.92±0.01 bcd 3.43±0.15 bc 20.90±19.03 f 10 4+1 1.03±0.01 ab 2.23±0.15 d 54.78±1.12 ab 4+2 1.04±0.01 ab 2.16±0.15 d 55.66±1.29 a 4+4 1.04±0.02 ab 2.46±0.46 d 56.12±2.49 a 20 0 0.90±0.01 bcd 4.00±0.10 ab 18.80±11.17 f 4+1 1.01±0.01 abc 2.66±0.12 d 52.31±2.06 abc 4+2 1.04±0.01 ab 2.60±0.20 d 53.83±4.36 ab 4+4 1.06±0.01 a 2.53±0.06 d 55.70±4.77 a 30 0 0.89±0.03 cd 4.26±0.21 a 7.38±2.89 g 4+1 1.01±0.01 abc 2.66±0.06 d 40.18±2.47de 4+2 1.02±0.01 abc 2.63±0.12 d 43.78±4.81 cde 4+4 1.03±0.01 ab 2.66±0.06 d 46.42±3.11bcd 40 0 0.86±0.03 d 4.36±0.40 a 6.95±1.38 g 4+1 1.00±0.01 abcd 2.63±0.12 d 38.06±1.09 de 4+2 1.02±0.02 abc 2.66±0.0 6d 38.61±1.31 de 4+4 1.03±0.02 ab 2.70±0.10 c 38.18±2.51 de table 3 effect of pre and postharvest treatments sa in ta, tss, apx of tomato fruit cv. baraka and stored at 10°c for up to 40 days adv. hort. sci., 2016 30(3): 183-192 188 ed fruits. the activity of apx in treated fruits was higher than the controls, and there was no significant difference between the three concentrations at the end of the experiment (p<0.01) (table 3). 4. discussion and conclusions the results of this study indicate that pre and postharvest treatment with sa produced the firmest fruits. softening of fruits is one of the most common physical parameters to assess the progress of ripening (srivastava and dwivedi, 2000; brummell, 2006) and softening is a major problem of tomato that limits the quality. key factors associating with fruit softening are the depolymerisation and degradation of cell wall components (brummell, 2006). srivastava and dwivedi (2000) reported polygalacturonase is primarily responsible for ripening associated pectin degradation and fruit softening. level of polygalacturonase activity has been positively correlated with fruit ripening and softening in banana and tomato fruits. application of salicylic acid is useful in inhibiting tissue softening in fruits by reducing cell wall hydrolases activities and maintaining cell membrane consistency (supapvanich, 2015). wei et al. (2011) reported that exogenous application of sa enhances defense mechanisms and production of antioxidants in fruits during storage that leads to a decrease in lipid peroxidation of the cell membrane and results in maintained cell membrane structure. this result was in agreement with the reports of babalar et al. (2007) and shafiee et al. (2010) that suggested pre and postharvest application of sa on strawberry could decrease the softening and keep them firm during storage. zhang et al. (2003) showed that sa effectively prevented kiwifruit softening during storage and rate of fruit ripening related to internal sa concentration as well as srivastava and dwivedi (2000) reported that salicylic acid treatment inhibited the process of banana fruit softening during ripening. srivastava and dwivedi (2000), zhang et al. (2003) and wang et al. (2006) reported that rapid softening of fruits during ripening was simultaneous with rapid decrease in endogenous sa of fruits. tomato fruit weight loss did not show changes in response to sa treatments (except in 30th days lower than the control set). the results of this study did not accord with the ones of babalar et al. (2007) and shafiee et al. (2010). the l* value decreased during storage. babalar et al. (2007) reported higher lightness in pre and postharvest sa treated strawberry fruits than control. shafiee et al. (2010) showed sa treatments were not effective on fruit lightness in comparison with the control set. fattahi et al. (2010) suggested that the decrease in l* value represented the formation of dark color in the pulp due to oxidative browning reactions or increasing in brown pigment concentrations. value a* increased during fruit ripening. the same results were obtained from pre and postharvest sa application on strawberry (babalar et al., 2007), but shafiee et al. (2010) reported that sa treatments were not effective on a* value in comparison with control. the a* value is a useful index of maturation and the degree of ripening in tomato (artes et al., 1999) and the external color is a key factor indicating the quality of tomato (supapvanich, 2015). changes in a* result increase the respiration rate during storage. the salicylic acid treatment causes a decrease in respiration and a delay in the appearance of the climacteric peak, which is concentration-dependent (srivastava and dwivedi, 2000). shafiee et al. (2010) reported that the effect of sa treatments might be due to the reduction of respiration, and it prevents from an increase in a* value, so it could have an advantage in delaying the senescence. the sa application did not affect b* value except 20th days in 4+2 mm and 40th days in 4+4 mm sa-treated fruit, which showed higher level than control set. there were not literature about the effect of pre and postharvest application of sa on b*changes. salicylic acid treatments lowered the levels of chilling injury compared to that of the fruits of control set, and the highest chilling injury was observed in control set. ding et al. (2001, 2002) reported that chilling injury was manifested in tomato fruit by some symptoms. severely injured fruit developed sunken areas (blemishes) an increased susceptibility to alternaria rot and decay. initially, ci affects the cell membrane with changes in the fatty acid of phospholipids. secondary damages are on the cell membrane t h a t l e a d s t o d i s r u p t i o n o f t h e c e l l s t r u c t u r e ( s o l e i m a n i a g h d a m e t a l . , 2 0 1 2 ) . a s g h a r i a n d soleimani aghdam (2010) suggested that treatment with sa prior to low-temperature storage induce heat shock proteins (hsps) biosynthesis and, at the same time, ci tolerance in tomatoes and peaches. accumulation of the heat shock proteins (hsps) in chilling-sensitive horticultural products with sa treatments would allow their storage at low temperatures without ci development. this membrane damage can be measured by the electrolyte leakage, which the results obtained from the present study showed that electrolyte leakage of control set was significantly baninaiem et al. effect of preand postharvest salicylic acid treatment on tomato during cold storage 189 higher than that of sa-treated fruits. therefore, these results indicate that sa can maintain membrane consistency through enhancing the antioxidant potential of the plant. reduction of electrolyte leakage and prevention of oxidative damage to cells under stress conditions has been mentioned as primary mechanisms of stress tolerance. the same results were obtained from postharvest treatment with sa to prevent chilling injury (sayyari et al., 2009; soleimani aghdam et al., 2012) and electrolyte leakage (sayyari et al., 2009; soleimani aghdam et al., 2012; orabi et al., 2015). fruits with sa treatments showed lower levels of decay as compared to that of the fruits of control set and in pre and postharvest treatments 4+4 mm, there were not any decay symptoms in tomato fruit after 40th days. salicylates are major components of the signal transduction pathways of plants playing an important role in disease resistance (asghari and soleimani aghdam, 2010). different researches show that sa had no direct effect on the decrease of decay in pear fruits (fruits were sprayed with sa), but it might reduce fungus development (shafiee et al., 2010). babalar et al. (2007) reported sa in a concentration dependent manner from 1 to 2 mm effectively reduced fungal decay in selva strawberry fruit. salicylic acid applied to either plant’s vegetative stage, fruit development stages or postharvest stage could completely control decay and increased fruit shelf life. yao and tian (2005) showed preharvest and postharvest treatments of sweet cherry fruit with sa showed significantly lower disease percentages in storage at 25°c than the control. at 0°c, the inhibitory effects of preharvest sa treatments on postharvest disease were better than those of the postharvest treatment. shafiee et al. (2010) also obtained similar results for pre and postharvest sa treatments on strawberry fruit. tomato fruits vitamin c content was decreased during storage and fruits treated with sa showed comparatively higher levels of ascorbic acid than the f r u i t s o f c o n t r o l s e t . s h a h k o o m a h a l l y a n d ramezanian (2014) reported that the utilization of ascorbic acid during later storage periods may be the reason for its decreased amounts. generally, when fruits become overripe, vitamin c content declines concurrently with the degradation of fruit tissues. the results obtained from this study indicate that the sa treatments were beneficial in delaying degradat i o n o f a s c o r b i c a c i d c o n t e n t d u r i n g s t o r a g e . therefore, the sa treated fruits exhibited higher maintenance of ascorbic acid as compared to that of control set. sa prevents vitamin c destruction by increasing the antioxidant ability and resistant of plants and fruits (wang et al., 2006; shafiee et al., 2010). also, exogenous sa could be effective in reducing the rate of respiration and ethylene production (renhua et al., 2008). thus, the results of this study confirm previous reports of postharvest treatment with sa to preserve vitamin c content in tomato (pila et al., 2010), orange (huang et al., 2008), rambutan fruit (supapvanich, 2015) and pineapple fruit (lu et al., 2011). titratable acidity content in treated fruits was higher than the control set. titratable acid depends directly on the concentration of organic acids in the fruit as an important factor in maintaining the quality of fruits (kazemi et al., 2011). therefore, any treatment that slows the metabolism and aging of the product can slow down the changes during storage to reduce titratable acid (zokaee khosroshahi et al., 2007). a correlation between enhanced respiration a n d a d e c r e a s e i n t a h a s b e e n s u g g e s t e d b y shahkoomahally and ramezanian (2014) to be due to the use of organic acids as respiratory substrates in the respiratory cycle in fruits. organic acids have a higher ratio of oxygen to carbon compared to carbohydrates or fatty acids; therefore, those are easier to consume as an energy source in the process of respiration. salicylic acid reduces respiration and ethylene production, leading to the reduction in consumption of organic acids as respiratory substrates (serrano et al., 2003). salicylic acid application in this experiment had a significant effect on soluble solids. therefore, soluble solids of control fruits were higher than of treated fruits. bal and celik (2010) revealed that after harvest and during storage and ripening of the fruits was increased the tss content. asghari and soleimani aghdam (2010) and bal and celik (2010) reported that cell walls contain large amounts of polysaccharides, mainly pectins and cellulose, and are digested due to the activity of the cell wall degrading enzymes leading to a significant increase in tss content. s a l i c y l i c a c i d e f f e c t i v e l y p r o t e c t s c e l l w a l l s b y decreasing the expression of degrading enzymes and as a consequence prevents from dramatic increase in tss content of the cells, and caused slow down of ripening. similar observation was reported with sa treated banana. salicylic acid treatments inhibited ethylene biosynthesis and delayed the senescence. this is because in the control fruits, due to the aging p r o c e s s ( r i p e n i n g ) , c e l l w a l l w a s d i g e s t e d a n d increased soluble solids. on the other hand, sa treatment reduces cellular metabolic activities, such as adv. hort. sci., 2016 30(3): 183-192 190 respiration and ethylene production, and thus maintains the membranes and cell walls, and prevents from an abnormal increase in the soluble solids (valero et al., 2006). an increase in tss content of fruits during storage due to the conversion of starch to be soluble sugars is one of the ripening indexes (fisk, 2006). this result was in agreement with babalar et al. (2007) who reported that the use of salicylic acid decrease tss of strawberry fruits and consequently, effectively delays fruit senescence process. treatment of kiwifruits maintained a lower tss content than the control fruits at the end of cold storage (soleimani aghdam et al., 2009). however, it is in disagreement with lu et al. (2011) results on pineapple fruit and shafiee et al. (2010), report on strawberry, who suggested that sa did not affect soluble solids content and titratable acidity. the activity of apx in treated fruits was higher than the controls. the study indicated the beneficial effects of sa by pre and postharvest treatments on t o m a t o f r u i t q u a l i t y . w a n g e t a l . ( 2 0 0 6 ) a n d soleimani aghdam et al. (2012) reported that sa might mitigate postharvest ci in fruits and vegetables v i a d i f f e r e n t m e c h a n i s m s . t h e s e m e c h a n i s m s include: a) enhanced alternative oxidase (aox) gene expression as an efficient ros avoidance gene, b) increased ascorbate peroxidase (apx) and glut a t h i o n e r e d u c t a s e ( g r ) a c t i v i t y , c ) e n h a n c e d reduced-to-oxidized ascorbate (asa/dhasa) and reduced-to-oxidized glutathione ratios (gsh/gssg) and d) improved heat shock proteins (hsps) gene expression in peach fruits. orabi et al. (2015) suggested that there is an important link between plant antioxidant ability and the applied doses of the sa. the observed variation (increase) activity of apx is due to s a acti vates the resi stance system and increases the cell antioxidant capacity. asadi et al. (2013) showed that exogenous application of salicylic acid lightened the toxic actions induced by stress and decreased lipid peroxidation rates with increasing antioxidant activity. there is not any report on the effects of pre and postharvest treatments with sa on fruits apx activity. as a whole, this study showed that pre and p o s t h a r v e s t t r e a t m e n t s o f s a a r e a n e f f e c t i v e method of extending storability and postharvest life of tomato fruits at 10°c. the most effective treatment in reducing losses of fruit quality was found to be sa 4+4mm treatments during the storage period of tomato fruit. it was determined that under these conditions baraka tomato could be stored for 40 days without losing much of its quality. acknowledgements financial support for this work was provided by vice chancellor of faculty of agriculture and natural resources of hormozgan university. references agamy r.a., elsayed e.h., tarek h.t., 2013 acquired resistant motivated by 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postharvest biol. technol., 28(1): 67-74. zokaee khosroshahi m.r., esna-ashari m., ershadi a., 2007 effect of exogenous putrescine on post-harvest life of strawberry (fragaria ananassa duch.) fruit, cultivar selva. sci. hortic., 114: 27-32. impaginato 105 adv. hort. sci., 2018 32(1): 105-111 doi: 10.13128/ahs-22807 volatile compounds from different fruit parts of two cultivars of cydonia oblonga c. taiti 1, e. giordani 1, e. palm 1, w.a. petrucci 1, g. bennati 2, g. gestri 2, e. marone 3 (*), e. azzarello 1, s. mancuso 1 1 d i p a r t i m e n t o d i s c i e n z e d e l l e p r o d u z i o n i a g r o a l i m e n t a r i e dell’ambiente, università degli studi di firenze, viale delle idee, 30, 50019 sesto fiorentino (fi), italy. 2 associazione oasi apistica le buche, via regina margherita, 26, 59016 poggio a caiano (po), italy. 3 facoltà di bioscienze e tecnologie agro-alimentari e ambientali, università degli studi di teramo, via r. bazarini, 1, 64100 teramo, italy. key words: f r u i t t i s s u e s , p r o t o n t r a n s f e r r e a c t i o n t i m e o f f l i g h t m a s s spectrometer, physicochemical fruit parameters, quince, vocs. abstract: quince is characterized as a fragrant fruit which, unlike other pomes (apple, pear), is not used for fresh consumption due to its astringency and compactness, but only in its processed form (jams, jelly, distillery products, and nutraceutical compounds). as a consequence, there is little knowledge currently available concerning the characteristics of the fruit, and in particular its aromatic and chemotaxonomic patterns. in this work, carpometric, chemometric and spectrophotometric measurements were performed on quince fruits. vocs emitted by different tissues or parts of the fruit were studied to describe its aromatic profile. the study was carried out on the fruits of an old, well-known cultivar (‘gigante di wranja’, commonly called ‘wranja’) and a new tuscan accession. intact, halved and solely pulp (cubed) samples were evaluated for each individual fruit. data obtained from voc analysis through proton transfer reaction time-of-flight mass spectrometer (ptr-tof-ms) were evaluated by multivariate statistical analysis. the spectra obtained from the intact fruit samples showed a higher amount of masses corresponding to terpenes or terpenoid compounds, which fundamentally characterize the aroma of this type of fruit; these substances were found to be much less present in the vocs emitted by the pulp, where high values of masses linked to the maturation processes were instead found. 1. introduction plants are not only a dietary source for both human beings and animals but also serve as medicinal, nutritional, and ornamental purposes (*) corresponding author: emarone@unite.it citation: taiti c., giordani e., palm e., petrucci w.a., bennati g., gestri g., marone e., azzarello e., mancuso s., 2018 volatile compounds from different fruit parts of two cultivars of cydonia oblonga. adv. hort. sci., 32(1): 105-111 copyright: © 2018 taiti c., giordani e., palm e., petrucci w.a., bennati g., gestri g., marone e., azzarello e., mancuso s. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 2 december 2017 accepted for publication 6 march 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(1): 105-111 106 (ashraf et al., 2016). the potential of plants as medicine is supported by abundant scientific evidence (lattanzio et al., 2009) and researchers are currently focused on isolating new aromas and active phytochemicals produced by different plant organs. quince is the fruit of an ancient tree, cydonia oblonga miller of the rosaceae family, and has a very wide origin area that ranges from anatolia to the caucasus. it was known to the greeks and romans, and it seems that the name of the genus cydonia comes from the ancient name of the town of chania on the island of crete (roversi, 1991). it is a climacteric, large-sized (up to 500 g) fruit, namely pome (false fruit), and generally oblong (pyriform types) or rounded (appleshared types) (bellini et al., 2007). the harvest is still carried out by hand, although the desire to mechanize this operation with systems similar to those used for the mechanical harvesting of the apple tree is increasing (fiorino et al., 2010). when ripening, the color of the fruit epidermis turns from greenish-yellow to bright yellow when fully ripe. the pulp is very firm, and not directly edible due to the presence of tannins and pectic substances (bellini et al., 2007). ripe fruits are commonly used as a source of pectin, and to produce jam or jelly, liquors, and distilled products with aromatic notes typical of quince (silva et al., 2004). the fruits can also be beneficial to human health thanks to their high phytochemical content (ashraf et al., 2016). many studies have reported cydonia oblonga as an excellent natural source of phenolic and flavonoid compounds which are considered potent antioxidants (silva et al., 2004; oliveira et al., 2007). quince fruit is considered an important dietary source of health-promoting compounds due to its antioxidant, antimicrobial and antiulcerative properties (magalhães et al., 2009). although the aroma is one its most important fruit-derived parameters, only a few studies have focused on the volatile emission by cydonia (tateo and bononi, 2010), and in particular, by different tissues of the fruit. indeed, the intense and pleasant aroma released by cydonia may represent an important starting point in the genetic improvement of this species. recently, proton transfer reaction-time of flightmass spectrometry (ptr-tof-ms) was proposed as an innovative analytical technology for volatile organic compound (voc) detection and quantification on fruit matrices due to its capacity to rapidly provide a comprehensive mass spectrum with high-time resolution and without sample treatment (mayr et al., 2003; taiti et al., 2017 a). the aim of the current study was to analyze the aroma profile of different fruit parts of two cydonia oblonga genotypes, both grown in italy, to increase the chemotaxonomic knowledge, fruit management and quince product trade. 2. materials and methods plant materials quince fruits belonging to an old, well-known cultivar (‘gigante di wranja’, commonly called ‘wranja’) and a new tuscan accession were harvested in tuscany (italy) in the last week of september 2017. for each cultivar, 12 homogeneous and healthy fruits were selected, gently brushed to eliminate the hair cover and washed in deionized water. fruits were stored in a climatic chamber (14±1°c, 85-90% relative humidity) for three days prior to laboratory analysis. for all the fruits, color, weight, maximum diameter and height were determined. six fruits were used for the spectrophotometric tests by ptr-tof-ms and the remaining six for the physicochemical measurements. for chemical analyses (lugol starch test and refractometric grade), fruits were divided in half so that one half of each fruit was used for the starch test and the other to evaluate refractometric grade. color and physicochemical fruit parameters peel color. the identification of the different peel color of quince fruits was assessed using a minolta cr-200 chroma-meter (minolta, ramsey, nj, usa) a c c o r d i n g t o t h e h u n t e r s c a l e , a s p r e v i o u s l y described by taiti et al. (2017 b). fruits dimensions and firmness. for each cultivar, the weight (g), maximum diameter (cm), and height (cm) of twelve samples were measured. the firmness of each fruit (expressed as kgf) was measured as the force needed to reduce fruit diameter by 2 mm using a penetrometer (model 53205d, turoni, italy). total soluble solids (tss). to evaluate tss concentration, six quince halved fruits for each genotype were squeezed to collect a few drops of juice; then tss levels were measured with an n1 atago refractometer (atago co., japan) and expressed as °brix. starch iodine test. starch-iodine test (lugol solution) was performed by visual evaluation on halved fruits and by scoring samples on a ‘golden delicious’ standardized 1-9 scale (smith et al., 1979). volatile compounds detection vocs analyses were carried out with a high-resotaiti et al. vocs from different fruit parts of two quince cultivars 107 lution ptr-tof-ms (ionicon analytik gmbh, austria) using h3o + as ion donor. for each genotype, six fruits were used; the same fruit was first analyzed as intact whole, then divided into two parts, analyzing the halves together; finally, two cubes were got from each one of the halved fruit respectively, without peel (5 × 5 × 5 cm, about 10 g each one); all the measurements were performed in triplicates on the same sample. all whole and halved samples were weighed, and the vocs data were based on 100 g samples for whole and halved samples to allow for comparisons between the obtained results. the drift tube of ptr-tof-ms instrument was set to 80°c and operated with a drift pressure of 2.30 mbar and a voltage of 550 v. these settings lead to an e/n ratio (e, electric field strength in the drift tube; n, buffer gas number density in the drift tube) e q u a l t o 1 3 5 t o w n s e n d s ( 1 t d = 1 0 1 7 v c m 2) . mass/charge ratio of peaks detected at m/z 21 (signal for h3o +) and m/z 37 [signal for water clusters (h2o)h3o +] were monitored during all measurements to check the instrument’s stability and cluster ion formation. to reach a good mass accuracy (up to 0.001 th), internal calibration was based on three points and was performed off-line. acquisition and post-processing data were performed as reported by marone et al. (2017). statistical analyses one-way analyses of variance (anova) were performed (1) to compare the physicochemical parameters of two quince genotypes, and (2) to compare the voc emission profiles whole, halved, and cubed fruit samples. separation of means was performed by the fisher’s lsd test (p= 0.05). computations were performed by statgraphics centurion xv v. 15.0.04. a principal component analysis (pca) was applied to the whole spectral data of 36 quince samples, previously submitted to a logarithmic transformation and mean centering as pre-processing. computations were performed by pls-toolbox v. 8.0.2 (eigenvector research inc., west eaglerock drive, wenatchee, wa) for matlab_ r2015b (mathworks inc., natick, ma, usa). a correspondent analysis (ca) was applied to the spectral data of the 36 quince samples to build up a simultaneous ordination of quince samples and protonated m/z, thus facilitating the evaluation of their reciprocal relationships. the analysis preserves the χ2-distances between the data matrix rows and columns. data were weighted using the symmetric option (module hierclus, syn-tax 2000 program package). 3. results and discussion physicochemical analyses the results of the physicochemical analyses on the different cultivars are reported in table 1, and are largely in agreement with a previous study on different cultivars (leonel et al., 2016). among the physicochemical parameters analyzed, some significant differences were observed between the two cydonia accessions, with regard to firmness, °brix values and starch contents. in particular, the °brix content was lower for ‘wranja’ (12.2±0.9) compared to tuscan accession (17.2±0.9); vice versa the starch content was higher for ‘wranja’. the starch and sugar contents measured for the tuscan accession indicate that these fruits may have been further along in the ripening process than the ‘wranja’ fruits. vocs headspace analysis samples (whole, halved and cubed) belonging to two different cydonia genotypes were analyzed by ptr-tof-ms detecting a total of 67 peak signals (fig. 1). these signals represent different groups of volatile compounds including hydrocarbons, esters, alcohols, terpenoids, aldehydes, ketones and lactones. these results show that, even if no different volatile compounds were identified, different emission intensities were found between the two genotypes (fig. 1). on the other hand, among fruit tissues, both differences in number and emission intensity of each detected compound were instead observed. figure 1 shows that for both cultivars, the intensity and number of compounds emitted were higher in table 1 one-way analysis of variance (anova) related to the quince fruits physicochemical parameters (average ± sd) different lowercase letters within a column indicate differences by the lsd test at the 95% confidence level (p= 0.05). cydonia accessions weight (g) ø (mm) high (mm) firmness (kgf) °brix starch overcolor background color l* a* b* l* a* b* wranja 268.9 82.8 77.8 6.1 12.2 4.25 77.7 -12.5 62.6 72.6 -15.9 58.4 ±54.5 ±7.0 ±4.5 ±0.9 a ±0.9 a ±0.5 a ±2.4 ±2.4 ±3.3 ±4.1 ±2.3 ±3.1 tuscan 320 86.5 85.4 7.6 17.2 2.75 76.9 -13.1 57.6 76.6 -12.8 60 ±92.6 ±9.2 ±9.7 ±0.4 b ±0.9 b ±0.5 b ±4.7 ±2.3 ±5.6 ±2.6 ±2.2 ±2.9 adv. hort. sci., 2018 32(1): 105-111 108 the whole fruit followed by halved fruit, and lowest in cubed fruit. these results are in agreement with a previous study of imayoshi et al. (1995) on nashi pear, where differences in the total number of vocs among pulp, peel, and whole fruit were found. indeed, as reported by paillard (1981) for many fruits species, voc production changes among different fruit tissues, being highest in the skin and nearby tissues. it is noteworthy that fruit’s aroma depends on the combination of vocs produced, and on the concentration and odor threshold of each in the blend. a further investigation of the spectral data was performed by a multivariate ordination. the pca (fig. 2) approach applied to the whole dataset (ppbv) of 67 detected protonated masses (data not shown) give a general view of the quince sample ordination, based on the analysis of defined fruits parts (intact, halved, and cubed fruits) related to the two chosen accessions (tuscan accession and ‘wranja’). the first three components accounted for 96.4% of the total variability (66.1%, 17.7%, 12.7%, respectively). a strong effect related to the type of the sample used rather than the cultivar is evident. the whole fruit samples of both cultivars, joined in the lower right quadrant of the diagram, strongly differ from those obtained from the head space of the pulp only, both correlated with the negative part of the x-axis (pc1), but clustered based on the cultivar, and respectively related to the positive part (tuscan accession) and to the negative part (wranja) of the y-axis (pc2). the halved fruit group is placed in the upper right quadrant, with the two cultivars partially overlapped, indifig. 1 typical ptr-ms mass spectrum (average, n=6) obtained by different fruit parts of two cultivars of cydonia. (a) tuscan accession, (b) ‘wranja’; (1) whole fruit, (2) halve fruit, (3) cube fruit. fig. 2 pca scores of the quince samples based on the full spectra distribution of the ‘tuscan accession’ and ‘wranja’. (a1) = ‘tuscan accession’ whole fruits; (a2) = ‘tuscan accession’ halved fruits; (a3) = ‘tuscan accession’ cube fruits; (b1) = ‘wranja’ whole fruits; (b2) = ‘wranja’ halved fruits, (b3) = ‘wranja’ cube fruits. taiti et al. vocs from different fruit parts of two quince cultivars 109 cating a greater similarity of masses with the intact whole fruit, even if positively correlated with pc2. to better evaluate the effect of specific chemical family compounds, the 20 masses with statistically significant (p= 0.05) discriminating power among the different parts of the fruit were selected by anova within t h e w h o l e s p e c t r u m ( t a b l e 2 ) , u p o n w h i c h a correspondence analysis was subsequently applied. the join plot from ca (fig. 3) simultaneously displays quince different sample and protonated m/z ordinations based on the results of the variable selection. the intact fruits of both cultivars are characterized by a group of masses in which terpenes and terpenoids are well represented. in particular, the highest presence of monoterpene (m/z 135, m/z 137), terpenoid (m/z 153) and sesquiterpene (m/z 205) compounds distinguished the whole fruit from the other two types of cut samples. according to taiti et al. (2017 b), the higher emission intensity and number of vocs detected in whole fruits was associated to the presence of the peel. it is well known that a great diversity of vocs linked to the pericarp tissue of the peel are emitted, such as esters, alcohols, a l d e h y d e s a n d t e r p e n e s ( c h e r v i n e t a l . , 2 0 0 0 ; rodriguez et al., 2013). the vocs linked to cutting, such as the green leaf volatiles (glvs) (i.e., hexenal, hexanal), are highest in halved and cubed fruits, compared to intact fruits where the intensities of these signals were very low (table 2), and confirmed by the fact that these compounds were well represented in pulp tissue. moreover, halved fruits occupy an intermediate position, while cubed fruit samples are shifted to the right in the diagram (fig. 3), with a differentiation along the axis of the y (axis 2) determined by the masses m/z = 45.033, m/z = 47.049 and m/z 33.033 (only for the tuscan accession). as reported by taiti et al. (2015) the methanol emission in fruits increase table 2 protonated selected masses discriminating whole, halved and cubed fruit samples, identified via ptr-tof-ms: mass/charge (m/z) ratios, chemical formula, tentative identifications, minimum and maximum values detected (ppbv) and voc, as previously reported in the literature (cydonia*; ptr-ms#) no. protonated m/z chemical formula tentative identification min detected value (ppbv) max detected value (ppbv) references 1 33.033 ch 5 o+ methanol 11.7 2724.1 khoubnasabjafari and jouyban (2011)* 2 45.033 c 2 h 5 o+ acetaldehyde 35 16416.9 umano et al. (1986)* 3 47.049 c 2 h 7 o+ ethanol 1.5 199.8 umano et al. (1986) 4 55.054 c 4 h 7 + c4 aldehydes fragment 65.3 433.1 taiti et al. (2017 b)# 5 57.033 c 3 h 5 o+ alkyl fragment (hexanal/hexyl acetate) 8.5 25.4 taiti et al. (2017 b)# 6 57.069 c 4 h 9 + alkyl fragment (hexanol/valeric acid) 18.1 75.2 taiti et al. (2017 b)# 8 75.044 c 4 h 11 o+ 2-butanol/2-methyl-1-propanol 0.5 19.7 umano et al. (1986)* 9 81.069 c 6 h 9 + terpenes and c6 fragments 49.8 203.5 maleknia et al. (2007) # 10 83.085 c 6 h 11 + c6 fragments /hexenol fragment 8.8 50.7 maleknia et al. (2007)# 11 85.064 c 5 h 9 o+ 2-methyl-2-butenal/1-penten-3-one 4.6 26.2 khoubnasabjafari and jouyban (2011)* 12 99.080 c 6 h 11 o+ (e)-2-hexenal 1.6 8.9 umano et al. (1986)* 13 101.096 c 6 h 13 o+ hexanal/(z)-3-hexen-1-ol 1.7 44.8 tateo and bonomi (2010)* 14 109.101 c 8 h 13 + terpene fragments 0.4 23.6 maleknia et al. (2007) # 15 117.091 c 6 h 13 o 2 + isobutyl acetate/ethyl butyrate/butyl acetate 1.5 176.7 tateo and bonomi (2010)* 16 135.116 c 10 h 15 + p-cymene 0.4 11.9 tateo and bonomi (2010)* 17 137.132 c 10 h 17 + monoterpenes (e.g. limonene, g-terpinene) 0 5.1 tateo and bonomi (2010)* 18 145.122 c 8 h 17 o 2 + ethyl hexanoate 0.5 153.9 tateo and bonomi (2010)* 19 153.127 c 10 h 17 o+ oxygenated terpenes (e.g. geranial) 0 0.4 tsuneya et al. (1983)* 20 205.195 c 15 h 25 + sesquiterpenes (e.g. bergamotene) 1.1 49.8 tateo and bonomi (2010)* fig. 3 join plot from correspondence analysis. (a1) = ‘tuscan accession’ whole fruits; (a2) = ‘tuscan accession’ halved fruits; (a3) = ‘tuscan accession’ cube fruits; (b1) = ‘wranja’ whole fruits; (b2) = ‘wranja’ halved fruits, (b3) = ‘wranja’ cube fruits. numbers correspond to the protonated m/z. adv. hort. sci., 2018 32(1): 105-111 110 steadily throughout the ripening process. on the other hand, ethanol is the product of anaerobic metabolism and, together with acetaldehyde, accumulates in pome fruit under imposed hypoxia and poor gas exchange in the pulp tissue (pinto et al. 2001). pome fruit contains ethanol and acetaldehyde as part of their aroma (ritenour et al. 1997) and as reported by rapparini and predieri (2003) these compounds increase together with the ripening process and their emission increased at a faster rate with the onset of fruit senescence. 4. conclusions the typical quince fruit aroma depends on a mix of volatile compounds which originate by different parts of the pome. by the analyses of different fruit tissues, differences in vocs number and in their emission intensity were observed, while little difference was observed between the two varieties analyzed. moreover, it has been shown that the intensity and number of compounds emitted was highest in the whole fruit, followed by halved fruit and cubed fruit. through multivariate analysis, some masses have been highlighted that determine the differentiation of the spectral composition between the vocs emitted by the pulp (halved and cubed fruit samples) and those obtained from intact fruit that could be specifically responsible for the particular aroma of quince fruit. the four masses referring to terpenes and terpenoids are prevalent in the peel, and are moderately related to the cultivar. further work should be done to increase the knowledge concerning the chemotaxonomic profiles of the different cultivars, and to understand how through this tool, innovations can be developed and moved to quince genetically improved and to the food industry. references ashraf m.u., muhammad g., hussain m.a., bukhari s.n., 2016 cydonia oblonga m., a medicinal plant rich in phytonutrients for pharmaceuticals. frontiers in pharmacology, 7: 163. bellini e., giordani e., giannelli g., picardi e., 2007 quince. cydonia oblonga mill., pp. 439-457. in: bellini e. 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khan 3 1 department of horticulture, agri-college, vali-e-asr university of rafsanjan, iran. 2 department of horticulture, vali-e-asr university of rafsanjan, iran. 3 nuclear institute for agriculture and biology (niab), faisalabad, pakistan. key words: nutrients, organic matter, pistachios, poultry manure, yield elements. abstract: the effectiveness of poultry manure application time was studied on pistachios (pistacia vera l.) trees. the experiment consisted of seven different poultry manure application time, including poultry manure application as one time in 1) last week of october 2) last week of december 3) last week of january 4) mid-march and dividing into two parts and use in fall or in winter, dividing into four parts and use in dormant seasons (fall and winter). based on the results, there were significant differences among treatments. the highest number of fruit per cluster (27.4) was found in poultry manure applied in last week of october. the highest nut splitting percent (84.3%) and the lowest nut blanking percent (8.6%) were obtained in poultry manure applied by dividing into four parts. half kernel nuts followed a similar trend with blanking percent. weight of 1000 nuts increased and responded positively and number of pistachios nut per ounce decreased by manure application when divided into two parts and used in the winter. application of poultry manure in the mid-march enhanced the nut protein (19.63%). 1. introduction production technologies of horticultural crops including pistachio tree have undergone vast changes recently, and led to the extension of innovative technologies about nutrient management. the critical factor of nutrient management of nut trees like pistachio is to elevate the net yield and improve the quality of nut fruits. pistachio blanking and flower bud abscission can be directly related to nutrition management (mahmoudi meimand and ghanbari odivi, 2013). nowadays, fertilizers play a key role in nutrition of fruit trees. because of harmful side effects of chemical fertilizers, (ljoyah and sophie, 2009) the use of bio fertilizers are increasing recently. organic fertilizers, such as animal manure, have a long history of use by men (baybordi and malakuoti, 2003). different animal manures such as sheep, cow and poultry manures have been used as natural crop (*) corresponding author: mmeimand@ut.ac.ir citation: mahmoudi meimand m.j., shamshiri m.h., roosta h.r., khan e.u, 2018 poultry manure application time on pistachio (pistacia vera l.) trees adv. hort. sci., 32(2): 177-183 copyright: © 2018 mahmoudi meimand m.j., shamshiri m.h., roosta h.r., khan e.u. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 10 october 2017 accepted for publication 12 january 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(2): 177-183 178 fertilizers for centuries. poultry manure, because of its high level of nitrogen which is absorbable for pistachio trees, has been recognized as one of the main favorable manures. it has been documented that poultry manures also supply other essential pistachio nutrients and act as soil amendant by raising organic matter content, which helps improve the moisture level of soil and nutrient maintenance. according to alimoradi (2011) nearly 5.3 million tons of poultry manure is consumed in rafsanjan and kerman pistachio orchards every year. these bio fertilizers are known as main source of manure for iranian pistachio growers. nutrients provided by poultry manures have been indicated to establish effects on different crops, including fruit crops (mitchell et al., 1993; miller, 1996). it has been reported that poultry manure is composed of essential elements for fruit crops containing about 3% of nitrogen nh4 +, 63.2% phosphorus p2o5 and 4.1% potassium k2o (reddy and reddy, 1995). the main form of nitrogen in poultry manures is nh4 +, which elevate the availability of nitrate to the plants for a longer period (burmester, 1993; crawford and chalk, 1993; touchton and bosewell, 1980). on the other hand, organic manures application to decrease the use of chemical fertilizers in pistachio orchards and other fruit trees is an important goal in fruit production (reganold et al., 2001; forge et al., 2002). organic manures increase the fertility of soil and the crop yield. therefore, they can be helpful to achieve sustainable agriculture. it has been demonstrated earlier that poultry manure improved growth parameters, yield and quality in different crops (ram and rajput, 2002; ingle et al., 2003; arancon et al., 2003). pimpini et al. (1992) revealed higher rate of ex-tractable sucrose by using 4 t ha-1of poultry manure in crops. increased content of total carbohydrate, protein and ascorbic acid were reported by abusaleha and dutta (1988) when poultry manure was used. improving photosynthesis, plant biomass and glycosides content of stevia were also demonstrated before (xiangyang et al., 2010). enhanced starch content, crude fiber, ash, crude protein, phosphorus, calcium and magnesium of d. bulbifera were reported by ezeocha et al. (2014). adekiya and agbede (2017) showed that poultry application increased soil organic matter, leaf n, p, k, ca, mg contents, growth and yield in tomato. they also showed that the application of poultry manure at 3 weeks before transplanting had highest effects on leaf nutrient concentrations, growth and yield in tomato (adekiya and agbede, 2017). now, pistachio growers are frequently using poultry manure as a source of plant nutrient. to our knowledge, there are no reports available to recommend favorable time for poultry manure application in orchard of pistachio trees. the aim of this investigation is to study the effect of poultry manure application time on nut yield and quality in pistachios trees. 2. materials and methods the experimental orchard selected for this study is located at khatam, yazd province, iran. it is located at 39.33°n latitude and 54.40°e longitudes, at an elevation of 1605 m above sea level. the average temperature of the zone is 18.5°c, the annual total chilling hours (≤7.2°c) is about 950; the average annual rainfall is 300 mm. the climate of this area is typically subtropical. just before poultry manures application, soil sample of the experimental orchard were collected and chemically analyzed (table 1). this research was conducted on 12 years old pistachio cv. akbari grafted on ‘badamii zarand’ rootstock. management factors such as irrigation regime, pruning practices, and weed control were followed according to local standards. trees were trained with a modified central leader system and distance of trees was 2x6 m. to achieve better results, uniform trees were selected with uniform vigor and age and three uniform shoots were selected from different sides of the tree for data collection. harvest index was considered when several nuts in the cluster were light colored and the hull was easily separated from shell. characteristics of yield, percentage of splitting, weight of 1000 nuts, blanking, pistachio weight in soil characteristics value ph 7.6 ec ds m-1 4 p ppm 28.1 k ppm 180 o.m. % 1.3 t.n.v. % 22.1 sand % 52 silt % 34 clay % 14 texture loam esp 17.9 table 1 soil fertility analysis report of the experimental orchard mahmoudi meimand et al. poltry manure application on pistachio trees 179 ounce, dry weight and some other characteristics related to kernel and leaf were recorded. the experiment consisted of seven different poultry manure application time, with three replications based on the randomized complete block design (rcbd). the treatments were poultry manure application as one time in 1) last week of october 2) last december 3) last week of january 4) mid-march and dividing into two parts and use in fall or in winter, dividing into four parts and use in dormant seasons (fall and winter). these treatments were conducted beneath the tree canopy and mixed well with surface soil, 10 kg per tree. data collecting was performed in next growth season. statistical analysis was conducted using the sas software (9.2) and means were compared by duncan’s multiple range test (p≤ 0.05). 3. results and discussion effect of poultry manure application time on yield parameters number of fruits per cluster. the study indicated that poultry manure application time had significant effects on number of fruits per cluster (p≤ 0.05) (table 2). the highest number of fruit per cluster was found by poultry manure application in last week of o c t o b e r ( 2 7 . 4 ) f o l l o w e d b y t h e l a s t w e e k o f december (26.9) (table 2). the minimum value (19.2) was observed in control treatment. the higher fruit per cluster with poultry manure application in last week of october (concurrently with tree deciduous) might be related with the positive role of nitrogen and other critical elements on cluster final development. burmester (1993) crawford and chalk (1993) touchton and bosewell (1980) demonstrated the positive role and proportion ratio of elements on reproductive growth parameters. nut splitting percent. also, our results demonstrated that poultry manure application time had significant effects on nut splitting percent (table 2). highest nut splitting percent (84.3%) was obtained in treatment of poultry manures divided into four parts and used in last weeks of october, december, january and mid-march followed by divided into two parts and used in fall (83.5%) and in winter (83.1%) (table 2). the minimum value for nut splitting percent was reported in control treatment. based on our results, the poultry manure application in four different times, thus involving a longer period of the year improved nut splitting percent. it has been demonstrated before that poultry manure was improved yield quality in different crops (arancon et al., 2003; ingle et al., 2003). number of pistachio nuts per ounce. table (2) indicates that the use of poultry manure increased the pistachio ounce index or decreased number of inshell pistachio nuts per ounce significantly (p≤ 0.01). the results showed also that when a portion of poultry manure is allocated at the end of dormant season, increase fruit size is achieved. previous findings showed that poultry manure improved yield component in different crops (ram and rajput, 2002; arancon et al., 2003). blanking percent. based on the results, the lowest value (8.6%) for nut blanking was observed when treatment consisted in dividing poultry manures into four parts and usi ng i n l ast weeks of october, december, january and mid-march; this was followed by treatment divided into two parts and used in winter (11%) (table 2). the maximum amount of blanking (%) was observed in control treatment (13.9%) with no significant difference with fall application. therefore, poultry manure application at over the year in four different times decreased nut blanking percent. yield component improving demonstratlwo= last week of october, lwd= the last week of december, lwj= the last week of january, mm= march mid, lwjm= divided into two parts and used in the winter, lwod= divided into two parts and used in the fall, lwodjm= poultry manures divided into four parts and used in the last week of october, the last week of december, the last week of january and march mid. means followed by the same letters are not significantly different (duncan test, p≤ 0.05). parameter\treatment lwo lwd lwod lwj mm lwjm lwodjm control number of fruit 27.4 a 26.9 b 22.6 c 21.2 e 21.05 e 22.1d 26.9 b 19.2 f nut splitting % 77.3 b 76.4 b 83.5 a 70 c 69.8 c 83.1 a 84.3 a 64.8 d fruit ounce 22.6 a 22.6 a 22 ab 22.6 a 22.3 a 20.6 b 20.6 b 23 a blanking % 13.3 ab 13.5 ab 13.1 bc 12.5 bc 12.8 bc 11 d 8.6 e 13.9 a half seed fruit % 1.3 c 1.2 d 0.7 e 4.4 b 3.1 c 0.9 e 0.7 e 5.0 a weight of 1000 nuts 1249 b 1249 b 1288 b 1249 b 1267 b 1372.1 a 1370 a 1231 b table 2 effect of poultry manure application time on yield parameters adv. hort. sci., 2018 32(2): 177-183 180 ed by ram and rajput (2002) and arancon et al. (2003). half kernel fruit percent. half kernel nuts followed a trend similar to blanking percent, it was decreased with dividing poultry manure into four parts (0.7%), followed by manure divided into two parts and used in fall (0.7%) or winter (0.9%) (table 2). the highest half kernel nuts percent was obtained in control (table 2). w e i g h t o f 1 0 0 0 n u t s . w e i g h t o f 1 0 0 0 n u t s increased and positively and significantly (p>0.05) responded to the treatments. highest weight of 1000 nuts (1372.1 g) were observed in poultry manure divided into two parts and applied in winter followed by treatment consisting in manure divided into four parts and used in last weeks of october, december, january and mid-march (1370 g). however, other treatments exhibited lowest weight of 1000 nuts (table 2). previous findings indicated that poultry manure improved yield component in different crops (ram and rajput, 2002; arancon et al., 2003). effect of poultry manure application time on nut quality parameters fruit stening. fruit stening was influenced by application of poultry manure in different times (table 3). maximum amount for fruit stening (7.23%) was noted at mid-march application (7.23%) and control (7.16%) and the lowest value for this parameter was obtained with treatment divided into two parts and used in fall treatment (table 3). it has been demonstrated earlier that poultry manure improved yield and quality in different crops (ram and rajput, 2002; arancon et al., 2003; ingle et al., 2003). n u t p r o t e i n . n u t p r o t e i n s p e r c e n t w a s a l s o enhanced by the application of poultry manure in all treatment versus control (table 3), but maximum value for nut protein percent was observed at midmarch (mm) application (19.63%) followed by the last week of january (19.5%) and in poultry manures divided into four parts and used in last weeks of october, december, january and mid-march (19.2%) with no significant differences (table 3). the minimum value for nut protein percent was observed in control treatment (17.4%). increasing amount of total carbohydrates, proteins and ascorbic acid content reported by abusaleha and dutta (1988) in a similar work that well described poultry manure application on increasing secondary metabolites. enhancing crude protein in d. bulbifera was reported by ezeocha et al. (2014). the results reported by adekiya and agbede (2017) showed that poultry application produced higher percent protein content, more growth and yield in tomato. effect of poultry manure application time on vegetative and reproductive parameters leaf fresh weight. according to obtained results, poultry manure application time had significant effects on leaf fresh weight (p≤ 0.05) (fig. 1). the highest amount of leaf fresh weight was obtained at poultry application in mid-march (2.1 g), followed by fig. 1 changes of leaf fresh (x) and dry weight (■) (gr) of pistachio (p. vera cv. akbari) grown in khatam, yazd, iran. duncan was calculated at p≤0.05. lwo= last week of october, lwd= last week of december, lwj= last week of january, mm= march mid, lwjm= divided into two parts and used in the winter, lwod= divided into two parts and used in the fall, lwodjm= poultry manures divided into four parts and used in the last week of october, the last week of december, the last week of january and march mid. parameter\treatment lwo lwd lwod lwj mm lwjm lwodjm control fruit stening (%) 5.6 d 6.56 b 4.2 f 6.8 b 7.23 a 5.9 c 4.96 e 7.16 a nut protein (%) 19.1 bc 19 c 18.9 c 19.5 ab 19.63 a 19.2 ab 19.2 ab 17.4 d table 3 effect of poultry manure application time on nut quality parameters lwo= last week of october, lwd= the last week of december, lwj= the last week of january, mm= march mid, lwjm= divided into two parts and used in the winter, lwod= divided into two parts and used in the fall, lwodjm= poultry manures divided into four parts and used in the last week of october, the last week of december, the last week of january and march mid. means followed by the same letters are not significantly different (duncan test, p≤ 0.05). mahmoudi meimand et al. poltry manure application on pistachio trees 181 application divided into two parts and used in winter (1.9 g) (fig. 1). the lowest rate of leaf fresh weight was occurred in control (1.5 g) and last week of october (1.5 g), respectively (fig. 1). based on our result, poultry manure application in mid-march at once increased leaf fresh weight. increasing photosynthesis and plant biomass of stevia was also demonstrated before (xiangyang et al., 2010). leaf dry weight. dry weight of leaves was signific a n t l y a f f e c t e d b y d i f f e r e n t t i m e s o f m a n u r e application (p≤ 0.05). maximum leaf dry weight was found in poultry application in mid-march (0.8 g) and divided into two parts and used in winter (0.8 g), whereas the minimum was observed in control (0.4 g) and last week of october (0.4 g) (fig. 1). this increase in leaf dry and fresh weights with application of poultry manures at the end of dormant season can be explained as following, this time of application cannot make the expected effect and causes to increase vegetative parameters, similar to reports published by xiangyang et al. (2010). leaf nitrogen content. results revealed that the leaf nitrogen content of pistachio trees fertilized with poultry manure in different times varied significantly (p≤ 0.05). leaf nitrogen content was the highest (3.1%) at poultry application in mid-march once at any time compared to the treatments divided into two parts and applied in winter (3.03%) and in last week of january (2.9%) (fig. 2). the higher leaf nitrogen content in mid-march application of poultry manures could be in relation with releasing more nitrogen nh4 + form which is useful for vegetative parameters and causes nitrogen accumulation in the leaves. previous findings emphasis on high level of nh4 + form in poultry manure. the application of poultry manure with high level of n, have influenced the growth and vegetative factors of tree and production of fruits (reddy and reddy, 1995). adekiya and agbede (2017) indicated that application of poultry manure increased percent of leaf n, p, k, ca, mg content, growth parameters and yield in tomato. poultry manure usage at 3 weeks before transplanting caused higher leaf nutrients concentrations and more growth and yield in tomato (adekiya and agbede, 2017). shoot length. based on our findings, shoot length was significantly affected by different application times of manure (p≤0.05). maximum shoot length was obtained when manure was divided into two parts and used in winter (44 cm) and mid-march application (43.3 cm). our results revealed that minimum value for this parameter was observed in control (26.6 cm) and in manure divided into two parts and used in fall (32.6 cm) (fig. 3). similar to the dry and fresh weights, application of poultry manures at the end of dormant season caused to increase shoot length. our results demonstrated that application of poultry manures during fall stimulates a least vegetative growth with no significant differences between various times in fall (fig. 3). increasing vegetative growth and similar findings by using poultry manure fig. 2 changes of leaf nitrogen content of pistachio (p. vera cv. akb a ri ) gro wn i n kh a t a m, y a z d , i ra n . d u n ca n wa s calculated at p≤0.05. lwo= last week of october, lwd= last week of december, lwj= last week of january, mm= march mid, lwjm= divided into two parts and used in the winter, lwod= divided into two parts and used in the fall, lwodjm= poultry manures divided into four parts and used in the last week of october, the last week of december, the last week of january and march mid. fig. 3 changes of shoot length of pistachio (p. vera cv. akbari) grown in khatam, yazd, iran. duncan was calculated at p≤0.05. lwo= last week of october, lwd= last week of december, lwj= last week of january, mm= march mid, lwjm= divided into two parts and used in the winter, lwod= divided into two parts and used in the fall, lwodjm= poultry manures divided into four parts and used in the last week of october, the last week of december, the last week of january and march mid. adv. hort. sci., 2018 32(2): 177-183 182 have been reported before (xiangyang et al., 2010). leafy bud number, floral bud number and leafy to flower bud ratio. results indicated that leafy and floral buds and leafy to flower bud ratio of pistachio trees fertilized with poultry manure was positively affected by treatments (p≤ 0.05). number of floral buds in the next growing season was the highest in last week of october and fall (in last weeks of october and december) (7.33) (fig. 4). minimum value for floral bud number was observed in control (fig. 5). results for leafy bud numbers showed that maximum value was obtained in treatment divided into two parts and applied in winter (5.6) and midmarch (5.3) (the letter two treatments have not any significant difference) (fig. 4). floral bud abscission ratio. it was revealed that floral bud abscission during the grow season by poultry manure usage was significantly less than control, which it might be due to the role of poultry manure in decreasing floral bud abscission totally. any treatment could not show significant effect on floral bud abscission, but minimum amount for this parameter was observed in treatment that was conducted in last week of december (0.32) (fig. 5). previous reports showed that poultry manure improves the yield component (ram and rajput, 2002; arancon et al., 2003), although there are no exact results about reproductive phase and parameters affected by poultry manure treatment, but flower bud abscission can b e d i r e c t l y r e l a t e d t o n u t r i t i o n m a n a g e m e n t (mahmoudi meimand and ghanbari odivi, 2013). 4. conclusions it is clear that application of organic fertilizers will improve the nut yield and quality, but application of poultry manure through dormant season can be effective on reproductive and vegetative parameters in pistachio. based on our findings, dividing poultry manures into four parts and applying in last weeks of october, december, january and mid-march, or using a single application in fall showed the best results for optimum nut yield, quality and floral bud emergence in pistachio. references abusaleha, dutta o.p., 1988 inter relationship of yield components in cucumber. veg. science, 15(1): 79-85. adekiya a.o., agbede t.m., 2017 effect of methods and time of poultry manure application on soil and leaf nutrient concentrations, 6 growth and fruit yield of tomato (lycopersicon esculentum mill). j. saudi soc. fig. 4 changes of leafy bud number (■), floral bud number (×) and leafy to flower bud ratio (○) of pistachio (p. vera cv. akb a ri ) gro wn i n kh a t a m, y a z d , i ra n . d u n ca n wa s calculated at p≤0.05. lwo= last week of october, lwd= last week of december, lwj= last week of january, mm= march mid, lwjm= divided into two parts and used in the winter, lwod= divided into two parts and used in the fall, lwodjm= poultry manures divided into four parts and used in the last week of october, the last week of december, the last week of january and march mid. fig. 5 changes of floral bud abscission ratio of pistachio (p. vera cv. akbari) grown in khatam, yazd, iran. duncan was calculated at p≤0.05. lwo= last week of october, lwd= last week of december, lwj= last week of january, mm= march mid, lwjm= divided into two parts and used in the winter, lwod= divided into two parts and used in the fall, lwodjm= poultry manures divided into four parts and used in the last week of october, the last week of december, the last week of january and march mid. mahmoudi meimand et al. poltry manure application on pistachio trees 183 of agric. sci., 16: 383-388. alimoradi m., 2011 the value of poultry by-products, livestock specialist. vtyvr blog. arancon n.q., galvis p., edwards c.a., yardim e., 2003 the trophic diversity of nematode communities in soils treated with vermicompost. pedobiología, 47: 736-740. baybordy a., malakouti m.g., 2003 effect of iron, manganese, zinc and copper on the yield and quality of wheat 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orissa j. hortic, 32(1): 10-13. ljoyah m.o., sophie v.l., 2009 effects of different level of decomposed poultry manure on yield of cabbage (brassica oleraceae l.). j. trop. agric., food, environ. extension, 8(1): 20-23. mahmoudi meimand m.j., ghanbari odivi a., 2013 new approach for cultivation pistachio trees, pub by nushe publisher, isbn:978-600-6376-18-8. miller d., 1996 poultry litter as a soil amendment in southeastern row crops: a feasibility study based on agronomics, environmental and economic factorssustainable agriculture research and education. southern region, as93-10, ls9139. final report. mitchell c.c., burmester c.h., wood c.w., 1993 broiler litter as a source of n for cotton. beltwide cotton conf., new orleans, la, natl, 1357-1359. pimpini f., giardini l., borin m., gianquinto g., 1992 effect of poultry manure and mineral fertilizers on the quality of crops. j. agric. sci. camb., 118: 215-221. ram a.a., rajput m.s., 2002 role of biofertiliser and manures in production of guava (psidium guajava l.) cv. allahabad safeda. haryana j. hortic. sci., 29(3-4): 193194. reddy t.y., reddy g.h., 1995 principles of agronomy. 2nd edition, kalyani publishers, new delhi, india, pp. 223. reganold j.p., glover d., andrews p.k., hinmam h.r., 2001 sustainability of three apple production systems. nature, 410: 926-929. touchton j.t., bosewell f.c., 1980 performance of nitrification inhibitors in the south east in j. j. meisinger (ed.) nitrification inhibitors: potential and limitation. asa spec. publ. asa and sssa, madison, usa, pp. 6374. xiangyang l., guangxi r., yan s., 2010 the effect of organic manure and chemical fertilizer on growth and development of stevia rebaudiana bertoni. energy procedia, 5: 1200-1204. impaginato 319 adv. hort. sci., 2017 31(4): 319-327 doi: 10.13128/ahs-20545 phenolic metabolism and antioxidant activity during endodormancy of kiwifruit buds e. abedi gheshlaghi 1 (*), v. rabiei 2, m. ghasemi 3, j. fattahi 3, f. razavi 2 1 horticulture crops research department, guilan agricultural and natural resources research and education center, areeo, rasht, iran. 2 department of horticulture, faculty of agriculture, university of zanjan, iran. 3 horticulture research institute, citrus and subtropical research center, agricultural research, education and extension organization (areeo), ramsar, iran. key words: actinidia deliciosa, biochemistry, chilling, enzyme. abstract: bud dormancy is an adaptability process in woody plants that enables them to survive in unfavorable conditions. in the present study, the phenols, antioxidant capacity, and activity of three enzymes were evaluated during endodormancy phases in two hayward and tomuri cultivars and two female and male golden genotypes of kiwifruit buds. the buds were collected from ten-year-old own-rooted vines from the end of october 2015 until the end of january 2016 in the north of iran. the results revealed that phenols, antioxidant capacity (rsa), phenylalanine ammonia-lyase (pal), and polyphenol oxidase (ppo) activities of buds significantly increased at the beginning of endodormancy and subsequently decreased at the end of the endodormancy. the pod activity increased in hayward and tomuri from the onset of endodormancy and continued for two weeks after the endodormancy release. the total phenol had a positive and significant correlation with rsa and pal enzyme activity. furthermore, higher antioxidant capacity and phenols in both male and female golden genotypes were attributed to the higher pal enzyme activity in both genotypes. this study proposes that the rsa%, pal activity, and phenol concentration could be employed as a biomarker to indicate bud dormancy phases in kiwifruit. 1. introduction the first axillary buds are initiated on the developing shoots of kiwifruit shortly after bud break in the first growing season (walton et al., 1997). similar to temperate fruits, the axillary buds of kiwifruit can be induced into endodormancy by short days and low temperatures at the end of the summer or the beginning of autumn (mcpherson et al., 1995). bud endodormancy is an adaptability process in woody plants that (*) corresponding author: eabedig@yahoo.com citation: abedi gheshlaghi e., rabiei v., ghasemi m., fattahi j., razavi f., 2017 phenolic metabolism and antioxidant activity during endodormancy of kiwifruit buds. adv. hort. sci., 31(4): 319327 copyright: © 2017 abedi gheshlaghi e., rabiei v., ghasemi m., fattahi j., razavi f. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 16 april 2017 accepted for publication 19 october 2017 ahs advances in horticultural science adv. hort. sci., 2017 31(4): 319-327 320 enables trees to survive in unfavorable conditions such as drought and extremely hot and cold weather (arora et al., 2003); in addition, more importantly, it encourages the reproductive processes such as the formation of flowers and fruit set to be accomplished in favorable condition and guarantees the reproductive growth and survival of the plants (campoy et al., 2011). endodormancy (winter dormancy) is a true dormancy in which the bud growth is prevented by an inhibitory system within the bud (horvath et al., 2003). overcoming endodormancy and maximum bud break and flowering in favorable environmental conditions are achieved by accumulating the minimum amount of chilling hours (mcpherson et al., 1997). after that endodormancy is released, the growth of buds is prevented directly by external environmental factors. this type of dormancy mainly occurs in late winter and is named ecodormancy (horvath et al., 2003). generally, horticultural specialists determine the t i m e o f b u d b r e a k b y c h i l l i n g a n d h e a t u n i t s . however, this method is based on ambient temperature, varies according to the environmental conditions, and cannot express the internal situation of buds (dennis, 2003). during bud endodormancy, there is no visible growth, but physiological changes in respiration, growth regulators, carbohydrate metabolism, the amount of water, and other compounds occur, influencing bud endodormancy control (ben mohamed et al., 2010). a series of the changes occurring in the biochemistry of the buds appear to indicate the shift from the endodormancy stage to the ecodormancy stage (pakish et al., 2009; szecskó et al., 2002). richardson et al. (2010) stated that high and stable sucrose concentrations are likely to be a good indicator of the true dormancy of the buds in kiwifruit (actinidia deliciosa). investigating the relationship between biochemical compounds and the beginning of the endodormancy in nine varieties of apricot revealed seasonal changes in phenolic compounds and peroxidase (laslo and vicas, 2012). these changes are caused by t h e a c c u m u l a t i o n o f c h i l l i n g u n i t s d u r i n g t h e endodormancy, that is necessary for the development of some phenological stages. pakish et al. (2009) studied peroxidase and oxidase activities in the varieties of pistachio buds during the winter and observed seasonal variation in the november-march interval. there is a significant difference in terms of the phenols of buds in different dormancy stages within cultivars of the same species. the total phenol of the flowering buds in peach (szalay et al., 2005) and apricot (laslo and vicas, 2012) increased at the beginning of endodormancy, showed a gradual increase in dormancy period, and disappeared during flowering. large variations in the content of polyphenols can be observed in certain varieties of pistachio (pistacia vera l.) in the november-march interval. the phenol of all cultivars was reduced in the swollen buds (pakish et al., 2009). factors controlling the onset, maintenance, and termination of endodormancy are varied and have not been studied much (luedeling et al., 2009). moreover, the starting point of endodormancy, the end of endodormancy, and the start of ecodormancy are not clearly defined in plants; therefore, the study of changes in biochemical compounds in this field could be useful (pakish et al., 2009; ben mohamed et al., 2010). a number of studies have been done on the changes in carbohydrates (richardson et al., 2007, 2010), nitrogen, and amino acid (walton et al., 1991) of kiwifruit in the endodormancy period; however, the activities of enzymatic and non-enzymatic antioxidants such as phenol and its metabolism have not been studied yet. thus, the aim of this study was to inspect the changes in antioxidant capacity, total p h e n o l , a n d a c t i v i t y o f t h r e e e n z y m e s d u r i n g endodormancy and the early ecodormancy in four cultivars and genotypes of kiwifruit. 2. materials and methods plant material and sampling bud samples of hayward and tomuri kiwifruit cultivars (actinidia deliciosa) and two male and female golden genotypes (a. chinensis; mass selection of golden kiwifruit cultivar seedlings) were collected from canes of ten-year-old own-rooted vines from the end of october 2015 until the end of january 2016 with a 7-day interval at 11 steps. from the first to the last sampling, the vines received 0, 222, 309, 443, 570, 740, 864, 1003, 1125, 1272, and 1450 chilling units, respectively (richardson et al., 1974). kiwifruit vines were located at the national citrus and subtropical research institute of iran (latitude 75.36° north and longitude 33.51° east) and were trained on a t-bar training system with a planting distance of 4×6 m. the samples were immediately frozen in liquid nitrogen and kept for subsequent analyses at -80°c. at every sampling date, 30 buds were collected from 1-year-old canes at nodes 6 to 20 starting from the basal end of canes. ten buds abedi gheshlaghi et al. phenolic metabolism and antioxidant activity during endodormancy of kiwifruit buds 321 were selected randomly in three replications for further analyses of biochemical compounds (richardson et al., 2010). the activities of phenylalanine ammonia-lyase (pal), peroxides (pod), polyphenol oxidase (ppo), antioxidant capacity (rsa), and total phenol content were determined in buds during endodormancy. estimation of endodormancy period chilling requirements and bud endodormancy period of kiwifruit cultivars and genotypes were estimated via single-node cuttings test. simultaneously, fifteen cuttings in three replications of each cultivar and genotype were collected and the buds were taken for biochemical measurements. cuttings from each treatment were transferred into a forcing chamber at 25°c, with 16 h of light (wall et al., 2008). the lowest mean time budburst (mtb) for half of the buds were considered as an endodormancy release (tisne-agostini et al., 1992). extraction and determination of total phenol and antioxidant capacity the total phenol of each extract was determined according to the folin-ciocalteu procedure reported by meyers et al. (2003). moreover, the spectrophotometric method introduced by wettasinghe and shahidi (2000) was employed for the chemical determination of antioxidant. extraction and assay of pal one hundred mg of kiwifruit buds powdered by liquid nitrogen were mixed with 2 ml of 1.0 mm borate buffer containing 1.0% of polyvinyl pyrrolidone. after homogenization by a homogenizer (model ika-t8, germany), the samples were centrifuged for 15 min at 4°c and 13,000 rpm. the supernatants were slowly transferred into the tubes by pipette. extracts for the ensuing measurements were maintained at -80°c. pal (ec 4.3.1.24) activity was determined according to the study carried out by yu et al. (2012). extraction and assay of ppo and pod activities the two hundred-mg samples of fresh buds, which were collected, were ground in liquid nitrogen and homogenated with 2 ml of potassium phosphate buffer (50 mm, ph= 7.0) containing 1% polyvinyl pyrrolidone (pvp) (w/v) and 0.05% edta at 4°c. the homogenate was centrifuged at 14000 rpm for 15 min at 4°c. the supernatant was used as a crude enzyme solution for assay and was maintained at -80°c for the following measurements. the activity of ppo enzyme (ec 1.14.18.1) was quantified by the method described by in et al. (2007). the activity of pod enzyme (e.c 1.11.1.7) was measured by the method employed by srivastava et al. (1983). the activities of these enzymes were calculated, using the beer-lambert law on the basis of a single enzyme unit (μmol) per mg of fresh weight according to the following formula: u/g fwmin = absorption changes per minute × reaction mixtur/ supernatant volume × extinction coefficient statistical analyses this study was conducted as a two-factor factorial in a completely randomized design. the first factor is 11 sampling dates, and the second factor is four cultivars having three replications in the period of 20152016. the anovas and standard errors of the mean (se) were generated, using sas 9. all significant means were separated, using the duncan (p≤0.01). the correlation between the total phenol and antioxidant capacity, pal, ppo, and pod activities was calculated via the software spss 22. 3. results and discussion the beginning date for the chilling accumulation was considered to be when a stable chilling accumulation occurred and the temperatures causing a negative effect were infrequent (richardson et al., 1974; guerriero et al., 1990). this date corresponded with 27th october 2015. the first samples were conducted on this date. mean time budburst was more evident on this date than on the date of the endodormancy release; thus, the kiwifruit axillary buds may inter endodormancy sooner than the end of the summer or the beginning of autumn (mcpherson et al., 1995). the results revealed that the maximum depth of endodormancy in this study was in late november. the duration of bud endodormancy was different in the cultivars and genotypes. the end of endodormancy was 21st december 2015 for female golden genotype (740-unit chilling), 28th december 2015 for male golden genotype (864-unit chilling), and 4th january 2016 for hayward and tomuri cultivars (1003-unit chilling) as shown in figure 1. the antioxidant capacity indicated a significant difference between the buds of genotypes and cultivars sampled on different dates (p≤0.01). the highest value in the antioxidant capacity was observed in female golden genotype on 28th december 2015 (fig. 2); however, there were no significant differences in the antioxidant capacity of all buds samples adv. hort. sci., 2017 31(4): 319-327 322 collected in december (i.e. 07th, 14th, 21st and 28th). the lowest value in the antioxidant capacity was observed in tomuri cultivar on 11th january after endodormancy release (fig. 2). the antioxidant capacity of buds changed considerably at the beginning and during the maintenance and release of endodormancy (p≤0.01) (fig. 2). the increase in antioxidant capacity from the end of october to the early november was simultaneous in four cultivars and genotypes; however, the peak period of the antioxidant capacity of buds was different between these cultivars and genotypes (fig. 2). the antioxidant capacity showed a significant reduction (p≤0.01) in the male and female genotypes at the end of december, being simultaneous with the end of bud endodormancy of both genotypes. the reduction in the antioxidant capacity in hayward and tomuri cultivars occurred about two weeks later, coinciding with the completion of their chilling requirements and the end of endodormancy in the buds (fig. 2). the activity of pal enzyme has been shown in figure 3 and demonstrated a significant difference in buds of all kiwifruit cultivars and genotypes (p≤ 0.01). the activity of this enzyme in the male and female g o l d e n g e n o t y p e s w a s m o r e t h a n t h a t o f t h e hayward and tomuri cultivars (fig. 3). female golden g e n o t y p e h a d t h e h i g h e s t p a l a c t i v i t y i n m i d december. the lowest activity of this enzyme was in tomuri cultivar on 30th november 2015. pal enzyme activity in all four kiwifruit cultivars and genotypes began an upward trend at the end of november (p≤0.01) (fig. 3). it remained relatively stable during endodormancy in cultivars and genotypes, but was associated with a fluctuation in male and female golden genotypes during ecodormancy. however, the enzyme activity of all cultivar showed a significant decrease (p≤0.01) at the end of endodormancy compared to the stable periods of endodormancy. p l a n t s r e l e a s e h y d r o g e n p e r o x i d e ( h 2o 2) i n response to the environmental stress. low temperature stress has also been shown to induce h2o2 accumulation in cells (okane et al., 1996). hydrogen peroxide, as the second messenger of the increase in the activity of pal enzyme, can activate pal enzyme activity, as a key enzyme in the phenylpropanoids pathway, ultimately leading to higher total phenol and accumulation of flavonoids (wang et al., 2015). o x i d a t i v e s t r e s s c a u s e d b y c h i l l i n g d u r i n g t h e endodormancy of kiwifruit buds increased the activity of this enzyme and the production of antioxidant compounds such as phenols, resulting in a higher antioxidant capacity in the buds during the endodormancy. it has been reported that cold acclimation of fig. 1 effect of sampling date of cuttings on average of 50% bud break and endodormancy end period in four kiwifruit cultivars and genotypes during dormant season in 2015-2016. the arrows show the endodormancy end date for each cultivar and genotype. fig. 2 effects of kiwifruit cultivars and different sampling dates on antioxidant capacity of axillary buds during dormant season in 2015-2016. each data point represents the mean of three replicates, each containing ten buds. moreover, ± the standard error of mean is shown on the vertical bar. fig. 3 effects of kiwifruit cultivars and different sampling dates on phenylalanine ammonia-lyase activity of axillary buds during dormant season in 2015-2016. each data point represents the mean of three replicates, each containing ten buds moreover, ± the standard error of mean is shown on the vertical bar. abedi gheshlaghi et al. phenolic metabolism and antioxidant activity during endodormancy of kiwifruit buds 323 plants leads to a remarkable increase in pal activity, depending upon the range of low temperature to which the plants are subjected (stefanowska et al., 2002). the amount of total phenol have been shown in figure 4 and varied between cultivars and genotypes of kiwifruit, and there were significant differences in their buds (p≤0.01). male and female golden genotypes had higher total phenol than hayward and tomuri cultivars (fig. 4). total phenol content showed substantial changes at the beginning, during the maintenance, and at the end of the endodormancy of kiwifruit buds (p≤0.01) (fig. 4). the amount of total phenol showed an increasing trend from the late october. in the late november-early december period, coinciding with the onset of true endodormancy, the amount of total phenol reached its maximum (fig. 4) and its value remained at a high level in the buds of cultivars and genotypes during this period. by reaching the end of endodormancy, the amount of phenol had decreased significantly at the end of december and at the beginning of january in the buds of male and female golden genotypes and hayward and tomuri cultivars, respectively (p≤0.01). phenolic compounds are a valuable piece of evidence used in determining the differences between diverse varieties of myrtus communis and pistacia lentiscus and have a key role in detecting the genetic differences in biochemical methods (tattini et al., 2006). thus, it appears that the significant differences in terms of the total phenol content between hayward and tomuri cultivars and male and female golden genotypes (p≤0.01) (fig. 4) are the result of their genetic differences. endodormancy is developed gradually after the cessation of growth; additionally, the severity of the endodormancy deepens in autumn and then gradually disappears by removing the physiological barriers of growth through the chilling process (dennis, 2003). total phenol concentration and antioxidant capacity enhance along with the development of endodormancy and reach their maximum value in the deepest stage of endodormancy (fig. 2, 4). the changes in these two variables are similar in the establishment, maintenance, and release of endodormancy. a high total antioxidant activity in male and female golden genotypes may be attributed to the high amount of phenol. phenolic compounds are synthesized in plant cells in favorable environmental conditions, but environmental stresses change their levels in cells (kliebenstein, 2004). mid-autumn cold and the start of endodormancy period causes an increase in oxidative stress in plants. this stress results from reactive oxygen species that affect the growth of plants (scalabrelli et al., 1991; mittler et al., 2004). plants possess a protective system composed by the enzymatic antioxidant system such as peroxidase and catalase (anderson et al., 1995) and the non-enzymatic systems (agarwal and pandey, 2004). phenols are non-enzymatic antioxidants and their antioxidant activities are mainly due to their redox properties which allow them to act as reducing agents, hydrogen donators, and singlet oxygen quencher (huda-faujan et al., 2009). phenols play an important role not only during c o l d r e s i s t a n c e , b u t a l s o d u r i n g b r e a k i n g t h e endodormancy of peach (siller-cepeda et al., 1992) and apricot (viti and bartolini, 1998) buds as an antioxidant. total phenol changes in kiwifruit buds are in agreement with the results of the studies on peach flower buds (szalay et al., 2005), apricot vegetative buds (laslo and vicas, 2012), and pistachio flower buds (pakish et al., 2009). correlation analyses showed a positive significant correlation between total phenol, antioxidant capacity and pal activity in kiwifruit cultivars and genotypes (table 1). reduction in antioxidant capacity (fig. 2) and total phenol (fig. 4) after receiving chilling and a stable period in winter could be considered as a biomarker of endodormancy release in the cultivars which were studied. in cultivars and genotypes of kiwifruit buds, ppo activity changed substantially (p≤0.01) as shown in figure 5. this enzyme had the highest activity in the tomuri variety on 7th january 2016 and the lowest activity in male and female golden genotypes at the fig. 4 effects of kiwifruit cultivars and different sampling dates on phenol content of axillary buds during dormant season in 2015-2016. each data point represents the mean of three replicates, each containing ten buds. moreover, ± the standard error of mean is shown on the vertical bar. adv. hort. sci., 2017 31(4): 319-327 324 first sampling date (fig. 5). at the end of october 2015 and contemporaneous with the development of bud endodormancy, the activity of ppo enzyme, in all cultivars except hayward, increased and reached its peak in mid-december and then decreased (p≤0.01; fig. 5). ppo activity in the hayward variety peaked a week earlier than the other cultivars. in tomuri cultivar, ppo activity was stable for three weeks in the dormancy period and reduced at ecodormancy. ppo is a copper-containing enzyme which catalyzes the oxidation of phenolic compounds to quinone or quinine-like compounds in the presence of molecular oxygen. a high ppo enzyme activity after endodormancy is probably due to the removal of some growth-inhibiting phenols (wang et al., 1991), and the phenolic substances such as inhibitors o r s t i m u l a n t s c h a n g e e n z y m e a c t i v i t y (thirugnanasambantham et al., 2013). the increased activity of the ppo enzyme at the early stage of endodormancy period and its declined activity at the end of the endodormancy of grape buds (scalabrelli et al., 1991), plums (szecskó et al., 2002), and pistachio flower buds (pakish et al., 2009) were reported, corresponding with the results of this experiment. it appears that the effect of antioxidant compounds is to inhibit free radicals and reactive oxygen species in cultivars and genotypes during stress. p o d a c t i v i t y w a s n o t a b l y d i f f e r e n t i n b u d s (p≤0.01). this enzyme had the highest activity in the tomuri cultivar after the endodormancy release (fig. 6). the lowest activity was observed at all cultivars and genotypes at the first sampling date (the 27th october). the pod activity pattern was not constant during the endodormancy of buds in the cultivars and genotypes which were studied (fig. 6). pod activity increased in golden genotypes later than that in the hayward and tomuri cultivars. the activity of this enzyme increased significantly in early december and mid-december 2015 in male and female golden genotype buds, respectively (p≤0.01). however, pod activity decreased significantly in both genotypes at t h e b u d e n d o d o r m a n c y r e l e a s e ( f i g . 6 ) . t h e increased activity of pod at the end of october, in ** correlation is significant at the 0.01 level. table 1 correlation coefficient of kiwifruit cultivars and genotypes between phenol, antioxidant capacity (rsa %), phenylalanine ammonia-lyase (pal), peroxidase (pod), and polyphenoleoxidase (ppo) fig. 5 effects of kiwifruit cultivars and different sampling dates on polyphenol oxidase activity of axillary buds during dormant season in 2015-2016. each data point represents the mean of three replicates, each containing ten buds. moreover, ± the standard error of mean is shown on the vertical bar. fig. 6 effects of kiwifruit cultivars and different sampling dates on peroxidase activity of axillary buds during dormant season in 2015-2016. each data point represents the mean of three replicates, each containing ten bud. moreover, ± the standard error of mean is shown on the vertical bar. cultivars and genotypes phenol pal rsa pod ppo hayward phenol 1 0.25 ns 0.95 ** -0.11 ns -0.34 ns pal 0.25 ns 1 0.17 ns 0.24 ns -0.18 ns rsa 0.95 ** 0.17 ns 1 -0.22 ns -0.27 ns pod -0.11 ns 0.24 * -0.22 ns 1 0.44 ns ppo -0.34 ns -0.18 ns -0.27 ns 0.44 ns 1 tomuri phenol 1 0.38 ns 0.94 ** 0.38 ns 0.28 ns pal 0.38 ns 1 0.42 ns 0.20 ns -0.22 ns rsa 0.94 ** 0.42 ns 1 0.27 ns 0.28 ns pod 0.38 ns 0.20 ns 0.27 ns 1 0.28 ns ppo 0.28 ns -0.22 ns 0.28 ns 0.28 ns 1 female golden phenol 1 0.76 ** 0.93 ** 0.20 ns 0.16 ns pal 0.76 ** 1 0.67 * -0.19 ns -0.16 ns rsa 0.93 ** 0.67* 1 0.19 ns 0.14 ns pod 0.20 ns -0.19 ns 0.19 ns 1 0.88 ** ppo 0.16 ns -0.16 ns 0.14 ns 0.88 ** 1 male golden phenol 1 0.75 * 0.83 ** 0.36 ns 0.26 ns pal 0.75 * 1 0.74 * 0.40 ns 0.37 ns rsa 0.83 ** 0.74 * 1 0.61 ns 0.49 ns pod 0.36 ns 0.40 ns 0.61 ns 1 0.95 ** ppo 0.26 ns 0.37 ns 0.49 ns 0.95 ** 1 abedi gheshlaghi et al. phenolic metabolism and antioxidant activity during endodormancy of kiwifruit buds 325 requires further investigation. this study indicated a significant and stable increase in the pal and rsa activities. furthermore, we found that phenol concentration could be associated with the transition to, and maintenance of, bud in true endodormancy. due to the positive and significant correlation between total phenol, antioxidant 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phytochemical screening. abstract: foliar application of 25% w/v crude aqueous extracts of calotrophus procera (aiton) w.t. aiton, canarium schweinfurthii (engl.) and bryscocarpus coccineus (schum. & thonn.) were evaluated for their insecticidal activity in reducing podagrica infestation on okra. results showed that the plants extracts significantly suppress podagrica spp. infestation and protect okra plant from severe leaves defoliation, with c. schweinfurthii (21.67 and 20.14) and b. coccineus (23.07 and 24.55) showing promising insecticidal activity for both cropping seasons. the yield attributes from okra sprayed with lambda cyhalothrin did not differ significantly compared to those sprayed with botanical insecticide despite having highest yield attributes. qualitative phytochemical screening revealed the presence of triterpenoids, steroids, flavonoids, phlobatanins, saponins, tannins, cardiac glycoside and anthraquinones. alkaloids and anthraquinones were not detected in c. procera and c. schweinfurthii while triterpenoids and phlobatanins were absent in c. schweinfurthii. the presence of these phytochemicals indicates that the plants possess insecticidal properties responsible for significant reduction in podagrica spp. infestation, severity of leaves damaged and improved okra yields. performance of the treatments is rated in the following order: lambda cyhalothrin > b. coccineus > c. schweinfurthii > c. procera, with b. coccineus and c. schweinfurthii having similar treatment means in all the parameters evaluated. in light of the foregoing, crude extracts of b. coccineus and c. schweinfurthii could be utilized as suitable alternative to synthetic insecticide in sustainable okra production. 1. introduction okra (abelmoschus esculentus l. moench) is an important fruit veg(*) corresponding author: moboladesina@yahoo.com citation: adesina j.m., rajashekar y., 2018 phytochemical composition and insecticidal potentials of some plant aqueous extracts in suppressing podagrica spp. (coleoptera: chysomelidae) infestation on okra (abelmoschus esculentus l. moench) adv. hort. sci., 32(1): 7178 copyright: © 2018 adesina j.m., rajashekar y. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 8 september 2017 accepted for publication 12 january 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(1): 71-78 72 etable crop in the diets of most people in the tropics and subtropical countries. it is grown mainly for its freshly immature pods and ranks fourth worldwide after pepper, tomato and onion on the basis of land area, production and value. despite the great demand for okra due to its nutritional and economic importance, okra production is being hampered by array of insect pest infestation r e s u l t i n g i n p o o r y i e l d a n d l o w m a r k e t v a l u e . d i f f e r e n t g r o w t h s t a g e s o f o k r a a r e g e n e r a l l y attacked by different insect pests (adesina and afolabi, 2014; kedar et al., 2014) and podagrica species (coleoptera: chrysomelidae) is one of the most prevalent and damaging insect pests considered as major constraint to cultivation of okra which defoliate or damage the plant leaves and flowers; thereby results in reduction of the photosynthetic capability of the crop (odebiyi, 1980). the insects equally act as vectors in transmitting mosaic viral diseases and ultimately reduce yield (fasunwon and banjo, 2010), mainly if control measures are not undertaken. important yield losses of 20-50% are reported in nigerian and ghana (obeng-ofori and sackey, 2003; ahmed et al., 2007; fajinmi and fajinmi, 2010). currently, synthetic insecticide is being used across the globe to control agriculturally important insect pests due to their quick action and lasting effect (alao et al., 2011). the failure of synthetic insecticides to ensure total insect pest control due to development of resistant and resurgence and couple with increasing concern over environmental pollution, carcinogenic effect and destruction of beneficiary insects (isman, 2008), level of pesticide residues in food had compelled the scientific community to explore the abandoned and neglected traditional and indigenous products that are cheap, environmental safe and easily biodegradable products as alternatives to synthetic insecticides for tackling agricultural insect pests (antonio, 2009; alao et al., 2011; aetiba and osekre, 2016). presently, in many parts of the world, attention has been focused on the utilization of plant products that possess both medicinal and aromatic properties which are abound in various agro-ecological zones of the world as novel chemotherapeutants in plant protection (dubey et al., 2010). the popularity of botanical pesticides is once again increasing and some plant products either in crude form or by processing into different formulations are being used globally as green pesticides (dubey et al., 2008). the shortcomings associated with the use of synthetic chemicals, necessitated the idea of developing effective, cheap and easily biodegradable alternative products. therefore, this study aims to screen and r e p o r t t h e e f f i c a c y o f a q u e o u s e x t r a c t s o f calotrophus procera (ait.) ait., canarium schweinfurthii (engl.) and bryscocarpus coccineus (shum and thonn.) in suppressing flea beetles podagrica spp. (coleoptera: chrysomelidae) infestation on okra. 2. materials and methods experimental location, designs and treatments the study was carried out on 168 m2 area of land manually cleared and prepared at the teaching and research farm, rufus giwa polytechnic (rugipo), owo, ondo state located in south western nigeria and lies on latitude 7° 12’ n and longitude 7° 35’ e. t h e e x p e r i m e n t w a s l a i d o u t i n a r a n d o m i z e d complete block design (rcbd) with three replications. the experimental land was divided into 4 blocks of 4x4 m to produce a total of 12 plots. the plot size was 2x1.5 m with 0.5 m between plots and 1x1 m between blocks to prevent pesticide drift and inter-plot interference, respectively. an early maturity okra cultivar (nh-47) obtained from ondo state agricultural development project (adp), akure, ondo state, nigeria was sown with two to three seeds per hole at a depth of 2-3 cm and spaced 50x60 cm and later thinned to one plant per stand. weeding was carried out manually as at when due to ensure clean plots free from weed competition. the treatments applied were 25% w/v b. coccineus, c. schweinfurthii, c. procera and 0.8 l/ha synthetic i n s e c t i c i d e ( l a m b d a c y h a l o t h r i n ) a s c o n t r o l . application of treatments commenced four weeks after planting (wap) with a 2-litre capacity hand-held sprayer. the spraying was carried out weekly till fruiting early in the morning to avoid photo decomposition and drifting of the extracts. synthetic insecticide was applied every two weeks. collection and preparation of plant extracts leaves of b. coccineus and c. schweinfurthii were collected from owo, ondo state, nigeria and c. procera were collected from ogbagi akoko ondo state, nigeria (7° 35’ n, 5° 43’ e) at full blooming and 25 kg were weighed separately using electronic balance (table 1). thereafter, the weighed plants were washed with water to remove dirt or other contaminants and each of the plant materials was pounded separately in a mortar into a fine soft paste form. the adesina and rajashekar plant aqueous extracts against podagrica spp. 73 paste (crushed plant leaves) was then put into a 10litre plastic bucket and the appropriate volume of distilled water added to make a 25% w/v crude aqueous extract solution (25% w/v represents an extract made with 25 g of crushed plant leaves per every 100 ml of water). the pastes were soaked overnight (approximately 14 hours) in a covered bucket with occasional stirring and filtered using muslin cloth with the filtrates stored in a 10 l keg in a cool dry place till use. phytochemical screening the qualitative phytochemical tests were performed on the crude aqueous extract to detect the presence of bioactive secondary metabolites in tested plant materials using standard laboratory methods following the procedure described by odebiyi and sofowora, 1978; sofowora, 1982; williamson et al., 1996; banso and ngbede, 2006; ngbede et al., 2008. data collection and statistical analysis all data were collected from five plants randomly tagged per treatment in the two middle rows of each plot. leaves damage index was determined by visual counting of the number of holes caused by the insect feeding activities. while insect count for estimation of the population densities of p. uniformis and p. sjostedti was made 4 wap between the hours 6-7 am when the insects are still inactive through visual counting. the fruits were harvested two months after planting (map), when the fruits were still fresh at the interval of 4 days. weighing balance was used to determine the weight of the fruits and the fruits length measured using ruler. data collected were analyzed by analysis of variance (anova) using gester version 1.2, insect count were subjected to square root (y =√) transformation before analysis to normalize the data. significant treatment means were compared using tukey test at 5% probability. 3. results podagrica spp. population before application of some plant aqueous extracts table 2 shows podagrica spp. population on okra before the application of the aqueous plant extracts treatments. the results showed that the insect populations were not significantly different. the population ranges between 4.07-4.93/plant for 2015 cropping season and 4.10-4.98/plant for 2016 cropping season. toxicity effect of some plant aqueous extracts in suppressing podagrica spp. infestation the utilization of plant aqueous extracts to suppress podagrica spp. infestation on okra was presented in tables 3 and 4 for 2015 and 2016 cropping season, respectively. application of the various treatscientific name common name family part used calotropis procera giant swallow wort or milkweed asclepiadaceae leaves canarium schweinfurthii african elemi or canarium burseraceae leaves byrsocarpus coccineus short-pod connaraceae leaves table 1 plant materials used for the experiment table 2 podagrica spp. population before application of some plant aqueous extracts treatments insect population 2015 2016 c. procera 4.07±0.77 a 4.98±0.15 a c. schweinfurthii 4.08±0.66 a 4.46±0.34 a b. coccineus 4.27±0.38 a 4.10±0.07 a lambda cyhalothrin 4.93±0.66 a 4.47±0.28 a treatments with the same letter in column are not statistically significant different from each other. treatments 4 wap 5 wap 6 wap 7 wap 8 wap 9 wap 3 das 7 das 3 das 7 das 3 das 7 das 3 das 7 das 3 das 7 das 3 das 7 das c. procera 2.93±1.86 a 0.33±0.31 ab 0.80±0.60 ab 0.87±1.17 a 1.80±1.51 a 3.27±1.36 b 1.33±1.03 a 0.67±0.12 a 0.20±0.20 b 0.60±0.35 a 0.33±0.31 a 0.80±0.31 ab c. schweinfurthii 1.13±1.14 ab 0.07±0.12 c 0.27±0.23 c 0.07±0.12 b 0.37±0.35 b 1.13±0.70 a 0.27±0.35 ab 0.20±0.35 a 0.00 0.53±0.61 a 0.18±0.12 a 0.73±0.51 ab b. coccineus 1.40±1.22 ab 0.6±0.87 a 1.0±1.40 a 0.40±0.53 a 0.22±0.06 b 0.47±0.53 a 0.4±0.69 ab 0.80±1.22 a 0.82±1.05 a 0.6±0.53 a 0.8±1.22 a 0.93±0.52 a lambda cyhalothrin 0.13±0.23 c 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00a 0.00 table 3 toxicity effect some plant aqueous extracts in suppressing podagrica spp. infestation 2015 wap = weeks after planting; das = days after spray. treatments with the same letters in columns are not statistically significant different from each other. adv. hort. sci., 2018 32(1): 71-78 74 ments resulted in significant reduction in the insect population, while synthetic insecticides completely suppressed the insect pest infestation. the results clearly indicate that insect population reduced in relation to the number of spraying regimes, with the exception of okra plants sprayed with c. procera aqueous extract, which slightly increased or resurgence at 6 wap for both seasons and 3 days after spraying (das) at 7 wap in 2015 cropping season only. in 2015 nonsignificant difference was observed at 3 das and 7 das in all the treatments at 7, 8 and 9 wap, while in 2016 non-significant difference was recorded 7 das at 6, 8 and 9 wap in 2016 cropping season, respectively. the insecticidal activity of the aqueous plant extracts treatments is rated in the following decreasing order: b. coccineus > c. schweinfurthii > c. procera, with b. coccineus and c. schweinfurthii having similar treatment means which is not significantly different in most instances but significantly differ compared to c. procera in both seasons. toxicity effect of some plant aqueous extracts on okra yield attributes the yield of okra plant in response to various plant aqueous extracts evaluated in reducing podagrica spp. infestation was presented in table 5. from the results, fruit weight and fruit diameter were not significantly different in both cropping seasons for all t h e t r e a t m e n t s s p r a y e d o n t h e o k r a a g a i n s t podagrica spp. infestation. also non-significant difference equally observed between okra plants sprayed with b. coccineus and c. schweinfurthii compared to lambda cyhalothrin for number of harvested fruit and fruit lengths for both seasons except for c. schweinfurthii in 2015 season. although, there was significant difference in the number of harvested fruits and fruit lengths between okra sprayed with b . c o c c i n e u s , c . s c h w e i n f u r t h i i a n d l a m b d a cyhalothrin compared to c. procera in both seasons. the yield attribute performance of the treatments is rated in the following decreasing order: lambda cyhalothrin > b. coccineus > c. schweinfurthii > c. procera, with b. coccineus and c. schweinfurthii having similar treatment means. effects of plants aqueous extracts on severity of leaves damage on okra by podagrica spp. the utilization of plants aqueous extract on severity of leaves damage on okra (a. esculentus l.) by podagrica spp. was presented in table 6. significant table 5 toxicity effect some plant aqueous extracts okra yield attributes treatments 4 wap 5 wap 6 wap 7wap 8 wap 9 wap 3 das 7 das 3 das 7 das 3 das 7 das 3 das 7 das 3 das 7 das 3 das 7 das c. procera 2.05±0.62 a 0.73±0.31 a 0.98±0.56 a 0.53±1.04 a 1.18±1.51 a 1.02±0.16 a 0.88±1.06 a 0.63±0.14 b 0.42±0.20 b 0.6±0.35 a 0.55±0.24 a 0.56±0.31 a c. schweinfurthii 1.89±0.14 ab 0.28±1.52 b 0.67±0.23 a 0.18±0.32 a 0.37±0.35 ab 0.63±1.08 a 0.41±0.05 ab 0.4±0.33 a 0.20±0.07 b 0.53±0.61 a 0.17±0.16 b 0.41±1.61 a b. coccineus 1.44±0.21 ab 0.92±0.87 a 0.82±1.03 a 0.46±1.07 a 0.08±0.11 b 0.82±0.12 a 0.2±0.09 ab 0.87±0.22 a 1.0±0.35 a 0.6±0.53 a 0.46±1.42 a 0.57±0.25 a lambda cyhalothrin 0.10±0.23 c 0.22±1.61 b 0.00 0.00 0.00 0.00 0.00 0.00a 0.00 12:00 0.00 0.00 wap = weeks after planting; das = days after spray. treatments with the same letters in columns are not statistically significant different from each other. table 4 toxicity effect some plant aqueous extracts in suppressing podagrica spp. infestation 2016 parameters c. procera c. schweinfurthii b. coccineus lambda cyhalothrin 2015 2016 2015 2016 2015 2016 2015 2016 number of pod 3.75±1.13 b 3.98 ±0.19 b 4.69±1.59 a 4.51±1.79 a 4.75±1.79 a 4.93±2.41 a 5.12±0.84 a 5.45±1.79 a fruit length 3.02±0.88 ab 3.47±1.66 ab 3.93±2.71 ab 4.00±1.34 a 4.12±1.76 a 4.70±0.28 a 4.83±2.02 a 4.67±1.17 a fruit weight 0.08±0.06 a 0.05±0.02 a 0.05±0.03 a 0.09±0.11 a 0.05±0.06 a 0.07±0.12 a 0.04±0.03 a 0.06±0.02 a diameter 5.51±1.89 a 4.80±0.43 a 4.9±1.45 a 4.64±0.93 a 4.44±1.72 a 4.03±1.06 a 4.72±0.69 a 4.46±0.82 a severity of leaf damage c. procera c. schweinfurthii b. coccineus lambda cyhalothrin 2015 2016 2015 2016 2015 2016 2015 2016 4 wap 3.47±1.08 e 4.51±0.71 e 4.73±0.43 e 4.02±0.66 b 3.40 ±1.83 c 3.27±1.10 d 1.67±0.76 a 1.44 ±0.83 a 5 wap 6.27±0.73 e 8.12±2.46 e 5.82±1.18 e 6.83±0.37 b 4.00±1.95 c 4.17±1.38 d 3.20 ±1.51 a 4.27 ±0.73 a 6 wap 12.23±1.09 d 12. 85±2.03 d 9.50±1.76 d 8.91±1.92 b 10.53±1.68 b 11.48±2.06 c 4.07 ±0.69 a 4.23 ±1.09 a 7 wap 19.93±1.93 c 20.14±1.17 c 11.97±0.86 c 13.25±1.81 ab 16.27 ±2.68 ab 17.04±1.39 b 4.07 ±0.39 a 4.93 ±2.93 a 8 wap 29.60±1.55 b 26.92±2.41 b 15.53±1.78 b 17.09±2.01 a 20.67±2.61 a 19.85±2.14 ab 4.80 ±1.70 a 5.60 ±1.55 a 9 wap 32.20±0.66 a 33.32±1.82 a 21.67±2.90 a 20.14±1.26 a 23.07±1.32 a 24.55±2.82 a 5.20 ±1.30 a 6.33 ±1.75 a table 6 effects of plants aqueous extracts on severity of leaves damage on okra by podagrica spp. treatments with the same letters in columns are not statistically significant different from each other. treatments with the same letters in columns are not statistically significant different from each other. adesina and rajashekar plant aqueous extracts against podagrica spp. 75 difference was not recorded between okra plant sprayed with the extracts at 4, 5, 6, 8 and 9 wap. however, there was significant difference in the severity of leaves recorded at 7 wap. significant difference was also recorded between c. procera and chemical insect controlled okra but c. schweinfurthii and b. coccineus were not statistically significant from each other. the highest severity was recorded at 9 wap followed by 7 wap which could be due to lack of rainfall in these periods. severity of leaves damage by podagrica spp. is presented in table 6. the least percentage leaves defoliation was observed from the plants treated with synthetic insecticide and c. procera treated plant recorded highest percentage of defoliation among the botanical insecticides. c. schweinfurthii and b. coccineus showed significantly promising e f f e c t i n r e d u c i n g s e v e r i t y o f l e a v e s d a m a g e . however, all botanical treated okra plants prevented the leaves from being severely defoliated as all the plants suffered below 33.32% defoliation at 9 wap. in general term, leaves damage increased with the increasing age of okra plants. phytochemical screening the result of phytochemical screening of crude leaf extracts revealed the presence of triterpenoids, s t e r o i d s , f l a v o n o i d s , p h l o b a t a n i n s , s a p o n i n s , tannins, cardiac glycoside and anthraquinones in the plants (table 7). alkaloids and anthraquinones were not detected in both c. procera and c. schweinfurthii while triterpenoids and phlobatanins were absent in c. schweinfurthii. the presence of these bioactive compounds in the plant materials is an indication that it may have some insecticidal potential against agriculturally important insect pest. 4. discussion and conclusions plants are rich sources of natural substances that can be utilized in the development of environmentally safe methods for insect pests control as veritable alternative to synthetic insecticide (sadek, 2003). moreover, growing awareness of health and environmental issues accompanied by the intensive use of chemical inputs has led to increased concerns and the need for alternate forms of crop protection in the world (adesina, 2013). the crude aqueous extracts evaluated in this study have been reported to contain insecticidal, termiticidal, antifeedant, ovacidal and larvacidal properties (ahmed, 1993; shaaya et al., 1977; david, 1989; jahan et al., 1991; abayeh et al., 1999; abbassi et al., 2003; umsalama et al., 2006; bakavathiappan et al., 2012; katunku et al., 2014; nagawa et al., 2015; okoli et al., 2016). the result of the study showed that the crude plant extracts were able to reduce insect population, but not significantly, compared to the synthetic insecticide, but were able to show some suppressing effect on the rate of feeding of the insects as the severity of the leaves damaged by the insects was not enough to cause significant reduction in yield compared to results obtained from okra plants sprayed with chemical. the target insects are notorious for leaves defoliation owing to their biting and chewing mouthparts resulting in reduction of photosynthetic ability of the infested plants which invariably affect the plant growth and yield (dent, 1999). the aqueous extracts were not effective at first two weeks of application. synthetic insecticide recorded a higher efficacy compared to aqueous extract. this might be as a result of active ingredients of aqueous extracts being easily volatilized especially in the sun, thereby leading to their limited efficacy (ware, 2000) and the delayed effect of aqueous extracts is reported to be one of t h e m a j o r p r o b l e m s o f b o t a n i c a l i n s e c t i c i d e s (oparaeke, 2006; isman, 2008). since the beetles live and feed on the vegetative parts, any chemical that shows a remarkable efficacy against them must have contact toxicity, repellent or anti-feeding action. several authors have reported that, the deleterious effects of crude plant extracts on insects are manifested in several ways, including toxicity (hiremath et al., 1997) and feeding inhibition (klepzig and schlyter, 1999; wheeler and isman, 2001). thus, reducing the level of beetle infestation and increasing okra pod carrying capacity; this confirmed the earlier report of thirumalai et al. (2003)+ = present, = absent. table 7 phytochemical screening of crude aqueous extract of c. procera, c. schweinfurthii and b. coccineus chemical constituents calotrophus procera canarium schweinfurthii bryscocarpus coccineus alkaloids + terpenoids + + flavonoids + + + anthraquinones + tannins + + + phlobatanins + + saponins + + + cardiac glycosides + + + steroids + + + adv. hort. sci., 2018 32(1): 71-78 76 who observed the effective reduction of mite population with application of neem seed kernel extract. the reduction in podagrica beetle population and increase in yield caused by the plant extracts treatment was comparable with those caused by synthetic insecticide. this supports the findings of katunku et al. (2014) and those of yusuf and mohammed (2009) who reported that c. schweinfurthii and monordica balsamina powder treatments were as effective as and comparable to permethrin and pirimiphos methyl in suppressing c. maculatus population and growth in cowpea storage. the high rate of podagrica spp. population reduction on exposure to the aqueous extracts treatments may be attributed to the chemical composition of the products. plant extracts often consist of complex mixtures of bioactive constituents which may act as antifeedants, disturb insect growth, development and inhibit oviposition (gerard and ruf, 1991; emimal victoria, 2010). thus, suggesting that most of the secondary metabolites such as terpenoids and alkaloids can be used as active insecticidal compounds that could be an effective alternative to synthetic insecticides for insect pest management. the toxicity of alkaloids was reported by abbassi et al. (2003) and david (1989) reported that tannic acid acts as toxin and feeding deterrent to insects. bernhoft (2010) reported that saponnins is found to affect the respiratory system of insects and causes emetic effect due to their detergent action. in the same vein, philip et al. (2009) reported that plant products have repellent properties and toxic effect on the heart muscles in insects. frazier (1986) reported that antifeedants can be found amongst all major c l a s s e s o f s e c o n d a r y m e t a b o l i t e s ( a l k a l o i d s , flavonoids, terpenoids and phenolics). plant metabolites may produce toxic effects if ingested leading to rejection of the host plant (russel and lane, 1993). the utilization of c. schweinfurthii and b. coccineus aqueous extracts had positive toxicity impact in decreasing the rate of podagrica spp. infestation and also increased the yield of okra plant thus could serve as alternative to synthetic insecticide to protect okra plant against podagrica spp. infestation to maximize the yield and income of the resource poor farmers. acknowledgements the authors are grateful to omowaye, iyanuoluwa daniel and umaru, muritala oluwateru of horticultural technology programme, department of crop, soil and pest management technology, rufus 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universidade federal do oeste da bahia, campus multidisciplinar da barra, 41100-000, barra, ba, brazil. 2 universidade federal de viçosa, departamento de biologia vegetal, viçosa, mg, 36570-900, brazil. 3 universidade federal de viçosa, departamento de fitotecnia, viçosa, mg, 36570-900, brazil. 4 universidade federal de viçosa, departamento de estatística, viçosa, mg, 36570-900, brazil. key words: carbohydrates, chlorophyll, cooling curve, lactuca sativa l., relative water content, weight loss. abstract: pre cooling is applied to remove the field heat of harvested horticultural produces. the goal of this work was to determine the cooling curve and the effects of hydro cooling on quality and shelf life of iceberg lettuce ‘lucy brown’ stored at 5°c and 22°c. through shelf life, it was determined the changes on accumulated fresh weight loss, leaf relative water content and total chlorophyll, total soluble sugars, reducing and non-reducing sugars, and starch. the field heat from iceberg lettuce heads was removed within the first 10 min when submerged into cooled water at 4°c. hydro cooled lettuce heads accumulated water over the leaf surfaces resulting in higher rate of fresh weight loss during storage when compared to control. lettuce stored at 5°c kept higher relative water content in the leaves throughout the shelf life. hydro cooling treatment delayed the wilting of the external leaves in three and two days when stored at 5 and 22°c, respectively. hydro cooling did not influence the decrease on total soluble sugars, reducing sugars, non-reducing sugars and starch throughout shelf life, but affected the leaf chlorophyll content. independent of the temperature in which the ‘lucy brown’ iceberg lettuce will be stored, hydro cooling is recommended to prolong quality and shelf life. 1. introduction senescence is a natural process common to all fresh vegetables, which is intensified after harvest, by handling and storage conditions. in addition, the rate of deterioration is quickly intensified if a vegetable or a fruit is stored under stressed conditions. storing fresh horticultural products under extremes of high temperature or under low relative humidity, results in intense water loss, triggers senescence and finally the death of the tissues. (*) corresponding author: ffinger@ufv.br citation: frança c.f.m., santos m.n.s., ribeiro w.s., cecon p.r., finger f.l., 2018 shelf life of iceberg lettuce affected by hydro cooling and temperature of storage. adv. hort. sci., 32(3): 319-324 copyright: © 2018 frança c.f.m., santos m.n.s., ribeiro w.s., cecon p.r., finger f.l. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 1 november 2017 accepted for publication 12 january 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(3): 319-324 320 the major important factor affecting the postharvest shelf life for most of fresh horticultural products is the temperature during storage or display. to preserve the quality and prolong the perishables commercial quality, it is necessary to rapidly remove the field heat to an optimum temperature for subsequent storage (brosnan and sun, 2001). independent of the pre cooling method to be used, the premises as an useful postharvest practice, is based on the quick reduction of product temperature. in addition, it is recommendable to reduce the temperature as soon as possible after harvest, which will increase the beneficial effects of the rapid cooling. among the many benefits of pre cooling, in keeping the quality of a produce, is the lower respiration rate, the reduction of water loss by the product, and less contamination by pathogenic microorganisms (brosnan and sun, 2001). the rapid loss of quality and limited shelf life of leafy vegetables, like lettuce, parsley and jute leaves is mainly due to their fast postharvest dehydration (tulio jr. et al., 2002; finger et al., 2008; aguero et al., 2011). in these products wilting of leaves occur even faster when the storage is done under high storage temperatures or without any refrigeration and inadequate packaging. there is a variety of pre cooling techniques available including cooling rooms, hydro cooling systems, air forced cooling, ice packaging, vacuum and cryogenic cooling (brosnan and sun, 2001). hydro cooling is relatively inexpensive and very effective method recommended to remove the field heat of several leafy vegetables including kale, green onions and spinach (sargent et al., 2007). álvares et al. (2007) determined that hydro cooled parsley leaves had less water loss, resulting in longer shelf life without the appearance of wilting symptoms compared to control. hydro cooling also proved to be a faster method for cooling peach pulp to 1°c compared to forced air and conventional cooling room methods (brackmann et al., 2009). the expansion of large cities in developing are pushing the vegetable farms farther away from the markets, making harder for them to deliver products with good quality to urban population. this situation demands the incorporation of appropriated postharvest handling, but most of the small farmers have no capital to purchase refrigeration systems. iceberg lettuce is the most popular leaf vegetable used in burgers, sandwiches and salads by fast food stores in most of the countries, including brazil. thus, there is the need to evaluate the influence of pre cooling on the shelf life of this lettuce. therefore, the objective of this work was to determine the cooling curve and the effects of hydro cooling on quality and shelf life of iceberg lettuce heads stored under cold and room temperature conditions. 2. materials and methods heads of iceberg lettuce ‘lucy brown’ were harvested from the field at federal university of viçosa (642 m asl, 20°45’ lat. s and 42°51’ long. w) in the morning between 7 and 7:30 hours. the heads of lettuce were taken to the laboratory quickly and the heads with external leaves with brown spots, wilted, or dirty leaves were discarded. the lettuce heads weighting between 300 to 400 g were subjected to the following treatments: 1) hydro cooling followed by storage at 5°c; 2) control without hydro cooling and storage at 5°c; 3) hydro cooling followed by storage at 22°c; 4) control without hydro cooling and storage at 22°c. hydro cooling was performed by submerging the heads in a mixture of tap water and crushed ice at proportion of 3:1 (v:v) kept at 4oc. temperature of lettuce heads was determined at every five minutes with the help of a digital infrared thermometer. the temperature of the heads before initiating the hydro cooling treatment was between 20 to 22°c. at every 5 minutes, two heads were quickly removed from the cold water to determine the changes in the temperature, repeating the procedure up to fifty minutes. at the end of hydro cooling, the heads were removed from the cold water and allow draining for 5 min in the air before storage in the plastic boxes. hydro cooled and control lettuces were kept in plastic boxes at 5 and 22°c for the whole experiment. the boxes (18 cm height, 25 cm wide × 48 cm length) were covered with perforated (12 holes 1.1 cm in diameter) low density polyethylene plastic sheets to protect from excessive dehydration. the relative humidity inside the boxes was always between 85 and 90%. loss of fresh weight of heads, leaves relative water content, chlorophyll, total soluble sugar, reducing sugar, non-reducing sugar and starch leaf contents were determined at every 12 h up to the first 48 h and then at every 24 h until the end of the lettuce shelf life. the end of shelf life was determined when the heads were wilted, yellowed or with signs of deteriofrança et al.storability of lettuce iceberg 321 ration, being unfit for commercialization. the wilting, yellowing or deterioration of 50% or more heads was used as the discard parameter. t h e a c c u m u l a t e d l o s s o f f r e s h w e i g h t w a s obtained in relation to initial fresh mass of heads and during storage period. the leaf relative water content (rwc) was determined as described previously by álvares et al. (2007) with modifications. fifteen leaf discs with 1.1 cm in diameter were removed from the external, middle and internal position in the lettuce head, which were kept between two layers of wet sponge sheets until to obtain the leaf turgid fresh weight and then, they were oven dried at 70°c to obtain the total dry mass. the fresh weight of the disc, the turgid weight and the dry weight were used to estimate the rwc according to the formula established by barr and weatherley (1962). total chlorophyll content was determined in a combine sample of leaf discs removed from the external, middle and internal position in the head, following the method described by inskeep and bloom (1985) using 5,5-dimethylformamide as extractor. the absorbance of the filtrate was determined in a spectrophotometer at 647 and 664.5 nm and the results expressed in µg cm-2. samples of five grams of leaves from the external, middle and internal position of the lettuce head were homogenized in 80% hot ethanol and centrifuged at 2000 rpm for 15 min. the pellet was then re-extracted twice with 80% ethanol, and the total soluble sugars were determined by the phenol-sulfuric acid reaction (dubois et al., 1956). from the same extract was determined the reducing sugars content by the somogyi-nelson method (nelson, 1944). for total soluble sugars analysis sucrose was used as standard a n d g l u c o s e f o r t h e r e d u c i n g s u g a r a n a l y s i s . afterwards, the pellet from the ethanolic extraction w a s d r i e d a t 6 5 ° c a n d t h e n t h e s t a r c h w a s hydrolyzed in 52% perchloric acid for 30 min with shaking (mccready et al., 1950). the procedure was repeated three more times. the quantification of starch was performed by the phenol-sulfuric acid reaction using sucrose as standard using the correction factor of 0.9. the total of non-reducing sugars was obtained by the difference between the total soluble sugars minus the content of reducing sugars. the experiment was conducted in a split-plot scheme, with the treatments in the plots and shelf life in the subplots in a randomized block design with four treatments and four replicates per treatment. each replicate was composed by one lettuce head. individual analysis of variance was performed to evaluate the effect of the hydro cooling and temperature of storage by using the saeg/ufv software, and the mean separation was done by scott knott test at 5% probability. the regression analysis was based on the regression coefficient using the t-test at 5% or 10% probability to establish the significance for the chosen regression model. 3. results and discussion initial temperature of the lettuce head showed a sharp drop within the first 10 min of hydro cooling time. the model that better explained the changes in temperature was exponential, with an estimated final temperature of 4.8°c after 10 min of hydro cooling (fig. 1). longer periods of cooling time did not remove additional field heat from the lettuce head. based on the lettuce temperature record data, the total amount of heat removed from the lettuce, by the cold water under this experiment conditions, corresponded to 71% from the initial temperature (fig. 1). the 87.5% or seven eighths cooling times recognized as the ideal theoretical reduction for the field heat presented by brosnan and sun (2001) was not achieved in this experiment, even after keeping the heads submerged in the cold water mixture for 50 min (fig. 1). using the same cooling technique of this experiment, álvares et al. (2007) reported the removal of only 43% of the initial temperature in bunched parsley leaves. the reason why the hydro cooling of lettuce was much more efficient in removing the field heat than parsley remains to be the subject of further studies. furthermore, the cooling time varied according to varieties of lettuce, as found by frança et al. (2015) working with butter lettuce where the ideal hydro cooling time was 5 min instead fig. 1 influence of hydro cooling time period treatment on the temperature of iceberg lettuce 'lucy brown'. adv. hort. sci., 2018 32(3): 319-324 322 of 10 min found for iceberg lettuce in this work (fig. 1). the difference of hydro cooling time between the two cultivars of lettuce may relay on the thickness and compactness of the whole head. the leaves from iceberg lettuce are thicker and more compact head than butter lettuce, which can restrict the access of cold water into the more internal leaves of the head. in others leaf vegetables, including peppermint, coriander and basil, the ideal time of hydro cooling was 20, 10 and 16 min, respectively (oliveira et al., 2015; barbosa et al., 2016; teixeira et al., 2016). the end of shelf was established when symptoms of wilting and discoloration appeared in the external leaves (data not shown). shelf life of hydro cooled lettuce and stored at 5°c was increased by 75% compared to not cooled lettuce, comprising a total of 168 h for the hydro cooled and 96 h for not cooled heads. for the lettuce that was hydro cooled and then stored at 22°c, the gain of shelf life was 50% or 72 h for hydro cooled and 48 h for not cooled lettuce. álvares et al. (2007) also found beneficial effects to the shelf life of hydro cooled bunched parsley leaves followed by cold storage. the result of this study shows the importance of keeping the cold chain for fresh vegetables, but also shows the contribution of hydro cooling on extending the shelf life even without further cold storage. because of the hydro cooling positive effects, the external leaves of the lettuce had a 72 h delay in showing wilt symptoms if stored at 5°c and 24 h delay for the lettuce stored at 22°c (data not shown). the increased shelf life of hydro cooled lettuce was determined by the higher leaf relative water content of hydro cooled lettuce (table 1). the smaller effect of hydro cooling in the lettuce kept at 22°c compared to 5°c may be due to the greater gradient of water vapor between the leaf surface and the atmosphere of storage at 22°c. but, in a similar experiment with butter lettuce, the beneficial effect of hydro cooling on shelf life was greater for the lettuce stored at 22°c compared to the shelf life of hydro cooled heads followed by storage at 5°c (frança et al., 2015). regardless the treatment, the rate of fresh weight loss was constant, resulting in linear accumulation up to the end of the lettuce shelf life (fig. 2). the lowest rate of weight loss was determined in the lettuce stored at 5°c without hydro cooling (0.109% h-1) and the highest for the heads hydro cooled and stored at 22°c (0.26% h-1), as previously observed in a similar experiment with butter lettuce by frança et al. (2015). the higher weight loss rate of hydro cooled lettuce was due to the water accumulated at surface and in between the leaves after being removed from the cooled water. regardless if the lettuce was hydro cooled or not, the lower rates of weight loss found for heads stored at 5°c was determined by the smaller gradient of water vapor compared to the storage room at 22°c (wills et al., 2010). like in this experiment, when coriander leaves were stored at 5°c also had lower rates of weight loss compared to leaves stored at 20°c, regardless if the leaves were submitted to hydro cooling treatment (oliveira et al., 2015). during the whole period of storage, the leaves of hydro cooled lettuce had higher relative water content when stored at 5°c (table 1). this higher content of water found in the hydro cooled lettuce leaves was clearly observed in the appearance of lettuces, which were more turgid than those not hydro cooled, which resulted in longer shelf life due to fresher appearance. similar results were found for hydro cooled butter lettuce heads, peppermint and coriander leaves, which also had higher relative fig. 2 accumulated fresh weight loss of iceberg lettuce 'lucy brown' submitted to the following treatments: t1) hydro cooling for 10 min + storage at 5°c; t2) control without hydro cooling + storage at 5°c; t3) hydro cooling for 10 min + storage at 22°c; t4) control without hydro cooling + storage at 22°c. te= time. table 1 influence of the hydro cooling and temperature of storage on the leaf relative water content during storage period of iceberg lettuce heads 'lucy brown' means followed by the same letter do not differ by the scott knott test at 5% probability. treatments rwc (%) hydrocooled + storage at 5°c 96.4 a storage at 5°c 92.8 b hydrocooled + storage at 22°c 94.0 b storage at 22°c 92.4 b cv % 3.2 frança et al.storability of lettuce iceberg 323 water content during their shelf life (frança et al., 2015; oliveira et al., 2015; barbosa et al., 2016). in conclusion, the hydro cooling treatment and storage 5°c resulted in higher level of water compared to not cooled heads, indicating the importance of field heat removal and followed by continuous cold chain. although hydro cooling reduced significantly the leaf chlorophyll content throughout storage for the lettuce stored at 5°c compared to the remaining treatments (table 2), and the coefficient of variation was high (35.7%). this reflects the positions internal, middle and external which the leaf samples were taken. because the relative water content present in the leaves of the hydro cooled lettuce stored at 5°c was higher, a much more favorable water status existed during storage. and at the same time, for the other treatments, the lower relative water content reflects a bigger dehydration rate of the cells, which resulted in higher chlorophyll concentration in the leaves (table 1). but, the same effect on chlorophyll content induced by hydro cooling was not present on parsley and ora-pro-nobis leaves (álvares et al., 2007; barbosa et al., 2015). these differences may be related to the lower trend of parsley and ora-pro-nobis leaves in loosing water from the cell to the environment during storage. hydro cooling had no effect on leaf carbohydrate changes during storage. however, there was significant decrease in the total soluble, reducing, nonreducing sugars and starch contents in the first 12 h of storage, either at 5 or 22°c (table 3). in the first few hours after harvest, a much greater amount of carbohydrate is required to maintain high respiratory demand, coinciding with the highest physiological activity (wills et al., 2010). in the study, during the first 48 h of storage there was a drop of 37.2, 24.5, 52.1 and 23.7% in the total soluble sugars, reducing, non reducing sugars and starch, respectively (table 3). in a similar work, frança et al. (2015) found similar decreases on non-reducing sugars and starch content of butter lettuce on the first 12 hours, but not on reducing sugars. the reduction of all carbohydrates found in this work, reflects the high demand of glucose and fructose to keep the respiratory activity even at low temperature of 5°c. since, leafy vegetables do not store large amounts of carbohydrates; their storage potential for longer shelf life is much smaller than tubers and fruits, which have large amount of stored carbohydrates. thus, further work with the use of controlled and modified atmosphere should be applied to increase iceberg lettuce shelf life. 4. conclusions regardless the temperature of storage, application of hydro cooling treatment removed most of the field heat with beneficial effects on quality, prolonging the shelf life of ‘lucy brown’ iceberg lettuce, by keeping higher water status in the cells and reducing discolorations in the leaves. hydro cooling had no influence on carbohydrate metabolism of the leaves throughout storage either 5 or 22°c. acknowledgements to cnpq, fapemig and capes for their financial support. references aguero m.v., ponce a.g., moreira m.r., roura s.i., 2011 lettuce quality loss under conditions that favor the wilting phenomenon. postharvest biol. technol., table 2 influence of the hydro cooling and temperature of storage on the total chlorophyll content during storage period of iceberg lettuce ‘lucy brown’ means followed by the same letter do not differ by the scott knott test at 5% probability. treatments total chlorophyll (µg cm-2) hydrocooled + storage at 5°c 6.00 b storage at 5°c 7.74 a hydrocooled + storage at 22°c 7.69 a storage at 22°c 7.81 a cv (%) 35.7 table 3 general mean values of total soluble sugars (tss), reducing sugars (rs), non-reducing sugars (nrs) and starch contents in 'lucy brown' icerberg lettuce stored at 5 or 22°c during the first 48 h of shelf life storage means followed by the same letter do not differ by the scott knott test at 5% probability. time (h) tss* rs * nrs * starch ** 0 2.72 a 1.73 a 1.25 a 3.36 a 12 1.84 b 1.12 b 0.71 b 2.65 b 24 1.82 b 1.21 b 0.60 b 2.55 b 36 1.62 b 1.06 b 0.55 b 2.32 b 48 1.71 b 1.11 b 0.59 b 2.57 b cv (%) 24.79 25.71 57.92 19.33 324 adv. hort. sci., 2018 32(3): 319-324 59: 124-131. álvares v.s., finger f.l., santos r.c.a., negreiros j.r.s., casali v.w.d., 2007 effect of pre-cooling on the postharvest of parsley leaves. j. food, agric. environ., 5(2): 31-34. barbosa c.k.r., finger f.l, casali v.w.d., 2015 handling and postharvest shelf life of ora-pro-nobis leaves. acta scientiarum agronomy, 37(3): 307-311. barbosa c.k.r., fonseca m.c.m., silva t.p., finger f.l., casali v.w.d., cecon p.r., 2016 effect of hydrocooling, packing, and cold storage on the postharvest quality of peppermint (mentha piperita l.). rev. bras. plantas med.: 18(1): 248-255. barr h.d., weatherley p. e., 1962 a re-examination of the relative turgidity technique for estimating water deficit in 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483-485. mccready r.m., guggolz j., silveira v., owens h.h., 1950 determination of starch and amylose in vegetables. anal. chem., 22: 1156-1158. nelson n.a., 1944 photometric adaptation of somogyi method for determination of glucose. -j. biol. chem., 135: 136-175. oliveira l.s., silva t.p., ferreira a.p.s., pereira a.m., finger f.l., 2015 efeito do hidroresfriamento na conservação pós-colheita de coentro. hort. bras., 33(4): 448-452. sargent s.a., ritenour m.a., brecht j.k., bartz j.a., 2007 handling, cooling and sanitation techniques for maintaining postharvest quality. vegetable production handbook, document hs719, uf/ifas: 97-109. teixeira d.a., gomes j.a.o., bonfim f.g.g., pardo p.i., mayobre m.t., 2016 técnicas de conservação póscolheita para o manjericão. rev. bras. plantas med., 18( 1): 168-171. tulio jr. a.z., ose k., chachin k., ueda y., 2002 effects of storage temperatures on the postharvest quality of jute leaves (coschorus olitorius l.). postharvest biol. technol., 26: 329-338. wills r., graham d., mcglasson b., joyce d., 2010 postharvest: an introduction to the physiology and handling of fruit, vegetables and ornamentals. 4th ed. cabi, new york, pp. 280. impaginato 157 adv. hort. sci., 2017 31(3): 157-163 doi: 10.13128/ahs-21954 salicylic acid improves salinityalkalinity tolerance in pepper (capsicum annuum l.) a.-a. amirinejad 1, m. sayyari 2, f. ghanbari 3 (*), s. kordi 3 1 soil science department, faculty of agriculture, razi university, kermanshah, iran. 2 horticultural science department, faculty of agriculture, bu-ali sina university, hamedan, iran. 3 young researchers and elite club, khoram-abad branch, islamic azad university, khoram-abad, iran. key words: alkali stress, pepper, salicylic acid, salt stress. abstract: salinization and alkalization of soils are agricultural problems in arid and semiarid regions of the world such as iran. in this experiment the effects of salicylic acid (sa) on resistance of pepper plants under salt stress (ss) and alkali stress (as) were evaluated. treatments include 0 and 150 mm of ss, 0, 50 and 100 mm of as and 0, 0.75 and 1.5 mm sa. results showed that ss and as imposed negative effects on pepper plant growth and productivity. reduction in growth and yield in ss was higher than as and maximum reduction occurred in high mixed stresses. sa application improved growth parameters and increased yield, relative water content (rwc) and chlorophyll of plants subjected to ss and as and provided significant protection against stress compared to non-sa-treated plants. for most traits, 0.75 mm of sa was more effective than 1.5 mm concentration. sa ameliorated the injury caused by ss and as by increasing chlorophyll and rwc and inhibiting proline accumulation and leaf electrolyte leakage (el). in general, results indicate that salinity and alkalinity have negative effects on growth and yield of pepper plants and these negative effects can be ameliorated by application of sa. 1. introduction salinity and alkalinity of soil seriously affect about 932 million hectares of land globally, reducing productivity in about 100 million hectares in asia (rao et al., 2008). in iran 12.5% of the agricultural lands in arid and semi-arid areas are alkaline. while salt stress (ss) in a soil generally involves drought stress and ion-induced injury (munns, 2002), alkali stress (as) exerts the same salt stress influences with the added effects of highph stress (shi and yin, 1993). the high-ph caused by as directly affects the mineral absorption and interferes with the re-establishment of ionic balance. it can strongly affect the absorption of inorganic anions such as (*) corresponding author: f.ghanbari92@basu.ac.ir citation: amirinejad a.-a., sayyari m., ghanbari f., kordi s., 2017 salycilic acid improves salinityalkalinity tolerance in pepper (capsicum annuum l.). adv. hort. sci., 31(3): 157-163. copyright: © 2017 amirinejad a.-a., sayyari m., ghanbari f., kordi s. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 15 septmeber 2016 accepted for publication 6 april 2017 ahs advances in horticultural science adv. hort. sci., 2017 31(3): 157-163 158 cl–, no3 – and h2po4 –, and can disrupt the selective absorption of k+-na+, and ph homeostasis in the plant tissues (yang et al., 2007). to resist as, plant not only have to regulate the intercellular ph to maintain ionic balance, but also have to spend material and energy to regulate ph in their root environment. thus osmo-regulation and ion balance play key influence in plants ss and as resistance (yang et al., 2007). there has been considerable study of ss on plants growth and development, however, relatively little attention has been given to as despite its importance. like to other environmental adverse conditions, as can lead to emerge reactive oxygen species (ros), as a result of closing stomata and reducing co2 into cells and consequently blocking photosynthesis activities (yan et al., 2011). as caused metabolic disturbance, lipid peroxidation and chlorophyll breakdown and proline accumulation in plant tissue (gao et al., 2012). various techniques have been tested to improve saline-alkali soils, including chemistry, physics, biology and engineering improvements to increase soil fertility and crop yield (li-ping et al., 2015). in recent studies a number of plant growth regulators (pgrs) have been under trial to alleviate the environmental stresses in plants. salicylic acid (sa) is a phenolic compound which is considered as a pgr and plays an important role in defensive mechanisms against biotic and abiotic stresses in plants. flowering induction, plant growth and development, synthesis of ethylene, opening and closure of stomata and respiration are some of the important roles of sa in plants (raskin, 1992). sa protects plants from damages caused by oxidative stresses through increasing antioxidants enzymes activities (el-tayeb, 2005; idrees et al., 2011). sa has received much attention due to its function in plants’ responses to environmental stresses. literature exists about some beneficial effects of sa on plants under drought (jafari et al., 2015), low temperature, high temperature (wang and li, 2006; sayyari, 2012), salinity (shakirova et al., 2003; el-tayeb, 2005; stevens et al., 2006; idrees et al., 2011), heavy metal (metwally et al., 2003) and biotic stresses (makandar et al., 2012). however, no information exists on the effects of the mentioned compounds on salt-alkali stress defense mechanisms, until now. thus the purpose of this experiment was to examine the possibility that application of sa would protect pepper plants from damaging effects of salt-alkali stresses. specific objectives of this research were: (1) to compare ss and as on pepper plant growth and development (2), to determine some physiological responses of pepper plant to ss and as and (3) evaluate the protective effect of a pre-treatment with sa. 2. materials and methods plant material and growing conditions seeds of capsicum annuum l. cv. plenty were obtained from pakan bazr co. (isfahan, iran) and cultured in bed to obtain seedlings for experiment. when the seedlings had 2-4 true leaves, the seedling with similar size were selected and transferred into plastic pots (20 cm height, and 23 cm diameter) which were filled with about 8 kg of 1:1:2 mixture of fine sand, leaf mound and garden soil. the pots were then transferred to the greenhouse with av erage temperature of 25.5/19.5°c (day/night) and natural light. salt stress, alkali stress and salicylic acid treatments when the seedlings have been established in pots (4-6 true leaves), they were sprayed with 0 (as control), 0.75 and 1.5 mm sa until both sides of the leaves were completely wet. three days later, plants were subjected to ss and as treatments until end of e a c h e x p e r i m e n t . t w o n e u t r a l s a l t s ( n a c l a n d na2so4) and two alkaline salts (nahco3 and na2co3) were selected for salinity and alkalinity stresses imposition. the two neutral salts were mixed in a 9:1 molar ratio (nacl:na2so4), and applied in 0 mm and 150 mm to the ss group, and two alkaline salts were also mixed in a 9:1 molar ratio (nahco3:na2co3) in 0 mm, 50 mm and 100 mm, and applied to the as group. ss and as treatment were conducted with daily watering of plants by mentioned treatment. control plants (0 mm ss and 0 mm as) were watered with distilled water (yang et al., 2007; rao et al., 2008). chlorophyll determination chlorophyll was assessed by taking fresh leaf samples (0.1 g) of plants in each replicate from young and fully-developed leaves. the samples were homogenized with 5 ml of ac etone (80% v/v) using pestle and mortar and centrifuged at 3000 rpm. the absorbance was measured with a uv/visible spectrophotometer at 663 and 645 nm and chlo rophyll were calculated (mg/g fw) using the equations pro posed by strain and svec (1966) given below: total chlorophyll = 20.2×(a645)+8.02(a663). amirinejad et al. salicylic acid improves salinity-alkalinity tolerance in pepper (capsicum annuum l.) 159 electrolyte leakage electrolyte leakage was determined according to lutts et al. (1995) method. ten leaf discs of randomly chosen plant were taken from the youngest fullyexpanded leaf. the leaf discs were then placed in test tubes containing 10 ml of distilled water. these samples were incubated at 25°c on a shaker for 24 h. electrical conductivity (ec) of bathing solution (ec1) was read after incubation. the same samples were then placed into a water bath (100°c) for 20 min and the second reading (ec2) was determined after cooling of the solution to room temperature. the el was calculated as ec1/ec2 and expressed as a percentage. proline content determination the proline was determined according to the method described by bates et al. (1973). the amount of 0.5 g of samples were homogenized in 10 ml of 3% (w/v) sulfosalicylic acid. after centrifuging at 10000 rpm, 2 ml of the supernatant was mixed with 2 ml of acid ninhydrin and 2 ml of glacial acetic acid in a test tube. the mixture was placed in a water bath (100°c) for 1 h. the reaction mixture was extracted with toluene and the chromophore-containing toluene was aspirated and cooled to 25°c. the absorbance was measured at 520 nm with a uv/visible spectrophotometer. proline concentration was expressed as μg/g fw. measurement of relative water content relative water content was measured according to costes et al. (2006). leaves sample in each treatment was weighed (fw) and then immediately floated on distilled water for 5 h in the dark. turgid weight (tw) of leaf disks was obtained after drying excess surface water with paper towels. dry weight (dw) of disks was measured after drying at 75°c for 48 h. the rwc was calculated using the following formula: relative water content = (fw-dw)/(tw-dw)×100 statistical analysis data were analyzed for significant differences using a factorial analysis of variance with ss and as levels and sa concentrations as main factors with three replications and 5 seedlings per each. statistical analysis was performed using sas program and the means compared using the duncan’s multiple range test at p=0.05. 3. results and discussion leaf area and total yield the leaf area and pepper yield displayed a significant reduction in response to the increasing levels of ss and as treatments. among all stress treatments tested, 100 mm as+ss exhibited the strongest reduction of leaf area and yield followed by 50 mm as+ss. f o l i a r s p r a y e d s a p l a n t s e x h i b i t e d s i g n i f i c a n t response to improve yield as compared to unsprayed ones in all stress levels (fig. 1 and 2). the general effect of soil salinity in plants is to decrease the growth resulting in smaller leaves, shorter stature, and sometimes fewer leaves. the primary effect of salinity, especially at low to moderate levels, is due to its osmotic stress (munns, 2002). the as exerts the same stress factors as ss but with the added effect of high-ph stress. the effect of ss and as in reducing the growth and yield of various plants was reported in (yang et al., 2007; chen et al., 2011) studies that are in agreement with the findings of this study. however, in the present study, adverse effects of as on yield and leaf area were lower than that of ss. this results implies not only that ss and as are different stresses, but also that resistance of pepper plant to as is stronger that to ss. the treatment of sa caused a significant increasing in yield and leaf area in comparison to control plants at all stresses levels (fig. 1 and 2). the positive effect of sa on plants under salinity stress have been reported (shakirova et al., 2003; stevens et al., 2006; idrees et al., 2011) which their upshots are in agreefig. 1 effect of salicylic acid (sa) on leaf area of pepper plant under salt stress (ss) and alkali stress (as). fig. 2 effect of salicylic acid (sa) on total yield of pepper plant under salt stress (ss) and alkali stress (as). adv. hort. sci., 2017 31(3): 157-163 160 ment with our findings. application of sa improved wheat plants under water stress by activities of cell division in apical meristem (shakirova et al., 2003). exogenous sa treatment also improved chlorophyll and photosynthesis rate and created stability of plant cell membrane, reduction of el in barely plants and finally tolerance plants to water stress (el-tayeb, 2005). the ability of sa to increase yield and growth parameters, ameliorating the adverse effects of stress, may have important implications in improving the plant growth and overcoming the growth barrier arising from ss and as conditions. chlorophyll content some of the environmental stresses symptoms in the plants are reduction of chlorophyll content and this reduction depends upon the plant species (colom and vazzana, 2001). in this experiment, the reduction of chlorophyll was occurred due to ss and as and 100 mm ss+as treatment resulted in having the lowest rate of total chlorophyll (fig. 3). in this respect, there are same reports over reducing chlorophyll due to exposing plants to ss and as (chen et al., 2011; gao et al., 2012). environmental stresses leads to increase the ros production in cells. these free r a d i c a l s c a u s e p e r o x i d a t i o n a n d c o n s e q u e n t l y destructing the photosynthesis pigments (schütz and fangmeier, 2001). because of these events, the growth of plant will be affected and declined. treated plants with sa as foliar spray increased chlorophyll content in all concentrations, but 0.75 mm sa treated plants showed significant increases in chlorophyll compared with control and 1.5 mm sa (fig. 3). these results are in agreement with those of sayyari (2012) who found that sa foliar and soil applications increased chlorophyll content in cucumber plants following chilling stress. salicylic acid by eliminating of ros may improve chlorophyll content in pepper plants under stressful c o n d i t i o n s . c h e n e t a l . ( 1 9 9 3 ) s h o w e d t h a t i n response to environmental stresses sa accumulates to high concentrations, and prevent antioxidant enzyme such as cat activity, thereby leading to an enhancement in hydrogen peroxide (h2o2) content, which could then induct the development of systemic acquired resistance (sar), induce activity of rosdetoxifying enzymes and antioxidant metabolites. also, idrees et al. (2011) reported that sa-induced salinity tolerance in periwinkle plants might be associated with an increase in the antioxidant activity. therefore, impact of sa on plants chlorophyll under stresses condition may be related to its effect on the antioxidative enzyme activities and h2o2 metabolism (idrees et al., 2011). proline content proline is sensitive physiological index of plants responding to some stresses. salinity and alkalinity treatments had significant effects on proline contents in pepper plant leaves. proline accumulation in leaves was markedly increased in salt and alkali stressed plants in comparison with that of the control plants. the results showed that mixed ss-as can cause heavy accumulation of proline content compared with ss and as single stresses (fig. 4). in general, the accumulation of proline, relates closely with osmotic stress intensity. osmo-regulation is a physiological phenomenon during which osmosis potential of stressed tissues are reduced due to the accumulation of some material such as elements, sugar, amino acids (proline) and organic acids. thus, turgor pressure of the cells is kept well (irigoyen et al., 1992). proline by osmosis control, maintaining enzymes activity and removal of hydroxyl radicals, increases t h e t o l e r a n c e o f t h e p l a n t s a g a i n s t s t r e s s e s (kuznetsov and shevyakova 1999). the results showed that proline accumulation increased not only with increasing ss, but also when as imposed (fig. 4). this suggested that the proline accumulation, as the major osmolyte, correlates closely with the intensity of the osmotic stress induced by ss and as. fig. 3 effect of salicylic acid (sa) on chlorophyll of pepper plant under salt stress (ss) and alkali stress (as). fig. 4 effect of salicylic acid (sa) on proline content of pepper plant under salt stress (ss) and alkali stress (as). amirinejad et al. salicylic acid improves salinity-alkalinity tolerance in pepper (capsicum annuum l.) 161 results showed that sa application increased proline content in pepper plants under ss and as. a highest amount of proline (44.67 μm/gfw) was achieved in 1.5 mm sa treatment and 100 mm as+ss, and lowest amounts (9.54 μm/gfw) were observed in 0.75 mm sa and 0 mm as (fig. 4). these results are in agreement with those of el-tayeb (2005) who showed that sa treatment increases the proline content in the leaves of barely plant subjected to salinity stress. in the present study, sa induced an accumulation of proline in the leaves under ss and as, and when sa was applied, a stress tolerance occurred in the pepper plant. thus, osmo-regulation can be considered to be one of the important phenomenon involved in sa induced protective mechanism in pepper leaves in response to salt and alkali stresses. relative water content one of the indices showed the water status of plants is rwc. measuring of relative water content is an important physiological parameter in evaluation of plant response to environmental stresses (nautiyal et al., 2002). results indicated that by increasing ss and as, rwc in pepper leaves decreased slightly. reduction under ss was greater than those under as and greatest rwc reduction was achieved in highest mixed stress level (100 mm as+ss) (fig. 5). these results are in agreement with yang et al. (2007) who found that salt-alkali stresses decreases the rwc in the leaves of kochia sieversiana. plants can reduce rwc as a quick and economical approach to osmotic adjustment in response to osmotic stress (lissner et al., 1999). reduction of rwc of the plants due to stress is related to the reduction of soil humidity; in these conditions, plants close the stomata to avoid more water waste. the reason of stomata closure is aba that is made in the root in stress conditions and is accumulated in stomata cells (chaves et al., 2002). therefore, maintaining a high rwc might be a key characteristic of plants that allow it to absorption osmolytes with minimum energy consumption. results showed that sa treatment increased leaf rwc in pepper plants under ss and as. a highest amount of rwc (89%) was achieved in 1.5 mm sa treatment and 0 mm as, and lowest (54%) were observed in 0 mm sa and 100 mm as+ss (fig. 5). parida and das (2005) reported that the rwc, water potential and osmotic potential of plants under stress become more negative with an increase in salinity. this study showed that sa treatments induced an increase in rwc of plants as compared to the un-treated sa plant. increases in rwc of plants treated with sa were also reported for other crops grown under stress including tomato (stevens et al., 2006) and barely (el-tayeb, 2005). increasing of rwc may be related to the role of sa in accumulation of compatible osmolytes in plants subjected to stress, as, this effect was observed in the results of proline. electrolyte leakage in order to assess membrane permeability, electrolyte leakage (el) was determined. its relative conductivity can be used to evaluate the damage on structure and function of cell membranes under stresses. results showed that el significantly higher under ss than as. on the other hand, both stresses increased the el but the extent of the increment under ss was much greater than under as and maximum of el was achieved in highest mixed stress level (100 mm as+ss) (fig. 6). the results of the present study are in agreement with gao et al. (2012) who determined that el of oat (avena sativa l.) was intensively increased by as. gao et al. (2012) showed that the el of alfalfa seeds gradually increased with increasing salinity and alkalinity, this was attributed to the damage seeds cell membranes resulting from mixed salt-alkali stress. these results suggested cell membrane structure of pepper leaves under ss and as received damage after treatment with salts. application of sa significantly decreased leaf el in salt and salt × alkali stressed plants. plants treated by sa foliar spray at 0.75 and 1.5 mm had shown significantly less el than control plants (fig. 6). these results indicated that, sa reversed the adverse effects of stress and caused a significant decrease in el. the results of the present study are in agreement with stevens et al. (2006) who showed that sa facilitated the maintenance of membrane functions in tomato under salinity stress. this effect could be attributed to the stimulation of antioxidant responses and elevated calcium absorption that protects thefig. 5 effect of salicylic acid (sa) on relative water content of pepper plant under salt stress (ss) and alkali stress (as). adv. hort. sci., 2017 31(2): 157-163 162 plant from the oxi dative damage (el-tayeb, 2005). also, jafari et al. (2015) showed that exogenous application of sa in cucumber (cucumis sativus l.) subjected to osmotic stress lead to a decrease in el and induced drought tolerance. these results suggested that pepper leaves cell membrane structure in during salt and alkali stresses received less damage after application of sa. 4. conclusions in summary, our study clearly showed that saline and alkaline stresses as two types of abiotic stresses, have negative effects on pepper plant growth and productivity. on the other hand, the negative effect of mixed salt-alkali stress is more severe than that of only salt or alkali. the results of this research demonstrated that treating plants with 0 mm as (control) is resulted in increasing plant growth parameters and rwc in comparison to other treatments. the harmful effect of saline stress on the pepper plants was significantly greater than that of alkaline; this harmful effect might have resulted from the higher concentration of salinity or higher sensitivity of pepper to ss c o m p a r e d t o a s . s a p r e t r e a t m e n t o f p e p p e r seedlings via foliar spray was effective in saline and alkaline resistance. in most of evaluated treatments employed in this research, they did not show significant differences on sa concentrations. the best protection was obtained in plants treated with 0.75 mm sa, however 1.5 mm sa is more effective in 100 mm as+ss in some trait for example proline content. this sa effect was associated with alter of physiological parameters such as increase of photosynthetic pigments and proline accumulation and decrease el of plants by addition of sa. further, the results indicate that sa can be considered as a potential growth regulator for improving plant growth and yield under ss and as, and it may be recommended in arid and semiarid regions. references bates l.s., waldren r.p., teare i.d., 1973 rapid determination of free proline for water-stress studies. plant and soil, 39(1): 205-207. chaves m.m., pereira 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balance traits of an alkali resistant halophyte kochia sieversiana during adaptation to salt and alkali conditions. plant and soil, 294(12): 263-276. impaginato 61 1. introduction oil pumpkins play a significant role in human nutrition and health. the nutritional value of oil pumpkin seeds is based on high protein and antioxidant content, and high energy potential due to the high percentage of oil (fruhwirth and hermetter, 2007; sari et al., 2008; fokou et al., 2009; lelley et al., 2009; urbanek krajnc et al., 2016). during the last decade, oil pumpkin cultivation declined regarding productivity and quality due to the outbreaks of the zucchini yellow mosaic virus (zymv), extremely high temperatures, radiation stress and prolonged periods of drought (lelley et al., 2009; seebold et al., 2009; gong et al., 2013). years 2013 and 2015 have been excessively hot and we have seen serious problems in fruiting pumpkins related to weather conditions especially high day/night temperatures and drought stress (yavuz et al., 2015). heat stress depends on intensity, duration, and rate of increase in temperature. the extent to which it occurs, in specific climatic zones, depends on duration and level of high temperatures occurring during the day and/or the night. in general, a transient elevation in temperature, 10-15°c above ambient, is considered as heat stress (wahid et al., 2007). it causes an array of morpho-anatomical, physiological and biochemical changes in plants, which affect plant growth and development and may lead to a drastic reduction in economic yield. on the morphological level, high temperature can cause considerable preand post-harvest damages such as scorching of leaves and stems, sunburns on leaves, stems and fruits, leaf senescence and wilting, shoot and root growth inhibition, fruit discoloration and damage, and reduced yield (wahid et al., 2007; ara et al., 2015; johnson, 2015). the physiological changes caused by high temperatures include the negative effect on photosynthesis, respiration, water relations, and modulated levels of hormones and primary and secondary metabolites. on the biochemical and subcellular level the direct injuries due to high temperatures include protein denaturation and aggregation, disorganization of cytoskeleton and increased fluidity of membrane lipids. indirect or slower heat injuries include inactivation of enzymes in chloroplast and mitochondria, inhibition of protein synthesis, protein degradation and loss of membrane integrity. these injuries eventually lead to starvation, inhibition of growth, reduced ion flux, production of toxic comadv. hort. sci., 2017 31(1): 61-73 doi: 10.13128/ahs-20727 the impact of fruit temperature dynamics on heat stress tolerance of selected oil pumpkin genotypes a. urbanek krajnc *, j. rakun, p. berk, a. ivančič faculty of agriculture and life sciences, university of maribor, pivola 10, hoče, slovenia. key words: breeding, cucurbita argyrosperma var. argyrosperma, cucurbita moschata, cucurbita okeechobeensis, cucurbita pepo, fruit temperatures. abstract: fruit temperature is a key parameter for fruit growth and quality which is affected by climate, plant vigorousity, solar exposure and fruit thermal properties. in the present study, the variability in temperature dynamics of styrian oil pumpkin fruits and selected interspecific hybrids involving cucurbita argyrosperma, c. moschata, c. pepo was analysed in two different periods of hot weather. the temperatures were measured with thermistors on (a) attached fruits, (b) detached fruits exposed to the sun and (c) artificially black coloured fruits. the highest average temperatures were determined in the styrian oil pumpkin, whereas the lowest temperatures were determined in genotypes with lighter fruit exteriors suggesting that those are less sensitive to heat stress conditions and may represent a good option for the improvements of adaptability to climatic changes. in order to combine lighter and harder pericarp, the most promising genotypes were crossed with wild cucurbita okeechobeensis. the histological analysis showed that c. okeechobeensis was a good source of genes for obtaining a thicker sclerenchymatic layer within pericarp. (*) corresponding author: andreja.urbanek@um.si received for publication 8 september 2016 accepted for publication 4 march 2017 copyright: © 2017 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2017 31(1): 61-73 62 pounds and reactive oxygen species (ros), which coincide with increased synthesis of antioxidants and activity of antioxidant enzymes. furthermore, enhanced expression of a variety of heat shock proteins and other stress-related proteins constitute major plant responses to heat stress (iba, 2002; howarth, 2005; wahid et al., 2007; ara et al., 2013 a, b, 2015). during the ontogenetic development of plants, flowering and fruit set are the most sensitive stages; fruit set of pumpkins is affected at day/night temperatures above 26/20°c and is severeliy affected above 35/26°c (schrader et al., 2004, 2011; saudreau et al., 2011; lei et al., 2014; johnson, 2015). the decrease in yield is due to the rapid decrease of photosynthesis, which reduces the amounts of sugars and other storage products that can go into fruits and developing seeds. on the other hand, hot night temperatures lead to greater cell respiration. high temperatures can also cause increased developmental disorders in fruiting vegetables due to the reduced pollen production leading to a reduction in fruit and seed sets, smaller pods, and split sets. another effect of heat stress in many plant species is induced sterility when heat is imposed immediately before or during anthesis (siddique et al., 1999; wahid et al., 2007). theoretical and experimental evidence shows that fruit temperature can be 10°c higher than the air temperature under sunny conditions (schrader et al., 2004; racskó et al., 2005; schrader, 2011; lei et al., 2014). sunburn of fruits is a surface injury caused by solar radiation which, during the initial phase, results in a light corky layer, golden or bronze discolouration. the damage occurs mainly in the surface and subsurface layers. there are two types of sunburn damage which may have effects on fruits and fruiting vegetables. the first, sunburn necrosis, appears due to the thermal death of cells on the sun exposed side of the fruit; cell membrane integrity is lost and cells start leaking their contents (schrader, 2011; johnson, 2015). the critical fruit tissues’ temperatures for sunburn necrosis vary with the type of fruit. the fruit surface temperature (fst) threshold for sunburn necrosis for cucumbers and pumpkins is between 37 and 42°c (rabinowitch et al., 1983, 1986; ara et al., 2013 a, b; 2015). the second type of sunburn injury is sunburn browning, which is caused by the combination of high fst and high solar radiation. it causes degradation of photosynthetic pigments resulting in yellow spots on the sun-exposed side of the fruit and occurs at a temperature about 5°c lower than sunburn necrosis (schrader, 2011; johnson, 2015). plants have three major ways in which they dissipate excess heat: (1) long-wave radiation, (2) heat convection into the air and (3) transpiration. if transpiration is interrupted by stomatal closure due to water stress, inadequate water uptake or other factors, a major cooling mechanism is not functioning. this will cause internal leaf/fruit temperatures to rise. without transpiration, the only way that plants can reduce heat is by heat radiation back into the air or wind cooling. under high temperatures, radiated heat builds up in the atmosphere around plants, limiting further heat dissipation (wahid et al., 2007; schrader, 2011; johnson, 2015). the adverse effects of high air and soil temperatures, and the high levels of solar radiation can be mitigated by developing plant genotypes with improved thermotolerance. some attempts to develop heat-tolerant genotypes via conventional breeding protocols have been successful (ehlers and hall, 1998; camejo et al., 2005). breeding of cultivated cucurbits was mainly focused in combinig good attributes of c. moschata and c. maxima. (balkaya and karaagac, 2005; balkaya et al., 2009, 2010 a, b, 2011; balkaya and kandemir, 2015). it is well known that c. moschata is best adapted to hot climate and is successfully cultivated in tropical and subtropical regions (balkaya et al., 2010 a, b; balkaya and kandemir, 2015). recently, a study was conducted to determine the extent of heat tolerance of newly developed interspecific squash hybrid named as ‘maxchata’ compared to its parents c. maxima and c. moschata (ara et al., 2013 a, b, 2015) under three different temperature regimes. results showed that various gas exchange and photosynthetic attributes dropped significantly with increasing temperature, while intercellular co2 concentration increased showing the nonstomatal limitations. these trends were more abrupt in c. maxima, reflecting that c. maxima was the most susceptible, while ‘maxchata’ showed intermediate response. c. moschata had the best photosynthetic attributes to sustain the heat regimes (ara et al., 2013 a, b). the ultramorphological, biochemical, and transcriptional analyses gave similar results. the electron microscopy highlighted the maximum degradation of the leaf ultrastructure of c. maxima. c. moschata and ‘maxchata’ exhibited lower degree of subcellular injury upon heat exposure. the antioxidant enzyme activities and their expression were found to be highest in c. moschata, moderate in ‘maxchata’, and lowest in c. maxima (ara et al., 2013 a, b; 2015). the authors concluded that the interspecific hybridization with c. moschata might significanturbanek krajnc et al. fruit temperature dynamics of selected oil pumpkin genotypes 63 ly contribute to heat tolerance (ara et al., 2013 a, b, 2015). the presented study is associated with creating heat tolerant pumpkins characterised by lighter exocarp. the work began in 1996 and is based on a modified recurrent selection approach. its aim is to create cultivars characterised by bushy growth, resistance to all major pests and diseases, tolerance to drought and high temperatures, and large and thick seeds having thin seed coats and high concentrations of high quality oil. the basic population (i.e., population of the cycle0) was established by inter-crossing all available sources of genes (i.e., numerous local and commercial cultivars, populations and hybrids of c. pepo, following the semi-diallel scheme). in 1997, the most valuable progenies were planted in new caledonia, at the cirad centre near pouembout (south pacific). due to the favourable semi-tropical climate, it was possible to execute three cycles per year. the problems, however, were seed germination within fruits and rotting fruits due to overheating. as the genetic resources within c. pepo were found to be insufficient for overcoming these problems, it was decided to incorporate interspecific hybridisation and change the exterior fruit colour. the main sources of genes for lighter fruit exterior were c. argyrosperma var. argyrosperma and c. moschata. cucurbita argyrosperma, which was the main source of genes for whitish exocarp and was also used as a genetic bridge between c. pepo and c. moschata. another interesting trait obtained by interspecific crosses was dark yellow fruit exterior which was associated with the same colour of mesocarp. some years later, a wild species c. okeechobeensis (small) l. h. bailey was added to the hybridisation programme, in order to improve the resistance to viruses and harder pericarp. the interspecific hybrids included in this study were indirect progenies developed within the recurrent selection programme which involved intraand inter-population crosses, self-pollinations, and backcrosses. the progress in breeding for heat stress tolerance strongly depends upon understanding the genetic and physiological mechanisms associated with stress tolerance of the whole plants well as at the molecular and cellular levels. our study involved thin-coated seed pumpkins with lighter exocarp because they were considered to have the highest level of tolerance to high air temperatures and high levels of solar radiation during summer. in order to investigate their tolerance to heat stress, pericarp tissue temperature profiles were monitored on the exocarp surface, as well as in the mesoand endocarp of fully developed attached and detached fruits during two different periods of hot weather. 2. materials and methods plant material four different plant materials with thin coated seed were used in the study: (1) styrian oil pumpkin cucurbita pepo subsp. pepo var. styriaca (o), (2) progeny with whitish fruit derived from the cross c. pepo (non-lignified seed coat, oil type) × c. argyrosp er m a va r. a r g y ro s p er m a ( o / a) , ( 3 ) a p ro gen y derived from crosses involving c. pepo (non-lignified seed type, oil type), c. argyrosperma and tropical c. moschata characterised by yellow fruits (a/mo x o/a) and (4) a three-species hybrid involving c. pepo (nonlignified seed coat, oil type), c. argyrosperma var. argyrosperma and c. okeechobeensis (oke × o/a) (table 1, fig. 1). the temperatures were measured on (a) attached fruits (1st period, 27th july to 5th august 2012), (b) detached fruits exposed to the sun (2nd period, 1st to 11th september 2012) and (c) artificially black coloured fruits (on both periods). the fruits were exposed to sunlight due to loss of foliage caused by drought stress and diseases. for each experiment, three fruits of each studied material were used. fruit temperature measurements the temperatures were measured during two different periods of hot weather, between the 27th july table 1 list of key plant materials with short explanations of abbreviations plant material abbreviations species cucurbita argyrosperma. var. argyrosperma a cucurbita moschata mo cucuribita pepo subsp. pepo var. styriaca o cucurbita okeechobeensis oke interspecific hybrids material obtained from the cross c. pepo (non-lignified seed coat, oil type) × c. argyrosperma. var. argyrosperma, followed by several seasons of intrapopulation recombinations and selection o/a three-species hybrid involving c. argyrosperma (used as a genetic bridge), c. moschata and c. pepo (non-lignified seed coat, oil type), after several seasons of intrapopulation crosses and selection a/mo x o/a a three-species hybrid involving c. pepo (non-lignified seed coat, oil type), c. argyrosperma var. argyrosperma and c. okeechobeensis oke x o/a adv. hort. sci., 2017 31(1): 61-73 64 to 5th august 2012 and 1st to 11th september 2012. during both periods, three pumpkins of each progeny (o, o/a, a/mo x o/a) were chosen and the thermistors were placed on exocarp the sun-exposed side, as well as inserted in meso(2 cm deep) and endocarp (10 cm deep) (fig. 1a, c, e). the temperatures were recorded every 15 seconds and stored as 10-min averages. the data represented in figures 3-6 show a diurnal and maximum day temperature as average of three fruits out of each progeny. in order to measure temperatures, 32 betatherm 10k3a542i thermistors connected to a campbell’s cr1000 datalogger (campbell scientific inc., logan, ut, usa) were used. the data logger recorded the current times for each iteration supply voltage and analogue input voltage. the thermistors’ readings were performed sequentially as they were all connected to the same adc converter through a multiplexor; so only one of the thermistors was connected to the converter at a time. the selected thermistors had accuracies of 0.2°c and were of negative-temperature-coefficient (ntk) type, which meant that their resistances decreased with the increases of temperature. as the data logger was unable to read the resistance of the thermistor directly, the thermistors were connected in a form of voltage divider, with an additional resistor with 1 kω±0.1% fixed resistance. as the resistive responses of the thermistors were nonlinear, the measured temperatures were calculated according to the temperature table from the datasheet (bethaterm). the results are represented by mean values (n=3), and were statistically evaluated by one-way analysis of variance (anova), using the spss 21 software (spss 21 software, spss inc., chicago, il, usa). significant differences between mean values were d e t e r m i n e d u s i n g t h e p o s t h o c d u n c a n t e s t . significant differences (α<0.05) between means were indicated by different letters. air/soil measurements furthermore, air/soil temperatures and relative humidity were measured on the sun-exposed side and within the canopy (5 and 20 cm below soil surface, on soil surface as well as 10 and 20 cm above soil surface using 215 and 107 temperature probe sensors (campbell scientific inc., logan, ut, usa). the temperatures were recorded every 15 seconds and stored as 10-min averages. histochemical evaluation of pericarp from each studied material, 3-6 fruits were taken for histological evaluation. out of each fruit six pieces (diameter 8 mm) of pericarp were sampled on equator of the fruit positioned around the pumpkin in regular intervals. the pieces were cut with a cryotom in order to evaluate the size of the lignified cell layer. an olympus microscope (provis ax 70) with a 100 w mercury arc lamp was used to take analogue images with a 3-chip-colour video camera (sony dxl 950 p, 3 ccd). fluorescence images were obtained through an uplanfi 40x dry objective (n.a., 0.75), a planapo 60x oil immersion objective (n.a., 1.40), and an uplanapo 100 x oil immersion objective (n.a., 1.35). lignified cells were visualised using an olympus filter set (umwu) with 330-385 excitation and 420 nm emission. fig. 1 fruits of selected genotypes with attached sensors, which were selected for lighter and harder fruit exterior and thin coated seeds: a, b) styrian oil pumpkin (o). c, d) three-species hybrid involving c. argyrosperma var. argyrosperma (used as a genetic bridge), c. moschata and c. pepo (non-lignified seed coat, oil type), after several seasons of intrapopulation crosses and selection (a/mo × o/a). e, f) genotype with lighter (i.e., whitish, white-green) fruits material obtained from the cross c. pepo (non-lignified seed coat, oil type) × c. argyrosperma, followed by several seasons of intrapopulation recombinations and selection (o/a). g) c. okeechobeensis. h) a three-species hybrid involving c. pepo (non-lignified seed coat, oil type), c. argyrosperma and c. okeechobeensis (oke × o/a). urbanek krajnc et al. fruit temperature dynamics of selected oil pumpkin genotypes 65 3. results and discussion our research was based on the hypothesis that interspecific hybridization aimed in creating lighter exocarp, thicker hypodermal sklerenchyma layer and cuticle would reduce the heat load on the fruit. air/soil measurements during the first period of measurements (jul.aug.), on the sun-exposed sides, the highest temperatures were registered on the ground surface reaching 45°c, whereas 20 cm above ground the maximum daily air temperatures were about 38°c. on the sides shaded by a canopy, the ground temperatures remained cooler and more stable, varying between 18 during the nights and 32°c on the hottest days. twenty cm above ground, the air temperatures reached 43°c. the underground temperatures (20 cm deep) remained stable with 20-22°c day/night variation (fig. 2 a). additionally, the ground temperatures were also measured within the proximities of the studied pumpkins. in the proximity of styrian oil pumpkins, from the 30th of july onwards, they increased from 27°c (daily maximum) to more than 47°c on the 1st august, and remained above 40°c for the next five days (fig. 3 a, b). the maximum day ground temperature in the proximity of white pumpkins were similar, reaching 45°c during the first three days of august (fig. 3 e, f). at the beginning of september, the minimum night temperature was 10°c. one day, on the 7th september, it was particularly hot and the air temperature 20 cm above ground, on the sun-exposed side, reached 45°c, whereas the temperature of the ground surface reached 58°c. a similar situation was observed within the canopy. at 20 cm height, the temperature reached 45°c (fig. 2 b). on attached styrian oil pumpkins (1st period), the maximum day temperatures measured on the exocarp varied between 38 and 50°c. in mesocarp the t e m p e r a t u r e s r a n g e d b e t w e e n 3 2 a n d 5 1 ° c . endocarp was more or less 2°c cooler than mesocarp (fig. 3 a, b). on detached styrian oil pumpkins (2nd period) the maximum day temperatures of exocarp varied between 20 and 54°c, whereas mesocarp heated up above the temperature of exocarp to 61°c on the hottest day (7th september), on later days, the maximum day temperatures were around 45°c (fig. 4 a, b). in the ‘yellow fruit’ pumpkin material (a/mo × o/a), the temperatures of exocarp ranged between 42°c and 50°c on attached fruits during the first period and 17°c and 50°c on detached fruits during the second period of hot weather. the temperatures of endocarp varied between 33°c and 42°c during the first period, whereas during the second period the temperatures of mesocarp of detached pumpkins ranged between 16°c and 45°c (fig. 3 c, d, fig. 4 c, d). ‘white fruit’ pumpkin material (o/a) was characterised by the lowest exocarp temperatures, which ranged between 30°c and 41°c during the first period, whereas during the second period the temperatures ranged between 17°c and 44°c. the mesocarp temperatures varied between 35 and 44°c, the endocarp temperatures were more or less 2°c above the mesocarp temperatures. during the second period, where temperatures were measured on detached fruits, the average temperatures of mesocarp were fig. 2 relative humidity and the average air/soil temperatures measured within canopy and on sun exposed side during the period between 27 july-6 august 2012 (a) and between 1-11 september 2012 (b). adv. hort. sci., 2017 31(1): 61-73 66 generally 2°c lower when compared to exocarp but the fruit flesh heated up to 56°c on the extremely hot days of the 6th and 7th september, similarly as was observed in the styrian oil pumpkin (fig. 4 a, b, e , f , f i g . s 1 s e e s u p p l e m e n t a r y m a t e r i a l ) . transpiration appeared to be vital for maintaining optimal growth temperatures in growing plants. detached fruits, however, lacked the protective effects of transpiration, and direct sources of heat, such as sunlight, can rapidly elevate the internal fruit fig. 3 average and maximum day temperatures measured in exocarp, mesocarp, endocarp of attached pumpkins and ground in the period between 27. july6. august 2012: a, b) styrian oil pumpkin (o). c, d) genotype with yellow fruits involving c. argyrosperma, c. moschata and c. pepo (a/mo × o/a). e, f) genotype with white fruits obtained from the cross c. pepo (non-lignified seed coat, oil type) × c. argyrosperma (o/a). 67 urbanek krajnc et al. fruit temperature dynamics of selected oil pumpkin genotypes temperatures to above that of exocarp and towards the thermal death points of their cells. in case of styrian oil pumpkin these lead to localised bleaching a n d n e c r o s i s ( s u n b u r n o r s u n s c a l d ) . s i m i l a r l y , r a b i n o w i t c h e t a l . ( 1 9 8 3 , 1 9 8 6 ) r e p o r t e d t h a t detached cucumbers (cucumis sativus l.) and peppers (capsicum annuum l.) had significantly higher surface temperatures and more serious sunburn injuries than attached fruit. comparing the measured results of the different fig. 4 average and maximum day temperatures measured in exocarp and mesocarp of detached pumpkins in the period between 1.11.september 2012: a, b) styrian oil pumpkin (o). c, d) genotype with yellow fruits involving c. argyrosperma, c. moschata and c. pepo (a/mo × o/a). e, f) genotype with white fruits obtained from the cross c. pepo (non-lignified seed coat, oil type) × c. argyrosperma (o/a). 68 adv. hort. sci., 2017 31(1): 61-73 varieties on attached fruits, we can conclude that styrian oil pumpkin was characterised by the highest temperatures within all three tissues (fig. s2), followed by yellow genotype, whereas white genotype had the lowest temperature, confirming the hypothesis that varieties with lighter fruit colour are less h e a t e d u p . o u r r e s u l t s o f t h e e x p e r i m e n t o n detached fruits, however, suggest that the most tolerant is a/mo × o/a which is a progeny derived from the interspecific cross involving the styrian oil pumpkin, c. argyrosperma and c. moschata. it is well known that c. moschata is best adapted to hot climate and is successfully cultivated in the tropical and subtropical regions (balkaya et al., 2010 a, b; balkaya and kandemir, 2015). heat tolerance of c. moschata can be very useful in breeding involving interspecific hybridisation. one of the successful examples, mentioned earlier in the text, is interspecific hybrid of squash named as ‘maxchata’. the authors concluded that the interspecific hybridization involving c. moschata might significantly contribute to heat tolerance (ara et al., 2013 a, b; 2015). in order to test further the resistance of selected genotypes to heat stress, the selected pumpkins were sprayed with black colour and exposed to the sun in order to induce bleaching by the excess heat. the exocarp of the black coloured styrian oil pumpkin fruit heated up to 52°c whereas the mesocarp temperature rose up to 43°c in the afternoon hours between 3 and 4 p.m. during both periods. when analysing the daily temperature curves of attached pumpkins during the july-august period, a depression in the curve was observed at around 12 a.m., reflecting a higher transpiration of fruits in response to heat stress; later, a rapid increase in temperature, may indicate stomata closure (fig. 5a). it is wellfig. 5 average and maximum day temperatures measured in exocarp and endocarp of black coloured attached pumpkins measured in the period between 27 july6 august 2012: a, b) styrian oil pumpkin (o). c, d) genotype with yellow fruits involving c. argyrosperma, c. moschata and c. pepo (a/mo × o/a). 69 urbanek krajnc et al. fruit temperature dynamics of selected oil pumpkin genotypes known that if transpiration is interrupted by stomatal closure a major cooling mechanism is lost and the internal fruit temperatures raise (wahid et al., 2007; johnson et al., 2015). the temperatures of coloured fruits were measured for three days since a general collapse and tissue death of the inner pericarp layers was observed later on. the fruits were too much damaged by sunburn and measurements of temperatures no longer made sense. in the september perio d , w h e n t h e e x p e r i m e n t w a s p e r f o r m e d o n detached fruits, the exocarp of styrian oil pumpkin heated up to 51°c, similarly as on attached fruits. mesocarp temperature raised to a higher level on detached fruits (61°c) in comparison to attached fruits (43°c) (fig. 6a, fig. 6b, fig. s2). i n t e r e s t i n g l y , d u r i n g t h e 1 s t e x p e r i m e n t o n fig. 6 average and maximum day temperatures measured in exocarp and mesocarp of black coloured detached pumpkins measured in the period between 1.-11.september 2012: a, b) styrian oil pumpkin (o). c, d) genotype with yellow fruits involving c. argyrosperma, c. moschata and c. pepo (a/mo × o/a). e, f) genotype with white fruits obtained from the cross c. pepo (non-lignified seed coat, oil type) × c. argyrosperma (o/a). 70 adv. hort. sci., 2017 31(1): 61-73 attached pumpkins the exocarp of the black coloured ‘yellow’ pumpkin heated up to 64°c, whereas the endocarp up to 48°c during the july-august period (fig. 5c, d). compared to the styrian oil pumpkin the temperatures were higher. a rapid increase of temperature was determined in the late morning hours until noon. later, an abrupt decrease of temperature followed and maintained at around 38°c between 3 and 6 p.m. after that, during the night hours, the temperature dropped below 20°c (fig. 5c). similarly, in september, during the early afternoon hours of the hottest day (7th september), the maximum day temperatures on exocarp reached 64°c, whereas in mesocarp the temperatures reached 57°c. on later dates the daily maximum temperatures of exocarp constantly reached 42°c, whereas mesocarp was heated up to 57°c (fig. 6 c, d). the ‘white fruit’ genotype (o/a) was characterised by the highest mesocarp temperatures during the hottest days of the 2th period when compared to o and a/mo × o/a progenies. during the first five days, the maximum exocarp temperatures were around 40°c, whereas the mesocarp reached maximum day temperatures between 48°c and 55°c. during the following two days, the exocarp temperatures reached 60°c and 63°c, respectively, whereas the mesocarp 62°c and 71°c. later, the exocarp maximum temperature was comparable to that of the black coloured ‘yellow’ pumpkin, reaching 42°c in the early afternoon hours, whereas the mesocarp was heated up to approx. 55°c (fig. 6 e, f). summarising the results of temperature measurements within the black coloured pumpkins, we can conclude that for the attached pumpkins, the ‘yellow’ genotype was heated up more than the styrian oil pumpkin. by analysing the results of detached pumpkins during the first days of measurements, the highest temperatures of exoand mesocarp were determined in the styrian oil pumpkin, whereas the maximum day temperatures of both ‘yellow’ and ‘white’ genotypes did not differ significantly and were about 5°c lower than those of the styrian oil pumpkin. h o w e v e r , o n t h e h o t t e s t d a y s ( 6 t h a n d 7 t h september), the temperatures of exocarp within ‘yellow’ and ‘white’ genotypes were 8°c higher than those of the styrian oil pumpkin reaching 64°c. consequently, when compared to the styrian oil pumpkin, higher temperatures were also determined in fruit tissues of both studied interspecific hybrids. during the last two centuries, pumpkins have been selected for their softer pericarp in order to ease the laborious and time-consuming hand-harvesting of seeds. however, the resulting pumpkins’ genotypes became more susceptible not only to diseases but also to various types of abiotic stress such as heat stress and drought. in order to create a variety with harder pericarp and also better abscission of the fruit peduncle, those pumpkins with lighter exocarp were crossed with the wild species c. okeechobeensis (fig. 1 g, h), which is characterised by hard perikarp, good abscission of the pedincle and resistance to viruses. the selected hybrids were histologically evaluated and compared to the styrian oil pumpkin and o/a hybrids with lighter pericarps. the styrian oil pumpkin (o) was characterised by its thick cuticle, a 15-cell layer of chlorenchyma cells and a 23 cell layer of more or less isodiametric lignified cells between the chlorenchyma and parenchyma of the mesocarp (fig. 7 a, b). both the ‘yellow’ (a/mo × o/a) as well as the ‘white’ (o/a) genotypes were characterised by a similar 2-3 cell layer of thick lignified cells although the cell walls of this layer as well as the cuticle were thicker (fig. 7 c, f). crosses of o/a genotypes with c. okeechobeensis (oke × o/a) became characterised by a thicker scklerenchymatic layer of 4-5 oblong cells in a radial direction, which were approx. 100 µm long (fig. 7 g, h). this progeny represents a good material for the future breeding for increased heat tolerance. furthermore, c. okeechobeensis has been recognised as promising for interspecific hybridization due to its central position in the genus cucurbita (gong et al., 2013). we may conclude that the genetic breeding of oil pumpkins for heat tolerance is still in its infancy stage and warrants more attention than it has been gi ven i n th e p a s t . c o n s i d era b l e i n f o rma ti o n i s presently available regarding the physiological and metabolic aspects of plant heat-stress tolerance (ara et al., 2013 a, b, 2015). furthermore, attempts have been made to include molecular marker technology for genetic characterization and/or development of plants with improved heat tolerance. gong et al. (2013) analysed ssr polymorphisms on a large collection of cucurbita materials in order to obtain an improved insight into the relationships amongst most of the species of the genus. wild species c. foetidissima has been identified as resistant to numerous pathogens and pests but is most distant to other cucurbita species. cucurbita pepo and c. ficifolia were the most outlying of the mesophytic species. the clusters of the six remaining species form three pairs, c. maxima with c. ecuadorensis, c. okeechobeensis with c. lundelliana, and c. moschata with c. argyrosperma. due to the genetic distances 71 urbanek krajnc et al. fruit temperature dynamics of selected oil pumpkin genotypes amongst the cucurbita species, various breeding strategies and biotechnological approaches have been employed (merrick, 1995; lebeda et al., 2007; orti z-al ami l l o et al. , 2007; l el l ey et al. , 2009; karaağaç and balkaya, 2013) but the success in introgressing desirable traits from one species to another have been limited. cucurbita pepo excels in plant earliness and productivity but lacks genetic resources for disease resistance. cucurbita moschata, on the other hand, carries resistance to various pathogens and is adapted to humid tropics but lacks earliness and productivity (lebeda et al., 2007; lelley et al., 2009; karaağaç and balkaya, 2013). relatively high successes and fertilities have been observed for the c r o s s c o m b i n a t i o n o f c . a r g y r o s p e r m a a n d c . moschata (montes-hernandez and eguiarte, 2002; ortiz-alamillo et al., 2007) and c. maxima × c. moschata (ara et al., 2013 a, 2015). however, despite all the complexity of heat tolerance and difficulties encountered during the genetic transfer of tolerance, few heat-tolerant inbred lines and hybrid cultivars with commercial acceptability h a v e b e e n d e v e l o p e d a n d r e l e a s e d ( m o n t e s hernandez and eguiarte, 2002; ortiz-alamillo et al., 2007; ara et al., 2013 a, b, 2015). 4. conclusions the presented study suggests that the colour of exocarp is probably one of the key parameters of tolerance to high temperatures. darker colours are generally associated with higher fruit temperatures. in general, for attached pumpkin fruits higher temperatures were measured on exocarp, followed by mesoand endocarp. however, for the detached fruits on extremely hot days in september, the temperatures within mesocarp increased above those of exocarp. it is well known that if transpiration is interrupted by stomatal closure due to water stress and heat stress, a major cooling mechanism is not functioning. this was the reason of rising the internal fruit temperatures in case of detached fruits. one could expect that the genotypes with whitish exocarp (i.e., o/a progenies derived from the cross styrian oil pumpkin × c. argyrosperma) would be the most tolerant to heat stress. the experiment on detached fruits, however, suggest that the most tolerant is the a/mo x o/a progeny derived from the cross involving the styrian oil pumpkin, c. argyrosperma and c. moschata, which is characterized by yellow exocarp. one of the reasons for this could be the introgression of desirable traits of c. moschata, which is more adapted to high temperatures. this parental species was brought from the island of espiritu santo, vanuatu (tropical pacific) and was obviously more tolerant to heat stress than the other two involved pumpkin species. the second important parameter appears to be the histological structure of fig. 7 histochemical analyses of perikarp. a, b) styrian oil pumpkin (o) characterised by its thick cuticle, a 15-cell layer of chlorenchyma cells and a 1-2 cell layer of isodiametric cells and one layer of oblong cells with thick cell walls. c, d) genotype with yellow fruits (a/mo × o/a), with two layers of isodiametric cells with lignified cell walls. e, f) genotype with white fruits (o/a) and three layer of lignified cells below the chlorenchyma. g, h) a three-species hybrid involving c. pepo (non-lignified seed coat, oil type), c. argyrosperma and c. okeechobeensis (oke × o/a) exhibiting 4-5 cell layers of oblong sklerenchymatic cells below the chlorenchyma. 72 adv. hort. sci., 2017 31(1): 61-73 the pericarp. the genetic improvement of this comp l e x t r a i t s h o u l d c o n s i d e r b o t h p a r a m e t e r s . nevertheless, to accelerate such progresses, major areas of emphasis in the future should be: (1) development of accurate screening procedures at each stage of plant development; 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( e d ) . proceedings of the ixth eucarpia meeting on genetics and breeding of cucurbitaceae. avignon, france, may 21-24th, pp. 21-32. saudreau m., marquier a., adam b., sinoquet h., 2011 modelling fruit temperature dynamics within apple tree crowns using virtual plants ann. botany, 108(6): 1111-1120. schrader l., sun j., zhang j., seo j. h., jedlow l., felicetti d., 2004 fruit skin disorders. proc. wash. tree fruit postharvest conf. schrader l.e., 2011 scientific basis of a unique formulation for reducing sunburn of fruits. hort. sci., 46: 6-11. seebold k., coolong t., jones t., strang j., bessin r., 2009 an ipm scouting guide for common problems of cucurbit crops in kentucky. in: kaiser c (ed.) an ipm scouting guide for common problems of cucurbit crops i n k e n t u c k y . u n i v e r s i t y o f k e n t u c k y c o l l e g e o f agriculture, lexington, usa. siddique k.h.m., loss s.p., regan k.l., jettner r.l., 1999 adaptation and seed yield of cool season grain legumes in mediterranean environments of south-western australia. aust. j. agric. res., 50: 375-387. urbanek krajnc a., janzekovic i., sober a., ivancic a., 2016 the impact of interspecific hybridization on the chemical compositions of oil pumpkin seeds. phyton, 56: 61-75. wahid a., gelani s., ashraf m, foolad m.r., 2007 heat tolerance in plants: an overview. environ. exper. bot., 61: 199-223. yavuz d., seymen m., yavuz n., turkmen o., 2015 effects of irrigation interval and quantity on the yield and quality of confectionary pumpkin grown under field conditions. agric. water manag., 159: 290-298. sm i adv. hort. sci., 2017 31(1): 61-73, sm i-ii doi: 10.13128/ahs-20727 the impact of fruit temperature dynamics on heat stress tolerance of selected oil pumpkin genotypes a. urbanek krajnc *, j. rakun, p. berk, a. ivančič faculty of agriculture and life sciences, university of maribor, pivola 10, hoče, slovenia. supplementary material copyright: © 2017 author(s). this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2017 31(1): 61-73, sm i-ii sm ii fig. s1 comparison of the maximum day temperatures measured in the period between 27 july 6 august 2012 in: a) exocarp of styrian oil pumpkin, yellow and white genotype; b) endocarp of styrian oil pumpkin, yellow and white genotype; c) exocarp of black coloured styrian oil pumpkin and yellow genotype; d) endocarp of black coloured styrian oil pumpkin and yellow genotype. fig. s2 comparison of the maximum day temperatures measured in the period between 1-11 september 2012 in: a) exocarp of styrian oil pumpkin, dark yellow and white genotype; b) mesocarp of styrian oil pumpkin, yellow and white genotype; c) exocarp of black coloured styrian oil pumpkin, yellow and yellow genotype; d) mesocarp of black coloured styrian oil pumpkin, yellow and yellow genotype. impaginato 535 adv. hort. sci., 2018 32(4): 535-540 doi: 10.13128/ahs-22474 effect of a novel hydrogel amendment and seedling plugs volume on the quality of ornamental/miniature tomato r.a.c. melo (*), m.h. jorge, n. botrel, l.s. boiteux brazilian agricultural research corporation, embrapa vegetables, brasilia df, brazil. key words: copolymer, pot plant, solanum lycopersicum l., transplants. abstract: the market for ornamental/miniature plants values aesthetic morphological characteristics, which give harmony to the potted plant. these traits depend on the growing media capacity to maintain quality and plant longevity. the use of hydrogels has increased recently in order to achieve visually attractive and long-lasting plants. thus, this study aimed to evaluate the effects of a novel hydrogel (h) amendment and seedling plugs volume (spv) on the quality of ornamental/miniature tomato. seedlings of tomato cv. brs finestra were produced in 200 and 162 plugs plastic trays with 18 cm³ trapezium-shaped plugs and 50 cm³ conically shaped plugs, respectively. 18 cm³ plugs and h amendment presented several significant responses for plant characters height, soluble solids, number of leaves, stem diameter, shoot dry matter and fruits weight per plant; making evident the advantages of using a growing medium of smaller volume and this copolymer amendement. although consistent results were obtained, a combination of both these factors in terms of an optimal aesthetic value and considering all the morphological traits could not be accomplished. therefore it’s necessary to study other elements such as plant nutrition and the use of plant growth regulators to complement them, aiming to promote better quality. 1. introduction miniature tomatoes can be grown singly in small pots, or more plants in larger hanging pots. they are also ideal for window boxes or garden borders because their plant canopy diameter is little (scott and harbaugh, 1995). these plants combine ornamental aspects of a well-proportioned, diminutive, tomato plant with tasting fruits that can be eaten. small plant sizes are ideal for commercial growing, shipping, and retail selling (scott and harbaugh, 1995). the commercial success of the cultivation of miniature/ornamental potted plants such as tomatoes and peppers depends on consumer appeal conferred by the plant beauty, quality, vigor, color, shape, and size of leaves and fruits. in addition to that, the cultivars must present canopy harmony and be able to develop in relatively small pots (costa et al., 2015). (*) corresponding author: raphael.melo@embrapa.br citation: melo r.a.c., jorge m.h., botrel n., boiteux l.s., 2018 effect of a novel hydrogel amendment and seedling plugs volume on the quality of ornamental/miniature tomato. adv. hort. sci., 32(4): 535-540 copyright: © 2018 melo r.a.c., jorge m.h., botrel n., boiteux l.s. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 8 january 2018 accepted for publication 28 june 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(4): 535-540 536 non-miniature tomato cultivars tend to overgrow small containers, and their plant size is restricted by the container size (scott and harbaugh, 1995), producing commercially unviable plants, requiring the use of plant growth regulators (pgr) to achieve attractive compact potted grown plants (moraes et al., 2005). another important feature of ornamental plants is the maintenance of interior quality and longevity, a never-ending effort by producers (wang, 1992). growing medium ability to prevent drying out is desirable, especially in peat-based substrates. some growers report a benefit when plants are watered with a wetting agent prior to shipment, thus making it easier for retail clerks and consumers to thoroughly rewet the medium. interest has increased recently among growers and mass market buyers in using water-absorbing gels (nell, 1991). however, studies investigating the effects of hydrogel (h) application o n o r n a m e n t a l p l a n t s a r e s c a r c e a n d l i m i t e d (ljubojević et al., 2017). particularly to ornamental/ miniature vegetables, this scenario can be considered negligible. in regard to this matter, a promising nanocomposite hydrogel developed by an innovative technique using calcium montmorillonite showed great swelling degree, higher than 2000 times in water. the formulated h with high calcium montmorillonite content (approximately 50.0% wt) as well as featuring high loading capacity and individual and simultaneous release, denotes an interesting material for agricultural applications (bortolin et al., 2016). thus, the present study has the objective of evaluating the effects of a novel hydrogel amendment and seedling p l u g s v o l u m e ( s p v ) o n t h e q u a l i t y o f ornamental/miniature tomato. 2. materials and methods plant material and growth conditions an experiment was conducted from july 18 (sowing) to october 25 (harvest), 2017 at embrapa vegetables 996 m altitude, 15° 56’ s, and 48° 08’ w brasília-df, brazil, in a glass-glazed greenhouse. a p h o t o s e l e c t i v e s h a d i n g n e t ( a l u m i n e t ® i c 5 0 ginegar polysack®) was installed 2 m above the benches. the greenhouse presented an air temperature of 15°c minimum and 44°c maximum, average dli (daily light integral) of ≈ 11 mol.m-2.d-1 considering a 12h period of sunlight and 81% maximum and 11% minimum of humidity. measures were taking after seedlings transplantation 15 minutes apart by sensors connected to a datalogger (watchdog 1000 series micro station spectrum technologies®). seedlings were produced in plastic trays with 200 and 162 plugs (jks® 18 cm³ trapezium shaped plugs and 50 cm³ conically shaped plugs, respectively) using a peat moss based substrate (carolina soil®) (fig. 1 a). solanum lycopersicum l. cv. brs finestra was selected, being the first brazilian ornamental/miniature tomato cultivar released by embrapa as a product for a very specific and demanding market (giordano et al., 2001). they were transplanted at 36 das (days after sowing) to 1 dm³ pots (nutriplan® np14) filled with a pine bark-based substrate (rohbacher®) with the following characteristics: ec 0.4; ph 6.0; water holding capacity 50%; humidity 60%; density 185 kg m³. filling were complemented with 5 g of bokashi compost per pot (korin® garden bokashi). nutrients were supplied weekly during the experiment, with a solution developed for ornamental peppers containing 14.4, 1.95, 12.92, 2.5, 1.0, 2.44 mmol/l of n, p, k, ca, mg, s and 30, 5, 50, 40, 2 and fig. 1 (a) seedling plugs volume and visual comparison 50 cm³ (left) and 18 cm³ (right); (b) plants from 50 cm³ plugs with hydrogel amendment (left) and without (right); (c) plants from 18 cm³ plugs with hydrogel amendment (left) and without (right). melo et al. quality of ornamental, miniature tomato 537 0.1 mol/l of b, cu, fe, mn, zn and mo, respectively, according to costa et al. (2015). watering from seeding to the transplantation was performed twice daily with enough water to start the run off at the bottom of the trays. all other cultural practices were performed using technical recommendations for controlled environment miniature tomato cultivation (schwarz et al., 2014). plant morphological characteristics and fruit quality analysis all plant morphological characteristics were analyzed when a commercial stage was reached, meaning that when 50% of the plant population of each treatment had at least 30% of fully ripe fruits or visually marketable fruits with the maximum size and shape typical of growth for miniature/ornamental tomato. these agronomic characteristics are based on the morphological descriptors suggested by ipgri (1995) and were validated by costa et al. (2015) for ornamental peppers: (p) precocity expressed by the number of days between transplantation and commercial stage; dry matter content of shoot (sdm) parts were dried in oven at 70°c until constant weight and values were obtained by equation: sdm% = {dry weight (g)/fresh weight (g) x 100}; (ph) plant height (cm) measured using a ruler, from the stem bottom until the last fully expanded leaf; (sd) stem diameter (mm) measured above the cotyledon leaves using a digital caliper; (cr) plant canopy ratio obtained from between the longitudinal (ld) and transverse diameters (td), where the closer the value to 1, more circular is the canopy. (nl) number of leaves expressed by counting the number of leaves per plant; number of fruits per plant (nfp); number of fully ripe (nr); (fw) fruit weight per plant (g) expressed by the sum of different fruits ripening stages per plant; (fd) ripe fruits diameter (cm) obtained from the longitudinal diameter of 4 fruits from each treatment. the following fruit quality basic parameters were a n a l y z e d b y a o a c ( 2 0 1 0 ) a n d m c g u i r e ( 1 9 9 2 ) methodologies, utilizing 4 fruits from each treatment: (ss) soluble solids (°brix); (ac) acidity; ss/ac ratio; color (c) evaluated by means of ripe fruits with the measurement of the colorimetric parameters l*, a*, b* c* and angle hue (h°) in fruits. the l * coordinate expresses the degree of clarity of the measured color (l = 100 = white; l = 0 = black), c* the intensity of the color and h ° the saturation of the color. statistical design and analysis the trial was conducted in a 2x2 factorial design in a complete randomized scheme, with six replications. the presence (amendment) and absence of h represented the first factor. spv18 cm³ plugs and 50 cm³ plugs represented the second factor. each replication was composed of 10 plants. the rate of h per substrate consisted of 2.0% (on volume/volume basis) and followed previous studies recommendations (bortolin et al., 2016). data were subjected to an analysis of variance (anova). all computations were performed with assistat® software (silva and azevedo, 2016). preliminary analysis indicated that ph, and npp presented a skew and overdispersed distribution, and it was required a transformation to normalize data. thus, their means were evaluated after square root transformation. normality of residuals was tested using shapiro-wilk test (alpha 5%) and the distribution presented as normal subsequently. 3. results plants grown utilizing 50 cm³ plugs required a shorter period of time to reach the ideal commercial stage precocity (p). when 18 cm³ plugs were used, this stage was reached at the same time, independently of h amendment (table 1). this differentiates the 50 cm³ plugs in 5 days (earlier) when compared to 18 cm³ plugs. a difference of 9 or 14 days when 50 cm³ plugs were amended or not with h, respectively, was also observed when comparing to 18 cm³ plugs. 50 cm³ plugs use resulted in plants with a very long stem (fig. 1 b). table 1 ornamental/miniature tomato precocity (p) stage reached according to hydrogel amendment and seedlings plugs volume p= when 50% of the plant population had at least 30% of fully ripe fruits or visually marketable fruits with the maximum size and shape typical of growth for miniature/ornamental tomato. plug volume p (days after transplantation) with hydrogel without hydrogel 18 cm³ 57 57 50 cm³ 43 48 adv. hort. sci., 2018 32(4): 535-540 538 ph values were significant for both factors and their interaction (tables 2 and 3), with 18 cm³ plugs and h combination reaching 19.63 cm. the general mean height value achieved in this trial (17.1 cm) is typical of the cv. brs finestra and is within the values quantified by scott and harbaugh (1995) evaluating different miniature tomatoes, ranging from 9 cm with cv. micro-tom to 25 cm with cv. micro-gold. characters of aesthetic significance, such as plant architecture, number, position, and color of fruits, leaves shape and density are some of the reasons that ornamental species of the solanaceae family are admired, being strictly related to plant longevity and to facilitate cultural handlings (neitzke et al., 2016). one of these traits, nl, is consistent with the cultivar and growing conditions, presenting a significant response for all the studied factors. the interaction of 18 cm³ plugs and h enhanced the nl to 51.6 (table 2) which can be perceived by the observation of leaves density in fig. 2 d, but this response was not converted into a greater nfp. cr represents the aspect of the aerial parts of the plant, where the closer the value to 1, more circular it is the canopy. therefore, when 50 cm³ plugs were used, a more circular shape was attained (ld/td = 1.16) (table 2). this format has a greater visual appeal, which is often decisive in the choice of the consumer to become more attractive and makes it easier to handle (costa et al., 2015), although with plants being long-stemmed, a visual aspect uncommon for miniature tomatoes was observed, resembling a palm tree shape (fig. 1 b). sdm and sd values were significant for both treatments (table 3). costa et al. (2015) evaluated the quality of ornamental pepper using two substrates and genotypes, founding distinct sdm content responses, with values ranging from 20.58% to 26.54%, corroborating with the best results here found. sd results presented in this study (5.53 mm with h amendment and 5.55 mm from 18 cm³ plugs) table 2 interaction between hydrogel and seedling plugs volume for plant ornamental/miniature tomato characters means followed by the same lowercase letters in the columns and capital letters in the lines do not differ by tukey test at 5% probability. fig. 2 ornamental/miniature tomato plants visual aspect when precocity stage was reached. (a) from 50 cm³ plugs with hydrogel amendment; (b) from 50 cm³ plugs without hydrogel amendment; (c) from 18 cm³ plugs without hydrogel amendment; (d) from 18 cm³ plugs with hydrogel amendment. table 3 hydrogel amendment and seedlings plug volume effect on ornamental/miniature tomato plant characters means followed by the same lowercase letters in the columns and capital letters in the lines do not differ by tukey test at 5% probability. plug volume 50 cm³ 18 cm³ plant height (cm) with hydrogel 16.49 ab 19.63 aa without hydrogel 16.30 aa 16.02 ba soluble solids (°brix) with hydrogel 4.65 ab 5.35 aa without hydrogel 4.50 ab 5.20 aa number of leaves (per plant) with hydrogel 37.41 ab 51.60 aa without hydrogel 37.45 ab 43.88 ba canopy ratio with hydrogel 1.16 ab 1.32 aa without hydrogel 1.23 aa 1.26 aa plant characters hydrogel plug volume with without 50 cm³ 18 cm³ stem diameter (mm) 5.53 a 5.31 b 5.29 b 5.55 a shoot dry matter (%) 19.49 a 18.37 b 18.31 b 19.55 a fruit weight per plant (g) 68.39 a 67.17 b 77.39 a 58.17 b number of fully ripe fruits 3.08 a 2.43 b number of fruits per plant 9.90 a 6.01 b melo et al. quality of ornamental, miniature tomato 539 (table 3) are inferior but consistent with the ones found in the work of backes et al. (2007) with ornamental pepper. they obtained an sd of 6.42 mm as the best result using controlled release fertilizer mixed to a commercial substrate. even though sd values in this study were significant to h and spv, only a slight increase was observed amongst treatments. nfp, nr, and fw values were superior with the use of 50 cm³ plugs (table 3). with a compact canopy and well-distributed leaves, the photosynthetic process that depends on the interception of light energy most likely was converted into chemical energy in an efficient way, resulting in this positive response. ss and ac values were significant for spv and also for the interaction of 18 cm³ plugs with h. the values of 5.35 and 0.61 achieved for ss and ac, respectively, are comparable to 5.37 and 0.65 from cv. micro-gold bred by scott and harbaugh (1995), ensuring that cv. brs finestra produces mild tasting fruits that can be appreciated/consumed. ni (general mean value of 1.52), fd (general mean value of 3.1 cm), ss/sc ratio (general mean value of 8.38) and c (general mean value of l* 40.67; c* 51.05 and h° 48.51) were not significant (ns) to both treatments. for c values, although being ns, ripe fruits exhibited an intense red coloration (fig. 1b). this color is favored by consumers, a point which is believed to possess the highest carotenoids content such as β-carotene and lycopene (kader et al., 1977). 4. discussion and conclusions plants grown with 50 cm³ plugs, in the presence or absence of h amendment, demanded a shorter period of time to reach the ideal commercial stage precocity (p). precocity is a very important attribute, as it would allow the grower to commercialize plants earlier and to reutilize the spaces emptied in the greenhouse benches. but for its recommendation, the fact that ph in this plug volume resulted in an undesired visual aspect displaying a very long stem, needs to be taking in to account. the 50 cm³ plugs were significant for nfp, nr, ng, and fw characters as well. an efficient interception of light with plants produced using this plugs made most likely converted chemical energy into fruits, being a response of a compact canopy with well-distributed leaves. 18 cm³ plugs were significant for sd, sdm and fw, making evident the advantages of using a smaller volume of substrate, which can be fully employed for the growing of ornamental/miniature tomatoes in 1 dm³ pots. the use of h resulted in higher sd, sdm and fw values, considered fundamental in the aspect of the plant canopy and its longevity. h and spv interaction responded differently to the analyzed plant characteristics, with 18 cm³ plugs and h amendment together showing significant responses only for ph and nl. in conclusion, although several consistent results for plant characters were obtained when 18 cm³ plugs and h were used solely, a combination of both in terms of an optimal aesthetic value and considering all the morphological traits could not be accomplished. therefore it’s necessary to study other elements such as plant nutrition and the use of pgr to complement them, aiming to promote better quality. references aoac, 2010 international official methods of analysis of aoac. 18th ed. association of analytical chemists, gaithersburg, md, usa. backes c., fernandes f.m., krohn n.g., lima c.p., kiihl t.a.m., 2007 produção de pimenta ornamental em função de substratos e doses de adubação com fertilizantes de liberação lenta e tradicional. scientia agraria paranaensis, 6: 67-76. 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(folder in portuguese). ipgri, 1995 descriptors for capsicum (capsicum spp). international plant genetic resources institute, rome, italy, pp. 110. kader a.a., stevens m.a., albright-holton m., morris l.l., algazi m., 1977 effect of fruit ripeness when picked on flavor and composition in fresh market tomatoes. j. amer. soc. hortic. sci., 113: 724-731. l j u b o j e v i ć m . , o g n j a n o v v . , m a k s i m o v i ć i . , čukanović j., dulić j., szabò z., szabò s., 2017 effects of hydrogel on growth and visual damage of ornamental salvia species exposed to salinity. clean soil, air, water, 45(2): 1-12. mcguire r.g., 1992 reporting of objective color measurements. hortscience, 27: 1254-1255. moraes p.j., saraiva grossi j.a., de araújo tinoco s., 540 adv. hort. sci., 2018 32(4): 535-540 henriques da silva d.j., cecon p.r., barbosa j.g., 2005 ornamental tomato growth and fruiting response to paclobutrazol. acta horticulturae, 683: 327-332 neitzke r.s., fischer s.z., vasconcelos c.s., barbieri r.l., treptow r.o., 2016 pimentas ornamentais: aceitação e preferências do público consumidor. horticultura brasileira, 34: 102-109. nell t.a., 1991 how to make long-lasting top performers out of your pot mums. grower talks, 54: 67-80. schwarz d., thompson a.j., kläring h.p., 2014 guidelines to use tomato in experiments with a controlled environment. front. plant sci., 5(625): 1-16. scott j.w., harbaugh b.k., 1995 ‘micro-gold’ miniature dwarf tomato. hortscience, 30: 643-644. silva f.a.s., azevedo c.a.v. 2016 the assistat software version 7.7 and its use in the analysis of experimental data. african j. agric. res., 11(39): 3733-3740. wang y.t., 1992 hydrophilic polymer and wetting agent had limited effect on growth and postproduction performance of poinsettia. subtr. plant sci., 45: 32-35. impaginato 363 adv. hort. sci., 2018 32(3): 363-369 doi: 10.13128/ahs-22320 assessment of vase life and postharvest quality of cut rose (rosa hybrida cv. angelina) flowers by application of cumin (cuminum cyminum l.) essential oil and 8-hydroxyquinoline sulfate s.a. mirjalili 1 (*), b. kavoosi 2, y. peyro 3 1 imam khomeini higher education center, agriculture research, education and extension organization, teheran, iran. 2 fars agricultural research and education center, agriculture research, education and extension organization, teheran, iran. 3 faculty of agriculture, science and research branch, islamic azad university, tehran, iran. key words: cumin, essential oil, hydroxyquinoline sulfate, rosa hybrida. abstract: natural preservatives such as herbal essential oils have potential ability for extending postharvest vase life of cut flowers. in this study, application effect of cumin (cuminum cyminum l.) essential oil and 8-hydroxyquinoline sulfate on vase life and postharvest quality of cut rose (rosa hybrida cv. angelina) flowers were investigated. a factorial experiment with three levels of each in different time after harvesting was conducted. results showed that usage of different level of cumin essential oil and hydroxyquinoline sulfate had significant effects on rose attributes at the level of 0.05. the results showed that the interaction effect of cumin essential oil and hydroxyquinoline sulfate in measuring time was significant (p<0.05) on all of parameters except for anthocyanin content in rose petals in a way that the highest amount for measured traits was obtained with treatment of 150 mg l-1 cumin essential oil and 400 mg l-1 8-hydroxyquinoline sulfate. 1. introduction roses have a critical role in the manufacturing of various medicinal and nutritional products. rosa, known as the symbol of affection and elegance in iran, is one of the leading cut flower in global floriculture trade including our country (butt, 2005; zamani et al., 2011). the genus rosa belongs to the family rosaceae and includes 200 species and more than 18,000 cultivars (ahmad et al., 2013). cut flower trading is the prime purpose of rose cultivation, but short vase life is the most crucial problem. commercially, post-harvest longevity of cut flowers is of importance. many studies have therefore focused on its quality both in pre and postharvest periods (mirjalili, 2015). the most common physiological and morphological responses after harvesting are wilting or bent neck caused by pathogens (*) corresponding author: abmirjalili@gmail.com citation: mirjalili s.a., kavoosi b., peyro y., 2018 assessment of vase life and postharvest quality of cut rose (rosa hybrida cv. angelina) flowers by application of cumin (cuminum cyminum l.) essential oil and 8-hydroxyquinoline sulfate. adv. hort. sci., 32(3): 363-369 copyright: © 2018 mirjalili s.a., kavoosi b., peyro y. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 19 december 2017 accepted for publication 18 april 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(3): 363-369 364 especially bacteria, resulted in decreasing the vase life of cut rose flowers (leiv and hans, 2005; thwala et al., 2013). the development of such symptoms is resulted from vascular occlusion, mainly located in the basal stem end (lü et al., 2010; farahi et al., 2013). study on effects of natural plant products, including essential oils as preservatives hasted during last decades (elgimabi and ahmed, 2009). in nature, essential oils play an important role in the protection of the plants as antibacterial, antiviral, antifungal and insecticides (bakkali et al., 2008). cumin (cuminum cyminum l.) is an aromatic plant in the family apiaceae. cumin seeds are rich of essential oil especially cumin aldehyde, used as a stimulant as well as carminative and therapeutics (iacobellis et al., 2005; asghari marjanloo et al., 2009). there are reports on preservative effects of plant essential oils on other plants pathogens, such as tea essential oil on the botrytis in grape (jobling, 2000) and antifungal effect of persian thyme essential oil on strawberry (nabigol and morshedi, 2011). positive effects of plant essential oils have been reported on longevity of cut flowers’ vase life (deans and ritchie, 1987; dudai et al., 1999). thwala et al. (2013) used cumin essential oil for decreasing degradation and vessel boring in orchids resulted in delay of senescence. 8-hydroxyquinoline sulphate (8-hqs) as a very important germicide in preservatives is used in floral industry. hqs acts as an anti-microbial agent and increases water uptake (ali and hassan, 2014). the positive effect of 8-hqs and calcium chloride alone or in combination with 4% sucrose as chemical preservative solutions to improve postharvest quality of cut gerbera flowers has been shown (soad et al., 2011). it reported that hqs extended the vase life of rose cut flowers, whereas sucrose can promote the effect of hqs (ichimura et al., 1999). it documented that vase life and postharvest quality of different cut flowers were enhanced by 8-hqs treatment through improving water uptake, fresh weight and carbohydrate content (kim and lee, 2002; hassan et al., 2003; 2004; ali and hassan, 2014). despite the valuable reports on successful use of various phytochemicals for improving longevity of fresh cut flowers, screening for introducing and developing an exact, cheap and easy-to-use preservative is of importance for floriculture (wu et al., 2016). the objective of this study was to investigate the effect of different cumin essential oil concentrations and 8-hydroxyquinoline sulphate (8-hqs) on vase life and postharvest quality of cut rose flowers in different measuring time. 2. materials and methods cut rose (rosa hybrida cv. angelina) flowers were obtained from the commercial greenhouse around shiraz. cut rose flowers were harvested when florets were not opened but sepals were turned back and separated from petals during september 2014 and immediately transported to the laboratory. prior to insert in solutions, flowering stems of plants were cut under water to prevent air entrance into the xylem conduits that were opened by cutting. this factorial experiment was conducted in randomized complete blocks design with three replications. treatments were 8-hydroxyquinoline sulfate (8-hqs) at four level (0, 200, 400 and 600 mg·l-1) and cumin essential oil at three level (0, 100, 150 mg·l-1) in three measuring time (1st day, 8th day and 16th day) after treatment. after the duration of treatments, the flowers were placed in beakers containing 400 ml distilled water during the vase life evaluation period. the control flowers were kept in distilled water. replications included five flowers per treatment. vase life room conditions was 12 hours day length, 18±2°c, 60±5% rh and 12 μmol s-1 m-2 light intensity and measured traits were vase life determination (days), petal fresh weight/dry weight rate (%), flower diameter (mm), anthocyanin content (mg 100 g-1 f.w.), relative water content (rwc) (%), leakage of ions (%), catalase enzyme activity (cat) (ua·mg-1 pro), peroxidase enzyme activity (pod) (ua·mg-1 pro), membrane stability index (msi) (%). vase life determination in this study, vase life was considered as the time during which cut-flower can keep its marketability quality and before senescence symptoms including bending of petal margins and wilting are appeared (singh, 1994). cut-flower durability was evaluated from cut flower treatment till their ornamental value has disappeared. leakage of ions floret samples from each treatment were taken on first day and were repeated on day 7 for determining ions leakage by using the method of sairam et al. (1997). two florets samples (0.2 g) were taken and placed in 20 ml of double distilled water in two different 50 ml flasks. the first one was kept at 40°c for 30 min while the second one was kept at 100°c in boiling water bath for 15 min. the electric conductivity of the first (c1) and second (c2) samples were measured with a conductivity meter. the leakage of ions was expressed as the membrane stability index according mirjalili et al. vase life and postharvest quality of cv. angelina cut rose 365 to the following formula (ezhilmathi et al., 2007): membrane stability index (msi)=[1-(c1/c2)] × 100 (eq. 1) petal anthocyanin the amount of 200 mg petal samples was pulverized in 3 ml 99:1 (v/v) methanol and hydrochloric acid and obtained extracts were centrifuged at 12000 rpm for 20 min at 4°c. supernatants were kept in 4°c and under darkness condition for 24 h. after that, light absorption was estimated by spectrophotomet e r i n 5 5 0 n m w a v e l e n g t h a n d u s i n g s i l e n c e coefficient (ɛ =33000 mol2 cm-1) (krizek et al., 1993). petal membrane stability index for determining petal membrane stability, two samples of petals each including 200 mg of each replication were weighted and dipped in 10 ml double distilled water. one of them was placed in 40°c b e n m a r y f o r 3 0 m i n a n d s e c o n d o n e a t 1 0 0 ° c benmary for 15 min. after reaching to the room temperature, electrical conductivity of the solutions was measured with a ec meter and the stability percent o f t h e m e m b r a n e w a s d e t e r m i n e d a c c o r d i n g ezhilmathi et al. (2007), as equation 1. enzymes assays peroxidase (pod) enzyme was extracted from 200 mg homogenized samples in 25 mm na-phosphate buffer (ph 6.8) followed by centrifugation at 12000 rpm for 30 min at 4°c. for assay, a mixture consisting of 25 mm na-phosphate buffer (ph 6.1), 28 mm guaiacol, 5 mm hydrogen peroxide and crude extract was prepared and its absorbance at 470 nm was detected during 1 min, using spectrophotometer (bio-rad). enzyme activity was expressed as absorption delta of 470 nm per mg protein (chance and maehly, 1995). catalase (cat) enzyme was extracted from 200 mg samples homogenized in 25 mm na-phosphate buffer (ph 6.8) followed by centrifugation at 12000 rpm for 30 min at 4°c. the supernatant was transferred to 15 ml tubes and referred to enzyme extract. for assay, a mixture consisting of 25 mm na-phosphate buffer (ph 6.1), 10 mm hydrogen peroxide and crude extract was prepared and its absorbance at 240 nm was detected using a spectrophotometer (bio-rad). enzyme activity was described by measuring the conversion rate of hydrogen peroxide to water and oxygen molecules, as the decrease of absorbance per time per mg of protein (8). enzyme activity was expressed as absorption delta of 240 nm per mg protein. all steps of enzyme extraction were performed on ice. cumin essential oil and 8-hydroxyquinoline sulphate (8-hqs) were purchased from z a r d b a n d p h a r m a c e u t i c a l s m e d i c i n a l p l a n t s production co., yasuj, iran and were used. statistical analysis all data were analyzed for significant differences using analysis of variance (anova) using the sas (statistical analysis system) statistical package (sas institute, cary, nc, usa). data were then subjected to mean separation by the least significant difference test (lsd) at p<0.05. 3. results according to results of variance analysis, interaction effects of cumin essential oil (ceo), 8-hydroxyquinoline sulfate (hqs) application and measuring times was significant (p<0.05) on measured traits of vase life, petal fresh/dry weight rate, flower diameter, relative water content (rwc), leakage of ions, catalase enzyme activity (cat), peroxidase enzyme activity (pod), membrane stability index (msi) except for anthocyanin content. interaction effect of hqs and measuring times was insignificant on anthocyanin content too, while main effects of each factor and interaction effects of ceo × hqs and hqs × t were significant (p<0.05) (table 1). s.o.v df mean squares vase life petal dry weight flower diameter anthocyanin content relative water content leakage of ions cat pod msi ceo 2 16.02 * 0.52 * 2.686 * 0.0020 * 17.33 * 37.31* 26.45 * 23.30 * 69.35 * hqs 3 45.99 * 0.30 * 1.542 * 0.0016 * 48.76 * 44.97* 51.20 * 54.21 * 47.07 * time 2 11.33 * 0.78 * 1.033 * 0.0011 * 93.32 * 52.47 ns 21.30 * 23.22 ns 99.33 * ceo×hqs 6 24.35 * 0.89 * 2.037 * 0.0034 * 58.25 * 41.25 * 35.15 * 35.81 * 49.99 * ceo×t 4 20.46 * 0.55 * 2.432 * 0.0037 * 48.39 * 39.56 * 28.14 * 38.92 * 58.23 * hqs×t 6 21.32 * 0.53 * 3.321 * 0.0061 ns 59.41 * 45.81 * 32.18 * 40.25 * 63.28 * ceo×hqs×t 12 25.41 * 0.24 * 1.421 * 0.0061 ns 39.99 * 21.34 * 45.23 * 39.48 * 48.49 * table 1 analysis of variance for measured traits in cut rose (rosa hybrida cv. angelina) flowers treated by cumin (cuminum cyminum l.) essential oil and 8-hydroxyquinoline sulfate in different measuring times *,**, shows significant differences at 5%, 1%, respectively. ns= not significant. ceo = cumin essential oil. hqs= 8-hydroxyquinoline sulfate adv. hort. sci., 2018 32(3): 363-369 366 concerning the mean comparison, the maximum vase life was obtained by application of 100 mg·l-1 cumin essential oil and 600 mg·l-1 8-hydroxyquinoline s u l f a t e . h o w e v e r , t h e m i n i m u m v a s e l i f e w a s observed in control treatments (fig. 1). the greatest petal fresh/dry weight rate was evident in the treatment of 150 mg·l-1 cumin essential oil and 400 mg·l-1 8-hydroxyquinoline sulfate and the least with control treatments (fig. 2). the results indicated that the highest flower diameter was found in 150 mg·l-1 cumin essential oil and 400 mg·l-1 8-hydroxyquinoline sulfate and the lowest diameter in control treatments (fig. 3). relative water content showed the maximum and minimum value in 150 mg·l-1 cumin essential oil and 400 mg·l-1 8-hydroxyquinoline sulfate and control treatment, respectively (fig. 4). the greatest amount of ions leakage in control treatments and the lowest amount in 150 mg·l-1 cumin essential oil and 400 mg·l-1 8-hydroxyquinoline sulfate were found (fig. 5). according to results of mean comparison, the highest catalase enzyme activity was attained in 150 mg·l-1 cumin essential oil and 400 mg· l-1 8-hydroxyquinoline sulfate, while the lowest of that was reported in control treatments (fig. 6). the greatest peroxidase enzyme activity was observed in 150 mg·l-1 cumin essential oil and 400 mg·l-1 8hydroxyquinoline sulfate and the least activity in control treatments (fig. 7). according to the obtained results, the highest membrane stability index was obtained in 150 mg·l-1 cumin essential oil and 400 mg l-1 8-hydroxyquinoline sulfate (fig. 8). fig. 1 changes of vase life under different levels of cumin essential oil (ceo) and 8-hydroxyquinoline sulfate (hqs). fig. 2 mean comparison for interaction effects of cumin essential oil (ceo) and 8-hydroxyquinoline sulfate (hqs) different levels on petal dry weight (g) in different measuring times. fig. 3 mean comparison for interaction effects of cumin essential oil (ceo) and 8-hydroxyquinoline sulfate (hqs) different levels on flower diameter (mm) in different measuring times (days). fig. 4 mean comparison for interaction effects of cumin essential oil (ceo) and 8-hydroxyquinoline sulfate (hqs) different levels on relative water content (rwc) (%) in different measuring times (days). fig. 5 mean comparison for interaction effects of cumin essential oil (ceo) and 8-hydroxyquinoline sulfate (hqs) different levels on leakage of ions (%) in different measuring times (days). mirjalili et al. vase life and postharvest quality of cv. angelina cut rose 367 quality of cut rose (rosa hybrid cv. angelina) flowers in different measuring time. application of 100 mg·l-1 cumin essential oil and 600 mg·l-1 8-hydroxyquinoline sulfate increased vase life of cut rose flowers. this result was in accordance with results of hussein (1994) and knee (2000). the application of 8-hqs may prevent the accumulation of microorganisms in xylem vessels and suppressed the xylem occlusion due to its role as anti-microbial agent and hence, it might reduce stem plugging. essential oils like ceo play an important role in the protection of the plants as antibacterial, antiviral, antifungal, insecticides and also against herbivores by reducing their appétit for such plants (bakkali et al., 2008). petal fresh/dry weight rate was improved significantly by application of 100 mg·l-1 cumin essential oil and 600 mg.l-1 8hydroxyquinoline sulfate. these results are in line with results of ali and hassan (2014) on strelitzia cut flowers with application of 8-hydroxyquinoline sulfate and gibberlic acid treatments. the application of 8-hqs may reduce the plasmolysis of cells which occurred when the rate of cellular water loss is too rapid. the cut rose flowers reached to the highest diameter with application of 100 mg· l-1 cumin essential oil and 600 mg·l-1 8-hydroxyquinoline sulfate. these findings are in according to reports of kim and lee (2002). hqs not only prevents the vascular obstruction caused by the microorganisms, but also prevents the blockage stimulated by the plant itself. the highest relative water content in cut rose flowers was related to treatment of 150 mg·l-1 cumin essential oil and 400 mg·l-1 8-hydroxyquinoline sulfate. these results are similar to knee (2000) findings on cut carnation flowers. leakage of ions was o c c u r r e d i n c o n t r o l t r e a t m e n t s i n t h e h i g h e s t amount. essential oil of cumin mainly conjugated to compounds that have known as phenolic compounds, are responsible for pathogen control in plants (plotto et al., 2003). these compounds prevent senescence and wilting by their antibacterial property and reducing the ph of the environment (elgimabi and ahmed, 2009). catalase and peroxidase enzymes activities increased significantly by treatments of 150 mg·l-1 cumin essential oil and 400 mg· l-1 8-hydroxy quinoline sulfate. these results are consistent with results of ranjbar et al., 2015. catalase is an important biological factor with major function in superoxide metabolism and plays an important role in releasing oxygen and hydrogen peroxide free radicals and prevents creation of hydroxyl radicals (spanou et al., 2012). peroxidase has different biologfig. 6 mean comparison for interaction effects of cumin essential oil (ceo) and 8-hydroxyquinoline sulfate (hqs) different levels on catalase enzyme activity (cat) (ua mg-1 pro) in different measuring times (days). fig. 7 mean comparison for interaction effects of cumin essential oil (ceo) and 8-hydroxyquinoline sulfate (hqs) different levels on peroxidase enzyme activity (pod) (ua mg-1 pro) in different measuring times (days). fig. 8 mean comparison for interaction effects of cumin essential oil (ceo) and 8-hydroxyquinoline sulfate (hqs) different levels on membrane stability index (msi) (5) in different measuring times (days). 4. discussion and conclusions the results showed that the application of cumin essential oil (ceo) and 8-hydroxyquinoline sulfate (hqs) had positive effect on vase life and postharvest 368 adv. hort. sci., 2018 32(3): 363-369 ical functions such as detoxification of hydrogen peroxide, lignin biosynthesis, hormonal signaling and response to stress (gao et al., 2010). maybe the treatment of 150 mg·l-1 cumin essential oil and 400 mg·l-1 8-hydroxyquinoline sulfate decreases oxidative stresses in cut rose flowers (hassan and ali, 2014). membrane stability index showed the highest percent in treatment of 150 mg·l-1 cumin essential oil and 400 mg·l-1 8-hydroxyquinoline sulfate. these findings are compatible to results of kazemi and ameri (2012). they showed the positive effect of herbal essential oils of thyme and lavender on the stability of the membrane and reduction of mda. the senescence of cut flowers with hormonal regulatory mechanism is involved in changing the physical and b i o c h e m i c a l f e a t u r e s o f c e l l u l a r m e m b r a n e (buchanan wollaston, 1997). oxidative membrane injury allows the mixing of the normally separated enzyme (ppo) and oxidizable substrates (polyphenols), which lead to browning (hodges, 2003). according to palma et al. (2002), the herbal essential oils by preventing the activity of oxygen species reduce the lipid peroxidation in cell membrane and the concentration of mda. plant essential oils are bioactive in the vapor phase, and this makes them fumigants for postharvest rotting fungi control in fruits and grains (paster et al., 1995; hammer et al., 1999; feng and zheng, 2007). different studies showed postharvest disease control in different fruit species by using biological agents including essential oils (bishop and thompdon, 1997; feng and zheng, 2007; amiri et al., 2008). a limiting factor in cut flower marketing is postharvest senescence. there are many reports used different materials for extending rose cut flower vase life. we studied application of cumin essential oil and 8hydroxyquinoline sulfate. they had positive effects (p<0.05) on vase life and postharvest quality of cut rose (rosa hybrida cv. angelina) flowers. results showed they affect some growth and development parameters such as relative fresh weight, flower and stem diameters, anthocyanin and chlorophyll contents as well catalase and peroxidase activities that cause improving vase life of rose cut flowers. acknowledgements t h e r e s e a r c h w a s f u n d e d b y h o r t i c u l t u r e department of agriculture faculty of islamic azad university in yasuj, iran. references ahmad i., aslam khan m., qasim m., ahmad r., ussamad t., 2013 growth, yield and quality of rosa hybrid l. as influence by nacl salinity. j. ornam. plant, 3(3): 143-153. ali e., hassan f., 2014 postharvest quality of strelitzia reginae ait. cut flowers in relation to 8-hydroxyquinoline sulfate and gibberelic acid treatments. scient. agri., 5(3): 97-102. amiri a., dugas r., pichot a.l., bompeix g., 2008 in vitro activity of eugenol oil (eugenia caryophylata) against four important postharvest apple pathogens. inter. j. food micr., 126: 13-19. asghari marjanlo a., mostofi y., shoeibi s.h., fattahi m., 2009 effect of cumin essential oil on 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glutamin. world appl. sci. j., 12(9): 1621-1624. impaginato 343 adv. hort. sci., 2018 32(3): 343-351 doi: 10.13128/ahs-22380 water spectral pattern as a marker for studying apple sensory texture m. vanoli 1 (*), f. lovati 1, m. grassi 1, m. buccheri 1, a. zanella 2, t.m.p. cattaneo 1, a. rizzolo 1 1 consiglio per la ricerca in agricoltura e l’analisi dell’economia agraria, centro di ricerca ingegneria e trasformazioni agroalimentari (creait), via venezian, 26, 20133 milano, italy. 2 centro di sperimentazione laimburg, laimburg 6, 39040 ora (bz), italy. key words: aquaphotomics, crispness, juiciness, malus x domestica borkh, mealiness, nir. abstract: aquaphotomics is a scientific discipline which investigates the waterlight interactions in biological systems by using nir spectroscopy and multivariate analysis to relate water absorption patterns to bio-functionalities. this work aimed at evaluating the feasibility of aquaphotomics to study apple fruit sensory texture. ‘braeburn’, ‘gala’ and ‘kanzi®’ apples were analyzed by a micronir spectrometer and for mechanical, structural and texture sensory characteristics. cluster analysis on sensory texture attributes showed four different profiles for each cultivar having different water spectral patterns ( w a s p ) . o n a v e r a g e , t h e w a s p o f m e a l y a p p l e s s h o w e d t h e h i g h e s t absorbance values at 1364, 1372 and 1382 nm and the lowest in the 1438-1492 nm range suggesting a preponderance of water structures with weak-hydrogen bonds; the opposite was found in crispy and juicy apples indicating the presence of more organized water structures with medium-strong hydrogen bonds. this wasp difference could be due to a different softening rate: apples clustered as firm/crispy/juicy had the highest firmness and the lowest intercellular spaces, while mealy apples had low firmness and high intercellular spaces indicating a more advanced softening. the chemical changes due to the pectin hydrolyzation could affect the water structures. the aquaphotomics approach could be a useful tool for studying the sensory texture of fruits as water structures actually change in apples with different textural characteristics whatever the cultivars. 1. introduction the preferences of apple consumers are generally based on a combination of texture and flavor (harker et al., 2003). textural properties can be considered as the main factors responsible for fruit freshness, having crunchiness and juiciness a positive effect and mealiness a negative effect on consumer choice (péneau et al., 2006). texture consists of a number of different properties perceived by human senses and depends on mechanical and structural characteristics of the fruit pulp. texture changes with (*) corresponding author: maristella.vanoli@crea.gov.it citation: vanoli m., lovati f., grassi m., buccheri m., zanella a., cattaneo t.m.p., rizzolo a., 2018 water spectral pattern as a marker for studying apple sensory texture. adv. hort. sci., 32(3): 343-351 copyright: © 2018 vanoli m., lovati f., grassi m., buccheri m., zanella a., cattaneo t.m.p., rizzolo a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 21 december 2017 accepted for publication 17 may 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(3): 343-351 344 fruit softening due to degradation and modifications o f t h e c e l l w a l l a n d m i d d l e l a m e l l a s t r u c t u r e s (gwanpua et al., 2014). during softening, pectins undergo extensive structural modifications so that cell wall polymers are less bound together and become highly hydrated (goulao and oliveira, 2008). the hydrolysis of pectin requires water as substrate and occurs in the intercellular water (de smedt et al., 2002). all these changes influence the organization of water molecules (free water, dimers, trimers, solution shells) in the fruit tissue depending on the strength of hydrogen bonding, and produce changes in the water absorbance pattern. thus, when an aqueous system, such as apple fruit, is measured by n e a r i n f r a r e d ( n i r ) s p e c t r o s c o p y , t h e l i g h t absorbance reflects the water vibrations and the contribution (concentration and structure) of the other molecules interacting with water. tsenkova (2009) proposed the aquaphotomics approach to relate water absorption patterns to bio-functionalities. in a series of experiments, it was found that vis-nir spectra acquired in living systems under various perturbations (temperature, ion concentrations, oxidative stress, illumination, disease, damage, etc) are characterized by twelve common water bands repeatedly occurring in different combinations in the spectral regression or classification models predicting the investigated perturbations. these water absorption bands (6-20 nm width, each) called water matrix coordinates (wamacs) corresponded to 1344 (c1), 1364 (c2), 1372 (c3), 1382 (c4), 1398 (c5), 1410 (c6), 1438 (c7), 1444 (c8), 1464 (c9), 1474 (c10), 1492 (c11) and 1518 (c12) nm (tsenkova, 2009). the variation of wamacs defines the water spectral pattern (wasp) which can be visualized by star-charts called ‘aquagrams’ (tsenkova, 2010). changes in water absorbance pattern have been used as biomarkers to monitor water quality (kovacs et al., 2016), to reveal the presence of bioactive compounds from propolis in smart packaging materials (barzaghi et al., 2017), to detect honey adulteration with high fructose corn syrup (bázár et al., 2016), to distinguish the effects of various coatings on the quality of different types of cheese and of winter melon (cattaneo et al., 2016). aquaphotomics has also been shown to be a useful tool for discriminating fresh and stored apples and for distinguishing apples stored under different atmospheres. differences between fresh and stored apples involved c5, c7, c9 and c12 water matrix coordinates, while differences between apples stored in normal and in controlled atmosphere activate the c 8 a n d c 1 0 w a m a c s ( b a r z a g h i e t a l . , 2 0 1 4 ) . significant relationships were found between vis/nir wavelengths and some sensory attributes of apples, involving water absorption bands. absorption measured at 720, 1440, and 2338 nm was positively correlated and at 1940 nm negatively related to mealiness, while the opposite was found for crunchiness and chewiness. the absorbance in the 630-700 nm range and at 1940 nm was positively correlated with juiciness, while negative correlations were found at 940 and 1450 nm (mehinagic et al., 2004). the aim of this work was to evaluate the feasibility of aquaphotomics to study the texture sensory profiles of apple fruit belonging to three cultivars having different texture characteristics. 2. materials and methods fruit ‘braeburn’ apples (808 fruits) picked in 2014, ‘gala’ apples (270 fruits) picked in 2015 and ‘kanzi®’ apples picked in 2014 (540 fruits) and in 2015 (270 fruits) at the experimental orchard of laimburg (bz) were stored for 4 (‘gala’) or 6 months (‘braeburn’ and ‘kanzi®’) under controlled atmosphere (‘gala’: 1% o2, 1.5% co2; ‘braeburn’: 1.8% o2, 1.3% co2; ‘kanzi®’: 1% o2, 1.5% co2) and analyzed after 1 and 7 days of shelf life at 20°c (‘kanzi®’2014) or after 1, 7 a n d 1 4 d a y s a t 2 0 ° c ( ‘ b r a e b u r n ’ , ‘ g a l a ’ , a n d ‘kanzi®’2015). all fruits of the experiment were individually measured by a micronir spectrometer and individually analyzed for texture sensory properties (firmness, crispness, mealiness, juiciness), mechanical characteristics and relative internal space volume (risv). sensory analysis sensory analysis was carried out separately per cultivar according to eccher zerbini et al. (1999) and rizzolo et al. (2010). each sensory session corresponded to a time of analysis, that was 9 sampling times for ‘braeburn’, ‘gala’ and ‘kanzi®’2015 apples and 6 sampling times for ‘kanzi®’2014 apples. as for ‘braeburn’, ‘gala’ and ‘kanzi®’2015 apples, in each session 9 fruits were presented to each panelist, while for ‘kanzi®’2014 apples 3 fruits per panelist were used. when 9 fruits were presented to each panelist, two sensory sessions were scheduled, the first with 5 fruits and the second with 4 fruits, at 10:30 and at 12:00 am, respectively. a total of 810 (‘braeburn’), 540 (‘kanzi®’ 2014) and 270 (‘gala’, ‘kanzi®’ 2015) apples were tested. the sensory tests vanoli et al. water spectral pattern and sensory texture 345 were performed by a panel of ten short-term-trained judges from research centre for engineering and agro-food processing of the council for agricultural research and economics (crea-it, milano), who had participated in prior studies on sensory evaluation of apples, in a sensory lab equipped with six computerized individual booths under white artificial lighting at room temperature (20°c). the fizz-network 2.47b (biosystemes, couternon, france) software was used for test implementation and results collection. fruits (one peeled slice/apple) were presented to the judges at most 1 h after the cut to avoid the browning process, coded with three-digit random numbers and placed on a white flat-bottomed dish in randomized order for each assessor. at the beginning of each session, a peeled slice of a fruit not included in the experimental plan was tasted to eliminate the first tasting effect. drinking water was provided as a palate cleaner between samples. as the quality of consumer perception is influenced by extrinsic attributes (e.g., price, dimension, size, origin, ripening stage), none of these attributes were mentioned to the judges (taiti et al., 2017). each sample was evaluated for the intensity of four sensory attributes related to fruit structure: firm, crispy, mealy and juicy. evaluation was based on a form with a continuous open linear scale, consisting of 40 characters, each panelist rated the intensity of each attribute on the open linear scale anchored to 0 (no presence) and 120 (maximum intensity). definitions of the sensory attributes are as follows: firm, the resistance to mastication perceived at the first and successive bites; crispy, the textural property perceived at the first bite when the fruit yields suddenly with a characteristic sound; mealy, the textural property consisting of the presence of lumps formed during mastication; juicy, the textural property giving the sensation of progressive increase in the free fluids in the oral cavity during mastication (eccher zerbini et al., 1999). prior to statistical analyses, the rating scores of each attribute were standardized by panelist in order to remove the variability due to panelists using different parts of the scale according to bianchi et al. (2009). mechanical properties and relative internal space volume individual fruit were analyzed for flesh firmness, compression and intercellular spaces (risv). flesh firmness and compression were measured on two opposite sides of each fruit (the blush side and the opposite one) in the equatorial region and data were averaged per fruit. firmness was measured with an 11 mm diameter cylindrical plunger mounted on a ta-xt plus texture analyzer (stable micro systems, godalming, uk) at the cross-head speed of 3.33 mm/s to a depth of 8 mm. mechanical properties were also nondestructively assessed by a uniaxial compression test. in this test, each apple was compressed between two steel parallel plates to a fixed deformation of 1 mm at a speed of 25 mm/min on an instron universal testing machine and the modulus of deformability (ed) was computed according to eccher zerbini (1981): ed = f 1-µ2 (d l /2)3/2 d1/2 where f is the force at 1 mm of compression (n), dl is the total deformation (mm), d is the fruit diameter (mm); µ is the poisson’s ratio. as poisson’s ratio was not measured in this work, it was fixed at a value equal to 0.3 (ahmadi et al., 2016). relative internal space volume (risv) was computed according to risv=100×[1−(d f −d j )] where d f is the density of the fruit (i.e. ratio fruit mass to fruit volume under water), and d j is the density of the fruit juice (baumann and henze, 1983). nir analysis nir spectra were acquired on each intact fruit in reflectance mode using a diode array spectrometer (micronir 1700 viavi, diessechem instrument, italy) over the 900 to 1670 nm range (50 scans, 128 reading points) on two opposite sides of each fruit (the blush side and the opposite one) in the equatorial region and data were averaged per fruit. the spectra were pretreated using multiplicative scatter correction (msc) to remove scatter effects, followed by savitsky-golay second derivative (15 points, secondorder polynomial) after converting the spectra from r e f l e c t a n c e t o a b s o r b a n c e ( t h e u n s c r a m b l e r software, ver. 10.0.1, camo process as, norway). aquagrams were built using the 12 characteristic water absorption wavelengths (1344, 1364, 1372, 1382, 1398, 1410, 1438, 1444, 1464, 1474, 1492 and 1518 nm) within each cluster sensory profile of each cultivar (see sensory analysis paragraph). the values of the aquagrams (aq) were obtained according to: aqλ = aλ µλ σλ where aλ is absorbance, µλ is the mean value of all adv. hort. sci., 2018 32(3): 343-351 346 spectra and σλ is the standard deviation of all spectra at wavelength λ (cattaneo et al., 2016). aquagrams were calculated using ms excel 2010 (microsoft, usa). the aquagram displays normalized absorbance values from different sample groups at several water bands on the axes originating from the center of the graph. absorbance values at the wamacs were placed on the respective radial axes. statistical analysis risv, mechanical and texture sensory data were submitted to analysis of variance (anova) considering apple cultivar as factor and means were compared by bonferroni’s test at p≤0.05. data of texture sensory attributes were also analyzed by using an agglomerative hierarchical clustering of observations. ward’s clustering method and squared euclidean distance were applied to create four data sets having distinctive sensory texture profiles. to form the clusters, the procedure begins with each observation in a separate group and then combines the two observations which are closer together to form a new group. after re-computing the distance between groups, the two groups then closest together are combined. this process is repeated until only the n fixed groups remained. as cultivars had different texture characteristics, cluster analysis on sensory texture attributes was carried out within each cultivar and year. data of firmness, compression and risv were submitted to anova considering sensory cluster as factor and means were compared by bonferroni’s test at p≤0.05. all the statistical analyses were performed using statgraphics version 7 (manugistic inc., rockville, md, usa) software package. 3. results mechanical and sensory analyses showed that the three cultivars had different texture characteristics (tables 1 and 2). ‘braeburn’ apples had very low firmness, intermediate risv and the highest ed, along with the lowest scores for firm, juicy and crispy and the highest scores for mealy. ‘gala’ apples had the highest risv, the lowest ed and firmness, showing intermediate scores for juicy, crispy and mealy . ‘kanzi®’ fruit had the highest firmness, the lowest risv, intermediate ed and were perceived as the most firm, juicy and crispy and the least mealy; kanzi®’2015 showed lower firmness and ed, higher risv and higher scores for mealy than ‘kanzi®’2014. from an exploratory analysis carried out by dividing the standard score of each texture sensory attribute into five arbitrary classes according to: very low (<20), low (21-39), medium (40-59), high (60-80) and very high (>80) intensity of the attribute, and by pairing the classes of all attributes for every fruit, it was found that the minimum number of combinations of intensity classes between attributes was four. so clustering analysis was applied with the aim of creating the four data sets having distinctive texture sensory profiles, according to the descriptions and centroid values reported in table 3. profile w1 corresponded to a very firm, juicy and crispy texture for ‘kanzi®’ apples, and to a very firm/juicy texture with a medium crispness for ‘braeburn’ and ‘gala’. profile w2 of ‘kanzi®’ apples was very similar to profile w1 of ‘braeburn’ and ‘gala’ apples, while that of ‘gala’ and ‘braeburn’ was characterized by a medium juicy and crispy texture, with low scores for mealiness in ‘braeburn’ fruit. profile w3 of ‘kanzi®’ apples showed a soft (2014) or a medium texture (2015) w i t h o u t m e a l i n e s s , w h i l e t h a t o f ‘ g a l a ’ a n d ‘braeburn’ was quite firm with low (‘gala’) or medium scores (‘braeburn’) for mealiness. profile w4 was characterized by a soft, dry and mealy texture with h i g h s c o r e s f o r m e a l i n e s s i n ‘ b r a e b u r n ’ a n d ‘kanzi®’2014. considering the cluster distribution in relation to storage time and shelf life period, profile w1 was found in 65% of fruit at harvest and at storcultivar firmness (n) ed (n/mm2) risv (%) 'braeburn' 56.2 bc 10.5 a 17.6 b 'gala' 55.2 c 6.5 d 19.6 a 'kanzi®'2014 61.5 a 9.8 b 15.0 d 'kanzi®'2015 57.6 b 7.5 c 15.9 c table 1 mechanical properties and intercellular spaces (risv) of 'braeburn', 'gala' and ' kanzi®' apples means in the same column followed by different letters are statistically different at p≤0.05 (bonferroni's test). table 2 mean scores of texture sensory attributes of 'braeburn', 'gala' and 'kanzi®' apples means in the same column followed by different letters are statistically different at p≤0.05 (bonferroni's test). cultivar firm juicy mealy crispy 'braeburn' 47.0 b 40.9 c 46.2 a 33.3 c 'gala' 55.0 a 50.0 b 29.0 b 40.0 b 'kanzi®'2014 57.2 a 62.0 a 18.1 d 53.4 a 'kanzi®'2015 59.5 a 59.4 a 24.5 c 51.5 a vanoli et al. water spectral pattern and sensory texture 347 age removal in ‘braeburn’ and in ‘gala’, and in 84% of ‘kanzi®’ apples; profile w2 of the three cultivars and profile w3 of ‘kanzi®’2014 were equally distributed among the storage times and the days of shelf life; p r o f i l e w 3 w a s f o u n d i n 7 5 % o f ‘ k a n z i ® ’ 2 0 1 5 , ‘braeburn’ and ‘gala’ apples kept at 20°c; profile w4 was characteristic of fruit held for 14 days at 20°c and was found in 75% of ‘kanzi®’2015, 80% of ‘gala’ and 90% of ‘braeburn’ apples. each cluster showed different mechanical properties and intercellular spaces (fig. 1). firmness significantly decreased from w1 to w2, w3 and w4 profiles in ‘braeburn’ and in ‘kanzi®’ 2014 and 2015 apples, while in ‘gala’ had t h e s a m e v a l u e s i n w 1 a n d w 2 p r o f i l e s a n d decreased in w3 and w4, where showed the lowest values. risv significantly increased from w1 to w2, w3 and w4 profiles in braeburn and in ‘kanzi®’ 2015, while in ‘gala’ had the same values in w1 and w2 and increased in w4; in ‘kanzi®’ 2014, risv showed the lowest values in w1 and the highest in w3 and w4. ed distinguished only profile w1 from the other ones, except for ‘kanzi®’2014 (fig. 1). nir spectra of the three cultivars (fig. 2) show high variability in the 1344-1518 nm range corresponding to the first water overtone considered in the aquaphotomics approach. aquagrams showed a different water organization according to the different texture sensory profiles and to the cultivars (fig. 3). ‘braeburn’ apples table 3 texture sensory profiles of the four clusters within each cultivar cluster number and texture sensory profile firm juicy mealy crispy % obs 'braeburn' w1 very firm/juicy, medium crispy, not mealy 66.0 59.8 21.2 52.3 26.7 w2 medium firm/juicy, quite crispy/mealy 52.4 40.7 35.4 37.3 23.9 w3 quite firm/juicy/crispy, mealy 38.5 36.6 55.5 24.1 29.0 w4 very soft, not juicy, not crispy, very mealy 28.0 22.4 78.2 16.9 20.4 'gala' w1 very firm/juicy, medium crispy, not mealy 75.5 67.9 19.2 58.6 31.9 w2 very firm, medium juicy/crispy, not mealy 65.1 46.4 24.5 45.0 20.0 w3 medium firm/juicy, quite crispy/mealy 43.4 44.4 32.5 30.6 30.4 w4 very soft, not juicy, not crispy, mealy 26.7 31.6 45.7 17.1 17.8 'kanzi®'2014 w1 very firm/juicy/crispy, not mealy 75.5 85.3 7.0 82.7 32.3 w2 very firm/juicy, medium crispy, not mealy 61.6 64.5 17.0 54.7 34.8 w3 low firm/juicy/crispy, not mealy 34.1 35.9 16.8 24.1 25.1 w4 very soft, not juicy, not crispy, very mealy 35.5 37.8 73.8 20.8 7.8 'kanzi®'2015 w1 very firm/juicy/crispy, not mealy 80.2 78.5 15.2 74.2 23.3 w2 very firm/juicy, medium crispy, not mealy 67.2 61.7 20.0 55.6 36.7 w3 medium firm/juicy/crispy, not mealy 51.9 48.9 25.7 41.7 20.0 w4 very soft, not juicy, not crispy, mealy 28.8 43.4 42.3 27.5 20.0 for each cluster are reported: the description of the texture sensory profile, the values of centroids for each descriptor and the percentage of observations (% obs) grouped in the cluster. fig. 1 mechanical properties and intercellular spaces of ‘braeburn’, ‘gala’ and ‘kanzi®’ apples according to the texture sensory profiles described in table 3. 348 adv. hort. sci., 2018 32(3): 343-351 s h o w e d t h e g r e a t e s t c h a n g e s i n t h e w a t e r absorbance pattern (wasp) according to the different texture profiles, while ‘kanzi®’2014 the lowest ones. mealy apples belonging to ‘braeburn’ w3 and w4, ‘gala’ w4 and ‘kanzi®’2015 w4 profiles were associated to very high values in the 1364-1382 nm range and to very low values in the 1410-1518 nm range, while mealy apples belonging to ‘kanzi®’2014 w4 profile showed a balanced distribution of water structures with highest absorbance at 1344 nm and fig. 2 raw nir spectra of ‘braeburn’, ‘gala’ and ‘kanzi®’ apples. fig. 3 aquagrams of ‘braeburn’, ‘gala’ and ‘kanzi®’ apples according to the texture sensory profiles described in table 3. 349 vanoli et al. water spectral pattern and sensory texture lowest absorbance values at 1518 nm. in low mealy apples (‘braeburn’ w2, ‘gala’ w3) the absorbance decreased at 1344-1382 nm while increased at 14101492 nm comparing to mealy apples of the same cultivars. in contrast, the wasp of firm, juicy and crispy sensory profiles (‘kanzi®’2014 and 2015 w1 and w2, ‘gala’ w1) were characterized by a regular distribution of the different water structures with lower absorbance values at 1344-1382 nm and higher absorbance values at 1410-1492 nm comparing to the other textural profiles, while the wasp of ‘braeburn’ w1 showed the lowest values at 1344 and at 1364 nm and the highest in 1398-1438 nm range. 4. discussion and conclusions ‘braeburn’, ‘gala’ and ‘kanzi®’ apples showed different texture characteristics and were clustered according to the texture sensory attributes in four different profiles, ranging from a very firm/crispy/ juicy texture to a mealy or a very mealy one. each texture profile was characterized by specific mechanical and structural properties and showed different water spectral patterns. the differences in the textural profiles could be due to different softening rates occurring in apples. it is well known that fruit softening involves degradation and modifications of the cell wall and middle lamella structures, loss of turgor pressure, starch degradation and modification in the symplast/apoplast relations affecting the textural characteristics of the pulp: when cell-to-cell adhesion is weaker than the individual cell walls, cell separation occurs and the intact cells are responsible for the mealy texture, while when the individual cell walls are weaker than cell-to-cell adhesion, cell wall breakage occurs and the cellular content is released producing a juicy texture (goulao and oliveira, 2008; harker et al., 2002; vanoli et al., 2009). in this work it was found that apples clustered as very firm and firm had the highest firmness and the lowest risv, whereas apples clustered as mealy showed low firmness and high risv indicating a more advanced softening as reported by ting et al. (2013), vanoli et al. (2011) and rizzolo et al. (2016). moreover, mealy texture mainly belongs to apples held at 20°c for 7-14 days, that are fruits in which the softening process has already occurred, while the firm texture was typical of apples just picked or at storage removal, when softening was only at the beginning and apples had a rigid cellular structure with intact and adherent cell walls. the chemical changes occurring with softening also affected the water structures with a similar pattern for the three cultivars and for the two seasons in ‘kanzi®’ apples. the wasp of mealy apples were characterized by a preponderance of water structure with weak-hydrogen bonds (1364-1382 nm), whereas the wasp of crispy and juicy apples indicated the p r e s e n c e o f m o r e o r g a n i z e d w a t e r s t r u c t u r e s (dimers, trimers) with medium-strong hydrogen bonds (1410-1492 nm). peirs et al. (2005) found that when cell walls deteriorate, water molecules may relocate into the intercellular spaces, and this phenomenon changes the refractive indices at the cell walls. møller et al. (2013) reported that apples treated with 1-mcp clearly differed in water state and dynamics compared to untreated fruit, as water in the cytoplasm and extra-cellular compartments and water in the vacuole were less restricted in treated apples, suggesting that a high firmness is associated with a low amount of vacuole water. the involvement of the first overtone of the oh vibration and pectin metabolism was observed by boeriu et al. (1998) who found that the absorptions in the 14401445 nm range varied with the percentage of the degree of pectin esterification in green beans. similarly, sirisomboon et al. (2007) found a correlation between the absorption in the 1418-1464 nm range and the oxalate soluble pectin fractions in the ais of intact pears and between the absorptions at 1368 and at 1452 nm and the total pectin content in pear juice. barzaghi et al. (2014) observed that in stored apples the organization of water molecules involved more hydrogen-bonded water than in fresh fruit. this is contrary to our results; this difference could be due to the fact that in this work intact fruits were measured while barzaghi et al. (2014) measured apple slices. in conclusion, the aquaphotomics approach could be a useful tool and the water spectral pattern could be a marker for studying the texture sensory profiles in apple fruit as water structures actually change along with texture characteristics whatever the cultivar. furthermore, being aquaphotomics based on nir spectroscopy it could be possible to discriminate apples with different texture sensory properties in a nondestructive way. however, further studies are needed to better understand the relationships between the water spectral pattern and pectin metabolism and the different water structure organizations and the sensory profiles. 350 adv. hort. sci., 2018 32(3): 343-351 acknowledgements the present work was carried out in the framework of the project ‘monitoring key environmental parameters in the alpine environment involving science, technology and application’ (monalisa), funded by the autonomous province of bolzano (italy). references ahmadi a., 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a., 2011 time-resolved reflectance spectroscopy nondestructively reveals structural changes in ‘pink lady®’ apples during storage. procedia food science, 1: 81-89. impaginato 471 adv. hort. sci., 2018 32(4): 471-477 doi: 10.13128/ahs-21989 essential oil production of murraya paniculata (l.) jack at different harvest times c.i.m. semarayani, s.a. aziz (*), m. melati department of agronomy and horticulture, faculty of agriculture, bogor agricultural university, 16680 bogor, indonesia. key words: β-methylesculetin, caryophyllene, murralongin, solvent extraction. abstract: murraya paniculata (l.) jack has a fragrant flower, from which the fragrance is due to the essential oil. the study aimed to investigate the production of essential oil and its chemical compounds at different harvest times. the research was conducted at an organic experimental farm, bogor agricultural university, bogor, indonesia (6°30’-6°45’ s, 106°30’-106°45’ e) from october 2016 to february 2017 using randomized complete block design. the experiment consisted of one factor, namely the harvest times, comprised of harvest at 05.00-07.00 and 07.00-09.00 a.m. m. paniculata flowers were collected at three different flower ages, comprised of two days before anthesis, one day before anthesis and the day of anthesis (blooming). the different flower ages indicated by the flower size. ethanol extraction method was used to extract the essential oil of the flowers from different harvesting times and then chemical compounds were analyzed by gas chromatography-mass spectrometry. the result showed that flower number and weight were not affected by harvesting times. the flower collected on the day of anthesis had the highest flower number and weight. harvesting flowers at anthesis can be done at 05.00-09.00 a.m. the highest quantity and quality of essential oils were obtained by harvesting the flowers at anthesis. β-methylesculetin and murralongin were the primary compounds in m. paniculata flowers that harvested at 05.00-09.00 a.m. 1. introduction murraya paniculata (l.) jack well known as orange jessamine is an ornamental plant and belongs to family rutaceae (shah et al., 2014), it has white flowers with sweet fragrance (gilman, 1999). the plants are native to southeastern asia, i.e. cambodia, laos, myanmar, thailand, vietnam, indonesia, malaysia, and philippine (dosoky et al., 2016). m. paniculata has been used in traditional medicine because the plant has anti-amnesic, anti-inflammatory, anti-diabetic, anti-fungal, anti-bacterial, anti-helminthic, anti-cancer, and anti-oxidative properties (sharma and arora, 2015). beside as a source for perfumery, m. paniculata is also used as a source of flavors (el-sakhawy et al., 1998) because the flowers are highly aromatic and contain sufficient amount of essential oil (naseem et al., 2015). plants essential oils are aromatic components that composed (*) corresponding author: sandra.a.aziz@gmail.com citation: semarayani c.i.m., aziz s.a., melati m., 2018 essential oil production of murraya paniculata (l.) jack at different harvest times. adv. hort. sci., 32(4): 471-477 copyright: © 2018 semarayani c.i.m., aziz s.a., melati m. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 3 november 2017 accepted for publication 18 may 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(4): 471-477 472 of different chemical compounds (younis et al., 2011). different compositions of chemical compound of m. paniculata essential oils have been studied. rout et al. (2007) found that pentane extraction was the best method to obtain the highest amount of essential oil of m. paniculata flowers in india. this study also found that manool and (e)-nerolidol were the major component of essential oil (rout et al., 2007). the chemical compounds of m. paniculata flowers can also be extracted with liquid co 2 (rout et al., 2010). different compounds from m. exotica flowers w e r e f o u n d , n a m e l y ( e , e , e ) α s p r i n g e n e , ( e ) nerolidol, (e,e)-α-farnesene, methyl palmitate and germacrene b (raina et al., 2006). the variation of chemical composition in the essential oil from murraya flowers is affected by the place where the plants are planted (el-sakhawy et al., 1998) and harvesting time. concerning the latter, harvesting time may influence the quantity and quality of essential oils of m. paniculata flowers; this has been reported in four rosa cultivars (younis et al., 2009) and jasminum sambac flowers (younis et al., 2011). younis et al. (2011) reported that the best time to collect j. sambac flowers was in the morning before sunrise because highly volatile of jasmine oil. therefore, the purpose of this research was to investigate the effect of harvest time on the production and chemical compounds of m. paniculata essential oil. 2. materials and methods plant material t h e f i e l d e x p e r i m e n t w a s c o n d u c t e d f r o m october 2016 to february 2017. the experiment used 30 (62 month-old) plants that were planted in 1 m x 1 m on latosol soil, at the organic experimental farm, bogor agricultural university, bogor, indonesia (6°30’-6°45’ s, 106°30’-106°45’ e) at 250 m above sea l e v e l . a v o u c h e r s p e c i m e n w a s d e p o s i t a t t h e herbarium bogoriense, bogor, indonesia. type-a climate based on schmidt-ferguson with the average monthly rainfall, temperature and humidity of 305 mm, 26°c and 85%, respectively (mcga, 2017). before treatment, each plant was fertilized with 3.0 kg rice-hull ash, followed eliazar and aziz (2015). experimental design the experiment was arranged in randomized complete block design, with single factor (petersen, 1994) with harvest times as treatments, comprised of harvest at 05.00-07.00 and at 07.00-09.00 a.m. the flowers were collected at three different ages, comprised of two days before anthesis, one day before anthesis, and at the day of anthesis. the two ages of flower before anthesis were indicated by the size of flower buds which have been observed in the preliminary study. the anthesis of the flower bud with 1.00±0.06 cm length and 0.48±0.06 cm width will occur two days later; while flower bud with 1.16±0.06 cm length and 0.56±0.04 cm width will occur in the next day. the observations include developmental stages, number, fresh weight, the content of essential oils and chemical compounds of m. paniculata flowers. data were analyzed using t-student with α= 5% (petersen, 1994). essential oil extraction the analysis of essential oils was conducted at t r o p i c a l b i o p h a r m a c a r e s e a r c h c e n t e r , b o g o r agricultural university, bogor, indonesia. all harvested flowers (from different harvesting times) were extracted with solvent extraction method of ethanol. the flowers were immersed in ethanol until all samples were submerged, for two days. this mixture was filtered, then, the extract was separated from the solvent by rotary evaporator with a temperature of 40°c. the oil yield percentage was calculated from the weight of extract (g) divided by weight of fresh flower (g). gas chromatography-mass spectrometry analysis chemical compounds of essential oils were analyzed at health laboratory of dki jakarta province. t h e e s s e n t i a l o i l s w e r e i n j e c t e d i n t o g a s chromatography-mass spectrometry (gcms). an agilent technologies 7890 gas chromatography instrument with an auto sampler and 5975 mass selective detector and chemstation data system, equipped with a 30 m x 0.25 mm hp ultra 2 capillary column with 0.25 µm film thickness. helium was used as the carrier gas. the initial temperature was programmed at 80°c and then increased at the rate of 3°cmin-1 to 150°c held for 1 min and finally raised to 280°c at the rate of 20°c min-1 held for 26 min. the injector and interface temperatures were 250°c and 280°c, respectively. the ionization voltage was 70ev and a sample injection volume 5 µl. the individual peaks were identified by retention times, compared with those of compounds in health laboratory of dki jakarta province database. the percentage of each compound detected from samples was calculated according to the area of the chromatographic peaks. semarayani et al. essential oil production of murraya paniculata l. 473 3. results the results showed that the average of flower number (regardless the flower ages) harvested at 05.00-07.00 am was not significantly different to those harvesting at 07.00-09.00 a.m. (table 1). at both harvest times, it was found the following order of flower number based on flower stages: flowers at anthesis, flowers that would bloom two days later, and the least was flowers that would bloom in the next day. the differences in flower number between flower stages were not always significant. the difference of flower number between harvesting time was 8.96%. the average flower weight (regardless the flower stages) of m. paniculata was not significantly different between the times of harvesting (table 2). a similar trend as of flower number was also found in flower weight based on flower stages. the highest flower weight was blooming flowers (flowers at anthesis), and the lowest was flowers at the stage of one day before anthesis. the difference in flower weight between harvesting time was 16.59%. the extraction of m. paniculata flowers at different harvesting times with ethanol resulted in a yellowish-brown solution called concrete. these results were in line with that of paibon et al. (2011), that reported how the extraction of j. sambac flowers with ethanol produced a solution of yellowish brown to reddish. on the other hand, m. paniculata flowers that extracted with pentane produced a deep yellow waxy residue (rout et al., 2007). there was an indication that the essential oil percentage between harvest times was different. the percentage of essential oils from flowers harvested at 07.00-09.00 was higher than that at 05.00-07.00 a.m., regardless the flower stages, the difference was 0.38% (table 3). comparing among flower stages, it was found that anthesis flowers harvested at 05.0007.00 am had the highest percentage of essential oils. on the other hand, the highest percentage of essential oils at 07.00-09.00 am was obtained from flowers at the stage of one day before anthesis. from this calculation, the production of essential oils from flowers harvested at 07.00-09.00 was higher than that from 05.00-07.00 am. based on flower stages, blooming flowers (at anthesis stage) produced the highest amount of essential oils at both harvesting times, this related to the highest fresh flower weight. the analysis of chemical compounds showed 41 types that were contained in m. paniculata flowers at different harvesting times. the highest number of chemical compound types was found in flowers at anthesis when they were harvested at 07.00-09.00 am. this result was in line with younis et al. (2011), where j. sambac flowers harvested at anthesis had table 1 number of orange jessamine flowers at different harvesting times if p-value>α; α= 0.05, then means between the treatments were statistically equal; (k) = average of flower number at three flower age criteria; x= comparison between the flower 2 days before anthesis with 1 day before anthesis; y= comparison between the flower 2 days before anthesis with anthesis; z= comparison between the flower 1 day before anthesis with anthesis. harvesting time average of flower number/plant p-value percentage of flower number (%) at 05.00-07.00 am, flower ages 29.40 (k) 2 days before anthesis 24.67 x: 0.3349 ns 27.97 1 day before anthesis 14.07 y: 0.1742 ns 15.95 at anthesis 49.47 z: 0.0397 * 56.08 at 07.00-09.00 am, flower ages 31.11 (k) 2 days before anthesis 35.53 x: 0.0150 * 38.82 1 day before anthesis 12.87 y: 0.5056 ns 14.06 at anthesis 44.93 z: 0.0282 * 47.12 table 2 weight of orange jessamine flowers at different harvesting times if p-value>α; α= 0.05, then means between the treatments were statistically equal; k: average of flower number at three flower age criteria; x: comparison between the flower 2 days before anthesis with 1 day before anthesis; y: comparison between the flower 2 days before anthesis with anthesis; z: comparison between the flower 1 day before anthesis with anthesis. harvesting time average of flower weight (g/plant) p-value percentage of flower weight (%) at 05.00-07.00 am, flower ages 3.43 (k) 2 days before anthesis 2.05 x: 0.4572 ns 19.92 1 day before anthesis 1.34 y: 0.0884 ns 13.21 at anthesis 6.88 z: 0.0355 * 66.86 at 07.00-09.00 am, flower ages 4.72 (k) 2 days before anthesis 5.70 x: 0.0819 ns 40.35 1 day before anthesis 1.33 y: 0.4398 ns 9.38 at anthesis 7.13 z: 0.0029 ** 50.27 table 3 the percentage and production of essential oil of orange jessamine flowers at different harvesting times data were not analyzed statistically; (k ) = average of essential oils at three flower age criteria; * = production of essential oils based on the weight of the harvested flowers. treatment essential oil (%) production of essential oil (g/g fresh flower) * at 05.00-07.00 am, flower ages 3.13 (k) 4.83 2 days before anthesis 2.06 0.63 1 day before anthesis 3.40 0.69 at anthesis 3.94 4.07 at 07.00-09.00 am, flower ages 3.51 (k) 7.47 2 days before anthesis 3.49 3.00 1 day before anthesis 3.80 0.76 at anthesis 3.25 3.47 adv. hort. sci., 2018 32(4): 471-477 474 more chemical compounds than those in flower bud. the analysis on m. paniculata flowers revealed the presence of coumarins, esters, fatty acids, phenolics, triterpenes, sesquiterpenes and other compounds (table 4). coumarin was the most common compound found in all treatments. harvesting time at 0 5 . 0 0 0 7 . 0 0 a . m . g a v e t h e h i g h e s t n u m b e r o f coumarins, esters, fatty acids, and sesquiterpenes. coumarin was the highest amount of bioactive compound found in m. paniculata essential oils, ( t a b l e 4 ) a n d t h e d o m i n a n t c o m p o u n d s i n t h e coumarin group were β-methylesculetin and murralongin (table 5). the amount of β-methylesculetin from flowers harvested at 07.00-09.00 am was higher than those from 05.00-07.00 a.m. different flower s t a g e s h a d a d i f f e r e n t d o m i n a n t c o m p o u n d . harvesting flowers at the stage of one day before anthesis gave the highest percentage of β-methylesculetin at both harvesting times, but harvesting at anthesis delivered the highest percentage of murralongin. the analysis of chemical compounds showed that terpenoid groups found in the essential oil of m. paniculata flowers were triterpenes and sesquiterpenes (table 4). the percentage of triterpenes was higher than sesquiterpenes, this finding was different from the previous study which showed that sesquiterpenes were the main compound in essential oils. the current study showed that sesquiterpenes derivatives found in the essential oils of m. paniculata flowers and found at both harvesting times were αzingiberene, α-bergamotene, and caryophyllene (table 6). flowers harvested at 05.00-07.00 am had a higher percentage of α-zingiberene, α-bergamotene, and caryophyllene compare to those harvested at 07.00-09.00 a.m. there was an indication that different flower ages have different compositions of chemical compounds, except caryophyllene that was found at the same flower age at both harvesting times. 4. discussion and conclusions the above results showed that there were no significant differences between the number and weight of flowers harvested at 05.00-07.00 and 07.00-09.00 am. the different harvesting times reflected the positable 4 chemical compounds of orange jessamine essential oils at different harvesting times data were not analyzed statistically; k= average of chemical compounds at three flower age criteria. treatment % peak area coumarin ester fatty acid phenolic triterpene sesquiterpene other compound at 05.00-07.00 am, flower ages 71.29 k 1.80 k 9.12 k 4.34 k 3.27 k 0.77 k 9.42 k 2 days before anthesis 69.68 3.16 12.39 5.52 1.55 0.47 7.24 1 day before anthesis 74.33 1.41 10.09 4.43 1.33 0.55 7.87 at anthesis 69.85 0.83 4.89 3.06 6.93 1.30 13.14 at 07.00-09.00 am, flower ages 68.07 k 1.60 k 10.40 k 5.09 k 1.93 k 0.72 k 12.20 k 2 days before anthesis 66.98 2.15 11.91 6.20 1.03 0.41 11.33 1 day before anthesis 70.52 1.21 5.87 5.13 1.21 0.71 15.34 at anthesis 66.72 1.44 13.41 3.94 3.55 1.04 9.92 table 5 coumarins compounds of orange jessamine essential oils at different harvesting times data were not analiyzed statistically; k: average of chemical compounds at three flower age criteria. treatment % peak area β-methylesculetin murralongin at 05.00-07.00 am, flower ages 60.65 (k) 7.36 (k) 2 days before anthesis 59.46 5.36 1 day before anthesis 61.59 7.76 at anthesis 60.89 8.96 at 07.00-09.00 am, flower ages 62.07 (k) 5.71 (k) 2 days before anthesis 61.00 5.12 1 day before anthesis 65.51 5.01 at anthesis 59.71 7.01 table 6 sesquiterpenes compounds of orange jessamine essential oils at different harvesting times data were not analyzed statistically; k: average of chemical compounds at three flower age criteria. zbn, zingiberene; bgn, bergamotene; cp, caryophyllene. treatment % peak area α-zbn α-bgn cp at 05.00-07.00 am, flower ages 0.75 (k) 0.55 (k) 0.27 (k) 2 days before anthesis 0.47 0.00 0.00 1 day before anthesis 0.00 0.55 0.00 at anthesis 1.03 0.00 0.27 at 07.00-09.00 am, flower ages 0.71 (k) 0.41 (k) 0.17 (k) 2 days before anthesis 0.00 0.41 0.00 1 day before anthesis 0.57 0.00 0.15 at anthesis 0.85 0.00 0.19 semarayani et al. essential oil production of murraya paniculata l. 475 tion of sunrise where higher light intensity was found at 07.00-09.00 am. the current study showed that the anthesis of m. paniculata flowers occurred before 05.00 am, therefore there was no increase in the number and weight of flower after that time. de souza et al. (2004) reported that the anthesis of metrodorea nigra st. hill. flowers, belonging to rutaceae family, occurs in the morning. time of harvesting is important because it is related to the amounts of essential oils produced. filho et al. (2006) reported that harvest at 08.00 am resulted in the highest yield of the essential oil from fresh leaves of basil (ocimum basilicum l.). the importance of harv e s t i n g t i m e i s a l s o s h o w n b y d o b r e v a a n d kovacheva (2010) where the essential oils content of rosa damascena mill. and r. alba l. drops dramatically when the flowers collected after noon. besides investigating the effect of harvesting time, this study also observed the essential oil production at different flower developmental stages of m. paniculata. the results showed that harvesting flowers at anthesis stage (blooming flower) yielded the highest percentage of essential oils. flowering plants released diverse blends of volatile to attract pollinator and seed disseminators. the floral scent is a signal, which pollinators can use to discriminate a particular flower. it may contain from one to 100 volatile substances, but most species emit between 20 and 60 different compounds, so there won’t be any identical floral scents (dudareva et al., 2006). therefore, the presence of essential oil at anthesis will ensure the reproductive success. azam et al. (2013) also reported that the highest amounts of volatile compounds were present in fully opened flowers of citrus reticulata blanco, c. unshiu marc., c. sinensis (l.) osbeck, c. limon (l.) burm., c. medica (l.), and c. changshanensis chen et. fu. in general, essential oils are a mixture of compounds belonging to different chemical entities such as terpenes, phenols, aliphatic compounds, benzenoid, and heterocyclic compounds (shakeel-urehman et al., 2018). chemical compounds found in m. paniculata flowers in india were monoterpenes, sesquiterpenes, benzenoids, diterpenes, and fatty acids (rout et al., 2007). different from those study, the current experiment showed that coumarins were the dominant compounds in m. paniculata flowers. coumarins are also present in leaves of m. paniculata in indonesia (kinoshita and firman, 1996) and taiwan (kinoshita et al., 1996). furthermore, from the curr e n t s t u d y , i t w a s f o u n d t h e p r e s e n c e o f t w o c o u m a r i n s d e r i v a t i v e s i n e s s e n t i a l o i l s o f m . paniculata, β-methylesculetin and murralongin. βmethylesculetin compounds can function as antioxidants (kontogiorgis and hadjipavlou-litina, 2005) and anti-inflammatory (kontogiorgis and hadjipavloulitina, 2005; zuoqi et al., 2008). murralongin is thought to be a chemical compound identifier of m. paniculata essential oils. harvesting at anthesis produced the highest percentage of murralongin. the previous study reported that murralongin was found in essential oils from leaves and flowers of m. paniculata (gill et al., 2014), and leaves of m. omphalocarpa in taiwan (chen et al., 2003). terpenoids compounds that were identified in this study were triterpenes and sesquiterpenes. in general, terpenoids are the dominant compounds in essential oils (sangwan et al., 2001). the current study showed that the percentage of triterpenes compounds is higher than sesquiterpenes (table 4). this was not in line with butu et al. (2014) who reported that the basic compound in the essential oil was sesquiterpenes. terpenoids have many volatile compounds that have high enough vapor pressures at normal atmospheric conditions to allow significant release into the air (dudareva et al., 2004). therefore, despite the same plant, may have different types of compounds. sesquiterpenes compounds that could be identified in this current study were α-zingiberene, α-bergamotene, and caryophyllene. there were similarity and difference between this finding and the previous study. the similarity was reported by raina et al. (2006) where those three compounds were also found in m. exotica essential oils from flowers. raina et al. (2006) found that caryophyllene had the highest percentage, on the contrary, the current study showed that caryophyllene had the lowest percentage. the different finding indicates that the chemical composition and yield of essential oils are affected by many factors, such as provenance, weather, soil conditions, time of harvest, and the extraction method (boira and blanquer, 1998). caryophyllene is one of the compounds in perfume ingredient (salvadorcarreno and chisvert, 2005), but it also used as a mixture of spices, citrus scents, soaps, detergents, lotions as well as in various food products (sabulal et al., 2006). flamini et al. (2007) and darjazi (2012) reported that caryophyllene is also present in flower of c. limon and c. nobilis lour var. deliciosa swingle. it can be concluded that the harvest of m. paniculata flowers can be done at 05.00-09.00 am to obtain the highest quantity and the best quality of essential oil. flowers must be harvested at anthesis stage to reach the highest production of essential oil with β476 adv. hort. sci., 2018 32(4): 471-477 methylesculetin and murralongin, as the main compounds of m. paniculata flowers. references azam m., song m., fan f., zhang b., xu y., xu c., chen k., 2013 comparative analysis of flower volatiles from nine citrus at three blooming stages. int. j. mol. sci., 14: 22346-22367. boira h., blanquer a., 1998 environmental factors affecting chemical variability of essential oils in thymus piperella l. biochem. syst. ecol., 26: 811-822. butu m., butnariu m., rodino s., butu a., 2014 study of zingiberene from lycopersicon esculentum fruit by mass spectometry. dig. j. nanomater biostruct., 9(3): 935-941. chen k.s., wu c.c., chang f.r., chia y.c., chiang m.y., wu y.c., 2003 bioactive coumarins from the leaves of murraya omphalocarpa. planta med., 69: 654-657. darjazi b.b., 2012 a comparison of volatile component of flower, leaf and peel of citrus reticulata blanco (citrus nobilis lour var. deliciosa swingle). j. med. plants res., 6: 2365-2372. de souza l.a., moscheta i.s., mourao k.s.m., da rosa s.m., 2004 morphology and anatomy of the flower and anthesis of metrodorea nigra st. hill. 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zhengtao w., 2008 anti-inflammatory effect of scopoletin and underlying mechanisms. pharm. biol., 46: 854-860. impaginato 487 adv. hort. sci., 2018 32(4): 487-493 doi: 10.13128/ahs-22569 in vitro activity of some essential oils against penicillium digitatum f. khorram, a. ramezanian (*), m.j. saharkhiz department of horticultural science, school of agriculture, shiraz university, shiraz, iran. key words: cinnamon, citrus, decay, postharvest, savory, summer. abstract: natural plant essential oils (eos) can be used instead of synthetic fungicides because of human health concerns and environmental protection. in this study, the in vitro activity of some plants eos against penicillium digitatum, the cause of citrus green mold was evaluated during 8 days of incubation at 25°c. the eos extracted from sweet orange (citrus sinensis), lemon (citrus limon), lime (citrus aurantifolia), and sour orange (citrus aurantium) fruit peel (500, 1000 and 2000 µl l-1 concentrations), cinnamon (cinnamomum cassia) bark and summer savory (satureja hortensis) aerial parts (400, 500 and 600 µl l-1 concentrations) were used on penicillium digitatum mycelium. none of the eos extracted from tested citrus in this study could inhibit mycelial growth completely even at concentration of 2000 µl l-1. the best results were obtained with cinnamon and summer savory eos at concentration of 500 and 600 µl l-1. so, based on the results, cinnamon and summer savory eos can be ideal candidates to replace the synthetic fungicides to control postharvest green mold of citrus fruit. gc-ms analysis showed that the most abundant of all constituents in eo extracts were carvacrol and γ-terpinene in summer savory and (e)-cinnamaldehyde in cinnamon. 1. introduction citrus spp. are the most important produced fruits in the world (sharma and saxena, 2004), due to their good taste, useful nutrients, and widespread availability (liu et al., 2012). nevertheless, the high water content and nutrient composition make them also very susceptible to decay by pathogens after harvest (tripathi and dubey, 2004). one of the most common diseases that infects citrus fruit is green mold caused by penicillium digitatum (zheng et al., 2005). the yield losses and the worsening of the quality caused by the fungus are economically important. this pathogen infects the fruit through wounds on the peel inflicted during harvest, transportation, handling or commercialization. penicillium digitatum is one of the most important pathogen in citrus industry, because one generation of green mold complete during 7-10 days in rotten fruit at 20-25°c, and the large amounts of spores are disseminated easily by air currents (palou, 2014). currently, the use of synthetic fungicides is the primary and most sim(*) corresponding author: ramezanian@shirazu.ac.ir citation: khorram f., ramezanian a., saharkhiz m.j., 2018 in vitro activity of some essential oils against penicillium digitatum. adv. hort. sci., 32(4): 487-493 copyright: © 2018 khorram f., ramezanian a., saharkhiz m.j. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 25 january 2018 accepted for publication 21 may 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(4): 487-493 488 ple method for the control of postharvest diseases of citrus fruit (palou et al., 2008). however, fungicides consumption is strongly becoming restricted because of residual toxicity, carcinogenicity, long degradation p e r i o d a n d i n c r e a s i n g h u m a n h e a l t h c o n c e r n s (tripathi and dubey, 2004; palou et al., 2008). recently, researchers have been interested in development of alternative methods to manage postharvest decay. the essential oils (eos) are one of non-chemical and useful control options for the management of fungal postharvest diseases (sassi et al., 2008). essential oils are complex compounds that are natural and environmentally friendly, having antioxidant, antimicrobial and medicinal properties (bakkali et al., 2008). so, they can be ideal candidates to replace synthetic antimicrobials for maintenance of harvested horticultural crops (tripathi and dubey, 2004). many studies reported the beneficial effects of eo treatments for the control of postharvest decay caused by p. digitatum, such as thymus vulgaris at concentration of 1000 ppm (fatemi et al., 2012), mentha spicata and lippia scaberrima at concentrations of 1000 and 3000 μl l-1, respectively (du plooy et al., 2009), bubonium imbricatum at concentration of 1000 ppm (alilou et al., 2008), citrus spp. at conc e n t r a t i o n o f 1 0 % ( b a d a w y e t a l . , 2 0 1 1 ) , a n d cinnamomum zeylanicum at concentration of 0.5% (kouassi et al., 2012), thereby enhancing shelf life of fruits and vegetables. the purpose of this study was to investigate the in vitro activity of eos obtained from sweet orange (citrus sinensis), lemon (citrus limon), lime (citrus aurantifolia), and sour orange (citrus aurantium) fruit peel, cinnamon (cinnamomum cassia) bark and summer savory (satureja hortensis) aerial parts for the control of green mold caused by p. digitatum as a preliminary study to find a suitable and effective eo as alternative to synthetic fungicides to control green mold in citrus postharvest management. 2. materials and methods extraction of essential oils plant materials used in this study are shown in table 1. the air-dried plants material (300 gr) were cut into pieces, grounded into powder by blender, then the eos extracted through hydro-distillation for 3-4 hours using a clevenger apparatus (miquel et al., 1976). then the eos were dehydrated with anhydrous sodium sulfate and stored in dark bottles at -20°c before using for antifungal study. isolation of fungus the fungus used throughout this study was p. digitatum, the cause of citrus green mold. for isolation of fungus colony, p. digitatum spores were isolated from a decayed orange and cultured on potato dextrose agar (pda) by the single spore procedure at 25°c. the isolates were maintained on pda until needed. in vitro antifungal assay the antifungal assay was performed on pda plates amended with three concentrations (500, 1000 and 2000 µl l-1) of sweet orange, lemon, lime and sour orange eos and three concentrations (400, 500 and 600 µl l-1) of cinnamon and summer savory eos. tween 80 (merck-kgaa, germany) as an emulsifier was mixed with 80 ml of sterilized and molten pda media, cooled to about 45°c, and then enriched with eos. there were four 80 mm plates/replicates per treatment. after one day, the mycelia of p. digitatum from 4-days-old cultures were put in the center of amended pda petri plates with a cork borer. all of the plates were sealed with parafilm. inoculated plates were kept at 25°c for 8 days. colony diameter was determined daily by measuring the average radial growth (obagwu and korsten, 2003). in order to evaluate its effect on fungal growth, tween 80 (emulsifier) was also considered as a treatment in the experiment. table 1 plant materials used for eos extraction name family used part origin sweet orange (citrus sinensis cv. thomson navel) rutaceae fruit rind tissue (flavedo and albedo) fars-iran lemon (citrus limon cv. lisbon) rutaceae fruit rind tissue (flavedo and albedo) fars-iran lime (citrus aurantifolia cv. mexican lime) rutaceae fruit rind tissue (flavedo and albedo) fars-iran sour orange (citrus aurantium cv. amara) rutaceae fruit rind tissue (flavedo and albedo) fars-iran cinnamon (cinnamomum cassia) lauraceae tree bark china summer savory (satureja hortensis) lamiaceae aerial parts fars-iran khorram et al. essential oils against penicillium digitatum 489 inhibition percentage (ip) of fungal growth was calculated as the radial growth of treated fungus (t) relative to the growth in control (c) treatment (plates without eo and tween 80) according to the following formula: essential oils analysis at the end of the study, the main components of the most effective eos on p. digitatum were analyzed by gas chromatography (gc) and gas chromatography-mass spectrometry (gc-ms). the gc analysis was c a r r i e d o u t b y t h e u s e o f a g i l e n t g c ( 7 8 9 0 a , perkinelmer, usa) and a flame ionization detector. it was done on fused silica capillary hp-5 column. the temperatures of injector and detector were held at 250°c and 280°c, respectively. nitrogen was selected as carrier gas; oven temperature was 60-210°c at a rate of 4°c/min, which was then increased to 240°c at a rate of 20°c/min, and finally, kept for 8.5 min. the gc-ms analysis was performed using an agilent gc series 7890-a (perkinelmer, usa) with a fused silica capillary hp-5ms column and 5975-c mass spectrometer (unico, usa). carrier gas was helium. ion source and interface temperatures were set at 230°c and 280°c, respectively. mass range was programmed from 45 to 550 amu. oven temperature was 60-210°c at a rate of 4°c/min. n-alkanes was used as a standard to determine the retention indices for all constituents. the constituents were recognized by comparing their retention indices with literature reports, and their mass spectra comparison with the wiley, adams and mass finder 2.1 library data (adams, 1997). statistical analysis the experiment was distributed according to a split plot in time design. the analysis of variance (anova) was performed. mean comparisons were conducted by lsd (least significant difference) at p≤ 0.01. data were analyzed by sas software (v. 9.1). 3. results and discussion i n h i b i t o r y e f f e c t s o f d i f f e r e n t t r e a t m e n t s o n penicillium digitatum growth the in vitro activity of the tested eos on colony diameter of p. digitatum during 8 days of incubation is summarized in table 2. our results indicated that colony radial growth of p. digitatum was inhibited completely (100%) under in vitro condition by both cinnamon and summer savory eos at 500 and 600 µl l-1 concentrations during 8 days of incubation. also, the mycelial growth table 2 inhibition percentage (%) of plant essential oils on in vitro radial growth of penicillium digitatum * for each column, similar letters (lower case, superscript) are not significantly different according to lsd (p≤0.01) test. ^ means followed by similar letters (subscript), are not significantly different according to lsd (p≤0.01) test. treatment eo concentration (µl l-1) time (day) 1 2 3 4 5 6 7 8 control 0.00 g * s^ 0.00 g s 0.00 h s 0.00 g s 0.00 e s 0.00 g s 0.00 e s 0.00 e s tween 80 0.80 g q-s 0.43 g rs 0.38 h rs 1.29 g p-s 1.24 e p-s 5.09 fg n-s 7.08 e l-s 8.13 d e k-s sweet orange 500 22.42 f w-i 5.81 fg l-s 10.34 gh i-s 12.73 fg d-q 6.59 e l-s 9.37 fg j-s 6.55 e l-s 0.00 e s sweet orange 1000 23.81 f w-g 5.75 fg m-s 18.02 e-g b-k 23.07 ef w-h 12.56 e d-r 16.26 e-g c-n 11.55 e g-s 0.00 e s sweet orange 2000 75.80 b b 54.74 b e-i 48.24 c i-o 50.72 c g-k 32.09 cd r-b 38.42 cd l-s 41.76 c j-s 36.56 bc o-v lemon 500 32.81 ef r-a 17.41 d-g c-m 20.79 e-g z-j 23.18 ef w-h 13.03 e d-q 12.79 fg d-q 12.19 e f-s 0.00 e s lemon 1000 46.91 c-e l-p 24.46 c-f v-e 38.18 cd m-s 41.15 cd j-s 17.42 c-e c-m 22.27 d-f x-i 20.14 de b-k 12.50 c-e e-r lemon 2000 61.38 b-d d-h 23.54 c-f w-h 45.84 c i-q 50.87 c g-k 34.39 c q-x 44.26 c i-r 34.66 cd p-w 25.31 b-e t-c lime 500 36.98 ef m-u 20.71 d-f a-i 21.95 e-g y-i 17.98 f c-m 15.74 de c-n 16.98 e-g c-n 17.25 de c-n 0.00 e s lime 1000 54.54 b-e e-i 30.49 c-e s-b 44.03 c i-r 48.19 c i-o 30.72 cd s-b 33.27 c-e r-y 33.22 cd r-y 30.85 b-d s-b lime 2000 70.43 bc b-d 42.48 bc i-s 49.22 c h-m 50.62 c g-l 34.11 c q-y 41.72 c j-s 40.39 c k-s 32.50 b-d r-a sour orange 500 48.92 c-e i-n 36.82 b-d n-u 14.64 fg c-o 12.61 fg d-r 11.36 e h-s 16.16 e-g c-n 17.89 de c-m 0.00 e s sour orange 1000 50.94 c-e g-k 24.00 c-f w-f 30.77 de s-b 33.67 de q-y 13.36 e c-p 15.97 e-g c-n 14.85 de c-o 9.37 de j-s sour orange 2000 61.44 b-d d-h 33.01 c-e r-z 44.12 c i-r 53.39 c f-j 33.12 c r-y 37.28 cd m-t 41.08 c j-s 40.94 b k-s cinnamon 400 40.52 d-f k-s 12.63 e-g d-r 24.77 ef u-d 22.22 ef x-i 3.34 e o-s 6.48 fg l-s 9.40 e j-s 3.44 e o-s cinnamon 500 100.00 a a 100.00 a a 100.00 a a 100.00 a a 100.00 a a 100.00 a a 100.00 a a 100.00 a a cinnamon 600 100.00 a a 100.00 a a 100.00 a a 100.00 a a 100.00 a a 100.00 a a 100.00 a a 100.00 a a savory 400 100.00 a a 89.61 a a 74.01 b bc 70.91 b b-d 66.01 b b-e 65.25 b b-f 61.83 b c-g 51.25 b g-k savory 500 100.00 a a 100.00 a a 100.00 a a 100.00 a a 100.0 0 a a 100.00 a a 100.00 a a 100.00 a a savory 600 100.00 a a 100.00 a a 100.00 a a 100.00 a a 100.00 a a 100.00 a a 100.00 a a 100.00 a a adv. hort. sci., 2018 32(4): 487-493 490 was decreased by cinnamon and summer savory eos at concentrations lower than 500 µl l-1, but it was not suppressed completely. the main activity of the eos in the postharvest fruit are derived from their ability to inhibit pathogen growth (periago et al., 2004). cinnamon eo has the potential to be employed as a natural antifungal agent for fruit disinfectation, as cinnamaldehyde is its main constituent (xing et al., 2010). furthermore, it has been reported that summer savory contains some substances with antibacterial properties (deans and svoboda, 1989). in this research, cinnamon and summer savory eos have the strongest effect on p. digitatum growth (table 2). it has been reported that eucalyptus and cinnamon (cinnamomum zeylanicum, blume) oil vapour (500 ppm) reduced decay almost by 50% in tomatoes after 10 days of storage (tzortzakis, 2007). moreover, win et al. (2007) presented that eos from cinnamon at the concentration of 5.0 g l-1 completely inhibited conidial germination and mycelial growth of all fungi on banana (colletotrichum musae, fusarium spp. and lasiodiplodia theobromae). in addition, lopez-reyes et al. (2010) showed that summer savory, oregano and thyme eos at 10% showed significant inhibitory effect (similar to chemical control) against p. expansum and botrytis cinerea on four cultivars of apples. the mechanism by which eos suppress the microbial growth is not fully understood, but a number of possible explanations have been postulated. essential oils are lipophilic and this property enables them to preferentially move from an aqueous phase into fungi membrane. this action leads to membrane expansion, increasing in membrane fluidity and permeability, membrane proteins disorder, respiration rate control, change of ion transportation in fungi and induced cellular contents leakage (burt, 2004; oonmetta-aree et al., 2006; khan et al., 2010; fadli et al., 2012). in the present study, the lowest inhibition was observed in control plates that contained only pda (0%); however, this was not significantly different from plates containing pda and the tween 80 without the eos during 8 days. so, results indicated that the tween 80 used as an emulsifier had no effect on the mycelial growth (table 2). we observed an increase of antifungal effects of the tested citrus fruits peel eos such as sweet orange, lemon, lime and sour orange as the eos concentration increased, but the fungi growth was not inhibited completely even at concentration of 2000 µl l-1. so, as the results showed, none of the tested concentrations of citrus fruits eos in this study could inhibit radial growth completely (table 2). essential oils are present in great quantities in the flavedo of citrus fruit (caccioni et al., 1998). the citrus fruits eo consists a mixture of components such as terpenes, hydrocarbons, ketones, aldehydes, alcohols, acids, and esters. the amount of them depends on the citrus cultivar, the extraction and separation techniques (fisher and phillips, 2008). the positive effect of the volatile components of citrus fruit essential oils on p. digitatum and italicum growth has been reported (caccioni et al., 1998). the s p o r e g e r m i n a t i o n a n d m y c e l i u m g r o w t h o f p . italicum and digitatum were stimulated by the essential oil of citrus reticulata blanco at concentration of more than 2.5 μl ml-1 (wang et al., 2012). moreover, badawy et al. (2011) reported that citrus aurantifolia eos had the antifungal effects against p. digitatum pathogens at concentration of 10% (v/v). however, in our study the application of citrus spp. could not provide acceptable control of green mold disease. analysis of the summer savory and cinnamon eos the analysis of the volatile profiles in summer savory and cinnamon eos are listed in table 3 and 4, table 3 chemical composition of the summer savory essential oil * retention index number component ri* (%) 1 αthujene 924 1.15 2 α-pinene 932 0.64 3 camphene 946 0.06 4 hepten-1-ol 958 0.05 5 sabinene 969 0.01 6 β-pinene 974 0.21 7 3myrcene 988 1.15 8 phellandrene 1002 0.23 9 α-terpinene 1014 3.75 10 p-cymene 1020 2.19 11 sylvestrene 1025 0.37 12 e-βocimene 1044 0.07 13 γ-terpinene 1054 31.98 14 terpinolene 1086 0.05 15 trans-α sabinene hydrate 1098 0.07 16 isoborneol 1155 0.06 17 terpinene-4-ol 1174 0.2 18 α-terpineol 1186 0.1 19 carvacrol methyl ether 1241 0.09 20 thymol 1289 0.8 21 carvacrol 1298 55.66 22 thymol acetate 1349 0.03 23 carvacrol acetate 1370 0.07 24 caryophyllene 1417 0.36 25 aromadendrene 1439 0.08 26 α–humulene 1454 0.01 27 bicyclogermacrene 1500 0.18 28 bisabolene 1505 0.21 29 unknown 0.01 30 spathulenol 1577 0.04 khorram et al. essential oils against penicillium digitatum 491 respectively. a total of 30 different components of summer savory, and 37 components of cinnamon were identified and isolated by gc and gc-ms from the eos. the principal components of the summer savory eo were carvacrol (55.66%), γ-terpinene (31.98%), α-terpinene (3.75%), p-cymene (2.19%), 3myrcene (1.15%), and α-thujene (1.15%). the major components of the cinnamon eo were (e)-cinnamaldehyde (70.04%), α-copaene (10.82%), δcadinene (5.35%), α-muurolene (4.23%), and γmuurolene (1.25%). other constituents which were less than 1% have been shown in table 3 and 4. as shown in table 2, both summer savory and cinnamon eos were equally effective in inhibiting the growth of p. digitatum. this is in accord with the reported in vitro inhibitory effect of carvacrol against pathogens (periago et al., 2004). in fact, the main c o m p o n e n t o f s u m m e r s a v o r y e o i s a p h e n o l (sacchetti et al., 2005), and its most important mechanism of antimicrobial activity is connected with the phenolic ring in its chemical structure (ultee et al., 2002). furthermore, it has been reported that the toxicity rate of the phenol ring is due to the site (s) and number of hydroxyl groups (cowan, 1999). concerning cinnamon eos, its major volatile compound is cinnamaldehyde. moreover, it has been reported that cinnamon eo had potent anti-bacterial and anti-fungal activities due to cinnamaldehyde (ooi et al., 2006), because it acts as membrane irritants (nabavi et al., 2015). 4. conclusions in this study, the in vitro activity of plants eos against p. digitatum were tested at different concentrations during 8 days of incubation at 25°c. as showed by the results, the stronger inhibitions were obtained by cinnamon and summer savory eos at concentration of 500 and 600 µl l-1. none of the citrus eos could inhibit fungus radial growth completely compared with cinnamon and savory eos. gc-ms analysis showed that the most abundant of all constituents in eo extracts were carvacrol and γ-terpinene in summer savory and (e)-cinnamaldehyde in cinnamon. acknowledgements we thank shiraz university research council for financial supports, fars agricultural and natural resources research and education center, shiraz, iran for technical assistance. references adams r.p., 1997 identification of essential oil components by gas chromatography/mass spectroscopy. j. am. soc. mass spect., 6(8): 671-672. alilou h., akssirar m., hassani l.m.i., chebli b., el hakmoui a., mellouki f., rouhi r., boira h., blázquez m.a., 2008 chemical composition and antifungal activity of bubonium imbricatum volatile oil. phytopathol. mediterr., 47(1): 3-10. badawy f.i., sallam m.n., ibrahim a., 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deficit stress on growth, yield, fruit quality and physiological traits of melon cv. khatooni, field experiments were conducted in split plot randomized complete block design with three replications. in 2014, irrigation treatments consisted of two deficit irrigation regimes, 33% and 66% of etc (crop evapotranspiration), and 100% etc as the control (di33, di66 and i100). in 2015, irrigation treatments applied were: 40, 70 and 100% etc (di40, di70 and i100). the results showed that plant height and leaf area decreased from treatment i100 to di40 and di33. the highest average fruit weigh and yield were obtained from irrigation 100% etc for both years. the water use efficiency (wue) significantly increased in response to increase water deficit stress. deficit irrigation treatments significantly decreased leaf relative water content, vitamin c and fruit firmness, whereas antioxidant enzymes activity, proline and total soluble solid contents increased. these results suggest that the crop is sensitive to water deficits, that moderate water stress (di70 and di66) reduced yield by about 28.5-38.2% and severe water stress (di40 and di33) had a much more marked effect, reducing yield by 48.1-61.4%. 1. introduction melon (cucumis melo l.) is an important horticultural crop in iran, generally cultivated in arid and semi-arid regions. iran is the third largest melon-producing country in the world with more than 1476801 tonnes (fao, 2014) of production. melon plants are highly productive under adequate irrigation conditions; however water for irrigation is not always available at the time and amount needed by the crop, so water scarcity is a major constraint to horticultural production in arid and semiarid regions (sharma et al., 2014). deficit irrigation regime, a practice that supplies water below evapotranspiration (et) demands, can optimize water productivity when full irrigation is not possible (fereres and soriano, 2007). when water supply is limited, plant growth and yield is reduced and plant structure is modified by decreasing in leaf size (kirnak et al., 2002; chaves et al., 2003). the effect of deficit irrigation on fruit yield and quality has been reported by numerous researchers with different results. in melon, deficit (*) corresponding author: tbarzegar@znu.ac.ir citation: barzegar t., heidaryan n., lofti h., ghahremani z., 2018 yield, fruit quality and physiological responses of melon cv. khatooni under deficit irrigation. adv. hort. sci., 32(4): 451-458 copyright: © 2018 barzegar t., heidaryan n., lofti h., ghahremani z. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 6 december 2017 accepted for publication 31 january 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(4): 451-458 452 irrigation reduced marketable fruit number and yield, average fruit weight, fruit diameter and did not affect rind thickness and seed cavity, but increased total soluble solids content (sharma et al., 2014). although deficit irrigation reduce crop yield, may be able to save a significant amount of irrigation water (sharma et al., 2014). fabeiro et al. (2002) stated that deficit irrigation during blooming stage affected mainly fruit yield, at setting stage both quantity and quality, and the deficit imposed at ripening stage affected sugar content. rouphael et al. (2008) indicated that water deficit significantly reduced yield, biomass production and leaf water status of mini-watermelon, but increase the water use efficiency. the soluble solids concentration (ssc) is probably the most important quality parameter that is commonly evaluated by consumers (cabello et al., 2009). water deficit studies in melon have been reported to increase (sharma et al., 2014), decrease (long et al., 2006), or had no effect (hartz, 1997) on soluble solid content. vitamin c content, as a secondary metabolite of plants, did not change with deficit irrigation in watermelons (75% etc) (leskovar et al., 2004) and melons (50% etc) (sharma et al., 2014). oxidative stress is one of the major causes of cellular damage in plants during stress (miller et al., 2010). however, plants can avoid the drought damage by promoting antioxidant enzymes activity, such as superoxide dismutase (sod), peroxidases (pod), and catalase (cat), to scavenge for free radicals and, or accumulate osmotic regulators such as soluble sugar, and proline may play a role in protection of cellular machinery against photo-oxidation by reactive oxygen species (ros) that increase the drought resistance of plants under water stress (foyer and noctor, 2005; veljovic-jovanovic et al., 2006). although the effects of water stress have been studied on growth and yield of different crops during the last years, recent information on the response of iranian melon yield and quality to deficit irrigation remains limited, particularly about the results of restricted water distributions in arid and sub-arid environments. the main goal of this study was to evaluate the effect of controlled deficit irrigation on t h e p h y s i o l o g i c a l p a r a m e t e r s a n d y i e l d o f t h e khatooni melon cultivar. 2. materials and methods experimental site two field experiments were conducted during the growing season of 2014 and 2015 from june to september at research farm of agriculture faculty, university of zanjan (iran), to study the effect of water deficit on fruit yield and quality, antioxidant enzymes activities, water use efficiency (wue), proline and vitamin c content. the soil texture was silty loam with 7.8 ph. some soil characteristics and irrigation water chemical properties were showed in table 1 and 2. the daily climate data during the growing seasons (2014 and 2015) was shown in table 3. table 1 soil physical and chemical properties at the experiment site ph ec (ds m-1) n (%) ca (g kg-1) na (g kg-1) k (g kg-1) om (%) soil texture sand (%) silt (%) clay (%) 7.40 1.49 0.07 0.12 0.13 0.2 0.94 silt loam 25 38 37 table 2 irrigation water chemical properties at the experiment site om= organic matter. bicarbonate (mg l-1) carbonate (mg l-1) cl (mg l-1) mg (mg l-1) ca (mg l-1) k (mg l-1) na (mg l-1) ec (ds m-1) ph 195.2 0.0 582.2 103.7 258.45 0.0 50 2.35 6.5 table 3 climatic parameters during the growing seasons climatic parameters june july august sept 2014 2015 2014 2015 2014 2015 2014 2015 minimum air temperature (°c) 7.60 12.90 10.70 18.53 13.10 16.14 6.80 12.58 maximum air temperature (°c) 35.80 31.90 39.50 34.46 39.10 35.50 35.40 30.28 rainfall (mm) 7.30 0.33 17.30 1.13 0.10 0.00 4.00 2.93 relative humidity (%) 41.50 44.00 43.40 42.00 37.00 39.00 41.40 52.00 barzegar et al. deficit irrigation effects on melon quality 453 plant materials and irrigation treatments the experiment was done on a completely randomized block design whit three irrigation levels and three replications. ‘khatooni’, yellow-green netted skin color and chimeric stripes, an iranian melon from the inodorous group widely cultivated in iran, was selected for study. the seeds were sown on 1th july 2014 and 23th may 2015 at recommended spacing of 50 cm in row with 200 cm between rows. the irrigation system consisted of one drip line every crop row. fertilizers was delivered as a pre-plant base comprising 80 kg n/ha, 50 kg p/ha and 80 kg k/ha. at a very early stage, plants were pruned (removing the apex of the main stem), and trained to have two lateral branches. three irrigation levels were calculated, based on actual evapotranspiration (etc). in 2014, irrigation treatments were control or irrigation at 100% etc (i100), deficit irrigation at 66% etc (di66) and at 33% etc (di33) of control. according to 2014 results, when water deficit stress treatments strongly reduced fruit yield, in 2015 deficit irrigation treatments were changed, and the irrigation treatments were: 100% etc (i100), 70% etc (di70) and 40% etc (di40). before starting the differential irrigation at five-leaf stage, all treatments were supplied with similar amount of water to maximize stands and uniform crop establishment. all other necessary operations such as pests and weeds control were performed according to recommended package of practices during the crop growth. measurements. plant growth and leaf area after 30 days of irrigation treatments, the average of leaf area was recorded whit leaf area measurement (delta-t device ltd, england). after fruit harvest, vine length of each plant was measured. for estimate leaf dry weight, at first fresh weight of leaf was measured; then they were dried in a hot-air oven for 2 days at 72°c, after which the dry weights (%) of leaf was recorded. yield and productivity components the fruits were harvested when color changed from green to yellow and after the appearance of the netted pattern. each melon fruit was weighed to determine mean fruit weight (fw). the fruit number per plant and fruit yield per plant was measured to determine of total yield, expressed in t ha-1. fruit yield was calculated by the mean fruit weight (kg), fruit number per plant and the density (20,000 plants/ha). fruit quality immediately after harvest, flesh ratio (fr), fruit firmness (ff), total soluble solid (tss) and vitamin c (vc) were determined. the flesh ratios were calculated using the formulae: fr (%)=[(a+b)2-(a’+b’)2/(a+b)2]×100 where a is the fruit length, a’ is the seed cavity length, b is the fruit diameter and b’ is the seed cavity diameter. from the liquid extract obtained by liquefying the mesocarp of each fruit, tss content was determined by a handheld refractometer and expressed as °brix. fruit firmness was measured on the mesocarp tissue at three random locations per fruit using a digital penetrometer (mc cormic-ft 327) and recorded as kg cm-1. proline content proline content in leaf tissue was determined according to the method of bates et al. (1973). mature leaves of plant were sampled 30 days after the onset of the deficit irrigation treatments. proline was extracted from a sample of 0.5 g fresh leaves material samples in 3% (w/v) solution sulphosalycylic acid and estimated using the ninhydrin reagent. after reading the absorbance of fraction at a wave length of 520 nm, proline concentration was determined using a calibration curve and expressed as mg g-1 fw. catalase and peroxidase enzymes activity samples were taken from the fully expanded leaf and transferred to the laboratory in the ice. leaf sample (0.5 g) was frozen in liquid nitrogen and ground using a porcelain mortar and pestle. catalase (cat) activity was measured by following the decomposition of h2o2 at 240 nm with a uv spectrophotometer (havir and mchale, 1987). samples without h2o2 were used as blank. the activity of cat was calculated by the differences obtained at od240 values at 30 second interval for 2 min after the initial biochemical reaction. peroxidase (pod) activity was measured using modified method of the tuna et al. (2008) with guaiacol at 470 nm. a change of 0.01 units per minute in absorbance was considered to be equal to one unit pod activity, which was expressed as unit g-1 fw min-1. leaf relative water content the relative water content (rwc) in leaves was determined whit sampling fully expanded young leaves at noon according to yamasaki and dillenburg, (1999). leaf relative water content was calculated adv. hort. sci., 2018 32(4): 451-458 454 using the following formula: rwc -(%) =[(fw-dw)/(sw-dw)]×100 where fw stands for fresh weight, dw for dry weight, and sw for saturated weight. water use efficiency water use efficiency (wue) was calculated for all treatments based on total crop yield and amount of water applied during growth period. wue was estimated as the ratio of fruit yield (y, kg ha-1) and irrigat i o n w a t e r a p p l i e d ( w , m 3) ( s t a n h i l l , 1 9 8 6 ) . wue=y/w. statistical analysis all data were analyzed statistically using a oneway anova. because of differences in the treatments, the data for each year were submitted to anova separately. for data analysis, a completely randomized block design was used (3 irrigation levels × 3 replications × 10 observations per experimental unit). data were analyzed using the sas statistical program (sas institute inc., cary, nc, usa), and means were compared by duncan’s multiple range tests at the 5% and 1% probability levels. 3. results and discussion plant growth leaf area, vine length and leaf dry weight (ldw) data of the treatments were presented in table 4. leaf area significantly decreased in the water deficit stress treatments in both years, reduction 20.38% (di33) and 30.4% (di40) in 2014 and 2015, respectively. in 2014, deficit irrigation stress had no effect on ldw. on the contrary, in 2015, ldw was affected significantly by the irrigation treatments, decreasing 22.05% in i100 treatment. also, water deficit stress significantly reduced vine length in 2014, but no significant effect was observed by water deficit stress in 2015. these findings are similar the results obtained by pew and gardner (1983) and ribas et al. (2001) who found that vegetative growth was higher under full irrigation instead of limited irrigation. growth is an irreversible increase in volume, size, or weight, which includes the phases of cell division, cell elongation, and differentiation. a decrease in plant growth may be due to the limitation of cell division, cell enlargement caused by loss of turgor and inhibition of various growth metabolisms (farooq et al., 2012), and also decrease in photosynthesis (huang et al., 2011). yield, productivity components and water use efficiency fruit yield was affected significantly by the irrigation treatments in both years (table 4). the highest value of fruit yield (40.37 and 43.43 t h -1) was obtained in the irrigation 100% etc in 2014 and 2015, respectively. fruit number and fruit weight significantly reduced under deficit irrigation (table 4). the mean fruit number per plant was lower in 2014 (1.8, i100) compared to 2015 (2.7, i100). in contrast, fruit mean weight was higher in 2014 (2.18 kg) against 2015 (1.60 kg) that was obtained under irrigation 100% etc. the lowest fruit number and fruit weight (1.25 kg) was observed respectively, with irrigation 33% etc in 2014 and irrigation 40% etc in 2015. this result agrees with the findings of ribas et al. (2001), cabello et al. (2009) and sharma et al. (2014), who reported that limited irrigation reduced fruit yield of melon. table 4 effect of deficit irrigation on average leaf area (la), vine length (vl), fruit weight (fw), number of fruits per vine (fn), yield (y), and water use efficiency (wue) in 2014 and 2015 seasons i33, i40, i66, i70 and i100 represent the irrigation treatments that received 33, 40, 66, 70 and 100% of etc, respectively. values are the average of 10 observation of each replication per irrigation level. within each column, values followed by the same letters are not significantly different at p<0.05. year irrigation (% etc) la (cm2) ldw (%) vl (cm) fn fw (kg) y (t ha-1) wue (kg m-3) 2014 100 151.53 a 16.24 a 185.6 a 1.8 a 2.18 a 40.37 a 14.14 ab 66 130.11 b 16.45 a 133.3 ab 1.3 ab 1.91 ab 24.94 b 15.11 ab 33 120.64 b 17.23 a 116.33 b 1.1 b 1.36 b 15.55 c 17.65 a 2015 100 183.74 a 16.4 b 148.33 a 2.7 a 1.60 a 43.43 a 14.24 b 70 151.72 b 19.59 a 138.33 a 2.2 ab 1.42 ab 31.03 b 14.53 b 40 127.88 b 21.04 a 115.5 a 1.8 b 1.25 b 22.50 c 18.45 a barzegar et al. deficit irrigation effects on melon quality 455 the reduction in fruit yield under deficit irrigation t r e a t m e n t s c o m p a r e t o i 1 0 0 t r e a t m e n t c a n b e explained by the decrease in both mean fruit weight and numbers of fruits per vine (table 4). cabello et al. (2009) and sharma et al. (2014) also reported the reduction in fruit number and fruit weight under deficit irrigation. previous studies indicated that fruit weight in melon is more sensitive to water stress than fruit number (long et al., 2006; dogan et al., 2008). figure 1 presents the correlation between irrigation and fruit yield, fruit weight and fruit number per vine. correlation between irrigation and fruit yield (r2= 0.93) was stronger than the correlation with fruit weight (r2= 0.58) and fruit number per vine (r2= 0.51) which indicates that the reduction in fruit yield with deficit irrigation was attributed to the significant decrease in average fruit weight and fruit number per vine (fig. 1 b and c). wue is the relation between yield and the quantity of irrigation water (zeng et al., 2009). in both years, wue was lowest for irrigation 100% etc. overall; deficit irrigation resulted in 19.88% and 22.81% wue increased in di33 and di40, respectively (table 4). wue had negative correlation (r2 = 0.64) with irrigation water amount (fig. 2). higher wue has also been achieved in watermelon (leskovar et al., 2004), muskmelon (kirnak et al., 2005; zeng et al., 2009), mission and da vinci melon cultivars (sharma et al., 2014) in response to deficit irrigation. fruit quality fruit quality as indicated with fruit firmness, flesh ratio, total soluble solid (tss) and vitamin c was presented in table 5. in both years, fruit firmness decreased as the irrigation was restricted. the lowest fruit firmness was 1.49 kg cm-1 under irrigation 33% etc, although there was no significant difference fig. 1 relationship between irrigation by fruit yield (a), fruit weight (b) and fruit number per plant (c) in 2014 and 2015. values are the mean of 3 replications/10 observations each irrigation level, in two years. fig. 2 relationship between irrigation by water use efficiency (wue) in 2014 and 2015. values are the mean of 3 replications/10 observations each irrigation level, in two years. table 5 effect of deficit irrigation on fruit firmness (ff), flesh ratio (fr), total soluble solid (tss) and vitamin c (vc) in 2014 and 2015 seasons i33, i440, i66, i70 and i100 represent the irrigation treatments that received 33, 40, 66, 70 and 100% of etc, respectively. values are the average of 10 observation of each replication per irrigation level. within each column, values followed by the same letters are not significantly different at p<0.05. year irrigation (% etc) ff (kg cm-1) fr (%) tss (°brix) vc (mg 100 ml-1) 2014 100 2.38 a 49.55 a 10.06 b 10.002 a 66 1.79 ab 49.53 a 11 ab 8.082 b 33 1.49 b 48.29 a 12.06 a 6.98 c 2015 100 3.15 a 54.07 a 9.03 b 10.68 a 70 3.00 a 49.04 ab 10.7 ab 9.21 b 40 2.1 b 45.77 b 11.76 a 7.88 c 456 adv. hort. sci., 2018 32(4): 451-458 between i100 whit di66 and di70 in 2014 and 2015, respectively. these results was agreement with cabello et al. (2009) in melon, who reported that increasing irrigation water improved flesh firmness, but obtained a reduction in flesh firmness when irrigation water increased in following year. also, sharma et al. (2014) did not obtain a significant difference of irrigation treatments on fruit firmness with approximately positive effect of optimal irrigation. the flesh ratio was unaffected by the irrigation rates in 2014 season. however, flesh ratio varied significantly in 2015. the largest flesh ratio (54.07%) was obtained under irrigation 100% etc in 2015. these results are in agreement with the results of dogan et al. (2008) in melon. the results indicated that optimal irrigation water could increase flesh thickness while water stress has a negative effect on it. it is not in accordance with ribas et al. (2003) who reported that the flesh and skin ratios are not usually affected by the irrigation levels. tss is a very important index of quality in melon fruits (zeng et al., 2009). in both years, larger amounts of irrigation water resulted in lower tss. the highest tss was recorded with 12.06 and 11.76 °brix in irrigation 33 and 40% etc, respectively. the similar results were also observed by some other researchers (lester et al., 1994; fabeiro et al., 2002). dogan et al. (2008) showed that fruit sugar content affected positively by water stress. furthermore, other studies have shown that in muskmelon, tss decreased with the decrease in irrigation water levels (long et al., 2006; zeng et al., 2009; li et al., 2012). gonzalez et al. (2009) found no significant differences for watermelon fruit soluble solids between well-watered and regulated deficit irrigation treatments, although it was 9.5% higher, for the regulated deficit irrigation treatment. deficit irrigation markedly (p<0.05) reduced vitamin c content. the highest value of vitamin c was found in treatments i100 in both years (table 5), which high decrease value (30.21%) was recorded in irrigation 33% etc. the results indicated that vitamin c content was highly sensitive to deficit irrigation. our results are agreement with li et al. (2012) and wang et al. (2017) who showed that severe water deficit stress reduced significantly the fruit vitamin c content, but these results differ from the findings of cui et al. (2008) who stated that water deficit during the fruit growth and maturation stages increased significantly vitamin c content. proline accumulation the exposure to water deficit stress significantly (p<0.05) increased proline content (table 6). the maximum value of proline content was 1.97 and 1.8 mg g-1 fw under irrigation 33 and 40% etc, respectively. accumulation of proline plays an important role in plants to adaptive on environmental stresses, particularly low water stress (kavas et al., 2013). the proline that accumulated in the leaves under waterlimited environment is a cellular regulator that helping to sustain the activity of the cell and tissue in water deficit condition by preventing injuries in the internal apparatus of cell (ahmed et al., 2009). catalase and peroxidase enzymes activity significant differences among treatments were observed for cat enzyme activity (table 6). cat activity was the highest (7.47 and 6.97 µmol h2o2 g-1 fw min-1) with di33 and di40 treatments. similar to cat, the pod activity in both seasons increased in response to an increase in water deficit stress (table 6), which high pod activity was found by irrigation 33% etc in 2014. in present study, the antioxidant enzyme activates increased with the decrease of irrigation water table 6 effect of deficit irrigation on proline, catalase enzyme activity (cat), peroxidase enzyme activity (pod) and relative water content (rwc) i33, i440, i66, i70 and i100 represent the irrigation treatments that received 33, 40, 66, 70 and 100% of etc, respectively. values are the average of 10 observation of each replication per irrigation level. within each column, values followed by the same letters are not significantly different at p<0.05. year irrigation (% etc) proline (mg g−1fw) cat (µmol h 2 o 2 g-1 fw min-1) pod (unit g-1 fw min-1) rwc (%) 2014 100 0.77 b 4.52 b 0.422 b 78.63 a 66 1.5 a 5.62 b 0.5 b 67.45 ab 33 1.97 a 7.47 a 0.789 a 55.19 b 2015 100 0.97 c 4.4 b 0.356 b 73.13 a 70 1.302 b 5.19 b 0.486 a 64.26 ab 40 1.808 a 6.97 a 0.511 a 58.74 b 457 barzegar et al. deficit irrigation effects on melon quality applied. as found by kavas et al. (2013) in melon and huseynova (2012) in wheat, the antioxidant activity of cat significantly increased by drought stress. antioxidative enzymes like pod and cat play a major role in conferring drought tolerance and cta and pod activity of drought tolerance genotypes were higher than sensitive genotypes under drought stress (hameed et al., 2013). relative water content as applied irrigation water decreased, the relative water content of leaf decreased (table 4). the results showed that different irrigation treatments had similar effects on rwc in both seasons. the highest value of rwc was recorded in irrigation 100% etc. the decrease in rwc being respectively, 29.8 and 19.67% for di33 and di40 compared to i100. rwc decreased linearly in response to an increase in water deficit stress in melon (kavas et al., 2013), watermelon (kirnak and dogan, 2009) and mini-watermelon (rouphael et al., 2008). the results indicated that the rwc was improved by the increasing irrigation water. kirnak and dogan (2009) stated the higher leaf relative water content values are generally indication of enough soil water in root zone. 4. conclusions water deficit has been shown to adversely affect leaf area, yield, and leaf water status of melon, but led to increase the wue and tss. since the water scarcity is a key factor for plant production under arid and semi-arid regions, thus achieving great values of wue is more reasonable than maximum yield. wue in di40 and di33 was greater than full irrigation treatment. irrigation water increased yields not only by increasing the mean weight of the fruits, but also by increasing fruit number per vine. in both years, the physiological parameters showed significant differences. results indicated that the change of cat and pod activity and proline accumulation cooperated with water deficit; indeed cat and pod activities and proline content (60.1% and 46% in di33 and di40, respectively) increased with enhancement of drought intensity, and in stressed plants were significantly higher than full irrigated plants. the results suggested that antioxidant enzyme activities (cat and pod) as well as proline accumulation may play an important role in protecting ‘khatooni’ melon plants against drought stress. references ahmed c.b., rouina b.b., sensoy s., boukhris m., abdallah f.b., 2009 changes in gas exchange, proline accumulation and antioxidative enzyme activities in three olive cultivars under contrasting water availability regimes. environ. exp. bot., 67: 345-352. bates l., waldren r.p., teare i.d., 1973 rapid determination of free proline for water stress studies. plant soil., 39: 205-207. cabello m.j., castellanos m.t., romojaro f., martinez-madrid c., ribas f., 2009 yield and quality of melon grown under different irrigation and nitrogen rates. agric. water manag., 96: 866-874. c h a v e s m . m . , m a r o c o j . p . , p e r e i r a j . s . , 2 0 0 3 understanding plant responses to drought from genes to whole plant. fun. plant biol., 30: 239-264. cui n.b., du t.s., kang s.z., li f.s., zhang j.h., wang m . x . , l i z . j . , 2 0 0 8 r e g u l a t e d d e f i c i t i r r i g a t i o n improved fruit quality and water use efficiency of pearjujube trees. agric. water manag., 95: 489-497. dogan e., kirnak h., berekatoglu k., bilgel l., surucu a., 2008 water stress imposed on muskmelon (cucumis melo l.) with subsurface and surface drip irrigation systems under semiarid climatic conditions. irrigation sci., 26: 131-138. fabeiro c., martin d.e., santa olalla f., de juan j.a., 2002 production of muskmelon (cucumis melo l.) under controlled deficit irrigation in a semi-arid climate. agric. water manag., 54: 93-105. fao, 2014 faostat. faostat3.fao.org/home/index.html. farooq m., hussain m., wahid a., siddique k.h.m., 2012 drought stress in plants: an overview, pp. 1-33. in: aroca r. 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present study, the growth, productivity and fruit quality of tomato was investigated under foliar application of nitrogen from different sources. ammonium sulfate, urea and calcium nitrate with constant concentration of 100 mm n were weekly sprayed during four months under hydroponic culture system. a water spray treatment was considered as control. the results showed that vegetative growth parameters were significantly affected by n sources in different patterns. the factors such as plant height, leaf area, number of lateral shoots and shoot fresh and dry weight, as well as leaf nitrate reductase activity was significantly reduced by foliar application of ammonium sulfate and to less extent by urea, while there was improvement of these traits by foliar application of calcium nitrate compared to control. however, ammonium sulfate treated plants had the highest leaf spad value and leaf n concentrations. plant fruiting pattern was also influenced by treatments, as ammonium sulfate spray reduced the fruit yield, and fruit vitamin c content, while it increased fruit tss and titratable acidity. the highest value of yield and vitamin c was recorded in calcium nitrate sprayed plants. 1. introduction application of different fertilizers play important role in agricultural production of food commodities. supply of adequate essential nutrients can significantly improve plant growth, quality and their nutritional values (marschner, 2011). different sources of each nutrient element can be applied as fertilizer to meet plant’s need of that special element. generally for most nutrients, there is little difference among effects of various sources; however, regarding nitrogen there is significant different effect of n form and sources on many vegetative and reproductive traits of plants (souri and roemheld, 2009). (*) corresponding author: mk.souri@modares.ac.ir citation: souri m.k., dehnavard s., 2018 tomato plant growth, leaf nutrient concentrations and fruit quality under nitrogen foliar applications. adv. hort. sci., 32(1): 41-47 copyright: © 2018 souri m.k., dehnavard s. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 18 october 2017 accepted for publication 22 november 2017 ahs advances in horticultural science adv. hort. sci., 2018 32(1): 41-47 42 nitrogen fertilizers have important role in improving crop productivity; however low use efficiency rate of n fertilizers threatens sustainable plant production (souri, 2010). on market, various nitrogen sources exist for application under field and hydroponic culture. urea, ammonium sulfate and ammonium nitrate are the main nitrogen fertilizers for soil application, while calcium nitrate and potassium nitrate are the major nitrogen sources commonly are used in hydroponic systems (marschner, 2011). nitrogen forms (ammonium vs. nitrate) can have significant effect on morphology and physiology of plants particu l a r l y u n d e r h y d r o p o n i c c u l t u r e ( s o u r i a n d roemheld, 2009). in addition, ammonium instead of n i t r a t e ( s m o l e ń a n d s a d y , 2 0 0 9 ; s o u r i 2 0 1 0 ; mars ch n er, 2011) an d f o l i ar co mp l emen tati o n (kolota and osinska, 1999; dehnavard et al., 2017) supply of nitrogen can significantly improve n fertilizing efficiency in cropping systems. there may be several potential benefits of providing nitrogen to greenhouse crops via the foliage. these include: reduced nitrogen losses through denitrification and leaching, the ability to supply nitrogen when root activity is impaired e.g. in soil or water saline conditions, and luxury supply of plants with nitrogen. in cereals and some agronomic crops, late foliar application of urea generally results in higher grain protein and n content (fageria et al., 2009). the best quality parameters of plant growth and productivity of cabbage was reported when foliar versus soil application of fertilizers was applied (atanasova et al., 2007). tomato is one of the major vegetable crops that is cultivated in many parts of the world and consumed in many dishes. the application rate of n fertilizers in t o m a t o c u l t u r e i s g e n e r a l l y h i g h ( s o u r i a n d roemheld, 2009), with n efficiency of about 30-50% (zotarelli et al., 2009; souri, 2010). in many greenhouses due to continuous cultivation and fertilization, soil salinity level is generally much higher than the threshold. if some levels of required n could be applied on plants as foliar spray, it can improve n use efficiency with less soil salinity buildup and less environmental side effects. in literature the tomato responses to continuous foliar spray of nitrogen sources have not well been established. therefore, the aim of this study was to evaluate response of tomato plants to foliar application of various nitrogen forms and sources under greenhouse and hydroponic culture. 2. materials and methods experimental set up this study was conducted during 2012 under greenhouse conditions at faculty of agriculture, trabiat modares uni., tehran-iran. the experiment was done in hydroponic system with four treatments and four replications arranged in completely randomized design. tomato seeds (lycopersicon esculentum var. money maker) were germinated in quartz sands and after germination (in four-leaf stage) two homogeneous seedlings were transferred to pots containing a mixture of cocopeat and perlite in ratio of 3:1 (v/v). one week later one of them was removed and one week later foliar treatments were applied on plants. the nutrient solution composition was prepared following hoagland formula (dehnavard et al., 2017). black plastic pots as replication, with a volume of nearly 12 liter were used for plants cultivation. for first two weeks after seedling transplanting, plants were supplied with one daily application of 100-200 ml of nutrient solution. thereafter, pots were supplied two times per day with nutrient solution of a final quantity of 250-1500 ml until end of experiment. the amount of applied solution increased with plant size and reached the amount of 1.5 liter per day at full plant size. treatments were foliar spray of three nitrogen sources of ammonium sulfate (as), urea, calcium nitrate (cn) and a no spray control. all three n sources were applied in constant concentration of 100 mm n (equals to 1400 mg l-1 n). sprays were done on weekly basis during 4 months of active growth period from 25 march (first foliar spray) until the end of july 2012. distilled water was sprayed in control plants. spraying treatments were done in the early morning, one hour after sun rise. measurements during plant growth period for four months, various vegetative traits as well as fruit harvesting records were collected. the final harvest of plants was done at the end of july. chlorophyll index was measured two times by spad meter (model 502 plus, illinois, usa), each time with 30 readings on 3 different areas for 10 randomly selected leaves per pot that the average was presented as leaf spad value. spad readings were done at the middle of experiment and before final harvest, at 10 o’clock in the morning. p l a n t h e i g h t , n u m b e r o f l a t e r a l s h o o t s , souri et al. tomato plant growth, leaf nutrient concentrations and fruit quality under n-foliar applications 43 shoots fresh and dry weight were measured at final harvest. cumulative harvest of fruits was recorded as final yield. plant leaf area was measured by leaf area meter and calculated as average area of a single leaf. fruits after harvesting were transferred to laboratory for further quality assessment. fruit firmness was measured by penetrometer (model wagner) after removing fruit skin using a blade. fruit total soluble solids (tss), titratable acidity (ta) and ph were determined in fruit juice squeezed by a squeezer. fruit tss percentage was measured by a portable refractometer (atago, tokyo, japan). fruit ph was determined using a portable ph meter, and titratable acidity was determined with titration of 5 ml of fruit juice with naoh 0.1 n until end ph of 8.1. for determination of vitamin c (l-ascorbic acid), 50 g of fresh fruit tissue was crushed in a porcelain mortar in vicinity of 20 ml metaphosphoric acid 6%, and then the juice transferred into a 50 ml tube, then centrifuged at 4000 rpm for 10 min. five ml of the supernatant transferred into an erlenmeyer flask, and received 20 ml of metaphosphoric acid 3%. then titration of the extract was done by di-chloro phenol indophenols until appearance of a rosa color, which the amount of vitamin c (mg 100-1 g fw) was calculated accordingly and based on a standard curve of lascorbic acid concentrations. from each treatment and replicates 3 fruits were kept in room temperature (25±2°c) for one week, thereafter their weight loss percentage was calculated. total nitrogen of leaves was determined using kejeldahl method and the activity of leaf nitrate reductase enzyme (nr) was determined after grinding and homogenizing of leaf materials in a mortar containing liquid nitrogen. nitrate reductase was extracted in a buffer consisting of 100 mm hepes (ph 7.5), 1 mm edta, 7mm cystein, 3% polyvinyl polypyrolidone (pvpp), 10 μm leupeptin, and 1 mm phenyl methyl sulfonyl fluoride (pmsf). after preparation of extracts sulfanilamide (0.5%) and n-(1-naphthyl)-ethylenediamine dihydrochloride (0.01%) in 1.5 m hydrochloric acid (hcl) were used for color development and the amount of no2was determined spectrophotometrically at 540 nm and then nitrate reductase activity was calculated accordingly. statistical analysis excel software was used for calculating means and standard deviations and data were analyzed by spss software. comparison of means was performed at 5% by duncan’s multiple range test. 3. results the results of present study showed that plant vegetative growth parameters were significantly affected by nitrogen sources. plant height was significantly higher in calcium nitrate treated plants compared to ammonium sulfate and urea treated plants (table 1). the significant largest area of a single leaf and the highest number of lateral shoots were recorded in those plants which were treated with calcium nitrate, while the significant lowest records were in ammonium sulfate treated plants (table 1). spad value as a chlorophyll concentration index of plants were highest in ammonium sulfate treated plants (table 1), and there was no significant effects among other treatments. plant shoot fresh and dry weights were significantly affected by foliar spray of nitrogen sources. the significant highest shoot fresh and dry weight was obtained from plants treated with calcium nitrate and control plants. the significant lowest shoot fresh foliar spray treatment plant height (m) leaf area (cm2) lateral shoots (no.) spad value control (d-water) 1.98±0.16 ab 74.95±6.2 b 59.25±3.5 b 37.025±1.6 b ammonium sulfate 1.365±0.22 c 66.60±6.0 c 43.25±5.6 c 46.925±3.8 a urea 1.753±0.17 b 71.25±5.6 bc 51.00±9.7 bc 39.825±1.8 b calcium nitrate 2.225±0.22 a 80.60±2.7 a 70.50±5.2 a 39.275±1.8 b table 1 mean values of plant height, leaf area, number of lateral shoots and leaf spad of tomato plants. plants were grown in hoagland nutrient solution for 17 weeks all three n sources were applied in constant concentration of 100 mm n. spad readings were done two times at the middle of experiment and before final harvest, and the average was presented. data are average of 4 replications ± sd. in each column means with a common letter have no significant difference at 5% of duncan test. adv. hort. sci., 2018 32(1): 41-47 44 and dry weight was in plants treated with ammonium sulfate and urea (table 2). determination of leaf nitrogen concentration (table 2) revealed that plants treated with ammonium sulfate and urea had significantly higher amounts compared to control and calcium nitrate treated plants. foliar spray of nitrogen sources showed significant effect on leaf nitrate reductase enzyme activity (table 2). nitrate reductase is the key enzyme in nitrate assimilation that its activity depends on several factors including nitrogen and nitrate status of plant tissues. the significant highest activity of this enzyme was in leaf of plants treated with calcium nitrate followed by control plants and those treated with urea. the significant lowest nitrate reductase activity was in ammonium sulfate treated plants. fruiting pattern of plants was also influenced by sprays of nitrogen sources and forms (table 3 and 4). number of fruits per plant was highest in calcium nitrate treated plants; however, they had no significant difference with control and urea treated plants. those plants which were treated with ammonium sulfate produced significant lowest number of fruits (table 3). the amounts of fruit yield per plant was significantly higher in calcium nitrate treated plants (table 3), followed by control, urea and ammonium sulfate treated plants. fruit firmness was not affected by foliar spray of nitrogen sources; however the percentage of fruit weight loss was significantly influenced by n foliar treatments. the highest weight loss, during one week keeping fruits at room temperature, was in fruits of those plants which were treattable 2 mean values of shoot fresh and dry weight, leaf n concentration and nitrate reductase activity of tomato plants plants were grown in hoagland nutrient solution for 17 weeks. all three n sources were applied in constant concentration of 100 mm n. data are average of 4 replications ± sd. in each column means with a common letter have no significant difference at 5% of duncan test. foliar spray treatment shoot fw (g) shoot dw (g) leaf n concentration (%) leaf nitrate reductase activity (µ mol no 2 g fw h) control (d-water) 1581±122 ab 13.7±1.6 a 2.3±0.17 b 0.71±0.1 b ammonium sulfate 1213±59 c 11.5±0.8 b 3.2±0.30 a 0.17±0.01 d urea 1469.5±79 b 11.4±1.14 b 2.9±0.32 a 0.30±0.09 c calcium nitrate 1758.3±186 a 14.2±1.7 a 2.5±0.13 b 0.90±0.06 a table 3 mean values of fruit number, fruit yield, fruit firmness and fruit postharvest weight loss in tomato plants were grown in hoagland nutrient solution for 17 weeks. all three n sources were applied in constant concentration of 100 mm n. fruit weight loss was measured after one week in room temperature of 25±°c data are average of 4 replications ± sd. in each column means with a common letter have no significant diff erence at 5% of duncan test. foliar spray treatment number of fruits plant-1 fruit yield (g plant-1) fruit firmness (kg cm2) fruit weight loss (%) control (d-water) 19.7±2.7 a 2210.7±253 b 1.24±0.16 a 5.15±0.83 b ammonium sulfate 17.2±2.6 b 1824.5±218 c 1.32±0.10 a 12.25±2.04 a urea 19.0±2.2 a 1896.0±230 c 1.23±0.21 a 6.35±0.79 b calcium nitrate 20.2±2.2 a 2994.5±306 a 1.38±0.11 a 5.40±1.01 b table 4 mean values of fruit tss, fruit ta, fruit ph, and fruit l-ascorbic acid content in tomato under different fertilization plants were grown in hoagland nutrient solution for 17 weeks. all three n sources were applied in constant concentration of 100 mm n. data are average of 4 replications ± sd. in each column means with a common letter have no significant difference at 5% of duncan test. foliar spray treatment fruit tss (%) fruit ta (%) fruit ph fruit l-ascorbic acid (mg 100 g fw-1) control (d-water) 5.50±0.16 ab 3.75±0.13 b 3.68±0.1 a 36.8±4.1 a ammonium sulfate 5.98±0.27 a 4.57±0.22 a 3.95±0.3 a 31.0±2.1 b urea 5.48±0.17 ab 3.85±0.21 b 3.90±0.2 a 33.5±2.9 ab calcium nitrate 5.35±0.18 b 3.60±0.18 b 3.78±0.2 a 37.1±3.4 a souri et al. tomato plant growth, leaf nutrient concentrations and fruit quality under n-foliar applications 45 ed with foliar spray of ammonium sulfate (table 3). fruit tss was also highest in ammonium sulfate treated plants and the significant lowest fruit tss was in calcium nitrate treated plants (table 4). similarly, fruit titrateable acidity values (ta) unchanged in urea and calcium nitrate treated plants compared to control, however sprays of ammonium sulfate resulted in significant higher amounts of fruit ta (table 4). fruit juice ph was not affected by foliar sprays of nitrogen sources (table 4). the significant highest l-ascorbic acid concentration was in calcium nitrate treated plants and control plants, while the significant lowest l-ascorbic acid was in ammonium sulfate treated plants (table 4). 4. discussion and conclusions the results showed that many growth and productivity traits of tomato plants were significantly affected by spray of n sources. plant biomass production and its different parameters including plant height, leaf area, number of lateral shoots, fresh and dry weight were significantly reduced by both ammonium sulfate and to less extent by urea treatments, while calcium nitrate sprays resulted in improvement of all these growth parameters compared to as and urea treatments. reduction in growth parameters of tomato due to foliar application of various concentration of ammonium sulfate have been reported by dehnavard et al. (2017). on the other hand, tomato is a distinct sensitive plant to ammonium nutrition particularly under hydroponic culture (loqué and von wirén, 2004; souri and roemheld, 2009). in present study, despite plants were fed by nitrate in nutrient solution; however foliar application of ammonium forms of nitrog e n ( a m m o n i u m s u l f a t e a n d u r e a ) r e s u l t e d i n reduced growth of plants. foliar absorption of nitrogen cannot be restricted by root medium n status, as there is always plant affinity to absorb nitrogen (marschner, 2011; dehnavard et al., 2017). foliar spray of ammonium sulfate in concentration of 100 and 200 mm with weekly application was resulted in significant growth restriction and less biomass production of tomato plants (dehnavard et al., 2017), while sprays of 50 mm improved tomato plant growth parameters, probably due to the fact that applied ammonium concentration and corresponding absorption was not in stressful level, but rather favored better photosynthesis and plant growth. daily foliar application of urea as the sole n source for tomato seedlings improved seedlings growth (nicouloud and bloom, 1996). despite the tomato tissue concentrations of ammonium increases significantly in 12-24 hour after foliar urea application (nicouloud and bloom, 1996), however from various studies it seems that plants can tolerate urea sprays better than ammonium sulfate (souri and roemheld, 2009). metabolism of malate and excretion of protons play important role in maintaining ph during ammonium assimilation in the shoot following ammonium sprays (peuke et al., 1998). urea in frequent applications and higher levels may have toxicity to plants (bowman and paul, 1992). however, although foliar spray of urea is common in some crops, but its physiological effects varies with season, cultivar and concentration (bowman and paul, 1992; fageria et al., 2009). in addition, it has been reported that foliar spray of urea compared to ammonium and nitrate may has less damage to leaves (bowman and paul, 1992). the absorption rate of urea is generally higher than calcium nitrate and ammonium sulfate in foliar spray (bowman and paul, 1992; fageria et al., 2009). however, within the tissues urea breakdowns to ammonium ions that similar to ammonium uptake can result in some toxicities (but with lesser extent) and restricted plant growth traits. spad values and leaf n concentration in ammonium sulfate treated plants were significantly higher compared to control and calcium nitrate sprayed plants. there are few studies reporting the effects of foliar application of nitrogen sources on vegetable crops. urea has been mainly used in one or limited applications with no negative side effect on plant growth (fageria et al., 2009; zhang et al., 2009). higher leaf spad value by ammonium sprays could be mainly due to higher chlorophyll concentration induced by restricted leaf expansion and higher n concentrations (souri and roemheld, 2009; dehnavard et al., 2017). the effect of foliar applications of ammonium sulfate and urea on chlorophyll readings in this study is in agreement with the results of foliar ammonium application on tomato (dehnavard et al., 2017), and urea spray on broccoli (yildirim et al., 2007) and onion (charbaji et al., 2008). ammonium spray probably by restriction of leaf area expansion has resulted in higher leaf n concentrations. in addition, ammonium absorption can take place by many nutrient specific and unspecific transporters, which results in less plant cell control over ammonium uptake and transport within the tissues (souri and roemheld, 2009). it seems that chlorophyll biosynthesis is less sensitive to ammonium rather than other leaf parameters adv. hort. sci., 2018 32(1): 41-47 46 such as leaf cell expansion and cell division, root and shoots growth and protein biosynthesis. it has been shown that foliar application of urea or soil application of a stabilized ammonium fertilizer (entec) can significantly increase n concentrations of leaves and root in carrot (smoleń and sady, 2009) and tomato (souri and roemheld, 2009) with less nitrate content. three foliar sprays of urea with 3 days interval on onion plants showed that bulb fresh and dry weight were increased by urea levels to 5000 mg l-1 without any damage to leaves (charbaji, et al., 2008). the best quality parameters of dry weight, total soluble sugars, vitamin c and low nitrate content in the cabbage leaves were achieved by foliar versus soil application of fertilizers (atanasova et al., 2007). foliar application of ammonium and then urea significantly reduced nitrate reductase activity of tomato leaves (table 2). this can be a negative factor when nitrate is actively taken up by plant roots and due to low activity of this enzyme most of nitrate accumulate in vacuoles resulting in higher nitrate accumulation in leaves and probably in fruits. tomato plant yield was significantly reduced by ammonium sprays, while it was increased by calcium nitrate treatment. this ammonium effect could be d u e t o r e s t r i c t e d v e g e t a t i v e g r o w t h , r e d u c e d hydraulic conductance, phloem translocation and less fruit set, while calcium nitrate had no effect on fruit number but it increased the average fruit weight resulted in higher yield compared to control. foliar nitrogen application generally increase plant yield. the maximum increase in marketable yield in cabbage, onion, and cucumber using supplementary foliar n fertilization was 20.3%, 10.8% and 7.3%, respectively (kolata and osinska, 1999). foliar fertilization significantly decreased the level of cucumber leaf infestation by downy mildew disease (kolata and osinska, 1999). c h a n g e s i n f r u i t q u a l i t y t r a i t s a r e g e n e r a l l y observed due to foliar n applications. in this study increase in fruit tss by ammonium spray can be due to higher chlorophyll content of leaves and higher photosynthetic rates. it is probably feasible that foliar sprays of ammonium induced stress signals leading plant to have more fruit sugars and tss content. guvenc et al. (1995) reported that foliar urea application improved some quality (vitamin c and titratable acidity) and growth properties of tomato. in onion plants foliar spray of urea increased n, p, k and ca concentration of leaves that resulted in higher photosynthesis and sugar production; however, other nutrients were not affected (charbaji et al., 2008). similar results in carrot were found by foliar application of nitrogen sources (smoleń and sady, 2009). in present study, ammonium spray reduced vitamin c content of fruits. this can be due to stress conditions induced by foliar ammonium spray and less hydraulic conductance of plant tissues (souri and roemheld, 2009). changes in quality parameters of tomato by foliar nitrogen sprays are in agreement w i t h s i m i l a r s t u d i e s o n o t h e r v e g e t a b l e c r o p s (guvenc et al., 1995; chaurasia et al., 2005; yildirim et al., 2007; dehnavard et al., 2014), which showed that increasing nitrogen application reduced the vitamin c content. in addition, foliar application of nitrogen compounds can significantly improve plants tolerate to heat stress (zhao et al., 2008). foliar n application can improve plant growth parameters of dry weight, relative water content and nitrate reductase activity under moderate water stress particularly with drought sensitive varieties (zhang et al., 2009). in this experiment, foliar application of ammonium sulfate and to less extent urea reduced normal plant growth and some quality traits, while calcium nitrate generally improved tomato growth and some quality factors. however, calcium nitrate can increase nitrate content of fruits that is not suitable, and therefore is not recommended in repeated applications. spray of ammonium sulfate increased fruit tss and titrateable acidity while it decreased vitamin c and fruit postharvest freshness. so, restricted growth through lower height and less lateral shoots in ammonium foliar sprayed plants may suggest benefits regarding labor requirement for plant pruning-training and management under greenhouse production of tomatoes. in a d d i t i o n , b y f o l i a r n a p p l i c a t i o n ( p a r t i c u l a r l y ammonium sulfate and urea) less soil salinity and less nitrate accumulation occur. nevertheless, it is revealed that tomato still show sensitivity to ammonium nutrition through the foliage similar to root ammonium nutrition via nutrient solution. references atanasova e., mitova i., dimitrov i., stancheva i., 2007 effect of different fertilizer sources on the quality of head cabbage. j. appl. hort., 9(1): 74-76. bowman d.c., paul j.l., 1992 foliar absorption of urea, ammonium, and nitrate by perennial ryegrass turf. j. amer. soc. hort. sci., 117: 75-79. charbaji t., arabi m.i.e., jawhar m., 2008 urea foliar fertilization affects onion weight and nutrient content. inter. j. vegetable sci., 14(3): 198-204. souri et al. tomato plant growth, leaf nutrient concentrations and fruit quality under n-foliar 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cultivars under heat stress. biol. plant., 52(1): 113116. zotarelli l., scholberg j.m., dukes m.d., muñozcarpena r., icerman j., 2009 tomato yield, biomass accumulation, root distribution and irrigation water use efficiency on a sandy soil, as affected by nitrogen rate and irrigation scheduling. agr. water manag., 96(1): 23-34. impaginato 299 adv. hort. sci., 2018 32(3): 299-309 doi: 10.13128/ahs-22260 postharvest melatonin treatment reduces chilling injury and enhances antioxidant capacity of tomato fruit during cold storage f. azadshahraki 1 (*), b. jamshidi 1, s. mohebbi 2 1 agricultural engineering research institute, agricultural research education and extension organization (areeo), karaj 31585-845, iran. 2 department of horticultural sciences, college of agriculture and natural resources, university of tehran, karaj 31587-77871, iran. key words: enzyme activity, lycopene, melatonin, proline, tomato fruit. abstract: in this study, tomato fruit was treated with 50, 100 or 200 µm melatonin and then stored at 5°c for 28 days to investigate the effect of melatonin treatment on chilling injury, nutritional quality and changes in the antioxidant system. tomato fruit developed chilling injury, manifested as surface pitting and irregular red color development during storage. these chilling injury symptoms, ion leakage and malondialdehyde content were significantly reduced, and proline and carotenoids contents were significantly increased by melatonin treatment. meanwhile, melatonin substantially reduced o2 production rate and h2o2 content, which result from significantly higher activities of superoxide dismutase, catalase, and peroxidase than control during the storage. these results suggest that melatonin treatment can effectively enhance chilling tolerance and reduce chilling injury. the reduction in chilling injury by melatonin may be associated with enhanced enzymatic and non-enzymatic antioxidants, in favor of membrane integrity and thus low cellular and tissue damage. 1. introduction cold storage is one of the most effective postharvest technologies to preserve the quality of fresh produces from the time of harvest until final preparation for human consumption in food chain (bourne, 2006). however, cold storage imposes great risk on postharvest commodities sensitive to chilling injury (ci). tomato (lycopersicon esculentum), as one of the most important tropical crops, is typically cold sensitive (hong and gross, 2006). the most common visual injury symptoms of ci depicted for tomato fruit include irregular ripening and red color development as well as surface pitting on the fruit. furthermore, as the chilled tissues are weakened, they become prone to decay and microbial spoilage. this phenomenon limits postharvest life and leads to significant degradation of (*) corresponding author: farzad_shahrakiazad@yahoo.com citation: azadshahraki f., jamshidi b., mohebbi s., 2018 postharvest melatonin treatment reduces chilling injury and enhances antioxidant capacity of tomato fruit during cold storage. adv. hort. sci., 32(3): 299-309 copyright: © 2018 azadshahraki f., jamshidi b., mohebbi s. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 6 december 2017 accepted for publication 31 january 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(3): 299-309 300 produce quality (wang, 1993). melatonin, first discovered in tomato in 1995, accumulates in the fruits as they mature (dubbels et al., 1995; hattori et al., 1995). melatonin content, as an endogenous signaling molecule, increases in response to abiotic and biotic stress, such as drought, salinity, chilling, and pathogens to protect against damage caused by them (zhang et al., 2014; arnao a n d h e r n á n d e z r u i z , 2 0 1 5 ; l i u e t a l . , 2 0 1 6 ) . accumulation of higher levels of melatonin in horticultural crops is beneficial not only for human health, but also for prolonging storability (tan et al., 2012). melatonin, a naturally occurring indoleamine, acts as endogenous elicitor and signaling molecule for plants growth and development, decreasing of biotic and abiotic stress, as well as a potent hydroxyl radical scavenger and antioxidant (zhang et al., 2014; zhang and zhang, 2014; manchester et al., 2015; zhang et al., 2015). melatonin contribution has been evidenced in a semilunar rhythm in macroalgae guarding this plant against high temperature stress (tal et al., 2011). melatonin treatment decreased apoptosis chilling-induced in carrot suspension cells. moreover, melatonin treatment alleviated chilling-induced shrinkage and disruption of carrot cell plasma membranes (lei et al., 2004). it has been reported that melatonin treatment reduced chilling injury in peach fruits by enhancement of chilling tolerance and provoking of defense response during cold storage (cao et al., 2016). soleimani aghdam and rezapour fard (2017) reported that melatonin treatment at 100 µm decreased strawberry fungal decay resulting from higher superoxide dismutase (sod) activity, associated with lower catalase (cat) and ascorbate peroxidase (apx) activities as well as higher phenylalanine ammonia lyase (pal) enzyme activity leading to higher total phenols and anthocyanins accumulation a l o n g w i t h h i g h e r d p p h s c a v e n g i n g c a p a c i t y . likewise, marssonina apple blotch caused by fungus diplocarpon mali decreased by melatonin treatment at 0.1 mm which is caused by higher h2o2 accumulation leading to enhancing pathogenesis related (pr) proteins accumulation such as peroxidase, chitinase and b-1,3-glucanase, and triggering phenylpropanoid pathway by enhancing phenylalanine ammonia lyase (pal) enzyme activity (yin et al., 2013). it has been reported that the attenuating of postharvest physiological deterioration in cassava roots by melatonin treatment, obtained by lower h2o2 accumulation as a result of increasing antioxidant enzymes; sod, cat and gr activities causing higher membrane integrity indicated by lower malondialdehyde (mda) accumulation (ma et al., 2016). gao et al. (2016) reported that lower o-2 and h2o2 accumulation in melatonin treated peach fruits resulted from higher antioxidant enzymes sod, cat, apx activities, concurrent with lower lipoxygenase (lox) enzyme activity leading to higher membrane integrity indicated by lower mda accumulation. in addition to antifungal and antioxidant activities, melatonin is useful in increasing postharvest sensory and nutritional quality of fresh produces (meng et al., 2015; cao et al., 2016; gao et al., 2016; liu et al., 2016; ma et al., 2016). it has been reported that preveraison melatonin-treated grape berries showed higher endogenous melatonin accumulation, which not only enhances berry size and weight, indicated by higher sugars accumulation and higher endogenous hormones ga/aba ratio, but also enhances synchronicity of berry ripening (meng et al., 2015). it has been observed that preharvest melatonin-treated tomato fruits showed higher fruits weight caused by higher sugars accumulation, as well as higher organic acids accumulation results in tomato fruits with favorable flavor. moreover, higher lycopene and ascorbic acid contents were observed in preharvest melatonin-treated tomato fruits (liu et al., 2016). since there is a lack of knowledge about the influence of melatonin on chilling tolerance of tomato fruit during low temperature storage, the present work was initiated to determine the efficacy of postharvest melatonin treatment on chilling demonstrations and enhanced fruits visual and nutritional qualities through augmenting antioxidant capacity of tomato fruits frequently encountered under cold storage. 2. materials and methods fruit and treatment tomato fruit (lycopersicon esculentum mill. cv banemi) were harvested at the mature green stage (i.e., liquefying locular tissue, seeds not cut with a knife) (saltveit, 1991) from a local producer in mohammad shahr, karaj (iran) and then immediately transported to karaj agricultural engineering and engineering research institute laboratory. the green stage of maturity with homogeneous size and randomly allotted into three groups (100 fruits per grop) for treatment in triplicate by dipping of fruits at 0 (control), 50, 100 and 200 µm melatonin solution for azadshahraki et al. postharvest melatonin treatment in tomato fruit during cold storage 301 5 min at 20°c. the selected concentrations were based on published effects of these compounds on peach, strawberries and cherry tomatoes (sun et al., 2015; cao et al., 2016; soleimani aghdam and rezapour fard, 2017). following immersion, the fruits were dried for 1 h at room temperature. the tomato fruits were then put in plastic baskets, covered with a perforated plastic bag to retard weight loss and stored at 5°c with 80-85% relative humidity for 4 weeks. the seven-day intervals during storage at 5°c followed by shelf life at 20°c for 1 and 3 days, the development of chilling injury and ripening characteristics as well as enhanced fruits nutritional quality through augmenting antioxidant capacity were measured, respectively (ding et al., 2002). measurements of chilling injury and ripening characteristics chilling injury (ci) of fruits was evaluated at 20°c for 1 day after the 7-, 1421 or 28-day cold-storage periods. symptoms of tomato fruit chilling injury were manifested as surface pitting and large green patches or blotchy yellow areas resulting from loss of full red color development ability (wang, 1993). the severity of the symptoms was assessed visually according to the following four-stage scale: 0= no pitting; 1= pitting covering <25% of the fruit surface; 2= pitting covering <50%, but >25% of surface; 3= pitting covering <75%, but >50% of surface and 4= pitting covering >75% of surface. the average extent of chilling-injury damage was expressed as a chillinginjury (ci) index, which was calculated using the following formula: ci index (%) = {[(ci level) × (number of fruit at the ci level)]/(total number of fruits) × 4} × 100. for determining the effect of different treatments on ripening, fruits following 28 days storage, were incubated in diffused light at 20°c for 3 days to full r e d c o l o r d e v e l o p m e n t ( d i n g e t a l . , 2 0 0 2 ) . measurement of full red color development in terms o f c a r o t e n o i d s a c c u m u l a t i o n w a s c o n d u c t e d . lycopene and β-carotene were determined by the method described by nagata and yamashita (1992). the amount of 0.1 g of fruit tissue was mixed with 20 ml of hexane:acetone solution (3:2). an aliquot was taken from the supernatant and measured at 453, 505, 645, and 663 nm in a spectrophotometer. the content of lycopene and β-carotene was estimated using the following equations: lycopene = -0.0458 a 663 + 0.204 a 645 + 0.372 a 505 0.0806 a 453 β-carotene = -0.216 a 663 1.220 a 645 + 0.304 a 505 + 0.452 a 453 the results were expressed in milligrams per 100 g fresh weight (mg 100 g-1 fw). measurements of ion leakage and malondialdehyde content ion leakage was measured at 20̊°c for 3 days after the 7-, 14-, 21or 28day cold-storage period according to the method of zhao et al. (2009). 3 mm thick of mesocarp tissues were excised from equator part of 5 fruits. disks were put into aqueous 0.1 m mannitol and shaken at 100 cycles/min for 2 h. the conductivity of the solution (l1) was measured with a conductivity meter. solutions were boiled for 10 min and then cooled to 20°c. the conductivity of tissues (l2) was measured. ion leakage was calculated as the ratio of l1 to l2. malondialdehyde (mda) content was measured at 20°c for 3 days after the 7-, 14-, 21or 28day cold-storage period using the thiobarbituric acid method described by zhao et al. (2009) w i t h m o d i f i c a t i o n . a b s o r b a n c e a t 5 3 2 n m w a s recorded and corrected for nonspecific absorbance at 600 nm. the amount of mda expressed as μmol mda per gram of pulp. measurement of proline content proline content was measured at 20°c for 3 days after the 7-, 14-, 21or 28day cold-storage period using the acid ninhydrin method described by shan et al. (2007). proline in tissues was extracted with 30 ml l-1 sulfosalicylic acid at 100°c for 10 min with shaking. the extract was mixed with an equal volume of glacial acetic acid and acid ninhydrin reagent and boiled for 30 min. after cooling, the reaction mix was partitioned against toluene and the absorbance of the organic phase was recorded at 520 nm. the resulting values were compared with a standard curve constructed using known amounts of proline and expressed as μg proline g−1 fresh weight (fw). measurements of o2 production rate and h2o2 content the o2production rate and h2o2 content were measured at 20°c for 3 days after the 7-, 14-, 21or 28day cold-storage period. o2production was measured using the method of elstner (1976) with modification. 4 g of fruit tissue was homogenized with 5 ml of 50 mm phosphate buffer (ph 7.8) and then centrifuged at 8000×g for 20 min at 4°c. the supernatant was used for the determination of o2production and expressed as nmol g-1 fw min-1. for h2o2 measurement, 2 g of fruit tissue was homogenized with 5 ml of cold acetone and then centrifuged for 15 min at 8000×g at 4°c, the supernatant was collected immediately for h2o2 analysis according to the method of patterson et al. (1984). adv. hort. sci., 2018 32(3): 299-309 302 h2o2 content was expressed as nmol g -1 fw. enzyme extraction and analysis enzyme activities were measured at 20°c for 3 days after the 7-, 14-, 21or 28day cold-storage period. 5 g of fruits tissue were homogenized with 50 mmol/l phosphate buffer (ph 7) containing 0.2 mmol/l edta and 2% pvp. the homogenate was centrifuged at 12,000×g for 20 min at 4°c and the supernatant was used. sod (ec 1.15.1.1) activity was determined according to giannopolitis and ries (1977) with modification. one unit of sod activity was defined as enzyme that caused 50% inhibition of nitro blue tetrazolium reduction by recording the absorbance at 560 nm. according to zhang et al. (2013) with modification, 1 unit of cat (ec 1.11.1.6) activity was defined as 0.01 decrease in absorbance at 240 nm per min. pod (ec 1.11.1.7) activity was determined according to maehly and chance (1954) with modification. one unit of pod was defined as 0.01 increase of absorbance at 470 nm as a result of guaiacol oxidation. statistical analysis experiments were performed using a completely randomized design. all statistical analyses were performed with sas 9.2 software package. data were analyzed by one-way analysis of variance (anova). mean comparisons were performed using hsd in tukey’s test for comparing treatment group at level of 1% (p<0.01) on three biological replicates. 3. results chilling injury and ripening characteristics the chilling injury (ci) symptoms were expressed on control group as surface pitting and large green patches or blotchy yellow areas resulting from loss of full red color development ability (wang, 1993), only after 7-day of cold storage and following shelf life at 20°c for 1 and 3 days, respectively (fig. 1). no significant difference in ci was observed between the control and 50 μm melatonin-treated fruit. whereas, melatonin-treated groups with 100 or 200 μm underwent normal ripening at 20°c and only few visual chilling-injury symptoms were observed after 14 days storage at 5°c (fig. 1). fruits treated with 100 or 200 μm melatonin maintained the same quality as before chilling-temperature storage except for developing a slight yellow pigmentation, and the effect of the used formulas increased with increasing their concentrations (fig. 2a). the results indicate that a 14-day storage was the maximum that could be tolerated by untreated mature green fruit. in this experiment, treatments with higher concentrations (100 or 200 μm) of melatonin were more effective in protecting against chilling injury than lower concentration (50 μm). for examining the effect of melatonin treatment on color development of fruit after cold storage, mature green tomatoes were transferred to 20°c for 3 days for ripening. treatment with 100 or 200 μm melatonin, prior to 5°c storage, was effective at alleviating chilling injury; this treatment category resulted in normally fruit ripening and uniform red color development caused by significantly (p<0.01) more lycopene and β-carotene accumulations, and the effect of the used formulas on fruit ripening and eventually visual quality increased with increasing their concentrations (fig. 2). however, control and melatonin-treated group with 50 µm failed to develop the normal red color, with lower lycopene and βcarotene values, demonstrated by large green patches or blotchy yellow areas. interaction effects and time of storage had no meaningful influence on these traits. ion leakage, malondialdehyde content ion leakage and mda, as a consequence of membrane damage, are credible parameters for ci development and degree for postharvest tomato fruit fig. 1 chilling injury (ci) index (%) of tomato fruits treated with 100 and 200 µm during storage at 5°c and after 1 day of shelf life. all data are presented as a mean of three biological replicates, and vertical bars indicated the standard errors. different letters indicate significant differences at p<0.01. azadshahraki et al. postharvest melatonin treatment in tomato fruit during cold storage 303 (zhao et al., 2009). in this experiment, significantly the highest ion leakage was detected in control group (p<0.01) (fig. 3a). however, no significant differences were statistically found in the ion leakage incidence between melatonin-treated groups with 100 or 200 μm. as shown in figure 2b, mda content showed a similar pattern of change during storage. mda content was significantly (p<0.01) lower in melatonin-treated groups with 100 or 200 μm compared with control at the same time of cold storage, and the highest level observed about 14 to 21 days (fig. 3b). proline content there was a peak of proline content appearing in 14-day in all groups, which suggested that low temperature induced the proline synthesis mechanism in fruits (zhao et al., 2009). however, proline accumulation was about 2 times high in the melatonin-treated groups with 100 and 200 µm compared to control from 14-day to the end of storage period, and the effect of the used formulas on proline content increased with increasing their concentrations (fig. 4). no significant difference in proline content was observed between the control and 50 μm melatonintreated fruit. fig. 2 fruit ripening uniformity (%), lycopene (a) and β-carotene (b) contents of tomato fruits treated with 100 and 200 µm during storage at 5°c and after 3 days of shelf life. all data are presented as a mean of three biological replicates, and vertical bars indicated the standard errors. different letters indicate significant differences at p<0.01. fig. 3 ion leakage (%) (a) and mda (b) content of tomato fruits treated with 100 and 200 µm melatonin during storage at 5°c and after 3 days of shelf life. all data are presented as a mean of three biological replicates, and vertical bars indicated the standard errors. different letters indicate significant differences at p<0.01. 304 adv. hort. sci., 2018 32(3): 299-309 o2 production and h2o2 content in figure 5, the measured levels of o2and h2o2 were shown as an influence of low temperature to ros generation in fruits exposure to chilling stress. c o n t e n t s o f o 2 a n d h 2o 2 r e m a i n e d r e l a t i v e l y unchanged in control and melatonin-treated groups within the first 14 days of cold storage. thereafter, both o2and h2o2 contents increased rapidly, treatment with 100 and 200 µm melatonin significantly (p<0.01) restrained the enhancement of o2and h2o2 contents, and again the effect of the used formulas on o2production rate and h2o2 content decreased with increasing their concentrations (fig. 4). no significant difference in o2and h2o2 contents was observed between the control and 50 μm melatonintreated fruit. sod, cat, pod activities as depicted in figure 6a, the sod activity in both control and melatonin-treated groups steadily increased during storage, nonetheless significantly the highest sod activity was observed in melatonin treated groups with 200 and 100 µm throughout the storage, respectively (p<0.01). the changes of cat and pod activities in tomato fruit showed a similar pattern during the cold storage. the activities of both enzymes in control and melatonin-treated groups oscillatory increased with storage time. melatonin treatment significantly promoted the increases in activities of cat and pod, the activities of both enzymes were significantly higher (p<0.01) in these groups than those in control group during the whole storage (fig. 6). 4. discussion and conclusions little information is available on melatonin treatment of horticultural commodities, even though there are many reports suggesting that melatonin is an endogenous signaling molecule for the activation of certain plant defense responses and the onset of the tolerance has often been correlated with the accumulation of defense-related enzymes and compounds (zhang et al., 2014; arnao and hernándezfig. 4 prolin content of tomato fruits treated with 100 and 200 µm melatonin during storage at 5°c and after 3 days of shelf life. all data are presented as a mean of three biological replicates, and vertical bars indicated the standard errors. different letters indicate significant differences at p<0.01. fig. 5 o2production (a) and h2o2 (b) content of tomato fruits treated with 100 and 200 µm during storage at 5°c and after 3 days of shelf life. all data are presented as a mean of three biological replicates, and vertical bars indicated the standard errors. different letters indicate significant differences at p<0.01. 305 azadshahraki et al. postharvest melatonin treatment in tomato fruit during cold storage ruiz, 2015; liu et al., 2016). exogenous melatonin application has been shown to result in an improved chilling tolerance and reduced incidence of chilling injury in peach and strawberry fruits (cao et al., 2016; soleimani aghdam and rezapour fard, 2017). in this experiment, we found that melatonin treatment could effectively not only reduce development of surface pitting on the fruit and irregular ripening and full red color development (large green patches or blotchy yellow areas), the typical chilling injury symptoms in tomato fruit, but also enhance fruits nutritional quality. this indicates that postharvest treatment with melatonin increased chilling tolerance in tomato fruit. since melatonin treatment is easy to set up, inexpensive and safe, even if higher amounts are accumulated in the plant (tan et al., 2012), it could be a functional method to decrease chilling injury, maintain quality and prolong shelf life of tomato fruit. carotenoids, highly characteristic phytochemicals, known to be potent ros scavengers and antioxidants, act as a cell proliferation inhibitor and hindering of cancer cell growing (tijskens and evelo, 1994; levi et al., 2001; giovannucci et al., 2002; stahl and sies, 2005). during maturation/ripening, the green pigment chlorophyll degrades and carotenoids are synthesized. carotenoids, particularly lycopene and β-carotene, represent the primary components of ripe fruit pigmentation in tomato pericarp and are responsible for the characteristic color of ripe tomatoes, conferring deep red and orange colors, respectively. these carotenoids largely influence flavor and nutritional qualities as well as commercial value and enhances consumer acceptance of fresh tomato fruit (tijskens and evelo, 1994). in this study, higher accumulation of lycopene and β-carotene in melatonin treated groups with 100 and 200 µm not only contributed to alleviate chilling injury to fruit, but also lead to normally fruit ripening with uniform red color development (panels b and c of fig. 2). it has been reported that in tomatoes, the contents of lycopene and β-carotene increase from the green to the fully ripe stage (fraser et al., 1994). melatonin may affect directly or indirectly other carotenoid genes and/or enzymes in tomato fruit. this could be the case for example of lycopene cyclases, which is responsible for the formation of β-carotene from lycopene, which its accumulation is a ripening-related event in tomato (giovannoni, 2001). the higher levels of these compounds in melatonin-treated red ripe fruits may be associated with a general acceleration in ripening and with some of the associated transcriptional events, leading to the color change of tomato fruit (guo, 2015; sun et al., 2015). therefore, the improved capability of full red color development in chilling-faced melatonin-treated group is one of the most important outcome of this study for the quality of tomato. proline, an important amino acid, has been considered as a cellular osmotic regulator, protein stabilizer, free-radical scavenger, and lipid peroxidation inhibitor in plant (sun et al., 2015). the elevated level fig. 6 superoxide dismutase (a), catalase (b) and peroxidase activities (c) of tomato fruits treated with 100 and 200 µm during storage at 5°c and after 3 days of shelf life. all data are presented as a mean of three biological replicates, and vertical bars indicated the standard errors. different letters indicate significant differences at p<0.01. 306 adv. hort. sci., 2018 32(3): 299-309 of proline found to be associated with improved cold tolerance in chilling-sensitive plants (zhao et al., 2009; shang et al., 2011; zhang et al., 2013; cao et al., 2016). our findings are in agreement with the above reports (zhang et al., 2010), because a signific a n t l y ( p < 0 . 0 1 ) h i g h e r c o n t e n t o f p r o l i n e w a s observed in melatonin-treated tomato fruits with 100 or 200 µm during the whole storage period along with the reduced ci incidence (fig. 4). cao et al. (2016) reported that higher transcripts of ppp5cs and ppoa were observed in melatonin-treated peach fruits which provokes proline accumulation. zhao et al. (2009) claimed that proline levels in a tissue may be an effective indicator for ci analysis in postharvest tomato fruits. the reduction of cell energy and/or induction of alterations in membrane integrity are occurred in chilling-sensitive horticultural commodities at low temperatures. reducing scavenging potency through such factors as chilling-related inactivation of antioxidants and/or obstructed antioxidant turnover may result in the enhanced ros generation. chilling temperatures destroy the balance between ros formation and defense mechanisms which cause oxidatively chilling injury and consequent cellular damage (hodges et al., 2004). it is figured that antioxidant enzymes, sod, cat, and pod are the primary enzymatic scavenging mechanism of ros that contribute to attenuate chilling injury to fruit (sala and lafuente, 2000; mondal et al., 2004; ding et al., 2007; imahori et al., 2008). thus, this balance between the generation and scavenging of ros is crucial to cell survival during cold storage and is thought to be a major mechanism of resistance to chilling stress. it has been reported that in harvested commodities enhanced e n z y m a t i c a n t i o x i d a n t a c t i v i t i e s r e s u l t i n t h e improved chilling tolerance. a higher antioxidant enzyme activity was indicated in the chilling-tolerant mandarins compared with the chilling sensitive cultivars (sala, 1998). in many other studies, enhancement of antioxidant enzyme activity through a number of postharvest treatments (e.g. heat shock, low temperature conditioning and superatmospheric oxygen treatment) provoked chilling tolerance and alleviated chilling injury to fruit (wang, 1995; sala and lafuente, 2000; zheng et al., 2008). neutralizing of the o2by sod is the initial step of cell defense against free radicals (bowler et al., 1992). cat is one o f t h e e n z y m e s t h a t p r o t e c t c e l l s a g a i n s t r o s because it catalyzes the decomposition of h2o2 to form o2and h2o2 (imahori et al., 2008). pod catalyzes h2o2 dependent oxidation of substrate (fu and huang, 2001). in the present work, the higher increases in activities of sod, cat, and pod concurrent with reduced o2and h2o2 content in melatonintreated groups than those in control group were indicated (fig. 6). while, the levels of h2o2 and o2significantly increased during the development of irreversible chilling injury symptoms surface pitting and irregular ripening and full color development in control group. treatment with melatonin significantly alleviated these chilling-induced damages and increased the activities of sod, cat, and pod under cold stress. the increased sod activity could enhance the ability of the fruit to dismutate superoxide radicals, while the increases in cat and pod activities would contribute to the stronger omitting of hydrogen peroxide (lukatkin, 2002), which may give an explanation for the lower levels of o2and h2o2 observed in melatonin-treated groups. these results suggest that effect of melatonin in reducing the incidence of chilling injury was correlated to enhanced enzymatic scavenging mechanism of ros. in melatonin-treated tomato fruits the continues functions of sod, cat, and pod may be associated with higher stress resistance and eventually extended shelf life. membrane lipid peroxidation may be one of the first events in the manifestation of ci, in which phase mda as a final product of polyunsaturated fatty acid oxidation was produced and damaged to cell membrane, resulted in ion leakage (lukatkin, 2002; imahori et al., 2008). as depicted in panels a and b of figure 3, the increase in ion leakage and mda from 14-day of storage period indicates that cold storage caused a distinct deterioration of membrane integrity and activation of lipid peroxidation in the non-treated control group, which could be attributed to the decreases in sod and cat activities as well as in antioxidant compounds including lycopene, βcarotene and proline. these reductions induced by chilling stress favor accumulation of o2and h2o2, which can result in lipid peroxidation. ion leakage and mda content may be a reflection of ci development and fruit cold tolerance (zhao et al., 2009). furthermore, posmyk et al. (2005) reported that ion leakage intensity and mda content in a tissue can be a reliable indicators of the structural integrity of the membranes of plants exposed to low temperature. given to these results, prevention of mda accumulation and subsequent ion leakage by melatonin treatment could be related to a low degree of lipid peroxidation, which could result from the maintenance of high enzymatic and non-enzymatic antioxidants. it has been reported that melatonin efficiently con307 azadshahraki et al. postharvest melatonin treatment in tomato fruit during cold storage tributes to membrane integrity maintenance, and in turn, alleviates symptoms and severity of ci (cao et al., 2016; soleimani aghdam and rezapour fard, 2017). treatment with melatonin attenuated chilling induced shrinkage and disruption of carrot cell plasma (lei et al., 2004). as a whole, the results of this study show that melatonin treatment can effectively enhance chilling tolerance and reduce chilling injury of 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fruits from 2 cultivars differing in chilling tolerance at cold storage.food chem., 74: 348-352. zheng y.h., raymond w.m.f., wang s.y., wang c.y., 2008 transcript levels of antioxidative genes and oxygen radical scavenging enzyme activities in chilled zucchini squash in response to superatmospheric oxygen. postharvest biol technol., 47: 151-158. impaginato 517 adv. hort. sci., 2018 32(4): 517-524 doi: 10.13128/ahs-20689 stability analysis of fruit yield of some olive cultivars in semi-arid environmental condition i. arji (*) crop and horticultural science research department, kermanshah agricultural and natural resources research and education center, areeo, kermanshah, iran. key words: adaptability, ammi, olea europaea l., olive, stability parameters, yield. abstract: this study was conducted to evaluate yield stability of 12 iranian and foreign olive cultivars in dalaho olive research station during 2006-2008. according to the variance analysis, significant variation (p<0.01) was observed between cultivars and years. classification based on duncan (p<0.05) showed that konservolia was superior variety and sevillano, koroneiki and zard were placed in the second group. cultivars were divided into 3 groups based on cluster analysis using ward method. the first principal component of the interaction between olive cultivars and the year’s show 69.25% of the variance and was statistically significant at 1% level based on ammi analysis. according to regression coefficient (bi) deviation from regression (s2di), wricke’s ecovalence (wi), coefficient of determination (ri 2) and shukla’s stability variance (δi 2) methods ‘mission’ and ‘zard’ had the higher stability. according to the ammi stability (asv) ranking, the following cultivars were the most stable, mission, amigdalolia and koroneiki, while the most unstable were ‘konservolia’, ‘sevillano’, ‘roghani’, ‘arbequina’ and ‘abou-satal’. ‘konservolia’ even showed the lowest stability but its stability in all parameters was significant different in terms of performance. generally ‘konservolia’, ‘sevillano’, ‘koroneiki’ and ‘zard’ were appropriate for fruit yield and will be introduced for breeding programs in semi-warm climate. 1. introduction o l i v e ( o l e a e u r o p a e a l . ) t r e e i s a n e v e r g r e e n n a t i v e t o t h e mediterranean region. some olive wild genotypes are present in different region of iran like kermanshah province in the west of iran. there are m o r e t h a n 4 0 n a t i v e o l i v e g e n o t y p e s i n s u b t r o p i c a l r e g i o n s o f kermanshah province like sarpool-e-zahab, gilan-e-gharb and paveh. marone and fiorino (2012) reported that olive (olea europaea l.) distributed across three continents from south africa to the central part of the africa and horn africa, from egypt and red sea to the mediterranean areas and asia from palestine, syria, mesopotamia and western and eastern areas of himalaya chain to the southwestern of china. this report (*) corresponding author: issaarji@gmail.com citation: arji i., 2018 stability analysis of fruit yield of some olive cultivars in semi-arid environmental condition. adv. hort. sci., 32(4): 517-524 copyright: © 2018 arji i. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 24 may 2017 accepted for publication 19 june 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(4): 517-524 518 revealed that there are some olive genotypes in three continents. in recent years, due to higher olive oil demand, the cultivation of olive has been expanded in various regions of iran. however, the cultivation of olive tree is limited because of harsh environmental conditions and water scarcity in most of the new olive plantation areas (arji and arzani, 2008). the limitation of water as well as long hot summers in the regions lead to poor fruit and oil quality (saadati et al., 2013; khaleghi et al., 2015). cheng et al. (2017) stated that low temperatures would be improved olive oil quality by increasing unsaturated fatty acid amounts in the fruit. temime et al. (2006) reported that more unsaturated fatty acid of chetoui olive variety was recorded in cooler regions than dry and warm regions. despite of good vegetative growth, some of the olive varieties do not show good performance as production in warm regions. this is due to lack of adapted and stable cultivars in such environmental conditions. check-adapted varieties and optimal stability are essential for the fruit yield. it is assumed that the stability of a genotype is very important over time in each region (finlay and wilkinson, 1963). homeostatic and agronomic are two genotypic stabilities. in homeostatic stability a certain genotype shows constant response under different conditions. but in agronomic stability, genotype yield is linked to p r o d u c t i v i t y p o t e n t i a l ( h a y w a r d e t a l . , 1 9 9 3 ) . generally, the stability is defined as the actual performance of a genotype under changing environmental conditions. reliable stability of production efficiency under environment changing is very important (kan et al., 2010). stability analysis methods are categorized in two parametric and non-parametric groups (sabaghnia et al., 2006). several methods such as regression coefficient (finlay and wilkinson, 1963), sum of squared deviations from regression (eberhart and russel, 1966), stability variance (shukla, 1972) and additive main effects and multiplicative interaction (ammi) (gauch and zobel, 1988) have been commonly used to parametric stability analysis. environmental sustainability of individual genotypes can also be estimated by regression analysis and cultivar will be stable when the deviation of regression was zero or at least (hayward et al., 1993). it is mentioned that regression analysis in bilinear models and analysis of variance in biadditive models have limitations in genotype and environment interaction. this restriction reduced by multiplicative components for interactions in generalized linear models (glm) such as additive effects and multiplicative interaction (ammi) (gauch, 1992). in this model the main additive effects was calculated by variance analysis and then genotypes and environment interaction, which is known as multiplicative interaction, are analyzed by principal components analysis (romagosa and fox, 1993). olive is one of the fruit trees with alternate bearing tendency in which it not bear regularly (lavee, 2007). this phenomenon is affected by different fact o r s l i k e g e n e t i c a n d p h y s i o l o g i c a l t r a i t s (goldschmidt, 2005). the degree of alternate bearing in olive is highly dependent on environmental conditions (lavee, 2007). fruit production in olive is more irregular by climate change where adverse environmental conditions are frequent (lodolini and neri, 2012). for this purposes stability of olive production is very important in new olive growing region like sarpool-e-zehab environmental conditions. ammi analysis was used to evaluate the stability of different crops (esmaeilzadeh-moghaddam et al., 2011), but there is lack of research in horticultural crops. weather conditions are variable during different years in new olive cultivation regions so that we need to find out more stable olive cultivars. in the present work, the year was considered as environmental variable. generally, the main goal of this study was the evaluation of yield stability of different olive cultivars in warm condition of kermanshah province. 2. materials and methods material, site characterization and experimental design this experiment was conducted in dalahv olive research station of sarpool-e-zahab (longitude: 45° 51´ e, latitude: 34° 30´ n, altitude: 570 m asl) to verify the yield stability of 12 iranian and foreign olive cultivars (table 1). two years old self-rooting plantlets were planted in the year 2000, with 6x6 m spacing table 1 name and codes of genotypes genotype name 1 amphisis 2 konservolia 3 zard 4 amigdalolia 5 koroneiki 6 roghani 7 manzanillo 8 abou-satal 9 mission 10 arbequina 11 sevillano 12 shenge arji olive fruit stability analysis 519 distance in a randomized complete block design with three replications. each experimental unit consisted of 5 trees so that 15 trees of each cultivar were evaluated. trees were pruned as vase shape and irrigated each three days with drip irrigation system. climate of sarpool-e-zahab is warm with relatively low humidity during summer as shown in figure 1. also soil and water analysis were reported (tables 2 and 3). data analysis methods fruit yield was measured during 5 years from 2004 to 2008. as fruit yield was low in the years 2004 and 2005, therefore 3 years (2006, 2007 and 2008) were analyzed to determine yield stability. spss, irristat and excel were used for statistical analysis and the mean comparison was done by duncan’s multiple range test at p<0.05. the model of ammi analysis is presented in equation (1). y ger = μ + α n + β e +σ n λ n α gn γ en + ρ ge + ε ger (1) where αn is the main effect of genotype; βe is the main effect of environment; n is the number of main components in ammi model; λn is a single value related to the n remained main components in the model; αgn is thespecific vector for the g genotype from n main component; γen is the specific vector for the e environment from n main components; ρ ge is the noise and ger is the error (clay et al., 1995). the following parameters were calculated to analyze yield stability, coefficient of variability (cvi) (francis and kannenberg, 1978), wricke’s (1962) ecovalance (wi2), shukla’s (1972) stability variance (σi2), pinthus’s (1973) coefficients of determination (r2), and finlay and wilkinson (1963) regression coefficient (bi). alternate bearing index (abi) was calculated during three successive years from 2006 till 2008, using t h e f o l l o w i n g e q u a t i o n ( 2 ) ( m o n s e l i s e a n d goldschmidt, 1982): (2) where n = number of years, and a1, a2... an = yields in the corresponding years. 3. results and discussion fruit yield analysis of variance the results of variance analysis for yield of olive (kg/tree) show that the genotype, environment ( yea r) a n d i n t era c t i o n ef f ec t s w ere s i gn i f i c a n t (p<0.01) (table 4). specific response of the cultivars to ecological factors over a 3-year period were confirmed by the results of duncan multiple range-test, which proved that cultivar and year interaction effect was also significant (table 5). it is evident from data in table 5, for 3 study years, ‘konservolia’ had thehighest mean yield, 24.69 kg/tree, while ‘roghani’ had the lowest mean yield, 4.87 kg/tree. fruit yield variability was depending on the year but olive varieties show different responses (table 5). so, this indicates that the genotypes present different behavior in that environment. this may be due to differences in genetic basis of cultivars (rakonjac and živanovic, fig. 1 precipitation, mean temperature and relative humidity during five years of experiment. table 2 physical and chemical soil characteristics soil depth (cm) particle-size distribution (%) oc (%) ph tnv (%) ava. k (mg/kg) ava. p (mg/kg) total n (%)clay silt sand 0-30 34 52 14 2.25 7.7 41 520 6.2 0.18 31-60 40 37 23 0.78 7.7 45 275 2.6 0.06 table 3 irrigation water chemical characteristics ec (ds/m) tds (mg/l) ph meq/l s.s.p (%) s.a.r co 3 -2 co 3 h clso4 -2 sum anions ca2++ mg2+ na+ sum cations 550 352 7.28 0 4.6 0.3 1.9 6.8 6.6 0.2 6.8 2.94 0.11 table 4 analysis of variance for olive fruit yield s.o.s df ss ms replication 2 2083 0.115 ns cultivar 11 3026.53 275.14 ** error 22 199.59 9072 year 4 1023.13 511.567 ** cultivar x year 44 1423.3 64.696 ** error 96 285115 5.94 cv% 19.18% adv. hort. sci., 2018 32(4): 517-524 520 2008). olive varieties with yield stability are important for sustainable production. stable cultivars have high yield with lower variation during the years. b a s e d o n t h e r e s u l t s , ‘ k o n s e r v o l i a ’ , ‘ z a r d ’ , ‘koroneiki’, ‘amigdalolia’, ‘arbequina’ and ‘sevillano’ have higher fruit yield with moderate yield fluctuation during the years. analysis of variance is only able to express the presence or absence of interaction and is not possible to interpret yield stability. for this reason, using univariate and multivariate nonparametric interpret better interaction of cultivars and years in the sustainability debate (gauch, 1992; falconer and mckay, 1996; arciniegas-alarcon et al., 2011; gauch, 2013). ammi analysis the anova for fruit yield using the ammi method is presented in table 6. there were significant differences among the genotypes, environments (years) and g × e interaction. in this experiment environments were the years based on citadin et al. (2013) method. combined analysis of variance (anova) for fruit yield of olive cultivars indicated that genotypes, year and genotype-by-year interactions (gei) were the most important source of fruit yield variation (table 6). the contribution of variation caused by the cultivar, year and gei were 52.56%, 17.77%, and 24.72%, respectively. this result showed that olive cultivars had different yield performance across years. the high share of interaction in the total sum of squares is very important to use stability analysis for fruit yield of olive varieties. similar results were reported in yellow passion fruit by oliveira et al. (2014) and peanuts (oliveira and godoy, 2006). maulión et al. (2014) stated that the significance of the environmental effect and gei were used as a starting point to study yield stability among peach accessions. ammi analysis indicated that two first ipca were significant (p<0.01). the ipca1 accounted for 69.25% of the ge interaction (table 6). however, based on these results most information can be graphically displayed using ipca1. biplot graph of the model (ipca1 vs. yield) is presented in figure 2. according to figure 2, ‘zard’ and ‘mission’ showed greater yield stability by values near the origin of the ipca1 axis. however, mean yield of ‘mission’ was lower than total mean yield. ‘konservolia’ with highest fruit yield and ‘roghani’ with the lowest fruit yield were unstable cultivars and the others were in the intermediate stability. one of the most important parameter in olive stability is alternate bearing. this index seems to be useful in determining the sustainability of production in f r u i t t r e e s . b a s e d o n b i p l o t a m m i 1 a n a l y s i s , ‘konservolia’ was more productive (fig. 2) in all years than the others and its alternate bearing index was low (table 5). so it is recommended to use this parameter in stability evaluation. in this experiment, variability due to the year was greater than variability table 5 fruit yield (kg tree-1), mean yield (kg tree-1) and alternate bearing index of olive cultivars during 20062008 cultivar 2006 2007 2008 mean alternate bearing index amphissis 7.03 hij 3.5 j 16.37 cdef 8.97 efg 0.16 konservolia 26.03 b 14.57 c-g 33.47 a 24.69 a 0.06 zard 17.37 cd 8.23 hij 16.67 cde 14.09 cd 0.01 amigdalolia 15.39 cdef 6.533 ij 10.43 fghi 10.78 def 0.1 koroneiki 23.8 b 13.33 d-h 16.7 cde 17.94 bc 0.08 roghani 7.1 hij 4.5 ij 3 j 4.87 g 0.21 manzanillo 24.53 b 7.86 hij 6.27 ij 12.89 de 0.31 abou-satl 7.78 hij 7.17 hij 9.1 ghij 8.02 fg 0.04 mission 14.53 c-g 8.07 hij 14.33 c-g 12.31 def 0.003 arbequina 7.36 hij 10.18 fghi 16.08 cdef 11.21 def 0.19 sevillano 25.13 b 10.8 e-i 20.48 bc 18.81 b 0.04 shenge 10.34 fghi 6.4 ij 7.13 hij 7.96 fg 0.09 mean 15.53 8.43 14.17 12.71 table 6 analysis of variance for fruit yield of 12 olive cultivars by ammi during 2006 -2008 s. o. v df ss ss% ms genotypes 11 3026.52 52.56 275.14 ** year 2 1023135 17.77 511.57 ** cultivar x year 22 1423305 24.72 64.7 ** ipc1 12 985614 69.25 82.13 ** noise 10 437688 30.75 43.77 ns error 96 285115 4.95 2.97 total 35 5758075 fig. 2 biplot ammi1 (means vs pc1) for the data on the yield of olive (ton ha-1) with 12 cultivars () and five years (δ). arji olive fruit stability analysis 521 caused by varietal effects based on scattered effect (fig. 2). ammi analysis method is highlighted to study g x e i n t era ct i o n wh i ch co mb i n es a u n i va ri a t e method for the additive effects of genotypes and years with a method for the multiplicative effects of the g x e interaction (zobel et al., 1988; citadin et al., 2013). gauch and zobel (1996) stated that this method can contribute to the identification of widely adapted genotypes with high yields, as to the agronomic zoning for regional cultivar recommendation. a genotype will be ideal with high yields and ipca1 values near zero. in general, according to the results of ammi analysis zard was the most stable cultivar w i t h h i g h y i e l d a n d i p c a 1 v a l u e s n e a r z e r o . ‘konservolia’ and ‘sevillano’ had high yield but higher ipc1 values than zero, therefore we recommend them as superior cultivars for pickling purpose. ferreira et al. (2006) reported that an undesirable genotype has low stability as well as low yields. cluster analysis according to the obtained dendrogram from cluster analysis using ward method, genotypes were divided in three groups (fig. 3). this result is confirmed by biplot ammi1 (fig. 2). stability analysis results eberhart and russell’s (1966) stated that a stable cultivar is considered to be the one that has regression coefficient approximating 1.0 and standard error of regression as low as possible. according to this model a genotype with the higher mean fruit yield has general adaptability. in the present research, regression coefficients ranged from 0.02 to 2.11 for fruit yield (table 7). this variation in regression coefficients indicates that cultivars had different responses to year’s fluctuations. a genotype would be adapted to favorable conditions when regression coefficient is higher than one and other would be adapted to unfavorable conditions when regression coefficient is less than one. a genotype with regression coefficient equal to one would have an average adaptation to all environments. according to table 7, ‘amphissis’, ‘mission’ and ‘amigdalolia’ with regression coefficients near to one are most stable all the years. ‘koroneiki’, ‘zard’, ‘manzanillo’, ‘sevillano’ and ‘konservolia’ with regression coefficients higher than one were stable (table 7, fig. 2), while other cultivars like abequina, table 7 mean yields (kg/tree) and various stability measurements and their ranking orders of 12 olive cultivars evaluated during five years 2006-2008 cultivar fruit yield (kg/tree) rank bi rank s2di rank wi rank δi 2 rank cvi rank ri 2 rank asv rank amphissis 8.97 9 1.01 6 59.32 11 59.32 9 33.44 9 74.14 11 0.33 3 0.291 4 konservolia 24.69 1 2.11 12 54.93 10 89.87 11 51.77 11 38.57 6 0.7 7 1951 12 zard 14.09 4 1.34 9 0.66 2 3.94 2 0.21 1 36.08 5 0.99 10 0.546 6 amigdalolia 10.78 8 1.08 7 6.27 6 6.44 3 1.71 2 41.14 9 0.84 9 0.032 2 koroneiki 17.94 3 1.21 8 15.34 8 16.62 5 7.81 5 29.78 4 0.73 8 0.184 3 roghani 4.87 12 0.18 3 7.67 7 26.73 8 13.88 8 42.63 10 0.11 2 1345 10 manzanillo 12.89 5 1.57 10 134.4 12 143.68 12 84.05 12 78.49 12 0.34 4 0.5 5 abou-satal 8.02 10 0.16 2 1.22 3 21.27 6 10.61 6 12.33 1 0.37 5 1239 8 mission 12.31 6 0.96 5 0.65 1 0.69 1 1.74 3 29.86 3 0.98 11 0.027 1 arbequina 11.21 7 0.02 1 39.64 9 66.81 10 37.93 10 39.73 8 0.0004 1 1265 9 sevillano 18.81 2 1.92 11 2.13 4 26.21 7 13.57 7 38.88 7 0.98 11 1349 11 shenge 7.96 11 0.43 4 3.57 5 12.85 4 5.56 4 26.36 2 0.59 6 0.937 7 bi = finlay and wilkinson’s (1963) regression coefficient; sdi2 = eberhart and russell’s (1966) deviation from regression parameter; wi = wricke’s (1962) ecovalence; δi2 = shukla’s (1972) stability variance; cv% = francis and kannenberg’s (1978) coefficient of variability; ri2= coefficient of determination; asv = ammi stability value fig. 3 dendrogram from cluster analysis based on ward method. 522 adv. hort. sci., 2018 32(4): 517-524 shenge, abou-satal and roghani with regression coefficients less than one were unstable (fig. 2). ‘konservolia’ (bi=2.11) was productive during 2006 and 2008 than the others. high yielding varieties were not found stable with regression coefficients (bi). similar results were found by maulión et al. (2014) in peach stability evaluation. as olives have alternate bearing, ‘konservolia’ had the highest fruit yield in non-bearing year (2007) in compare to the others (table 5). the most stable cultivars with the lowest s2di values were mission and zard. the most unstable cultivarswith the highest s2di values were manzanillo, amphissis and konservolia. according to the eberhart and russell’s (1966) model, regression coefficients (bi) approximating 1.0 coupled with s2di of zero indicate an average stability. ‘mission’ and ‘zard’ with regression coefficients near to 1 and s2di near to zero were most stable than the others. zard cultivar had higher mean yield so it has general adaptability all the years. concept of ecovalence was defined by wricke (1962), where the genotypes with low eco valence have smaller fluctuations across environments and therefore are stable. the most stable cultivars according to the ecovalence method of wricke (1962) were mission and zard. these cultivars were in the ranked 6 and 4 for mean yield, respectively. the most unstable cultivars according the eco valence method were manzanillo and konservolia with the mean yield rank of 5 and 1 respectively (table 7). this method would not be suitable to select high-yielding cultivars but it is useful to select cultivars with the same yield of the mean yield (table 5). for this reason, genotypes with a low wi value have smaller deviations from the mean across years and are thus more stable. shukla’s (1972) stability variance (δi2) revealed that ‘zard’, ‘amigdalolia’ and ‘mission’ had the smallest variance across the years and were stable, while manzanillo and konservolia cultivars had the largest δi2 and were unstable. the ‘konservolia’, ranked first for mean yield, showed insteadpoor stability based on shukla’s stability variance. the mean cv analysis was proposed by francis (1977) to study the physiological basis of yield stability. the stable cultivar is the one that provides a high yield performance and consistent low cv (crossa et al., 1990). according to this method, ‘abou-satal’, ‘shenge’, ‘mission’ and ‘koroneiki’ were the most s t a b l e ; ‘ z a r d ’ , ‘ k o n s e r v o l i a ’ , ‘ s e v i l l a n o ’ a n d ‘ a r b e q u i n a ’ w e r e i n t e r m e d i a t e s t a b l e , w h i l e amigdalolia, roghani, amphissis and manzanillo w e r e t h e m o s t u n s t a b l e c u l t i v a r s ( t a b l e 7 ) . moghaddam and dehghanpour (2001) stated that the main problem with this method is that low-yielding cultivars are placed into the category of stable cultivars. in this experiment high yielding varieties were in intermediate parts of classification. a greater coefficient of determination (ri2) value is desired because higher ri2 values indicate favorable responses to environmental changes (sayar et al., 2013). in our study, zard, mission and sevillano cultivars had higher ri2 values for fruit yield and ‘amigdalolia’, ‘koroneiki’, ‘konservolia’ and ‘shenge’ with medium ri2 values have high and medium stability in yield, respectively while others with low ri2 values were unstable cultivars (table 7). according to the asv ranking, the following cultivars were the most stable, mission, amigdalolia and k o r o n e i k i , w h i l e t h e m o s t u n s t a b l e w e r e ‘konservolia’, ‘sevillano’, ‘roghani’, ‘arbequina’ and ‘abou-satal’ . based on yield cluster analysis olive cultivars were classified into three categories. category 1 was cultivars having high yield and medium alternate bearing (‘konservolia’, ‘sevillano’ and ‘koroneiki’) (fig. 3). these cultivars are widely adapted around the world (barranco et al., 2000; therios, 2009). barranco et al. (2000) reported that ‘konservolia’ has a high productivity and alternate bearing but ‘sevillano’ is productive with constant production in mediterranean regions. also, therios (2009) stated that ‘sevillano’ is cultivated in warmer regions in spain and italy witho u t a n y p r o b l e m s . o u r r e s u l t s r e v e a l e d t h a t ‘sevillano’ had relatively constant production during the experiment. koroneiki is one of the most important olive oil cultivar in the greece with high fruit yield and good oil quality (barranco et al., 2000). our results indicated that its productivity was relatively high and constant but oil content (data not presented) was low. category 2 was cultivars having medium yield and m e d i u m o r h i g h a l t e r n a t e b e a r i n g ( ‘ z a r d ’ , ‘manzanillo’, ‘mission’, ‘arbequina’ and ‘amigdalolia’) (fig. 3). results showed that ‘arbequina’ had medium productivity with medium alternate bearing. our result was not confirmed by therios (2009) and barranco et al. (2000) findings, where ‘arbequina’ has a high productivity with constant yield and high oil content in the italy. therios (2009) stated that manzanillo is categorized as a good performance o l i v e c u l t i v a r i n t h e w o r l d . i n o u r r e s e a r c h , ‘manzanillo’ had medium productivity with high arji olive fruit stability analysis 523 alternate bearing. mission is a dual-purpose commercial olive cultivars in the world (therios, 2009). mission’s productivity was medium and alternate in our research. amigdalolia is an olive cultivar originated from greece with medium productivity and alternate bearing (barranco et al., 2000). our result represent that this cultivar show medium productivity and alternate bearing. category 3 was cultivars having low yield and low, medium or high alternate bearing (‘abou-satl’, ‘shengeh’, ‘roghani’ and ‘amfissis’) (fig. 3). we do not recommend these cultivars for planting in warm environmental condition. 4. conclusions in conclusion, one of 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weed species, under laboratory and greenhouse conditions. celery extract had a strong inhibitory effect on germination and seedling growth of corchorus olitorius, echinochloa crusgalli and portulaca oleracea seeds. from dose response curves of tested seeds, lc50 were calculated to be in the range from 6.3 to 8.3 g l-1 for germination percent, from 7.2 to 8.0 g l-1 for shoot length and from 1.7 to 3.6 g l-1 for root length. completely inhibition of root growth was exhibited c. olitorius and p. oleracea at 7.5 g l-1 corresponded with 15 g l-1 for e. crusgalli seed. total phenolics in celery extract at 20 g l-1 constituted 201 mg l-1. ten phenolic acids were identified in extract by hplc, among of them p-coumaric acid and p-hydroxybenzoic acid were presented in high amounts. aqueous extract was partitioning between three solvents, hexane, methylene chloride, ethyl acetate. generally, water residue after partitioning aqueous extract with the three solvents had the most phytotoxic effect on seedling growth of target seeds. in greenhouse trial, foliar spray of aqueous extract of celery (30, 60 and 90 g l-1) and its fractions did not produce any significant effect on growth of two-weeks-old c. olitorius, or e. crusgalli or p. oleracea weeds. 1. introduction worldwide, weeds caused about 34% yield losses among the major crops. herbicide application is the most reliable weed control methods. negative impacts of herbicides on environmental human health and herbicide resistant weeds were considered the two problems faced in weed management with herbicides (jabran et al., 2015). thus, manipulating the allelopathy can help to improve weed control in agriculture and increase the acceptance of agricultural products in today´s demanding consumer markets (trezzi et al., 2016). allelopathy has been used as the basis for identifying plant species which may contain phytotoxic chemicals. these natural compounds can (*) corresponding author: ragabelmergawi05@hotmail.com citation: el-mergawi r., el-desoki e.r., 2018 allelopathic activities of celery extract and its fractions against corchorus olitorius, echinochloa crusgalli and portulaca oleracea weeds. adv. hort. sci., 32(4): 503-510 copyright: © 2018 el-mergawi r., el-desoki e.r. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 22 november 2017 accepted for publication 28 may 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(4): 503-510 504 offer excellent potential for new herbicidal solutions, or lead compounds for new natural herbicides (duke et al., 2000; vyvyan, 2002). the main purposes of research on allelopathy include the application of the allelopathic effects to agricultural production, reduction of the input of chemical pesticides and consequent environmental pollution, and provision of effective methods for the sustainable development of agricultural production and ecological systems (han et al., 2013; jabran et al., 2015). knowledge concerning allelopathy can also be a key component in supporting organic farming, for which weed control is a major problem (trezzi et al., 2016) organic farming can involve reduced weed infestation by using plant extracts or intercropping plant species with an allelopathic potential (bajwa et al., 2015). plant extracts are the way of using allelochemicals for weed control in agroecosystems, as they have been already used as post-emergence natural herbicides in some countries. in pakistan, for example, an aqueous extract deriving from sorghum shoots with a 10% concentration was left to ferment for several weeks and was subsequently sprayed post emergence for weed control. this fermented water extract, known as “sorgaab”, reduced weed density a n d w e e d d r y w e i g h t u p t o 5 0 % i n f i e l d t r i a l s (cheema and khaliq, 2000; cheema et al., 2002). celery (apium graveolens l.; apiaceae) has been cultivated for the last 3000 years, notably in egypt, and was known in china in the fifth century bc. it has been used as a popular aromatic herb and spice (chevallier, 1998). earlier studies of a. graveolens led t o i s o l a t i o n o f s o m e p h e n o l i c c o m p o u n d s a s phthalides (tang et al., 1990; momin and nair, 2001) and furocoumarins (garg et al., 1979). these compounds are reported for their insecticidal, nematicidal, antifungal and phytotoxic activities (kato et al., 1977; momin and nair, 2001; pavela and vrchotová, 2013). recently, sbai et al. (2017) reported that the aqueous extract (10-50 g/l) of a. graveolens had great inhibitory effect on root growth of germinated seeds of lettuce (in the range between 80% and 90%). they isolated six compounds which included three phthalides [senkyunolide a, (3s)-butylphthalide and sedanolide], two furanocoumarins (bergapten and scopoletin) and one phenyl propanoid (p-hydroxyphenethyltrans ferulate). senkyunolide a compound was the most toxic on lettuce germination and shoot growth, however, p-hydroxyphenethyl trans-ferulate was the most toxic on root growth. however, in spite of the wide range of biological activity of celery extract, but information concerning the herbicidal activity of this extract is rarely available. therefore, the main objective of this study was to evaluate the herbicidal activity of the aqueous celery extract and its fractions against corchorus olitorius, echinochloa crusgalli and portulaca oleracea weeds, with the goal of developing an effective plant derived herbicide. moreover, the phenolic acids that considered the main source of all bioactive phenolic substances were identified in celery extract via hplc. 2. materials and methods plant material plants of celery (a. graveolens l. var. dulce) were purchased from a local market in cairo, egypt. identification of celery based on morphological traits that extensive observation of mature plants. the leaves located at the top of the leaf stalks were collected and dried in hot-air oven at 50°c for 72 h, powdered and used for extraction. preparation of aqueous extract different concentrations (w/v) of extracts were prepared by soaking known weight of dried leaves in known volume from distilled water at room temperature, and shaken for 24 h. the extracts were filtered through a whatman no. 1 filter paper and kept at 4°c in the dark until use. germination bioassay mature seeds of corchorus olitorius, echinochloa crusgalli and portulaca oleracea were collected from plants growing in fields of the experimental station of t h e n a t i o n a l r e s e a r c h c e n t r e ( r e s e a r c h a n d p r o d u c t i o n s t a t i o n , n u b a r i a r e g i o n , b e h a i r a governorate, egypt. uniform healthy seeds were selected. seeds were surface-sterilized with sodium hypochlorite (0.1%, w/v) for 2 min, washed under running tap water for 5 min followed by distilled water for 2 min, and stored for further use. twenty seeds were placed in a 9-cm plastic petri dish lined with a single whatman no. 1 filter paper, and then 4 ml of each extract concentration (2.5, 5, 7.5, 10, 12.5, 15, 17.5 and 20 g l-1) was added onto filter paper. distilled water (4 ml) was applied to a petri dish to serve as a control. four replicates of petri dishes of each treatment were placed, in a completely randomized manner, in a growth chamber at 25°c. after 5 days, germination percent, shoot length and root length of germinated seeds were determined. el-mergawi and el-desoki allelopathich activies of celery extract as weeds control 505 extract concentrations required to cause 50% inhibition of different germination criteria (germination percentage, root length and shoot length) were calculated by probit analysis. determination of total phenolics in aqueous extract extract of celery at 20 g l-1 was subjected to determ i n e t o t a l c o n t e n t s o f p h e n o l i c s u s i n g f o l i n ciocalteu reagent (singleton and rossi, 1965). analysis of phenolic acids in aqueous extract by highperformance liquid chromatography (hplc) phenolic acids in aqueous celery extract at a concentration 20 g l-1 were hydrolyzed with sodium hydroxide (mckeehen et al., 1999) and subjected to hplc analysis. approximately 15 ml of aqueous extract was added to 15 ml of 8 n naoh in a 50 ml pyrex centrifuge tube, purged with nitrogen, shaken for 2 h in the dark with a shaker and acidified with ice-cold 6 n hcl to reduce ph to 2. sample was centrifuged at 3000g, and the supernatant was decanted into separatory funnel. the supernatant was extracted with ethyl acetate (3 x 50 ml) with shaking for 10 s, and the mixture was allowed to settle for 5 min between extractions. the phenolic acids rich ethyl acetate fraction was dried by addition of anhydrous sodium sulfate and concentrated using rotary evaporator at 40°c to dryness. the residue was re-solubilized in 2.5 ml of methanol and filtered through a 0.2 μm ptfe filter prior to analysis. hplc analysis was performed using equipment from shimadzu (japan): a shimadazu lc-2010a liquid chromatograph, a shimadazu spdm10a diode array detector and a shimadzu class-vp v6.12 sp4 offline processing system. phenolics were analyzed using a luna rp-c18 (2) column (250×4.6 mm i.d, 5 μm, phenomenx). the mobile phase consisted of a mixture of acetate buffer: acetonitrile (9:1, v/v). acetate buffer was prepared by dissolving 6.35 g sodium acetate in one-liter h2o and 20 ml acetic acid. the detecting wavelength was 260 nm. standard phenolic acids (gallic, protocatechuic, p-hydroxybenzoic, syringic, ferulic and pcoumaric) were purchased from sigma aldrich and vanillic, caffeic, salicylic and cinnamic were purchased from fluka. greenhouse bioassay another experiment was performed to study the effect of the aqueous. celery extract on the growth of two-weeks-old c. olitorius, e. crusgalli and p. oleracea weeds grown under controlled conditions in an experimental greenhouse (25±3°c, 12 h photoperiod). aqueous extracts at four concentrations (0, 30, 60 and 90 g l-1) were prepared as previously mentioned and used in a greenhouse bioassay. plants were raised from the collected seeds in 12-cm diameter earthenware pots. the pots were filled with 750 g soil (sand:peat moss = 3:1, w/w) and 20 seeds of each plant species were sown per pot. two weeks after emergence, plants were sprayed with plant extracts at 0, 30, 60 and 90 g l-1 concentrations. extract solutions were applied to shoots of tested weeds using a epoca sprayer (italy). the solution was sprayed evenly over the entire surface of the plant, including the adaxial and abaxial surface of leaves. a total of 48 pots were maintained (i.e. 4 extract concentrations × 3 weeds × 4 replicates) in a completely randomized design. seven days after extract spraying, the plants were examined for visible injury levels and the percent of chlorotic and necrotic areas were recorded. fractionation of extract one hundred milliliter of high extract concentration that used either in germination trial (20 g l-1, w/v) or in greenhouse trial (90 g l-1, w/v) was subsequently partitioned with organic solvents with increasing polarity. the extract was partitioned three times with 200 ml aliquots of each solvent: hexane, methylene chloride (mecl2) and ethyl acetate (etoac) using separatory funnel. solvents were dried by addition of anhydrous, sodium sulfate, filtered and evaporated using buchi rotary evaporator at 40°c to dryness. the residues of hexane, mecl2 and etoac fractions were dissolved in 100 ml of dmso-water solution (0.1%, v/v). these three fractions and water residue (h2o fraction) were subjected to bioassay as previously mentioned. statistical analysis all data was subjected to anova analysis using costat software to evaluate the effect of extract rates upon germination and growth parameters of tested weeds. after anova, the parameters that were statistically significant (a. graveolens extracts with p≤0.05), were subjected to pro-bit analysis using ldp line. pro-bit curves were derived by plotting the extract concentration (on the x-axis) and inhibition % (on the y-axis). with the curves obtained, lc50 value was calculated for each parameter. this value represents the extract concentration at which 50% inhibiadv. hort. sci., 2018 32(4): 503-510 506 tion in different germination parameter occurs. 3. results effect of celery extract on germination percentage of seeds as shown in table 1, aqueous extract of celery had a strong inhibitory effect on germination percentage of the three tested seeds and the inhibition increased with concentration. increasing extract concentration above 5 g l-1 produced gradual decrease in germination percentage of c. olitorius. the highest reduction effect was produced by extract at highest concentrations, 17.5 and 20 g l-1 (89% and 93%, respectively). also, germination percentage of e. crusgalli and p. oleracea was reduced by the extract at all tested concentrations. the reduction percentage varied from 37% and 9%, respectively at the lowest concentration to 96% and 73%, respectively at the highest ones. effect of celery extract on seedling growth of seeds aqueous extract of celery exhibited a great inhibitory effect on shoot length of the three germinated seeds (table 2). the extract improved the shoot growth of e. crusgalli and p. oleracea at the lowest concentrations. the progressive increase in extract concentration followed by progressive reduction in shoot length of all tested seeds. more than 90% reduction in shoot length of three germinated seeds was obtained by soaking seeds in celery extract at 17.5 and 20 g l-1. an inhibition of root growth was observed in presence of all extract concentrations and roots more sensitive than shoots (table 2). root length of three target seeds varied in their response to different extract concentrations and e. crusgalli was less effective one. soaking seeds in celery extract at the lowest concentration produced great reduction in root elongation of all treated seeds ranged between 36% and 71%, relative to control. using extract at 7.5 g l-1 and above completely inhibited root growth of both p. oleracea and c. olitorius, whereas complete inhibition of e. crusgalli roots was obtained at 15 g l-1. extract concentrations required to cause 50% inhibition (lc50) inhibition percent and levels of lc50 of all germination parameters of tested weeds were calculated and dose-response curves were illustrated in figure 1. the three tested weeds varied in their inhibition percentages as affected with celery extract. among tested seeds, germination percent of p. oleracea was conconcentration (g l-1) germination % c. olitorius e. crusgalli p. oleracea control 100±0 a 100±0 a 100±0 a 2.5 95±5 a 63±7 b 91±9 b 5 93±2 a 67±4 b 84±4 b 7.5 49±5 b 52±2 c 73±3 c 10 41±4 c 43±5 c 74±7 c 12.5 23±3 d 39±7 c 60±2 d 15 11±5 e 25±4 d 40±2 e 17.5 11±5 e 14±5 d 37±2 e 20 7±2 e 4±1 e 27±3 f table 1 effect of aqueous celery extract on germination percentage of three weed species values are given as means of three replicates ± standard error. means with the same letters in a column are not significantly different at p<0.05. table 2 effect of aqueous celery extract on shoot and root lengths of three weed species values are given as means of three replicates ± standard error. means with the same letters in a column are not significantly different at p<0.05. rate (g l-1) shoot length (cm) root length (cm) c. olitorius e. crusgalli p. oleracea c. olitorius e. crusgalli p. oleracea control 2.56±0.14 a 3.35±0.35 a 2.17±0.13 b 2.41±0.08 a 1.83 ±0.26 a 1.95±0.23 a 2.5 2.74±0.25 a 2.91±0.34 b 2.50±0.23 a 0.81±0.17 b 1.17 ±0.12 b 0.57±0.07 b 5 2.21±0.19 b 2.43±0.19 c 2.19±0.60 b 0.51±0.11 c 0.65 ±0.20 c 0.29±0.06 c 7.5 1.38±0.40 c 1.73±0.33 d 0.58±0.40 c 0.00 d 0.51 ±0.17 c 0.00 d 10 0.76±0.28 d 0.79±0.20 e 0.60±0.06 c 0.00 d 0.25 ±0.06 d 0.00 d 12.5 0.36±0.06 e 0.77±0.20 e 0.56±0.31 c 0.00 d 0.22 ±0.01 d 0.00 d 15 0.10±0.02 e 0.53±0.31 ef 0.15±0.08 d 0.00 d 0.00 e 0.00 d 17.5 0.05±0.03 e 0.27±0.14 ef 0.14±0.14 d 0.00 d 0.00 e 0.00 d 20 0.00 f 0.06±0.03 f 0.11±0.06 d 0.00 d 0.00 e 0.00 d el-mergawi and el-desoki allelopathich activies of celery extract as weeds control 507 sidered a less sensitive to celery extract. since, value of lc50 of p. oleracea constituted 13.4 g l-1 corresponded with 8.3 and 6.3 g l-1 for c. olitorius and e. crusgalli, respectively. whereas, inhibition percent of shoot length did not produce a great variation among tested seeds and lc50 calculated to be 7.8, 7.2 and 8 g l-1 for c. olitorius, e. crusgalli and p. oleracea seeds, respectively. as shown in figure 1, root length exhibited the maximum inhibition effect as affected by celery extract when compared with germination percent and shoot length. moreover, root length of tested seeds varied in their inhibition percent as affected with celery extract. depending on lc50 levels, root length of p. oleracea (1.65 g l-1) was considered the most sensitive, followed by c. olitorius (1.8 g l-1) and e. crusgalli (3.6 g l-1). total phenolics and phenolic acids content in aqueous extract in this study, we determine total phenolics for celery extract at a concentration 20 g l-1 and found that the extract contained high amounts of phenolics config. 1 dose-response curves showing the effect of celery extract (inhibition % and lc50) on percent germination and seedling growth of three tested seeds. 508 adv. hort. sci., 2018 32(4): 503-510 stituted 201 mg l-1, as gallic acid. using hplc, ten p h en o l i c a c i d s w ere d et ec t ed i n t h i s a q u eo u s . extract, of which six compounds consisted of benzoic acid derivatives and four of cinnamic acid derivatives (table 3). among the identified phenolic acids, phydroxybenzoic acid, p-coumaric acid and ferulic acid were presented in high amounts (18.72, 30.32 and 11.12 mg l-1, respectively). except the minor quantity of cinnamic acid, moderate concentrations from other phenolic acids were determined in celery sample. effect of fractions on germination of weeds as shown in table 4, fractions obtained from the aqueous extract of celery varied in their effects on germination criteria of target seeds. as for germination percentage, the significant reduction effect was recorded only for mecl2 fraction on p. oleracea (15%) as well as for h2o residue on both c. olitorius (30%) and p. oleracea (36%). but, all examined fractions did not produce any significant effect on germination percentage of e. crusgalli seeds. whereas, h2o fraction induced a great reduction on shoot elongation of c. olitorius, e. crusgalli and p. oleracea seeds, reached 64%, 47% and 74%, respectively relative to control. with less extent, hexane and mecl2 fractions reduced shoot length of e. crusgalli. with few exceptions, all fractions reduced root elongation of target seeds. water fraction was the most toxic, inducing an inhibition of 99%, 94% and 50% in root length of p. oleracea, c. olitorius and e. crusgalli, respectively. whereas, the other fractions, hexane, mecl2 and etoac fractions reduced root length of e. crusgalli and p. oleracea between 17% and 43%, relative to control. effect of celery extract and its fraction on growth of weeds data presented table 5 revealed that spraying aqueous celery extract and its fractions did not produce any significant effect on growth of two-weeks old of either c. olitorius, or e. crusgalli or p. oleracea weeds. table 3 phenolic acids in aqueous extract of celery at concentration 20 g l-1 means with the same letters in a raw are not significantly different at p<0.05. table 4 effect of fractions obtained from aqueous extract of celery on germination of three weeds (% of control) acid derivatives concentration (g l-1) benzoic acid derivatives p-hydroxybenzoic acid 18.72 gallic acid 8.56 vanillic acid 8.8 syringic acid 6.4 protocatechuic acid 9.28 salicylic acid 7.84 cinnamic acid derivatives cinnamic acid 0.64 caffeic acid 9.52 p-coumaric acid 3.032 ferulic acid 11.12 table 5 effect of aqueous celery extract and its fractions on the growth of two-weeks-old of three weeds criteria weed species fractions control hexane mecl2 etoac h2o germination (%) c. olitorius 100 a 104 a 96 a 102 a 70 b e. crusgalli 100 a 98 a 104 a 102 a 95 a p. oleracea 100 a 107 a 85 b 109 a 64 c shoot length c. olitorius 100 a 97 a 102 a 102 a 36 b e. crusgalli 100 a 86 b 83 b 95 a 53 c p. oleracea 100 a 95 a 100 a 87 a 26 b root length c. olitorius 100 a 101 a 74 b 103 a 6 c e. crusgalli 100 a 57 c 83 b 79 b 50 c p. oleracea 100 a 61 b 76 b 63 b 1 c treatments injuries ( % of control) c. olitorius e. crusgalli p. oleracea 30 g l-1 2±0 a 0±0 a 1±0 a 60 g l-1 3±1 a 2±5 a 3±2 a 90 g l-1 3±2 a 3±1 a 3±1 a hexane fraction 2±1 a 3±2 a 4±2 a mecl 2 fraction 4±2 a 1±0 a 1±0 a etoac fraction 3±1 a 2±1 a 3±1 a h 2 o residue 4±1 a 1±0 a 1±0 a values are given as means of three replicates± standard error. means with the same letters in a column are not significantly different at p<0.05. 509 el-mergawi and el-desoki allelopathich activies of celery extract as weeds control 4. discussions and conclusions aqueous extract of celery was evaluated for its phytotoxicity against three plant species typically present as weeds in summer crops, under laboratory and greenhouse conditions. the extract displayed a great inhibition on germination percentage and seedling growth of target seeds. in line of these results, sbai et al. (2017) reported that lettuce germination was completely inhibited by celery extract at concentration above 20 g l-1. they extracted and identified the allelochemicals compounds that responsible of toxicity namely phthalides, among of them senkyunolide a was the most toxic in lettuce g e r m i n a t i o n . c u r r e n t s t u d y r e v e a l e d t h a t t h e inhibitory effect of celery extract on germination percent varied between tested seeds, and p. oleracea possessed the least sensitivity. variation between different plant species in their sensitivity to plant extracts was previously observed by many investigators (al-humid and el-mergawi, 2014; han et al., 2008). this study showed that root growth was the most sensitive to extract than shoot growth. since, lc50 of shoot length of three target seeds ranged between 7.2 g l-1 and 8 g l-1 whereas, values of lc50 for root growth are calculated to be in the range of 1.7 g l-1 and 3.6 g l-1. these results are in agreement with sbai et al. (2017), who reported that celery extract had more pronounced effects on roots of lettuce, rather than shoots. generally, in germination bioassay, water extract of allelopathic plants have more pronounced effect on root rather than shoot growth (inderjit and dakshini, 1995; muhammad et al., 2011). this may be attributed to the fact that roots are the first to absorb the phytotoxic compounds (turk and tawaha, 2002). in order to identify and distribute the chemical groups of toxic allelochemical constituents, celery aqueous. extract was partitioning between three organic solvents varied in their polarities. generally, the most phytotoxic compounds were represented water residue (h2o fraction). it can be suggested that the high toxicity compounds in celery extract may be related to presence of more polar compounds. these results are in general agreement with the results obtained by sbai et al. (2017). who observed that extraction of celery with non-polar solvents, petroleum ether or chloroform had no significant effect on lettuce germination, contrarily to the high toxicity effect of methanol extract (polar solvent). the high toxicity of celery extracts may be attributed to the present water soluble compounds as saponins, glycosides, hormones or enzyme which could affect growth directly or by altering the mobilization of storage compounds during germination (chaves and escudero, 1997; el-khatib, 1997). for the greatest inhibition effect of aqueous celery extract, we analyzed phenolics and phenolic acids in the extract at a concentration 20 g l-1. the results indicated the presence of high amount of phenolic compounds (201 mg l-1 as gallic acid) may explain the greatest effect of celery extract. in line of these results, jung et al. (2011) found high levels of phenolic in celery (51.09 mg g-1 dw, as gallic acid). phenolic acids are the precursor of all phenolic constituents in plants as well as the bioactive constituents in celery extract. identification and determination the concentration of the phenolic acids in the aqueous extract was conducted by using hplc. ten phenolic acids were identified in celery extract, among of them phydroxybenzoic acid, p-coumaric acid and ferulic acid were presented in relatively high amounts. in line of these results, yang et al. (2010) identified caffeic acid, p-coumaric acid, and ferulic acid in celery extract. presence of high concentration of total phenolic and phenolic acids in areal parts of celery was previously reported by many investigators (yang et al., 2010; sbai et al., 2017). in greenhouse trial, we evaluated the effects of foliar s pray of aqueous extract of celery and its fractions, hexane, mecl2, etoac and h2o-residue on growth of two-weeks-old c. olitorius, or e. crusgalli or p. oleracea weeds. celery extract at 30, 60 and 90 g l-1 as well as the obtained fractions did not produce any significant effect on growth of three examined weeds. in general, the growth of two-weeks-old weeds tended to be less sensitive to the test fractions than the weed germination process. these results are in agreement of those obtained by inderjit and weston (2000), they found that greenhouse bioassays do not adequately predict the responses observed in laboratory bioassay. hence, weed germination might be the most sensitive index with which to judge allelopathy of plant extracts and its fractions under natural conditions (corrêa et al., 2008). in conclusion, in this study aqueous extract of celery was evaluated for its phytotoxicity against three weed species under laboratory and greenhouse conditions. the extract displayed a great inhibition on germination percentage and seedling growth of target seeds. root growth of c. olitorius and p. oleracea was completely inhibited by extract at 7.5 g l-1, corresponded with 15 g l-1 for e. crusgalli. celery aqueous extract was fractionated by using three less polar sol510 adv. hort. sci., 2018 32(4): 503-510 v e n t s ; h o w e v e r , w a t e r e x t r a c t d i s p l a y e d t h e strongest inhibition effect on germination of three target seeds. celery extract had a relatively high amount of phenolics and phenolic acids. high concentrations of water celery extract 30, 60, 90 g l-1) and the obtained less polar fractions did not produce any significant effect on growth of two weeks-old of tested weeds. water extract of celery may be a useful source for the future development of pre-emergence bioherbicide. references al-humaid a., el-mergawi r., 2014 herbicidal activities of seven native plants on the germination and growth of phalaris minor, echinochloa crusgalli, portulaca oleracea and lactuca sativa. j. agric. sci. tech. a, 4: 843-852. b a j w a a . a . , m a h a j a n g . , c h a u h a n b . s . , 2 0 1 5 nonconventional weed management strategies for modern agriculture. weed sci., 63(4): 723-747. chaves n., escudero j.c., 1997allelopathic effect of cistus ladanifer on seed germination. functional ecology, 11: 432-440. cheema z.a., khaliq a., 2000 use of sorghum allelopathic properties to control weeds in irrigated wheat in a semi-arid region of punjab. agric. ecosyst. environ., 79: 105-112. cheema z.a., khaliq a., ali k., 2002 efficacy of sorgaab for weed control in wheat grown at different fertility levels. pak. j. weed sci. 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zhang y., hartman t.g., rosen r.t., ho c.t., 1990 free and glycosidically bound volatile compounds in fresh celery (apium graveolens l.). j. agric. food chem., 38: 1937-1940. trezzi m.m., vidal r.a., balbinot j.r., von hertwig bittencourt h., da silva souza filho a.p., 2016 allelopathy: driving mechanisms governing its activity in agriculture. j. plant interactions, 11(1): 53-60. turk m.a., tawaha a.m., 2002 inhibitory effects of aqueous extracts of black mustard on germination and growth of lentil. pakistan j. agron., 1(1): 28-30. vyvyan j.r., 2002 allelochemicals as leads for new herbicides and agrochemicals. -tetrahedron, 58: 1631-1646. yang y., sang w., zhou m., ren g., 2010 phenolic composition and antioxidant activities of 11 celery cultivars. j. food sci., 75(1): 9-13. impaginato 563 adv. hort. sci., 2018 32(4): 563-568 doi: 10.13128/ahs-22468 a preliminary investigation on developmental and biochemical responses of amsonia orientalis to ultraviolet-c irradiation a. acemi 1 (*), y. avcı duman 2, y. yüzügüllü karakuş 1, f. özen 1 1 department of biology, faculty of sciences and arts, kocaeli university, 41380 kocaeli, turkey. 2 department of chemistry, faculty of sciences and arts, kocaeli university, 41380 kocaeli, turkey. key words: antioxidant enzymes, european bluestar, plant development, tissue culture, uv-c irradiation. abstract: the present study aims to investigate the developmental and biochemical responses of ornamental amsonia orientalis irradiated with ultraviolet-c (uv-c). nodal explants of the species were exposed to uv-c irradiation on the first, 15th and the last days of the in vitro culture for 15 (3.47 kj m-2), 30 (6.94 kj m-2) and 60 min (13.87 kj m-2). in general, root lengths and numbers were negatively influenced by prolonged uv-c exposure. however, mean shoot numbers and lengths were slightly enhanced after 15 and 30 min of irradiation. high hydrogen peroxide (h2o2) and malondialdehyde (mda) levels were observed due to the uv-c exposure. activities of the antioxidant enzymes, pod (peroxidase) and cat (catalase) were found to be enhanced whereas sod (superoxide dismutase) was reduced. these results indicated that uv-c irradiation for shorter durations may be carefully used to improve in vitro shoot proliferation in a. orientalis. however, it should be noted that longer irradiation durations can trigger stress responses and lipid peroxidation-dependent cell membrane damage which will further result in the plant loss. 1. introduction ultraviolet (uv) irradiation is present in sunlight in three different wavelengths which are classified as uv-a (400-315 nm), uv-b (315-280 nm) and uv-c (280-100 nm). uv irradiation may affect growth and metabolic processes in plants due to its high quantum energy (kobashigawa et al., 2011). since wavelengths below 280 nm are absorbed by the ozone layer they do not reach to the surface of the earth (alexieva et al., 2001). however, reduction of stratospheric ozone and, a decrease in the ozone layer may lead to increase in uv-c irradiation reaching the biosphere. (*) corresponding author: arda.acemi@kocaeli.edu.tr citation: acemi a., avci duman y., yüzügüllü karakuş y., özen f., 2018 a preliminary investigation on developmental and biochemical responses of amsonia orientalis to ultraviolet-c irradiation. adv. hort. sci., 32(4): 563-568 copyright: © 2018 acemi a., avci duman y., yüzügüllü karakuş y., özen f. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 7 january 2018 accepted for publication 30 may 2018 ahs advances in horticultural science short note adv. hort. sci., 2018 32(4): 563-568 564 beside its germicidal activity, application of uv-c irradiation at 254 nm wavelength is used for several purposes including regulation of fruit ripening in tomato (tiecher et al., 2013), fruit quality stabilization during storage of fresh-cut watermelon (artés-hernández et al., 2010) and retardation of fruit decay in strawberry (erkan et al., 2008). its role in growth regulation and modulation of flowering time in ornamental plants was declared since uv-c light application increased branching and the number of flowers in salvia splendens and viola tricolor (bridgen, 2016). also, the antioxidative function was found to be enhanced a f t e r u v c a p p l i c a t i o n i n s p i n a c i a o l e r a c e a (kobashigawa et al., 2011). however, the application of uv irradiation can be hazardous in higher doses. therefore, to better understand the developmental and metabolic responses of horticultural plants to uv-c irradiation, uv-dose-dependent studies in controlled environmental factors should be also conducted on other plant species. amsonia orientalis decne. [syn. rhazya orientalis (decne.) a. dc.] which is also known as european blue star or eastern rhazya is an ornamental plant with medicinal properties. it is cultivated more commonly by gardeners in the usa than those in europe since it is deer-resistant due to its slightly toxic latex content (acemi et al., 2016). however, the natural populations of the species were taken under conservation by the european council in the frame of bern convention since they are quite limited in nature (acemi et al., 2017). in the current study, the effects of dose-dependent uv-c irradiation applications on in vitro development and antioxidative enzymes of a. orientalis were investigated to shed light the usability of this treatment in horticultural plant propagation. 2. materials and methods plant material preparation and in vitro uv-c treatments nodal explants were excised from mature individuals of amsonia orientalis growing in the garden of kocaeli university. the shoots were multiplied by following the protocol described by acemi et al., (2013 a). nodal explants excised from in vitro-raised shoots were inoculated into the ms (murashige and skoog’s) medium (1962) supplemented with 30 g l-1 sucrose and 7 g l-1 of plant agar. the ph of the medium was set to 5.7, and the cultures were incubated for 30 days under the same conditions defined by acemi et al., (2013 a). the applied uv-c doses were determined from previous reports (lópez-rubira et al., 2005; artés-hernández et al., 2010; bridgen 2014; castronuovo et al., 2014; bridgen, 2016). the cultures were subjected to uv-c irradiation at the first, 15th and the last days of the culture period. the irradiation was applied from 30 cm distance for 15, 30 and 60 min per application day by using sylvania g15w uv-c lamp (λmax 253.7 nm). uv-c treatments were applied from the top of the culture vessels in a sterile biosafety cabinet, and lids of the culture vessels were kept opened during treatments to ensure penetration of the irradiation energy. the total accumulated irradiation levels were estimated as 3.47, 6.94 and 13.87 kj m-2 for 15, 30 and 60 min of uv-c irradiation, respectively (kobashigawa et al., 2011; severo et al., 2015). biochemical assays the optimized method of acemi et al. (2017) was followed to determine malondialdehyde (mda) as lipid peroxidation product, and h2o2 contents. the crude extract for antioxidant enzyme activities’ determination was prepared by homogenizing the tissue samples (shoots and roots together) in extraction buffer of 50 mm sodium phosphate (ph 7.0) containing 0.1 mm ethylenediaminetetraacetic acid (edta). after centrifugation of the homogenate at 14,000 g for 15 min at 4°c, the resulting supernatants were collected and used for catalase (cat), peroxidase (pod) and superoxide dismutase (sod) activity assays. cat activity was determined according to the method of aebi (1974). pod activity was assayed using the pyrogallol oxidation method (kar and mishra, 1976) while sod activity was determined by following the method of dhindsa et al. (1981). data collection and statistical analysis the morphometric evaluation was done using 30 explants in each repeat. all assays were repeated t h r i c e a n d m e a n v a l u e s w e r e c o m p a r e d u s i n g duncan’s multiple range test at p<0.05 significance level. the enzyme assays and extract preparation were done on the same day to minimize the loss of enzymatic activities. 3. results plant growth and organ development at the end of the incubation period, the mean shoot length slightly increased in response to 15 and acemi et al. uv-c irradiation effects on amsonia orientalis 565 30 min uv-c exposure while this parameter was negatively affected by 60 min of exposure. however, increments in the mean shoot length were not statistically different than the control group (fig. 1a). the mean shoot numbers were influenced positively by 15 and 30 min uv-c exposure whereas only the result of 15 min exposure was found to be statistically different than the control group (fig. 1b). the mean root lengths were found to be decreased due to uv-c application even at the shortest duration. this reduction in root lengths was between 62.5% (15 min) and 95% (60 min) compared to the control (fig. 1a). in contrast to mean shoot numbers, the mean root numbers reduced gradually in response to the elevated uv-c exposure (fig. 1b). lipid peroxidation and h2o2 content application of uv-c irradiation significantly changed the lipid peroxidation level of the plant. fifteen and 30 min of uv-c exposures triggered mda a c c u m u l a t i o n s w i t h o u t s t a t i s t i c a l d i f f e r e n c e s between them. the highest accumulation level was observed from the cultures exposed to uv-c for 60 min (fig. 2a). h2o2 content increased following uv-c exposure maintaining similar levels within treatments (fig. 2b). antioxidant enzyme activities uv-c irradiation caused a decrease in sod activity without statistical differences between treatments (fig. 3a). pod activity significantly increased due to u v c e x p o s u r e s . t h e h i g h e s t p o d a c t i v i t y w a s observed in cultures exposed to 15 min of uv-c followed by a significant decrease at 30 and 60 min of uv-c exposures, which were not significantly different (fig. 3b). cat activity exhibited a similar trend with pod activity. the shortest uv-c exposure caused the highest increase in the cat activity while longer exposures induced statistically same results (fig. 3c). fig. 1 effects of uv-c irradiation on amsonia orientalis growth parameters. a= length of shoot and root, b= number of shoot and root. the values are represented as means ± sd, different letters denote significant differences (p<0.05). fig. 2 oxidative effects of uv-c irradiation on amsonia orientalis. a= mda and b= h2o2 contents. the values are represented as means ± sd, different letters denote significant differences (p<0.05). adv. hort. sci., 2018 32(4): 563-568 566 4. discussion and conclusions uv irradiation can damage dna and affect plant growth through various regulatory and/or stressmediated processes. as one of these processes, cell cycle modulation sourced by dna damage may particularly slow down the progress from g1 to s phase (jiang et al., 2011). therefore, in our results the decrease in the mean shoot length after 60 min uv-c irradiations might be due to the uv-induced dna damage and impaired cell cycle progress. differently, mean shoot number was found to be increased in a. orientalis after 15 min of uv-c exposure, which was in accordance with the previous report that found an increment of axillary branching in viola tricolor (bridgen, 2016). our results also showed adverse effects of uv-c on the root growth of a. orientalis. this negative effect can be explained by oxidative degradation and/or inhibited-synthesis of indole acetic acid (iaa); a phytohormone responsible for root proliferation (berli et al., 2013). this possible inhibition of iaa might also be a result of photo-oxidation of the plant growth regulator (ciurli et al., 2017). the oxidative degradation of iaa starts with a peroxidase-involved decarboxylation process on the side chain or oxidation of the indole ring (normanly, 2010). in the enzyme assays, elevated peroxidase activity against uv irradiation supports this discussion which is still in need of further experiments to be clearly proven. mda content indicates reactive oxygen species (ros)-mediated cellular damage considering damage to membrane lipids of stress-exposed plants. the observed increase in h2o2 levels coincided with enhanced mda levels in a. orientalis. also, pod and cat activities were found to be increased to remove r o s t o l i m i t m d a p r o d u c t i o n i n a . o r i e n t a l i s . degradation of the enzyme proteins and nucleic acids can be started after ros-induced peroxidation of the cell membrane lipids. at the same time, h2o2 could activate mitogen-activated protein kinases (mapks) in plants, leading to an enhanced antioxidant defense system (nie et al., 2013). however, excess accumulation of h2o2 causes cellular damage (gong et al., 2001). the increase in both h2o2 and mda levels indicates cellular damage in a. orientalis. excessive ros in uv-c-exposed plants may be produced because of disruption in metabolic activities or increased activity of membrane-localized nadph-oxidase (kalbina and strid, 2006). the increment in the activities of cat and pod enzymes did not seem to limit ros production in a. orientalis since h2o2 accumulation was consistently found at high levels. as a defense mechanism against environmental stress factors, plants favor the production of antioxidant enzymes (berli et al., 2013). maintenance of the fig. 3 effects of uv-c irradiation on enzymatic antioxidant activities in amsonia orientalis. a= sod, b= pod and c= cat activities. the values are represented as means ± sd, different letters denote significant differences (p<0.05). acemi et al. uv-c irradiation effects on amsonia orientalis 567 antioxidant defense system to cope with ros plays a significant role in keeping the cell membranes stabilized. the antioxidant enzymes sod, cat, and pod a r e w i d e l y d i s t r i b u t e d i n a l l h i g h e r p l a n t s a n d involved in decomposition of different forms of ros (foyer and noctor, 2000). excessive production of o2– triggers sod enzyme activity which converts superoxide radicals into either o2 or h2o2 while the excess accumulation of h2o2 is prevented by catalase and/or the ascorbate-glutathione cycle enzymes (ma et al., 2014). although the o2− content was not determined in our study, the inhibition of sod activity by uv irradiation might be due to increased o2− content. pod is involved in such processes like lignification and tolerance to environmental stresses in higher plants. during uv exposure, elevated pod and cat activities showed that activities of both enzymes can be triggered in a. orientalis. the bulk h2o2 removal activity of cat in the cell is followed by the scavenging action of pod on the h 2o 2 which is not taken by cat (willekens et al., 1997). in this report, this cooperation between both enzymes is also shown in a. orientalis. thanks to in vitro plant tissue culture technique, several conservation studies on a. orientalis were conducted and a high number of individuals were propagated thereby populations in turkey were conserved (acemi et al., 2013 a, b). the present study has revealed that although short-term application of uv-c irradiation can enhance shoot induction, it limits root growth and triggers oxidative stress at extended exposure durations. therefore, in further detailed studies on a. orientalis uv-c radiation should be applied for less than 30 min (6.94 kj m-2). however, it should be noted that short-term uv-c application can be considered as a promoter factor only in shoot multiplication phase in a. orientalis. the lower doses may be carefully applied to enhance branching in the horticultural industry. however, 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chamnongpol s., davey m., schraudner m., langebartels c., van montagu m., inzé d., van camp w., 1997 catalase is a sink for h2o2 and is indispensable for stress defence in c3 plants. embo j., 16(16): 4806-4816. impaginato 549 adv. hort. sci., 2018 32(4): 549-556 doi: 10.13128/ahs-23504 flower development and pollen vitality of moringa oleifera lam. grown in a humid temperate climatic condition s. radice 1, e. giordani 2 (*) 1 department of plant physiology, faycaum conicet, machado 914, university of morón, argentina. 2 d i p a r t i m e n t o d i s c i e n z e d e l l e p r o d u z i o n i a g r o a l i m e n t a r i e dell’ambiente, viale delle idee, 30, 50019 sesto fiorentino (fi), italy. key words: fertility, flower anatomy, microsporogenesis. abstract: moringa oleifera is a tropical tree cultivated in many countries. this species has acquired a great importance in human nutrition and it was recently indicated as a “novel food” by the european commission. recently, moringa plants have been introduced in humid temperate climatic areas, among which moreno (buenos aires province argentina). in such area, the cultivation is possible for the production of leaves, but plants need protection during winter time in order to overcome damages due to low temperatures and hence to produce capsules and seeds. the main objective of this research was to study flower morphology and anatomy of m. oleifera, as well as microsporogenesis and viability of pollen grains of plants cultivated in moreno in comparison with those produced in a humid sub-tropical climatic area of argentina (san miguel de tucumán). flowers grown in the temperate environment resulted similar for morphological parameters to those observed in the sub-tropical environment. nevertheless, pollen grain fertility depended directly on air temperature and it was negatively affected by the lower temperatures registered in the temperate site. according to the observed results, pollen viability increases with mean monthly temperatures above 16°c. 1. introduction moringa oleifera lam. (moringa) is a multipurpose small to mediumsized, evergreen or deciduous tree, native to northern india, pakistan and nepal. it has a spreading open crown with drooping, fragile branches, feathery foliage with tripinnate leaves, and a thick corky whitish bark (marcu, 2005). m. oleifera is utilised as animal fodder and employed in human nutrition due to its healthy properties (fuglie, 1999; palada and chang, 2003; ganatra et al., 2012; paula et al., 2017), as well as in the production of fuel (foidl et al., 2001), water sanitation (wilson, 1992; lekgau, 2009; padilla et al., 2012). moringa leaves are considered a “novel food” by the european commission, so confirming their valuable properties in terms of energy, nutrients, proteins and minerals, as reported by several authors (atawodi et al., 2010; tende et al., 2011; yameogo (*) corresponding author: edgardo.giordani@unifi.it citation: radice s., giordani e., 2018 flower development and pollen vitality of moringa oleifera lam. grown in a humid temperate climatic condition. adv. hort. sci., 32(4): 549-556 copyright: © 2018 radice s., giordani e. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 26 june 2018 accepted for publication 28 september 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(4): 549-556 550 et al., 2011; gopalakrishnan et al., 2016; vats and gupta, 2017). araujo et al. (2016) highlighted also the importance of m. oleifera in regions characterized by desertification and water deficit. moringa cultivation is expanding all over the world, including in climatic areas, which differ noticeably from those of its tropical origin. recently some experiments showed the feasibility of cultivating moringa in the humid temperate climatic conditions of buenos aires province (argentina) for leaf production. leaf extracts from trees grown in that conditions showed higher phenol content and antioxidant activity than those obtained from plants cultivated in typical tropical climates (arena and radice, 2016). nevertheless, flower differentiation, anthesis and fertility resulted negatively altered and the production of pods and seeds, both of them important source of nutrients, was very low. the main objective of this research was to study the effect of air temperature on flower morphology and anatomy of m. oleifera, as well as on the microsporogenesis and pollen grain viability, observed on trees cultivated at moreno (buenos aires province) in comparison with those grown in san miguel de tucumán (argentina), the first characterized by a humid temperate climate and the latter by a humid subtropical environment. 2. materials and methods plant material all plants were obtained from the same seed lot. homogeneous seedlings (n= 10) were grown in soil and in open air and cultivated in san miguel de tucumán (26° 49’59.00” s, 65° 13’00” w, elevation 456 m asl), while another similar set of seedlings (n=10) was planted in moreno (34° 39’ 0” s, 58° 47’ 0” w, elevation 14 m asl) in 25 l plastic pots under a glasshouse from april to september. successively pots were placed in open air. moreno has a humid temperate climate, with an average temperature of 23.4°c in january and 10.0°c in winter time (june); san miguel de tucumán, has humid subtropical climate (19.4°c the average annual temperature) with a hot and long summer and mild and dry winter. the precipitation pattern is monsoonal with an average of 997 mm (climate-data.org). a set of monthly air temperature parameters is reported in figure 1. flower morphology flowers (n = 100) on different phenological stages were observed on both groups of plants, and samples were collected monthly for further observations from september to december. button flower collected were used fresh and fixed in faa (formaldehyde, 100 ml; ethyl alcohol, 500 ml; acetic acid, 50 ml; distilled water, 350 ml). light microscopy button flowers (n= 10) were immediately frozen to -25°c and embedded in a medium consisting of p o l y e t h y l e n e g l y c o l a n d p o l y v i n y l a l c o h o l . successively they were cut frozen by microtome inside the cryostat. histologic slices were cut at 5 to 10 µm. a set of ten button flowers were dehydrated in an ethanol series and embedded in spurr’s resin. thin sections (75-90 nm thick) were stained with uranyl acetate and lead citrate. sections were observed with a leica dm 2500 microscope. fluorescent microscopy flowers in anthesis phase (n= 50) fixed in faa were shaved with distilled water and softened with naoh (8n) as described by martin (1959). then, they were stained with aniline blue to study pollen tube growth. squash material was observed by a leica microscope (dm 2500) using fluorescence with excitation filter bp: 450-490. scanning electron microscopy (sem) button flowers fixed in faa (n= 10) were dehydrated in an ethanol series and critical point-dried with liquid co2 was employed. then it was sputtercoated with gold-palladium (40% gold and 60% palladium) for 3 minutes. samples were observed with philips xl30 sem. fig. 1 mean (a) and average minimum (b) air monthly temperatures (°c) in san miguel de tucumán and moreno locations. radice and giordani moringa in temperate climate 551 pollen viability pollen viability was performed with fluorescent microscopy according to radice and arena (2016) on fresh anthers taken from button flowers of two localities (moreno and san miguel de tucumán). pollen evaluation was expressed in percentage. to determine the statistical significance of the hypothesis the chi-squared test (χ2) was used. 3. results flower development flower development starts with the appearance of the flower on a clustered inflorescence (fig. 2). these first buds are green reddish and about 1mm long (fig. 2a). they grow up to about 10mm and turn to white greenish (fig. 2a). anthesis takes place sequentially among the flowers of the inflorescence (fig. 2b). at anthesis, flower shows a zygomorphy symmetry. the larger transversal petal is bent upwards, while the others are reflexed downwards together with the sepals (fig. 2c). anthers are yellow and no dehiscent; flowers have odour and nectar in this phase. as the flower develops, anthers change colour to dark yellow (fig. 2c) and finally to brown (fig. 2d). it was observed that 1-3 anthers were not developed in flowers collected from moreno field. pistil, which at the time of the anthesis is below the anthers, continues to grow until it protrudes several millimetres above the androecium (fig. 2c). finally, petals wither and fall, while the ovary enlarges and turns to reddish colour regardless its fertilisation (fig. 2e). flower structure moringa oleifera plants grown in moreno and san miguel de tucumán experimental fields developed flowers with average values of ≈5 petals, ≈6 sepals, ≈6 stamens, ≈5 staminoides and 22 ovules (data not showed). in the observed flowers, the unique largest petal (referred as “primordium petal”) stands right; the others are folded (fig. 2c). female part of the flower shows the complete pistil with the style and a hairy ovary (fig. 3a). ovary is tricarpelar and ovules are located in parietal placentation (fig. 3b). stigma is just a hole (fig. 3c). some nectarostomata surround the gynophore (fig. 4). glandular hairs, with the function of expelling the nectar, are present on the nectarostomata surface (fig. 4a). nectarostomata produces nectar in sub epidermal cells, that accumulate it and transfer it through the intercellular spaces (fig. 4b). frozen section of a button flower just before the anthesis phase allows to appreciate the state of the fig. 2 flower development of moringa oleifera lam. a, from button flower to anthesis. b, beginning of anthesis. c, anthesis; d, flower senescence; e, pod formation. bar = 1 cm. fig. 3 pistil of moringa oleifera lam. a, external view of the pistil; b, internal view of the ovary with ovules; c, detail of the stigma. bars = a-c, 1mm. adv. hort. sci., 2018 32(4): 549-556 552 structures and their normal coloration (fig. 5). in fact, it is possible to see anthers with pollen grains already formed wrapped in a yellow substance similar to sporopollenin (fig. 5b). pistil shows developing ovules attached to the carpelar wall (fig. 5a). finally, all internal organs are enveloped by sepals and petals (fig. 5a). flowers studied by sem showed there is no defined stigmata structure. pistil is coronate by a smooth cell structure as a continuation of the style (fig. 6a). the internal cavity of the ovary is covered with hairs (fig. 6c) and ovules adhere to their walls on the connection of two carpels. ovules appear to be campylotropous (fig. 6d). microsporogenesis button flowers from 1mm to 10 mm (fig. 2a) have been used to observe different steps of pollen grain formation. different stages of pollen grain differentiation were observed: microsporocytes mother cells, tetrads, microsporocytes and mature pollen (fig. 7). development was very fast and it accomplished in less than one week. m o r i n g a f l o w e r s h a v e m o n o t h e c a l a n t h e r s . button flowers shorter than 5 mm contains inside the just formed pollen sac microspore mother cells (fig. 7a). these cells show a big and visible nucleus and a very dense cytoplasm indicating a high activity. fig. 4 nectary of moringa oleifera lam. (light micrographs). a, longitudinal section of the nectary; b, detail of the nectar cumulated in the intercellular spaces (arrows). bars = a, 100 µm; b, 10 µm. fig. 5 flower in pre anthesis phase of moringa oleifera lam. (frozen section). a, longitudinal section of flower with petals (pe), anthers (an) and ovary with ovules (arrows); b, mature pollen grain surrounded by sporopollenin. bars = a, 10 cm; b, 10 µm. fig. 6 sem micrographs of moringa oleifera lam pistil. a-b, style and stigma; b, detail of the stigma with pollen grain; c, ovarian cavity with ovules; d, detail of an ovule; e-f, pollen grains. bars = a, 50 µm; b, e, 20 µm; c, 500 µm; d, 100 µm; f, 10 µm. radice and giordani moringa in temperate climate 553 during this stage, the last inner layer of the anther wall corresponds to tapetal cells that are formed by large and binucleate cells (fig. 7a). as the buds lengthen, more advanced stages of microsporogenesis are observed. in fact, it was observed the tetrad (fig. 7b) and then the release of microsporocites (fig. 7c). at this point, tapetum degrades (fig. 7c). the young microspores show a central nucleus and a vacuolated cytoplasm. the last stage shows mature pollen grains and free orbicules (fig. 7d). when mature pollen grains are formed, tapetum disappears completely. mature pollen grains measure about 20 µm. it is possible to observe the exine of the grains well formed and the cytoplasm of the vegetative cell with a lot of amyloplasts (fig. 7d). pollen viability it was possible to differentiate green, red and orange yellowish pollen grains corresponding to viable, non-viable and sub viable pollen grains respectively. pollen viability among flowers at different dates (table 1) showed great variations on flowers collected in moreno; on the contrary, no differences were observed between flowers collected from san miguel de tucumán throughout the study period (table 2). moreno flowers showed a very low percentage of table 1 viability of pollen grains collected on different months and different locality values with different letters between the same column are significant different. tukey (p≤0.05). v i a b l e p o l l e n g r a i n s r e s p e c t t o s a n m i g u e l d e tucumán flowers during september to november. although the number of pollen grains per anther in source month viable non viable sub-viable moreno september 10 c 79 a 11 a moreno october 28 c 53 a 19 a moreno november 48 b 32 b 20 a moreno december 74 a 3 c 23 a s.m.tucumán september 68 a 22 b 10 a s.m.tucumán october 63 a 25 b 12 a s.m.tucumán november 70 a 22 b 8 a s.m.tucumán december 69 a 28 b 13 a table 2 viability of pollen grains collected on september from anthers of the same flower values are expressed on percentage. values with different letters between the same column are significant different. tukey (p≤0.05). source anther viable non viable sub-viable moreno 1-1 10 a 84 a 6 a moreno 1-2 7 a 91 a 2 a moreno 1-3 10 a 82 a 8 a fig.. 7 microsporogenesis of moringa oleifera lam (light micrographs). microsporangium with conspicuous microspore mother cells (mmc) and tapethal cells (tc); b, tetrads (td); c, microporocytes free (m) and tapetal cells (tc) in metabolization phase; d, mature pollen grains (arrows) with amyloplast (a) in the cytoplasm and orbicules (circles). bars = a 10 µm; b-d, 20 µm. 554 adv. hort. sci., 2018 32(4): 549-556 the flowers was not evaluated, it was observed that flowers collected from san miguel de tucumán had more amount of pollen grain in each anther. some anthers of moreno flowers contained immature pollen and a viscous substance. additionally, according to the harvesting period of the flowers, some anthers developed only dead pollen grains. according to these results, a more detailed analysis of pollen viability was performed between pollen grains derived from different anthers of the same flower and between anthers from different flowers collected from moreno trial. viability of pollen grains of different anthers collected from the same flower was not statistically different (table 2), while viability of pollen from different flowers of different trees collected on the same date resulted significantly different between flowers (table 3). viable pollen grains were contained on great proportion in some flowers but scarce in others, with a random distribution among trees. pollination during anthesis, pistils both with or without germinated pollen were observed. when pollination occurs, pollen grains fall freely into the stigma cavity and then germinate (fig. 6b). in effect, pistils treated by martin technique showed that a mass of pollen grains is housed in the cavity and that many pollen tubes germinated (fig. 8a) and later on reached the ovary and fertilized the ovules (fig. 8b). pollination was rarely observed on flowers collected from moreno during spring time (september to november) but it was very frequent in summer time. on the contrary, it was observed that flowers collected on san miguel de tucumán were profusely pollinated in both periods and fruit production was continuous throughout the year. fruit production on moreno plants started in summer until may, while it resulted continuous in san miguel de tucumán. 4. discussion and conclusions flower morphology and anatomical structure observed in flowers collected from moreno field and san miguel de tucumán did not show significant diff e r e n c e w i t h t h o s e d e s c r i b e d i n l i t e r a t u r e (ramachandran et al., 1980). furthermore, flowers from the two localities studied in argentina have a normal external and internal development. frozen sections allowed to see the internal anatomy with its natural colorations and to show more clearly the presence of orbicules, that, in the traditional cuts, appear very confused because of the different colorations applied. on the other hand, flowers studied by sem allowed clarifying some concepts. in fact, it was clearly showed how pollen germination begins even in the absence of any connection to any structure of the stigma or style. bhattacharya and mandal (2004) found that some extra proteins and esterases contribute towards the stigmatic receptivity; furthermore, the occurrence of intraovarian trichomes, which is not widespread in the angiosperms, could facilitate the growth of the pollen tubes. this assessment made by dickison (1993) is based on that the trichomes functionally resemble obturators. pollen grain formation seems to be very variable depending on the geographical location and the time of year. in fact, muhl et al. (2011) showed that low temperature regime induces flowering but provokes low pollen viability and this statement would explain the results obtained in moreno spring flowers, thus confirming that pollen grain viability is affected by air temperature. in fact, the lowest values of pollen fig. 8 fertilization of moringa oleifera lam (fluorescent light micrographs). a, pollen tube growing toward the style (arrows); b, ovules (ov) and pollen tube crossing the micropile and entering the embryo sac (arrows). bars = a-b, 100 µm. table 3 viability of pollen grains from different flowers of the same plant source flower viable non viable sub-viable moreno 1 a 7 b 78 ab 15 a moreno 1 b 6 b 82 a 12 a moreno 1 c 18 a 67 b 15 a moreno 1 d 2 b 95 a 3 b moreno 1 e 15 a 72 b 13 a values are expressed on percentage. values with different letters between the same column are significant different. tukey (p≤0.05). 555 radice and giordani moringa in temperate climate grain viability were observed on september and october when mean temperatures were lower than 16°c. additionally, the higher percentage of pollen viability observed in san miguel de tucuman is consistent with the air temperatures registered in that location, which resulted about 4/5°c higher than those observed in moreno (fig. 1). the difference of the quality of pollen grains collected in spring and summer could be related to the amount of orbicules or ubisch bodies. studies on ubisch body formation in brachypodium support the evidence that they are formed in the tapetum and are involved in exine synthesis (sharma et al., 2014). actually, during spring time, anthers with mature pollen grains brings many orbicules. this fact suggests that when the exine was not well formed the quantity of dead pollen was important. on the other hand, pollen viability seems to have an influence on the efficiency of pollination. as directly observed, all plants were very visited by insects during blooming on both experimental locations, but pollination was rarely observed in moreno flowers, while it was very frequent in flowers collected in san miguel de tucumán. although tropical climates are those considered ideal for m. oleifera according to muhl et al. (2011), good results obtained with san miguel de tucumán flowers confirm that sub-tropical climates are also suitable for this species. taking into account reproductive functions, and namely the microsporogenesis, moreno environment seems to be just below the threshold of good temperature regime for m. oleifera during spring time despite the climatological predictions made by falasca and bernabé (2008). in conclusion, the reported results demonstrate the possibility of moringa cultivation unusual non tropical climates. flowers of the trees grown in moreno field were normal from the morphological point of view only in some periods of the year and the quality of their development was related to air t e m p e r a t u r e s . p l a n t s g r o w n o n s a n m i g u e l d e tucumán have an un-interrupted production of flowers despite not being in a tropical climate. on the other hand, moringa cultivated in marginal areas, as the locality of moreno can be considered, could offer important advantages. acknowledgements the authors thank ing. oscar dantur for the collection of plant material in san miguel de tucumán and mrs. isabel farías for her assistance with the histology. this research was supported by ifund university of florence project. references araújo m., santos c., costa m., moutinho-pereira j., correia c., dias m.c., 2016 plasticity of young moringa oleifera l. plants to face water deficit and uvb radiation challenges. j. photochem. photobiol., b. biol., 162: 278-285. arena m.e., radice s., 2016 seasonal variation in leaf growth and antioxidant content of moringa oleifera cultivated at buenos aires, argentina. int. j. agric. biol., 18: 719-725. atawodi s.e., atawodi j.c., idakwo g.a., pfundstein b., haubner r., wurtele g., owen r.w., 2010 evaluation of the polyphenol content and antioxidant properties of methanol extracts of the leaves, stem, and root barks of moringa oleifera lam. j. of medic. food, 13(3): 710-716. bhattacharya a., mandal s., 2004 pollination, pollen germination and stigma receptivity in moringa oleifera lam. grana, 43: 48-56. dickison wc., 1993 floral anatomy of the styracaceae, including observations on intra-ovarian trichomes. bot. j. linn. soc., 112: 223-255. falasca s., bernabé m.a., 2008 potenciales usos y delimitación del área de cultivo de moringa oleifera en argentina. revista virtual redesma, 2(1). foidl n., harinder p.s., becker k., 2001 potentiel du moringa oleifera pour les besoins agricoles et industriels, pp. 45-78. in: fuglie l.j. 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31(3): 191-198 doi: 10.13128/ahs-21958 an examination into the effects of frozen storage of olive fruit on extracted olive oils m. asheri 1 (*), m.m. sharifani 1, g. kiani 2 1 faculty of plant production, gorgan university of agricultural sciences and natural resources, basij square, 49189-43464 gorgan, iran. 2 department of agronomy and plant breeding, sari agricultural sciences and natural resources university, po box 578, sari, iran. key words: fatty acids, freezing, olea europaea. abstract: this study was achieved to examine the effects of freezing olive fruits of the arbequina, koroneiki and mission cultivars (the most common olive oil producing cultivars in iran) on the standard indices used for assessing virgin olive oil quality. oil was obtained from olive fruits stored at -4˚c for 1 week and 3 weeks, and compared with oil obtained immediately after harvest (control). the quality indices of oils obtained from frozen fruit showed no significant degradation in quality compared with the control samples. in fact the peroxide value of the frozen fruits decreased compared to the control, which is considered to have a positive effect on oil quality. in addition, compositions of the main fatty acids are not altered by freezing which demonstrate frozen storage as a viable option. oil derived from frozen olive fruit is not of inferior quality to non-frozen fruit in the production of olive oil. 1. introduction conservation of foods prior to processing by means of cold and frozen storage has been a relatively recent technique coming to prominence over the last 50 years (poerio et al., 2008). olive oil consumption is increasing throughout the world, even in countries that traditionally have not used olive oil. this trend has been promoted due mainly to the nutritional value of the mediterranean diet (patumi et al., 2002). olive fruits (olea europaea l.) undergo some mechanical procedures (milling, malaxation and centrifugation) to extract extra virgin olive oil. the quality of virgin olive oil is intimately related to the characteristics and composition of the olive fruit at the time of its processing (inarejos-garcía et al., 2010). highest quality extra virgin oils require optimum harvest stage, reduction in the time between harvest and milling, high quality oil extraction procedures and optimum storage conditions. the storage time between harvest and processing is one of the most important postharvest factors in oil quality. this becomes a significant problem when the volume of olive fruit exceeds the capacity of the mill plants. olive fruits are often stacked (*) corresponding author: maisa.asheri@gmail.com citation: asheri m., sharifani m.m., kiani g., 2017 an examination into the effects of frozen storage of olive fruit on extracted olive oil. adv. hort. sci., 31(3): 191-198 copyright: © 2017 asheri m., sharifani m.m., kiani g. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 2 march 2017 accepted for publication 16 may 2017 ahs advances in horticultural science adv. hort. sci., 2017 31(3): 191-198 192 into large heaps at ambient temperature for several weeks prior to milling, which exceeds the storage limits (~48 hr) for the highest quality oil (garcia et al., 1996 a; ranali et al., 2000; angerosa, 2002). during this period of time fermentation may also occur, and pressure and heat within the piles provide a medium for fungi and bacteria growth (olias and garcia, 1997). anaerobic and aerobic processes take place inside and outside the piles of olive fruits, respectively, which causes deterioration of the fruit. this deterioration increases the acidity and reduces stability of the recovered oils (garcia et al., 1996 a). increase in volatile acids (acetic and butyric acid) during decomposition results in an unpleasant musty smell (olias and garcia, 1997). pigment content also decreases during this period, and additional refining needs to be done to clear all these unfavorable characteristics, increasing production cost and lowering market value (gutierrez-rosales et al., 1992). it is important to increase storage duration before milling the olive fruits to permit higher yields of better quality oil (petruccioli and parlati, 1987). studies have shown that cold storage can increase storage time without significantly affecting oil quality. clovodeo et al. (2007) stored ‘coratina’ olives used for oil production for 30 days at different temperatures and under different atmospheric conditions. they found that storage at 5°c, both under a flow of humidified air and a flow of 3% o2 + 5% co2, produced oils that maintained their initial chemical qualities until the end of the experimentation. however, the olives stored at room temperature deteriorated after 15 days of storage and the extracted oil had significantly reduced quality. poerio et al. (2008) frozed olives at -18˚c for 24 hours and extracted the oil with and without thawing the fruits. results were significantly different regarding peroxide value (pv), free fatty acids and polyphenols compared to control samples. oils from frozen olives had lower free fatty acids and pvs and freezing reduced the oxidative stability of the oil. the iranian government supports and subsidizes the expansion of olive cultivation to see about a sixfold increase in olive cultivated lands, i.e. from approximately 103,000 in 2014 to 600,000 ha by 2025, starting at 4,800 ha in 1993 at the launch of the expansion of olive cultivation plan (asheri et al., 2016). this has resulted in the spread of olive cultivation to areas where olive is not traditionally cultivated. therefore, many of the olive groves produce small amounts of fruit and have no olive processing and oil extraction facilities, and the fruit must be transported to an extraction facility. the ability to r e d u c e d e t e r i o r a t i o n d u r i n g t h e s t o r a g e t i m e between harvest and processing is important to maintain quality. hence, it is worthwhile to discover ways to preserve olive fruits during this critical period. the aim of this study is therefore, to find methods that allow olive fruits to be stored for longer periods of time, without compromising the quality of extracted oils. this study investigated the effect of freezing olive fruits at moderate temperatures (-4°c) on quality indices of extracted oil, to understand if it was possible to extract high quality oil from fruits frozen for extended periods of time. the study is aimed to examine how susceptible the oil quality of different cultivars is to freezing, and to establish if the fruit of a particular cultivar responds better to freezing. the study considered 3 freezing treatments for 3 cultivars: fresh samples (control), freezing for 1 week and 3 weeks. 2. materials and methods t h e o l i v e c u l t i v a r s m i s s i o n , a r b e q u i n a , a n d koroneiki were selected for study as they are commonly used for the production of olive oil in iran. fruits were hand-picked from fadak grove located near qom, iran (34° 30´ n, 51° 00´ e). the grove is located about 15 km south of qom, a city at 150 km south of tehran, in the central hyper-arid to arid parts of iran. the fruits selected were at similar stages of ripening according to their ripening index which were 3.93, 3.84 and 4.23 for ‘arbequina’, ‘koroneki’ and ‘mission’, respectively. the fruits were i m m e d i a t e l y t r a n s f e r r e d t o t h e u n i v e r s i t y l a b , washed and de-leafed. olives used to produce control samples were processed immediately, and the remaining olives stored in a freezer at -4°c. oil extraction for oil extraction, only sound and undamaged fruits were used. olives in the control samples were crushed using a hammer mill to form a paste. the paste was malaxed at 30˚c, then centrifuged at 4000 rpm for 10 minutes. olives used for treatment 1 were kept in freezer at -4˚c for one week and treatment 2 for 3 weeks at the same temperature. frozen samples were crushed and malaxed until they reached 25˚c (due to not thawing the samples), and then centrifuged at 4000 rpm for 10 minutes. extracted oil was collected using asheri an examination into the effects of frozen storage of olive fruit on extracted olive oils 193 a pipette and stored in dark glass jars in a refrigerator until analysis. determining oil content fifteen olive fruits from each replicate were placed in a petri dish and then placed in an oven at 105°c for 48 h. the dried olives were used to determine oil content in three replicates. olives were ground to a paste by mortar and pestle. the amount of 10 g of paste was placed in a soxhlet cartridge and oil extracted with 150 ml hexane at 70°c. after 6 h of extraction, hexane was collected and oil content was calculated on dry mass basis (agar et al., 1998). peroxide value the peroxide value of the oils was measured using the methods of garcia et al. (1996 b). a 5 g sample of t h e e x t r a c t e d o l i v e o i l w a s p l a c e d i n a 2 5 0 m l erlenmeyer flask. the sample was shaken and then dissolved in 25 ml solution of acetic acid and chloroform (2:1, v/v). one milliliter of saturated potassium iodide (ki) solution was added. the mixture was placed in darkness for 5 min and then 75 ml of distilled water was added to stop the reaction. half of one milliliter of freshly prepared starch indicator solution (0.5%) was added to each sample. finally, the mixture was titrated with 0.01 n sodium thiosulfate until the blue indicator color disappeared. the peroxide value was expressed as milliequivalents of active oxygen per kilogram of oil (meq o 2 kg-1) ( agar et al., 1998). coefficients of specific extinction values (k232 and k270) coefficients of specific extinction at 232 and 270 nm were measured by the methods reported in regulation eec/2568/91 of the european union commission (eec, 1991). the amount of 1 g of oil sample was diluted in 100 ml isooctane. the sample was then homogenized using a vortex and the solution was transferred to a 10 mm cuvette. absorbance at 232 and 270 nm was measured in a spectrophotometer using pure isooctane as a blank. fatty acid composition fatty acid composition in the oil was determined by the aocs official method (1997). methyl esters were prepared by vigorous shaking of a solution of oil dissolved in hexane (0.5 g in 7 ml) with 2 ml of 2n methalonic potash, and analyzed by gas chromatography. chromatographic analysis was performed on a trace gc (gas chromatograph), equipped with a flame ionization detector and split/splitless injector (trace gc, thermofinnigan, italy), using a silica capillary column, bpx-70 (30 m × 0.25 mm i.d. × 0.25 µm film thickness). the injector temperature was set at 250˚c and samples were injected manually (1 µl) with a split ratio of 1:80. the oven temperature was maintained at 175˚c for two min, and increased gradually to 230˚c at 3 ˚c/min and maintained for 10 min. nitrogen was used as carrier gas at a flow rate of 0.8 ml/min. the detector temperature was maintained at 270˚c. fatty acids were identified by comparing retention times with those of standard compounds. oxidizability (cox value) was calculated based on the fatty acid content of three unsaturated fatty acids [oleic acid (c18:1), linoleic acid (c18:2), and linolenic acid (c18:3)] using following relation (fatemi and hammond, 1980). oxidizability = [1 × (c18:1%) + 10.3 × (c18:2%) + 21.6 × (c18:3%)] determining oil chlorophyll and carotenoid content pigment contents were assayed using the spectrophotometric method of minguez-masquera et al. (1991). one gram of oil was dissolved in 10 ml of isooctane and the resulting solution transferred to a c u v e t t e . a b s o r b a n c e a t 4 7 0 a n d 6 7 0 n m ( f o r carotenoid and chlorophyll, respectively) was measured in a spectrophotometer (unico/2800 uv/vis) using pure isooctane as a blank. the results are expressed as milligram of carotenoid or chlorophyll per kilogram of oil. statistical analysis experimental layout was factorial with three levels of freezing treatments and three olive cultivars. data were analyzed by spss software. comparison of means is performed using duncan’s multiple range test at a 95% confidence level and tables for analysis of variance (anova) are provided. 3. results and discussion oil content in fruit from the koroneiki and arbequina cultivars, there is no statistically significant difference in oil content between the control and the frozen samples (table 1). however, the mission cultivar fruit frozen for 1 and 3 weeks showed a significant decrease in mean oil content compared with the control under duncan’s multiple range test at 5%. since the independent variables (i.e. cultivar and freezing) act independently, freezing had no significant overall effect on the content of extracted olive oil (table 2). however in interpreting the results, it needs to be adv. hort. sci., 2017 31(3): 191-198 194 considered that oil extraction of frozen treatments was performed at a different temperature (25°c) in respect to the control (30°c). peroxide value the peroxide value (pv) is a measure of primary oxidation. table 1 shows the measured mean pv (meq o2 /kg) of the oils obtained from olives stored for the different time periods. our data showed that freezing reduces the peroxide value. the reduction was significant when olive fruits were frozen for three weeks, but not significant when frozen for only 1 week. cultivar’s effect in reduction of pv was significant at a 95% statistical level, while freezing had a very significant effect at a 99% level (table 2). no oil had peroxide values above the limit for extra virgin olive oil (20 meq o2/kg). poerio et al. (2008) and gomez and escoda (2010) also found reduced peroxide values in oil from olive paste and fruits that had been frozen. the lower peroxide value of oil from frozen fruit could be due to reduced enzymatic activity during crushing-malaxation due to the initial low temperature. specific extinction coefficients at 232 nm and 270 nm the k232 and k270 values for all treatments were below the maximum permitted values for extra virgin olive oil [2.50 and 0.20 respectively, according to regulation eec (1991)]. across all cultivars there was a net decrease in the k232 values due to freezing, but the difference was statistically insignificant. no change was detected in the k270 values between treatments (table 2). between cultivars however, the difference in the k232 and k270 values was significant with the arbequina cultivar showing the highest values, ‘koroneiki’ showing the lowest k232 and intermediate k270, and ‘mission’ showing intermediate k232 and the lowest k270 (table 1). these differences were consistent with the variations between olive cultivars shown by asheri et al. (2016). gomez and escoda (2010) also showed freezing to have no effect on the k270 and k232 values independent of cultivar. pigment content (chlorophyll and carotenoid) t h e s l i g h t d e c r e a s e i n t h e p i g m e n t c o n t e n t observed between the frozen treatments and the control was not statistically significant (tables 1, 2). also different cultivars did not demonstrate different levels of chlorophyll content. carotenoid content, on the other hand, was significantly different among the cultivars at 99% statistical level. chlorophylls and carotenoids play crucial roles in health and also in the oxidative activity of processed food stuff, due to their antioxidant nature in the dark and pro-oxidant activitable 1 means of chemical characteristics of olive oils derived from fresh and frozen olive fruits and their comparison with duncan’s multiple range test at 5% m= mission; k= koroneiki; a= arbequina; c= control sample; t1= 1 week freezing sample; t2= 3 week freezing sample; pv= peroxide value. means within a column with the same lowercase letters are not significantly different. table 2 results of anova for chemical characteristics of olive oils derived from fresh and frozen olive fruits pv= peroxide value. ns= not significant; * significant at 5% level; ** significant at 1% level. samples oil content (% of dry matter) pv (meq o2 kg-1) k232 nm k270 nm chlorophyll (mg kg-1) carotenoid (mg kg-1) m c 47.21 a 7.33 a 1.13 bc 0.08 c 2.22 a 1.84 a m t1 43.08 b 6.63 ab 1.06 c 0.08 c 2.14 a 1.86 a m t2 42.73 b 5.47 bcd 1.05 c 0.08 c 2.06 a 1.79 ab k c 43.34 b 6.97 a 0.82 d 0.13 ab 2.20 a 1.53 bc k t1 41.80 b 6.53 ab 0.80 d 0.12 b 2.03 a 1.50 bc k t2 41.65 b 5.2 cd 0.76 d 0.12 b 2.02 a 1.43 c a c 43.57 b 6.53 ab 1.33 a 0.15 a 2.12 a 1.52 bc a t1 45.00 ab 5.63 bc 1.24 ab 0.14 ab 2.01 a 1.48 c a t2 42.82 b 4.47 d 1.22 ab 0.14 ab 1.98 a 1.48 c source mean square oil content pv k232 k270 chlorophyll carotenoid treatments 8.83 ns 2.63 ** 0.129 ** 0.002 ** 0.023 ns 0.091 * cultivar (a) 10.46 ns 2.11 * 0.50 ** 0.009 ** 0.024 ns 0.352 ** freezing (b) 12.14 ns 8.35 ** 0.16 ns 0.00004 ns 0.066 ns 0.008 ns ab interaction (cv *fr) 6.37 ns 0.04 ns 0.001 ns 0.000004 ns 0.002 ns 0.002 ns error 3.56 0.39 0.004 0.00009 0.058 0.026 asheri an examination into the effects of frozen storage of olive fruit on extracted olive oils 195 ty in the light (fakourelis et al., 1987). the higher the amount of these pigments, the higher the resistance to oil oxidation. our results indicated that freezing did not change olive oil resistance to oxidation based on pigment contents. kiritsakis et al. (1998) found that storage at lower temperatures reduced the pigment content of the extracted oils and morello et al. (2003) found slight decreases in chlorophyll and carotenoid concentration of oils obtained from frosted olives. these authors suggested that this could be due to the involvement of chlorophyllase and lypoxygenase. amongst cultivars, the mission cultivar shows significantly higher carotenoid content than ‘koroneiki’ and ‘arbequina’, as observed by asheri et al. (2016). fatty acid composition this paper reports 12 fatty acids detected in gc analysis including myristic acid (c14:0), palmitic acid (c 16:0), palmitoleic acid (c 16:1), heptadecanoic acid (c 17:0), heptadecenoic acid (c 17:1), stearic acid (c 18:0), oleic acid (c 18:1), linoleic acid (c 18:2), linolenic acid (c 18:3), arachidonic acid (c 20:4), arachidic acid (c 20:0), and erucic acid (c 22:1) (table 3). analysis of variance (anova) shows that freezing olive fruits did not have significant effects on the fatty acid composition of the oils, while cultivars had very significant influence on all the fatty acids except for myristic acid. in addition, freezing and cultivar variables acted independently on fatty acids level, except for myristic and palmitic acids in which the interaction of variables were very significant (at 99%) and significant (at 95%), respectively (tables 4, 6). comparison of the means demonstrate that ‘koroneiki’ and ‘mission’ had significantly higher levels of oleic acid (74.85 and 72.96% in fresh state, respectively) than ‘arbequina’ (60.76%). oleic acid is the most prominent fatty acid in olive oil and is a monounsaturated fatty acid (mufa) with demonstrated qualities in the stability of the oil. oleic acid levels did not change significantly during the storage except for the 1 week freezing of ‘arbequina’ (table 5). the other predominant fatty acids are palmitic acid [a saturated fatty acid (sfa)] and linoleic acid [a polyunsaturated fatty acid (pufa)]. ‘arbequina’ had the highest amounts of palmitic and linoleic fatty acids. this indicates lower quality of its oil, especially when it is considered together with its lower oleic samples myristic acid palmitoleic acid heptadecanoic acid heptadecenoic acid stearic acid linolenic acid arachidic acid arachidonic acid erucic acid m c 0.010 d 0.730 bc 0.0255 b 0.044 b 2.55 b 1.05 a 0.290 cd 0.409 a 0.068 c m t1 0.014 d 0.701 bc 0.024 b 0.042 b 2.44 b 1.00 ab 0.281 d 0.387 ab 0.065 c m t2 0.051 a 0.682 c 0.022 b 0.038 b 2.38 b 0.95 b 0.271 d 0.382 abc 0.063 c k c 0.030 b 0.798 bc 0.031 b 0.050 b 3.26 a 0.76 c 0.465 a 0.379 abc 0.137 a k t1 0.018 bcd 0.828 b 0.035 b 0.051 b 3.20 a 0.80 c 0.459 a 0.356 c 0.132 a k t2 0.011 d 0.817 b 0.032 b 0.050 b 3.16 a 0.77 c 0.448 a 0.359 bc 0.136 a a c 0.028 b 2.512 a 0.070 a 0.169 a 1.43 c 0.68 d 0.324 b 0.159 d 0.114 b a t1 0.027 bc 2.515 a 0.070 a 0.170 a 1.45 c 0.68 d 0.319 bc 0.151 d 0.103 b a t2 0.016 cd 2.579 a 0.072 a 0.169 a 1.44 c 0.69 d 0.319 bc 0.167 d 0.107 b table 3 fatty acid (myristic, palmitoleic, heptadecanoic, heptadecenoic, stearic, linolenic, arachidic, arachidonic and erucic acids) content (%) of olive oils derived from fresh and frozen olive fruits and comparison of the means with duncan’s multiple range test at 5% m= mission; k= koroneiki; a= arbequina; c= control sample. t1= 1 week freezing sample; t2= 3 week freezing sample. means within a column with the same lowercase letters are not significantly different. source mean square oil content pv k232 k270 chlorophyll carotenoid treatments 8.83 ns 2.63 ** 0.129 ** 0.002 ** 0.023 ns 0.091 * cultivar (a) 10.46 ns 2.11 * 0.50 ** 0.009 ** 0.024 ns 0.352 ** freezing (b) 12.14 ns 8.35 ** 0.16 ns 0.00004 ns 0.066 ns 0.008 ns ab interaction (cv *fr) 6.37 ns 0.04 ns 0.001 ns 0.000004 ns 0.002 ns 0.002 ns error 3.56 0.39 0.004 0.00009 0.058 0.026 table 4 results of anova for fatty acids content (reported in table 3) of olive oils derived from fresh and frozen olive fruits pv= peroxide value. ns= not significant; * significant at 5% level; ** significant at 1% level. adv. hort. sci., 2017 31(3): 191-198 196 acid content. palmitic and linoleic acids amounts in the mission cultivar and linoleic acid in ‘koroneiki’ show significant changes after three weeks of frozen storage (table 5). among the fatty acids of lower content, only linolenic and myristic acids demonstrate significant changes in their mean values (table 3). linolenic and myristic acid contents of the mission cultivar show significant increases after three weeks of frozen storage. myristic acid levels of ‘koroneiki’ and ‘arbequina’ decreased due to freezing. total sfas, mufas and pufas did not demonstrate any statistically significant change under frozen storage, and all the change is due to cultivar (table 6). cultivar had very significant effect on the ratios of mufa/pufa and oleic/linoleic acids, and the effect of freezing on these ratios was significant at 95% confidence level. also, the analysis indicated that cultivar and freezing have significant interaction during the storage of olive fruit on these ratios. cox value, however was not influenced significantly by freezing (table 6). m e a n t o t a l s f a l e v e l s f o r ‘ k o r o n e i k i ’ a n d ‘arbequina’ did not change, while ‘mission’ showed a significant reduction in total sfa after 3 weeks of freezing. this change could be due to the reduction of palmitic acid. means comparison of total mufa content did not detect any significant changes. ‘koroneiki’ shows a significant increase in the total pufa content after 3 weeks of frozen storage, which leads to a reduction of mean mufa/pufa and oleic/linoleic acids ratios. similar to mufa/pufa and oleic/linoleic acids ratios, cox values did not change significantly for ‘mission’ and ‘arbequina’, while ‘koroneiki’ experienced a significant decrease for 3 w e e k s f r e e z i n g t r e a t m e n t . t h e h i g h e r t h e mufa/pufa and oleic/linoleic acids ratios and the lower the cox values, the higher the oxidative stability of the oil. the implication of the reduction in mufa/pufa and oleic/linoleic acids ratios and increase in cox values after 3 weeks freezing for ‘koroneiki’ is that longer periods of freezing storage could reduce oxidative stability of its oil. however, it is important to note that despite the changes after 3 weeks of storage, ‘koroneiki’ oil still possessed signiftable 5 fatty acid (palmitic acid, oleic acid, linoleic acid, ∑sfa, ∑pufa, mufa/pufa, oleic/linoleic and cox value) compositions and comparison of the means with duncan’s multiple range test at 5% m= mission; k= koroneiki; a= arbequina; c= control sample. t1= 1 week freezing sample; t2= 3 week freezing sample. sfa= saturated fatty acids; mufa= monounsaturated fatty acids; pufa= polyunsaturated fatty acid; cox value: calculated oxidizability. means within a column with the same lowercase letters are not significantly different. table 6 results of anova for fatty acids (reported in table 5) composition of olive oils derived from fresh and frozen olive fruits sfa= saturated fatty acids; mufa= monounsaturated fatty acids; pufa= polyunsaturated fatty acid. cox value: calculated oxidizability; ns= not significant; * significant at 5% level; ** significant at 1% level. samples palmitic acid oleic acid linoleic acid ssfa smufa spufa mufa/pufa oleic/linoleic acid cox value m c 11.11 c 72.96 c 10.47 c 14.25 c 73.80 c 11.93 b 6.19 c 6.97 c 2.03 b m t1 10.73 c 73.49 bc 10.53 c 13.76 74.30 c 11.91 b 6.24 c 6.98 c 2.03 b m t2 10.23 d 73.60 bc 11.00 b 13.24 d 74.39 bc 12.34 b 6.03 c 6.69 c 2.07 b k c 12.48 b 74.85 a 6.51 e 16.51 b 75.83 a 7.65 d 9.92 a 11.52 a 1.58 d k t1 12.48 b 74.74 a 6.62 e 16.43 b 75.75 a 7.78 cd 9.74 a 11.29 a 1.60 cd k t2 12.47 b 74.15 ab 7.09 d 16.37 b 75.15 ab 8.22 c 9.14 b 10.46 b 1.64 c a c 16.63 a 60.76 e 15.96 a 18.73 a 63.56 d 16.80 a 3.78 d 3.81 d 2.40 a a t1 16.54 a 61.76 d 15.94 a 18.65 a 64.55 d 16.77 a 3.85 d 3.87 d 2.41 a a t2 16.96 a 61.53 de 15.68 a 19.05 a 64.39 d 16.54 a 3.89 d 3.92 d 2.38 a source mean square palmitic acid oleic acid linoleic acid ssfa smufa spufa mufa/pufa oleic/linoleic acid cox value treatments 14.46** 80.35 ** 31.51 ** 9.76 ** 58.30 ** 29.31 ** 12.70 ** 19.90 ** 0.23 ** cultivar (a) 57.37 ** 320.35 ** 126.63 ** 38.45 ** 232.15 ** 116.90 ** 50.43 ** 78.93 ** 0.93 ** freezing (b) 0.06 ns 0.338 ns 0.13 ns 0.13 ns 0.33 ns 0.10 ns 0.14 * 0.30 * 0.001 ns ab interaction (cv*fr) 0.21 * 0.365 ns 0.14 ns 0.24 * 0.36 ns 0.12 ns 0.11 * 0.19 * 0.001 ns error 0.03 0.142 0.04 0.05 0.12 0.05 0.03 0.04 0.0004 asheri an examination into the effects of frozen storage of olive fruit on extracted olive oils 197 icantly higher mufa/pufa and oleic/linoleic acid ratios and lower cox values than the other cultivars, and therefore indicated the highest resistance to oxidative stability among these three cultivars. 4. conclusions the oils obtained from olive fruits stored at -4˚c are demonstrated to maintain the same characteristics of the control. among the biochemical indices, the decrease in pv was the only factor that varied significantly from the control samples. this decrease is received positively, as less oxidation occurs. pigment content remained stable during freezing period which is considered to be good to maintain quality oil. no differences in characteristics evaluated in this study were observed between cultivars, suggesting that freezing the olive fruits of all three cultivars in this research did not have any negative effect on the studied characteristics of the extracted oil. based on this study, freezing could be a suitable method of preserving olive fruits in the waiting period between harvesting and processing, which could assist in the maintenance of characteristics evaluated in this study during olive oil production. however, it may be worthwhile extending this experiment with different cultivars and to test characteristics such as sensory quality evaluation and oxidative stability. nonetheless, our results suggest that olive fruit may be stored frozen before processing into oil. so the products could be harvested at the optimal stage of ripening and preserved frozen while shipping them to the mill plant stations. this preservation system could be extended to the pre-processing waiting period at the mill plant, so that the processing of olives could be optimized. acknowledgements this research was funded by gorgan university of agricultural sciences and natural resources. we thank ahmad bolandnazar, the owner and manager of fadak olive grove, for his permission to access the grove. we would like to express our sincere gratitude to justin parker for proofreading the manuscript and also to naser mostafavi for his assistance in improvement of the earlier drafts of the manuscript. references agar i.t., hess-pierce b., sourour m.m., kadar a.a., 1998 quality of fruit and oil of black-ripe olives is influenced by cultivar and storage period. j. agr. food chem., 46: 3415-3421. angerosa f., 2002 influence of volatile compounds on v i r g i n o l i v e o i l q u a l i t y e v a l u a t e d b y a n a l y t i c a l approaches and sensor panels. eur. j. lipid sci. tech., 104: 639-660. aocs, 1997 preparation of methyl esters of long-chain fatty acids from sampling and analysis of commercial fats and oils. aocs press, champaign, il, usa, pp. 266. asheri m., sharifani m.m., yamchi a., kiani g., ahmadi golsefidi m., hosseini-mazinani m., 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2000 the compositional quality and sensory properties of virgin olive oil from a new cultivar i-77. food chem., 69: 37-46. impaginato 205 adv. hort. sci., 2017 31(3): 205-214 doi: 10.13128/ahs-21959 effect of different physio-chemical factors on sex expression and fruit yield in greenhouse cucumber m. golabadi 1, p. golkar 2 (*), a.-r. eghtedari 3 1 department of agronomy and plant breeding, college of agriculture, isfahan (khorasgan) branch, islamic azad university, 81595158 isfahan, iran. 2 research institute for biotechnology and bioengineering, isfahan university of technology, 8415683111 isfahan, iran. 3 department of agronomy and plant breeding, college of agriculture, isfahan (khorasgan) branch, islamic azad university, 81595158 isfahan, iran. key words: cucumber, flowering, male, sex expression, silver ions. abstract: male flower expression is considered an important aim in greenhouse cucumber breeding for creating paternal lines as a base for hybrid progeny. the study was carried out to evaluate the effects of different treatments on sex expression and fruit yield of cucumber in two different season (autumn-winter and spring-summer), in particular this research focuses on the influence of 1) usage of two chemical agents: silver thiosulphate [ag(s2o3)2 -3] and silver nitrate (agno3) at different concentration, respectively 200 and 500 ppm and 100, 200 and 300 ppm 2) plant development stages at the moment of the treatment (5, 10, and 15-leaf growth stages) and 3) application of single or double sprayings. analysis of variance showed that season, chemical applications and number of spaying had significant effect on the induction of a higher number of male flowers. a positive significant effect of season suggested that longer days and higher temperature promote the formation of male flowers in cucumber. this study showed that male flower production was induced by all concentrations of silver ions, especially high concentrations. important traits related to change of sex expression such as the number of days to male flowering and the number of male flowers are more affected by different interactions of studied factors in contrast to vegetative and yield related traits. also, the quadruple interaction effects indicated that silver ion could change sex expression at higher temperatures and longer days (second season in summer) with high concentration when applied in 15 leaf stage for agno3 and 5 and 15 leaf growth stage for ag(s2o3)2 -3 with double spraying. consequently, female lines can be induced to male flowering with silver ions, thus increasing the feasibility of large scale seed production of gynoecious × gynoecious cucumber hybrid. 1. introduction commercial cucumber (cucumis sativus l., 2n= 2x= 14) is a member of cucurbitaceae that is indigenous to india (renner et al., 2007). it is one of (*) corresponding author: golkar@cc.iut.ac.ir citation: golabadi m., golkar p., eghtedari a.-r., 2017 effect of different physio-chemical factors on sex expression and fruit yield in greenhouse cucumber. adv. hort. sci., 31(3): 205-214. copyright: © 2017 golabadi m., golkar p., eghtedari a.-r. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 9 november 2016 accepted for publication 19 june 2017 ahs advances in horticultural science adv. hort. sci., 2017 31(3): 205-214 206 the most economically important cucurbit vegetable p l a n t s ( t a t l i o g l u , 1 9 9 3 , r o b i n s o n a n d d e c k e r walters, 1997) and among the most widely-grown vegetable crop in the world, after tomato, onion and cabbage (plader et al., 2007). cucumber is used in different types (harvesting, slicing and fresh eating) that are used as fresh or processed vegetable (shetty and wehner, 2002). the cucumber is a thermophilic and frostsusceptible crop, usually cultivated in fields during the spring-summer period. its high demand has also made it an important crop to be widely grown in glasshouses or plastic houses (sarkar and sirohi, 2011). sex expression is an important factor that has a positive effect on yield and that constitutes a major component of cucumber improvement programs (yamasaki et al., 2003). in gynoecious cultivars, all flowers are female, so, male flowers are required for new line production via female flowers crossing. the sex expression of cucumis sativus l. is determined by genetics as well as environment (e.g. photoperiod, temperature) (yamasaki et al., 2003; bano and khokhar, 2009). change from vegetative growth to reproductive stages is a complex process regulated by many factors, and could be influenced b y t h e a p p l i c a t i o n o f p l a n t g r o w t h r e g u l a t o r s (ainsworth, 1999; sure et al., 2013). growth regulators have tremendous effects on sex modification and flowering in cucurbits that leading to either suppression of male flowers or an increase in the number of female flowers (al-masoum and al-masri, 1999). some researchers have already reported the effects of plant growth regulators on modification of sex expression in cucumber flowers (vadigeri et al., 2001; rafee kher et al., 2002; bano and khokhar, 2009). line production is important in breeding programs, because many cultivars in cucumber are hybrids (golabadi et al., 2015). in gynoecious cucumbers, male flower induction is necessary for production of f1 hybrid seeds (yamasaki et al., 2003; wang et al., 2011). therefore, change of sex expression in gynoecious cucumber is necessary. in general, auxin, ethylene and cytokinins promote female sex expression in various monoecious and dioecious systems (mohan ram and sett, 1982). exogenous application of plant growth regulators could alter the sex ratio if applied at the twoor fourleaf stage, which is the critical stage at which the suppression or proportion of either sex is possible (hossain et al., 2006). the effect of ethylene can be antagonized by specific ethylene inhibitors (hirayama and alonso, 2000). silver ions (ag+) restrain the physiological effect of the ethylene by blocking the ethylene receptors (hirayama and alonso, 2000). it is known that silver ions (ag+) applied as silver nitrate (agno3) or as silver thiosulfate [ag(s2o3)2 -3] replace copper ions (cu+) which is a part of the ethylene receptor preventing the receptor from responding to ethylene (abeles et al., 1992). another inhibitor of the ethylene action, agno3, suppresses the development of female flowers and induces the male ones (kumar et al., 2009; stankovic and prodanovic, 2002). male flower formation increased proportionally with the concentration of agno3 applied (stankovic and prodanovic, 2002). according to previous reports, agno3 induced more male flowers than ga3 on gynoecious breeding lines of cucumber (kalloo and franken, 1978) and summer squash (yongan et al., 2002). the positive effects of agno 3 on male flower production in cucumber have reported by karakaya and padem (2011). thappa et al. (2011) used different plant growth regulators (ethephon, naphthalene acetic acid and maleic hydrazide) at the two, four and sixleaf and full-bloom stages in cucumber to induce male flowers. law et al. (2002) reported that silver thiosulfate, an ethylene inhibitor, enhanced stamen development in female white campion (silene latifolia). however, little effort seems to have been directed toward the study of the effects of silver ions on male flower production sprayed at different growth stages and if the numbers of spray applications could improve the effectiveness of the induction. on the other hand, it seems that there are few studies that correlate the effect of environmental conditions, especially season, to the number of spraying and leaf-stage growth on male flower induction of plant treated with silver ions. although silver is a toxicity ion, however the amount of its toxicity depends on species, silver concentration, times of application and plant growth stage spraying and exposure media. for exmple fuente et al. (2014) applied different concentrations of agno3 (0, 30, 60, 90, 200 mg l -1) at intervals of 8 days throughout the crop cycle (90 days) in watermelon. their results showed that silver accumulate in root more than other parts of plants and antioxidant in fruits increased in plants exposed to 30 mg l-1 agno3, but lycopene content decreased. therefore the main objectives of this study was to investigate the effects of chemical application [agno3 and ag(s2o3)2 -3], different leaf growth stages and number of spraying on flower and fruit characteristics of greenhouse cucumber in two different golabadi et al. effect of different physio-chemical factors on sex expression and fruit yield in greenhouse cucumber 207 seasons to identify the best condition and the optimal level of chemical application to boost the formation of male flowers. our findings will help to develop practical recommendations on these issues for improve male flowers production in cucumber breeding programs. furthermore, this work aimed to evaluate the stability of sex expression of a gynoecious cultivar adrian in different seasons. the present investigation was done as a first step to a hybridization program of cucumber. 2. materials and methods planting material and field evaluation the seeds of cv. adrian as a gynoecious genotype were sown at the mid autumn of 2014 and mid spring of 2015 at research greenhouse of agriculture department at islamic azad university isfahan branch, isfahan, iran (51° 36´ longitude and 32°63 latitude). the soil used was loam with ph 7.7 and electrical conductivity (ec) of 4.1 (ds/m). seeds were planted to soil directly and covered with peat moss and coco peat. plants were disposed in two rows, where the spaces between cucumber seedling was 50 cm and within the rows were 90 cm respectively, and 180 cm was left between every couple rows. the greenhouse air temperature at the growing period was maximum 29°c/18°c (day/night) in first season (autumn-winter) and 34°c/20°c (day/night) in second season (spring-summer) with a relative humidity of about 55% and 60%, respectively. nutrient levels in the irrigation solution water were n:216 (mg l-1), p:58 (mg l-1), k:286 (mg l-1), ca:185 (mg l-1), mg:185 (mg l1), s:43 (mg l-1), fe:5.59 (mg l-1), mn:1.97 (mg l-1), b:0.7 (mg l-1), zn:0.2 (mg l-1), cu:0.07 (mg l-1) and mo:0.05 (mg l-1). different fertilizers were used based on soil analysis that included: potassium nitrate, ammonium nitrate, magnesium nitrate, iron and other mineral elements such as sulphate dissolved in the irrigation water. dichlorvos, trigard, abamectin and organic neem oil were applied for insect control. the same fertilizers and pest management were used for all plants in the same time. drip irrigation was applied when needed. the source of water was urban water with electrical conductivity (ec) of 0.4 (ds/m). the ph of the irrigation water was adjusted to 6.5 by adding nitric acid. silver application different chemical treatments including agno3 [h1=100 ppm (0.59 mm), h2=200 ppm (1.18 mm) and h3=300 ppm (1.77 mm)], and ag(s2o3)2 -3 [h4=200 ppm (0.6 mm) and h5=500 ppm (1.5 mm)] and control (h6) were applied to induce changes in sex expression. a g n o 3 w a s p u r c h a s e d f r o m m e r c k c h e m i c a l company and ag(s2o3)2 -3 was synthesized according to law et al. (2002). the solutions were applied as a spray on the whole plant. water was sprayed on the control plants. spraying was accomplished at 5, 10, and 15-leaf growth stages (five leaves stage: ls1, ten leaves stage: ls2, and fifteen leaves stage: ls3). chemical treatments were employed in single or double spraying at one week interval. agno3 and ag(s2o3)2 -3 applications were conducted early in the morning (before sunrise) to avoid plant sunburn. each treatment was applied with adequate amounts of the solutions to assure that all the leaves were completely wetted in each spray event. the total time of experiment in every season was about 4 months after sowing and measurement of traits were done in this period. studied traits the traits of interest were measured on eight plants per replication and included: days to male flower expression (days to first male flower appearance after sowing) (dmf); the node number that the first male flower was appear (nnmf); the mean of internode length (the mean of five internode from node number 15 to 20) (il); the number of male flower (total male flower that opened in whole plant) (nmf), male flowering period (days from observation the first male flowering until the last male flowering observation (mfp); male flower diameter (was recorded at the widest points) (mfd); single fruit weight (from dividing total fruit weight to total fruit number in every harvesting) (sfh); fruit number per harvesting (fnh); fruit weight per harvesting (fwh) (the numbers and the weights of all the fruits harvested in each plot in each harvesting). statistical analysis the experiment was conducted as a combined analysis of variance with three different factors (chemical treatments, leaf growth stage, and number of spraying) under two different seasons. the data collected were subjected to analysis of variance (anova) based on a completely randomized block design with three replications using the general linear model (glm). statistical analysis system program (sas ver. 9) was used for data analysis. the differences between applications were decided on the basis of the least significant difference (lsd) test adv. hort. sci., 2017 31(3): 205-214 208 (p<0.05) according to their importance at the 0.05 confidence level. 3. results and discussion the result of analysis of variance based on a factorial experiment is presented in table 1. according to table 1, the season (s) had significant effect on all of the studied traits, except for single fruit weight. this result is logical, since phenotypic expression of sex is strongly modified by environmental and hormonal factors (mohan ram and sett, 1982). long days, high temperature, and silver ion promote formation of male flowers, whereas short days and low temperature promote the formation of female flowers (perltreves, 1999). clearly, chemical treatments (ch) had significant effects on all the traits studied, except for fruit number per harvesting (table 1). number of spraying (single or double) also had significant effects on the days to male flowering, the node number of first male flower, the mean of internode length, the number of male flower and male flowering period (table 1). hallidri (2004) reported that increased number of spraying events had significant effects on male flower production. he suggested that doses of 400 and 500 ppm of agno3 produced the highest number of male flowers. the leaf growth stage (lgs) of spraying showed significant differences on days to male flowering, the node number of first male flower, male flowering period, male flower diameter and fruit weight per harvesting (table 1). the interaction effect of environment × silver application had a significant effect on all traits with the exception of male flower period and fruit number per harvesting (table 1). interaction of season × leaf stage was significant for days to male flowering, the node number of first male flower, the number of male flower and male flower diameter. interaction of number of spray events × chemical application had a significant effect on days to male flowering, the node number of first male flower, the number of male flower and single fruit weight (table 1). interaction of chemical application × leaf stage of spraying showed significant differences on days to male flowering and the node number of first male flower (table 1). the interaction of environment × chemical application × leaf stage of spraying showed significant difference on days to male flowering, the node number of first male flower, the number of male flower and period of male flowering. study of these interactions df= degree of freedom; s= season; ch= chemical treatments [silver nitrate (agno3) and silver thiosulphate ag(s2o3)2 -3); sp= number of spraying (single or double); lgs= leaf growth stage (5, 10 and 15). dmf= days to male flowering; nnmf= the node number of first male flower; il= the mean of internode length (cm); nmf= the number of male flower; mfp= male flowering period; mfd= male flower diameter (cm); sfw= single fruit weight (g); fnh= fruit number per harvesting; fwh= fruit weight per harvesting (g). ns= non significant. *, ** significant at p<0.05 and p<0.01, respectively. table 1 analysis of variance for single and combined effects of different studied traits in greenhouse cucumber source of variation mean squares of studied traits df dmf nnmf il nmf mfp mfd sfw fnp fnh season (s) 1 ** ** ** * ** ** ns ** ** replication (season) 4 ns ns ** ns ns ns ** ns ns chemical treatments (ch) 5 ** ** ** ** ** ** ** ns ** number of spraying (s0) 1 ** * ** ** ** ns ns ns leaf growth stage (lgs) 2 ** 9** ns ns ** ** ns ns ns** sp × ch 5 ** ** * ** ns ** ** ns ns s×sp 1 ns ns ns * ns * ns ns ** s×lgs 2 ** ** ns * ns ** ns ns ns ch×sp 5 * * ns ** ns ns * ns ns ch ×lgs 10 * ** ns ns ns ns ns ns ns sp×lgs 2 ns ns ns ns * ns ns ns ns s×ch×sp 5 ns ns ns ns ns ns ns ns ns s×ch×lgs 10 ** ** ns ** * ns ns ns ns ch×sp×lgs 10 ns ns ns ns ns ns * ns ns s×sp×lgs 2 ns ns ns ns * ns ns ns ns s×chsp×lgs 10 ns ns ns ns ns ns ns ns ns residual 140 ns ns ns ns ns ns ns ns ns golabadi et al. effect of different physio-chemical factors on sex expression and fruit yield in greenhouse cucumber 209 showed that important traits related to change of sex expression such as days to male flowering, node number of first male flower, and number of male flower more affected by different interactions in contrast to vegetative and yield related traits. therefore selection of best treatments should be done based on other treatments. for example numbers of male flower are affected by silver application, leaf stage, number of spraying and all their interactions. there was no significant difference for other triple interactions and environment × chemical application × leaf stage of spraying × number of spray events interaction in all of the studied traits. effects of season on the studied traits the comparison between two seasons showed that the first season had superior mean on days to male flowering, the node number of first male flower, male flower diameter and single fruit weight. it could be concluded that in the first season male flowers expressed later than second season and therefore dmf and nnmf were increased (table 2). on the other hand, in second season the number of male flower was increased (from 36 to 100 male flowers) and also the time of male flowering was earlier that is related to high temperature and long days. this result showed that environmental conditions, especially low temperature, could retard male flower formation and confirmed also that cucumber is a thermophilic plant. accordingly, stankovic and prodanovic (2002) reported that sowing season affected the number of male flowers. the interaction effect of season × chemical application × leaf stage was significant for number of male flower, days to male flowering and period of male flowering that all of them are related to sex expression, even though this interaction was not significant for vegetative related traits (table 2). effects of silver application on the studied traits in this study, different doses of chemical treatments [agno3 and ag(s2o3)2 -3] were used. the mean comparisons showed that chemical application increased the mean values of days to male flowering (table 3). there was a narrow variation in days to male flowering between different chemical treatments. the least day to male flowering was at a high concentrations of agno3 and ag(s2o3)2 -3 (table 3). this result showed that high concentrations of different chemicals agent caused earlier male flowering. dmf= days to male flowering; nnmf= the node number of first male flower; il= the mean of internode length (cm); nmf= the number of male flower; mfp= male flowering period; mfd= male flower diameter (cm); sfw= single fruit weight (g); fnh= fruit number per harvesting; fwh= fruit weight per harvesting (g). means followed by the same letter in each column were not significantly different at 0.05 level using lsd test. table 2 the mean comparison of different studied traits in cucumber in different seasons season traits dmf nnmf il nmf mfp mfd sfw fnh fwh s1 23.08 a 20.17 a 7.13 b 36.24 b 12.20 b 5.46 a 73.17 a 1.93 b 138.80 b s2 19.89 b 11.96 b 7.63 a 99.78 a 13.35 a 4 74.95 a 3.60 a 272.96 a table 3 the effects of various chemical treatments application on studied traits in cucumber means followed by the same letter in each row were not significantly different at 0.05 level using lsd test. traits control chemical treatments agno3 ag(s2o3)2 -3 100 ppm 200 ppm 300 ppm 200 ppm 500 ppm days to male flowering 0.23 c 26.41 a 26.08 a 25.04 b 26.02 a 25.17 b node number of male flower 0.13 b 20.18 a 19.11 a 19.23 a 19.08 a 18.68 a the mean of internode length 6.25 b 7.38 a 7.8 a 7.64 a 7.28 a 7.65 a the number of male flower 0.18 d 20.89 cd 68.07 b 129.76 a 44.79 bc 144.4 a male flowering period 0.074 e 10.16 d 16.13 bc 17.366 ab 14.71 c 18.21 a male flower diameter 3.29 d 5.36 c 5.45 bc 5.65 ab 5.36 c 5.71 a single fruit weight 84.7 a 73.9 b 70.95 b 72.15 b 70.68 b 71.89 b fruit number per harvesting 2.69 a 2.71 a 2.65 a 2.88 a 2.84 a 2.84 a fruit weight per harvesting 219.74 a 205.88 a 190.87 a 212.9 a 199.72 a 206.22 a adv. hort. sci., 2017 31(3): 205-214 210 the highest value for male flower diameter observed at 500 (ppm) of ag(s2o3)2 -3 and 300 (ppm) of agno3 (table 3). therefore these concentrations of ag (s2o3)2 -3 and agno3 could affect both vegetative and reproductive growth of flower. however there was no significant difference between all treatments for internode length. hallidri (2004) reported that the greatest number of staminate nodes was produced on plants sprayed two or three times with 400 to 500 (ppm) of agno3. rafee kher et al. (2002) reported that 200 (ppm) of ga increased the length of internode in cucumber. according our results the highest value for the number of male flower was denoted at 300 (ppm) of agno3 and 500 (ppm) of ag(s2o3)2 -3 about 130 and 144 male flower, respectively. law et al. (2002) reported that application of ag2s2o3 produced stamens in female flowers of white campion (silene latifolia) with longer filaments and larger anther locules. yongan et al. (2002) reported that agno3 had more significant effects than ga3 on male flower production in summer squash. stankovic and prodanovic (2002) reported that increasing concentrations of agno3 from 0.01% to 0.04% enhanced the number of male flowers in gynoecious lines, which is similar with the results obtained in the present study. jadav et al. (2010) reported that ethrel (200 ppm) had the greatest effect on male flower production among the hormones they investigated (ga3, ethrel, naphthalene acetic acid, and absisic acid). jutamanee et al. (1994) reported that the effects of different doses of ga3 and agno3 depended on the genotype and photoperiod. similar to their results, agno3 in the present study induced the formation of male flowers in all concentration. hallidri (2004) reported the concentration of 100 (ppm) of agno3 was ineffective on male flower induction. kalloo and franklen (1978) reported that different doses of agno3 (50, 200, and 500 mg l-1) led to non-significant effects on male flower production, fruit weight and fruit number. they showed similar trends in all the treatments. however in this study all concentrations of agno3 (100, 200, 500 ppm) produced male flower with significant differences. one explanation which might account for disparities observed between experiments was the difference in average quanta of solar radiation received by the plants during two different environmental conditions in two seasons. this result confirms the inducing effects of different chemical compounds used on male flower production. the highest value for single fruit weight was observed at control. mean comparison showed that chemical application has reduced the mean of single fruit weight in comparison to control, because production of male flower in every treated plant will reduce female flower number in contrast to control. there were no significant differences between all treatment for fruit number per harvesting and fruit weight per harvesting (table 3). so, this result could demonstrated that chemical application has only effects on flower number and its sex expression. effects of number of sprays on the traits studied number of sprays (single or double) showed significant effects on days to male flowering, the node number of first male flower, the number of male flower and period of male flowering (table 4). according to table 4, double spraying treatment gave rise to higher mean values for days to male flowering, the node number of first male flower, the number of male flower and period of male flowering. on the other hand, double spray treatment was found to affect only sex expression while it had no effect on the traits related to morphology and fruit yield. effects of leaf stage on the studied traits the comparison of the mean values for the studied traits at different stages of leaf growth [five leaf dmf= days to male flowering; nnmf= the node number of first male flower; il= the mean of internode length (cm); nmf= the number of male flower; mfp= male flowering period; mfd= male flower diameter (cm); sfw= single fruit weight (g); fnh= fruit number per harvesting; fwh= fruit weight per harvesting (g). means followed by the same letter in each column were not significantly different at 0.05 level using lsd test. table 4 the effects of single and double spraying on studied traits in cucumber number of spraying traits dmf nnmf il nmf mfp mfd sfw fnh fwh single spraying (sp) 20.85 b 15.49 b 7.31 a 49.15 b 11.61 b 5.11 a 74.04 a 2.76 a 205.96 a double spraying (dp) 22.13 a 16.64 a 7.44 a 86.86 a 13.94 a 5.16 a 74.08 a 2.77 a 205.82 a golabadi et al. effect of different physio-chemical factors on sex expression and fruit yield in greenhouse cucumber 211 stage: ls1, ten leaf stage: ls2, and fifteen leaf stage: ls3] is presented at table 5. the highest mean for dmf and nnmf were observed at ls3 stage (table 5).this logical result is resulted from the most distance (days) between planting time to fifteen leaf stage. on the other hand, the highest dmf at 15 leaf growth stage belonged to 200 ppm ag(s2o3)2 -3 treatment (fig. 1). el-ghamriny et al. (1988) confirmed that sex differentiation in cucumber takes place at the 2-true leaf stage and that this was the best time for studying the effects of growth regulators on sex expression. there was no-significant difference between three leaf growth stages for number of male flower (table 5). therefore use of silver ions at different three growth stages had similar effects on m o d i f i c a t i o n o f f l o w e r s e x t y p e i n c u c u m b e r . therefore, if it is necessary to separate monoecious or androecious from gynodioecious plant types in breeding programs (for example in line production), breeder could apply these chemical agents at 15 leaf stage, when sex form of plant has been expressed. raymond (2004) proposed that 1000 ppm of ga3 at the 2-leaf stage had the best effect on male flower production with 3 hormone applications at 2-week intervals in cucumber. also, the highest values for period of male flowering and male flower diameter were obtained at the five leaf stage; that is logical, since the period between male flower observation and end of male flowering in ls1 was higher than ls2 and ls3. as already mentioned, male flower diameter was greater at the 5-leaf stage, which might be caused by the higher production of pollen and the increased capacity for crossing in the breeding programs. effect of different factor interactions on number of male flower as the number of male flower trait is the most important trait related to male flower induction in cucumber, the different significant interaction effects in this trait was assayed (table 2). the highest number of male flower was recorded at double spraying in 500 ppm of ags2o3 and double spraying at 300 ppm of agno3, respectively of 187.36 and 179 flowers (fig. 2 a). in the two chemical treatments at higher concentration, double spraying caused increasing o f m a l e f l o w e r n u m b e r , a l t h o u g h d i f f e r e n c e s between one and two spraying in low concentrations of chemical agents was not significant. the highest (119.2) and the least (24) number of male flower was observed at second season and the first season at fifteen leaf stage, respectively (fig. 2 b). in all stages, high number of male flower was observed in second season and pointed out significant differences with first season. also, the most number of male flowers was obtained in high concentration of chemical treatments at the second season, however in all chemical treatments the number of male flowers was more than at second season (fig. 2 c). again, the second season showed the highest number of male flower with double spraying in contrast to first season, although double spraying in two seasons was more than single spraying (fig. 2 d). the triple interaction effects of season, leaf growth stage and chemical treatment on male flower number showed that male flower number in all three leaf growth stages and all chemical treatments under second season were more than first season, specially high concentration dmf= days to male flowering; nnmf= the node number of first male flower; il= the mean of internode length (cm); nmf= the number of male flower; mfp= male flowering period; mfd= male flower diameter (cm); sfw= single fruit weight (g); fnh= fruit number per harvesting; fwh= fruit weight per harvesting (g). means followed by the same letter in each column were not significantly different at 0.05 level using lsd test. table 5 the mean comparison for some of the studied traits in different leaf growth stages in cucumber developmental stages traits dmf nnmf il nmf mfp mfd sfw fnh fwh five leaf stage (ls1) 19.44 c 8.73 c 7.55 a 68.94 a 14.21 a 5.40 a 74.63 a 2.88 a 216.06 a ten leaf stage (ls2) 21.40 b 17.10 b 7.40 a 63.47 a 12.40 b 5.05 b 73.00 a 2.69 a 195.85 a fifteen leaf stage (ls3) 23.62 a 22.38 a 7.18 a 71.60 a 11.71 b 4.95 b 74.55 a 2.73 a 205.76 a fig. 1 the interaction effect of chemical treatment × leaf growth stage on the days to male flowering in cucumber adv. hort. sci., 2017 31(3): 205-214 212 of chemical treatments in fifteen leaf growth stage under second season (table 6). these results demonstrated that the second season had progressive effect on induction and development of male flower and environmental condition could be having a significant effect on this phenomenon. as mentioned before, in second season the temperature of greenhouse was higher than first season and also days were longer. these two reasons changed sex expression in second season more than first season. 4. conclusions phenotypic expression of sex determining loci in cucumber is strongly modified by environmental, chemical and hormonal factors. the results of the experiment revealed that agno3 and ag(s2o3)2 -3 have similar and positive effects on the male expression of cucumber. this study showed that male flower induction was induced by all concentrations of silver ions. these two chemical agents could induce a high number of male flowers in 300 and 500 ppm concent r a t i o n [ a g n o 3 a n d a g ( s 2o 3) 2 3] , r e s p e c t i v e l y . chemical treatments with silver ions as agno3 and ag(s2o3)2 -3 application can, therefore, be recommended for enhancing male flower production in cucumber, if further study will confirm the accumulations of silver in the fruit is negligible for the human health. as the time of agno3 spraying in this experiment was only three times, so the toxicity of this ion was low in a short time of period. on the other hand, as this chemical agent only was applied in experimental greenhouse, therefore the fruits of treated chemical treatment seasons season 1 season 2 ls1 ls2 ls3 ls1 ls2 ls3 control 0.00 n 0.00 n 0.00 n 0.00 n 0.00 n 0.00 n agno3 (100 ppm) 6.83 mn 8.00 mn 9.17 mn 42.42 i-n 31.75 j-n 27.17 k-n agno3 (200 ppm) 43.00 i-n 20.33 lmn 14.67 mn 64.33 h-m 123.33 d-h 142.75 d-g agno3 (300 ppm) 122.33 d-h 91.00 e-j 19.33 lmn 145.33 def 147.42 cde 253.17 a ag(s2o3)2 -3 (200 ppm) 16.331 mn 26.00 k-n 19.00 lmn 84.58 f-k 38.00 i-n 84.67 f-k ag(s2o3)2 -3 (500 ppm) 77.33 h-l 97.17 e-i 81.83 g-k 224.83 ab 178.75 bcd 206.5 abc ls1= five leaf growth stage; ls2= ten leaf growth stage; ls3= fifteen leaf growth stage. means followed by the same letter were not significantly different at 0.05 level using lsd test. table 6 the effects of interaction between chemical treatment, seasons and leaf growth stage on male flower number in cucumber fig. 2 the effect of different interactions (a, b, c and d) on the number of male flower in cucumber. golabadi et al. effect of different physio-chemical factors on sex expression and fruit yield in greenhouse cucumber 213 plants were disposed from toxic effects of ag ion. also, the morphologic appearance of the cucumbers showed no abnormality after ion application. overall, this result indicates that silver ion could change sex expression in higher temperatures and longer days with high concentration and when applied in 15 leaf stage for agno 3 and in 5 and 15 leaf stage for ag(s2o3)2 -3, although this ion is able to modify sex form in lower concentration and earlier growth stage in cucumber. although double spraying led to late ma l e f l o w eri n g, h o wever t h i s t rea t men t c o u l d i n c r e a s e n u m b e r o f m a l e f l o w e r s i g n i f i c a n t l y . therefore, the double spray treatment is proposed for male flower production in greenhouse conditions. this can be beneficial for breeding programs that need high numbers of male flowers and flowering periods, especially for parents that have different periods of growth stage. the interaction effect showed that the highest number of male flowers was induced by applying ag(s2o3)2 -3 (500 ppm) in 5 and 15 leaf stage in high t e m p e r a t u r e a b o u t m a x i m u m 3 5 ° c a t d a y a n d minimum 20˚c at night. another finding of this study was that the number of male flowers showed significant increase with increasing doses of agno3 from 100 ppm to 300 ppm and ag(s2o3)2 -3 from 200 ppm to 500 ppm. therefore, high dosages of agno3 and ag(s2o3)2 -3 should be selected for inducing male flowers in cucumber. the highest dosage of chemical agents led to decrease in days to male flowering and the mean of internode length that is related to the effect of silver ion on sex form earlier than low dosage of this ion. leaf stage of the spray event was found to have a toxic effect on fruit. over all, this result indicates that high concentration of silver ion could change sex expression at higher temperatures and longer days in 15 leaf stage for agno3 and in 5 and 15 leaf stage for ag(s2o3)2 -3. because the highest level of number of male flower and male flower period and the lowest level of days to male flowering were obtained in these treatments. finally, male flowering started about 3 weeks after treatment and lasted for a period of up to 3 weeks thereafter. plants treated with silver ions did not elongate more than normal plants and grew normally; 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(eds.) genetic improvement of vegetable crops. pergamon press, ltd., tarrytown, ny, usa, pp. 833. thappa m., kumar s., rafiq r., 2011 influence of plant growth regulators on morphological, floral and yield traits of cucumber (cucumis sativus l.). kasetsart j., 45: 177-188. vadigeri b.g., madalgeri b.b., sheelavantar m.n., 2001 effect of ethrel and gibberellic acid on yield and quality of two cucumber varieties. karnataka j. agric. sci., 14: 727-730. wang y.h., behera t.k., kole c.h., 2011 genetics, genomics and breeding of cucurbits. crc press, enfield, nh, usa, pp. 425. yamasaki s., fujii n., takahashi h., 2003 characterization of ethylene effects on sex determination in cucumber plants. sex. plant reprod., 16: 103-111. yongan c.h., bingkuii z., enhui z., zunlian z., 2002 control of sex expression in summer squash (cucurbita pepo l.). cucurbit genet. coop. rep., 25: 51-53. impaginato 389 adv. hort. sci., 2018 32(3): 389-398 doi: 10.13128/ahs-23229 postharvest control of aspergillus niger in mangos by means of essential oil s. javadpour 1, a. golestani 2, s. rastegar 2 (*), m.m. dastjer 2 1 food and cosmetic health research center, hormozgan university of medical sciences, bandar abbas, iran. 2 department of horticultural science, college of agriculture, university of hormozgan, hormozgan, iran. key words: aspergillus niger, essential oil, mango, postharvest. abstract: the use of essential oil as an alternative mean to synthetic fungicides has been considered in the past years for management of the postharvest decay of fruits in order to ensure more safe and long storage life of these perishable commodities. aspergillus niger is one of the most dangerous fungal pathogen which can cause postharvest diseases in fresh mangos. the aim of this study was to assess the effectiveness of essential oil from four aromatic plants (thymus vulgaris, salvia mirzayanii, artemisa persica, and rosmarinus officinalis) in comparison to fungicide ‘mancozeb’ against a. niger under in vitro and in vivo conditions. after inoculation of mango fruits with an isolate of a. niger followed by curative treatments with essential oil, the main physical and chemical attributes of mangoes were determined under postharvest condition. the in vitro results showed that colonies of a. niger were totally inhibited by application of essential oil of t. vulgaris (at all the tested concentrations) and a. persica (1500 μl/l). while, s. mirzayanii showed the lowest effect at 1000 μl/l if compared with the other essential oils. the results of the in vivo experiments showed that treatments with t. vulgaris and s. mirzayanii essential oil had significant (p<0.05) effects in preventing fruit decay at 1000 μl/l after 10 days of storage, while, r. officinalis essential oil significantly (p<0.05) reduced deterioration of mango fruits at 500 μl/l, followed by a. persica. rosemary also showed the highest fruit firmness in comparison with other treatments. also, the essential oils maintained higher chlorophyll content. the results of this work showed that application of essential oil on mangos assurance both a significant preservation on their quality attributes by controlling, at the same time, decaying caused by a. niger during the postharvest phase. 1. introduction postharvest diseases are among the major causes of losses of mangos (mangifera indica l.) fresh produce throughout the supply chain. the incidence of the postharvest diseases can also affect the quality of mangos limiting their shelf life up to 3-4 days. in literature is reported that about 17-37% of fresh mangos is wasted after harvesting and marketing (madan (*) corresponding author: rastegarhort@gmail.com citation: javadpour s., golestani a., rastegar s., dastjer m.m., 2018 postharvest control of aspergillus niger in mangos by means of essential oil. adv. hort. sci., 32(3): 389-398 copyright: © 2018 javadpour s., golestani a., rastegar s., dastjer m.m. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 14 may 2018 accepted for publication 12 september 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(3): 389-398 390 and ullosa, 1993). sharma et al. (1994) have reported that about 17.7% of this fresh produce is lost during the storage and marketing. mango decay caused by the plant pathogenic fungus aspergillus niger is one of the most dangerous postharvest diseases, leading t o t h e l o s s e s o f f r u i t q u a l i t y d u r i n g s t o r a g e (duamkhanmanee, 2008). it well known from published reports as more negative effects associated to use of chemical fungicides for controlling postharvest diseases have been reported on the human’s health and environment (wightwick et al., 2010). furthermore, consumers believe that fruits not treated (or minimally-treated) with fungicides are safer for fresh consumption (du plooy et al., 2009). in the past 20 years there has been a great interest in using essential oils (eos) to control postharvest diseases, such increasing shelf life of stored fruits (tripathi and dubey, 2004). several studies have also reported on the antifungal activity of thymus vulgaris against different strains of colletotrichum gloeosporioides, rhizopus stolonifer, p e n i c i l l i u m d i g i t a t u m ( a b d o l a h i e t a l . , 2 0 1 0 ; s e l l a m u t h u e t a l . , 2 0 1 3 ) , a s p e r g i l l u s f l a v u s , a s p e r g i l l u s n i g e r , a s p e r g i l l u s f u m i g a t u s , a n d alternaria alternata (kumar et al., 2008). different species of salvia are yet used as antimicrobial agents (fiore et al., 2006; kelen and tepe, 2008), however salvia mirzayanii is an endemic plant which grows only in some parts of iran, and thus there is no an exhaustive information regarding its effect on a. niger (rechinger, 1986). centeno et al. (2010) have reported that extracts from rosmarinus officinalis and t. vulgaris could have a significant effect on the control of fungal decaying. previous studies have also confirmed the interesting antimicrobial activity of r. officinalis eo against spoilage and pathogenic foodrelated fungi (abdolahi et al., 2010). de sousa et al. (2013), for instance, detected the strong effect of the origanum vulgare and r. officinalis eos in controlling a. flavus. on the other hand, fewer papers report issues regarding to postharvest control of a. niger on mango fruits using eos as an alternative mean to synthetic fungicides. this study was focused to evaluate the effectiveness of eos derived from four aromatic plants (t. vulgaris, s. mirzayanii, artemisa persica and r. officinalis) to in vitro and in vivo suppress the growth of one pathogenic strain of a. niger by preserving the mango fruit quality attributes under postharvest condition. 2. materials and methods plant material and extraction of essential oil s. mirzayanii and a. persica samples were collected from lar region of fars province, iran (lat. 27°41’ 3’’ n and long. 54°2’ 10’’e). a t. vulgaris sample was collected from geno region of hormozgan province, iran (lat. 25° 38’ 37.9’’ n and long. 57° 46’ 28’’ e) and r. officinalis samples from kerman province, iran (lat. 30° 17’ 2.1’’ n and long. 57° 5’ 0.1’’ e). samples were harvested in vegetative stage (before flowering). the leaves of samples were cut into small pieces and shade-dried at room temperature. the material was then ground to fine powder. the 80 g of plant material were subjected to extraction of eos by hydro-distillation method for 6 h using a clevenger’s apparatus (moghaddam et al., 2011). the eos were separately collected, dehydrated using sodium sulphate (na2so4), and finally stored in a dark bottle at 4°c until tested. in vitro experiments fungi were isolated from mango and their identity was confirmed the aspergillus niger (ptcc 5010) was supplied by iranian research organization for science and technology (irost). culture of the pathogen organism was maintained on potato dextrose agar (pda) medium. stock cultures were grown at 25°c for 7 days to allow for sufficient sporulation. antifungal effects of eos were carried out by the solution method (sm) according to pitarokili et al. (1999). inhibitory effects of eo extracted from s. mirzayanii, a. persica, r. officinalis and t. vulgaris were determined by in vitro antifungal assays. to measure the direct fungal inhibition of each eo on mycelial growth of a. niger, three different concentrations of them (1000, 1200 and 1500 μl/l) were added to potato dextrose agar (pda; provided by scharlau) media before solidification into petri dishes (8 cm diameter) at 45-50°c. fungal disks with 5 mm diameter were placed on the middle of petri dishes and incubated at 25°c for 10 days. three replicates per each treatment (4 eo × 3 concentrations) including control plate without eo were prepared. inhibition percentage was determined at the end of incubation time by the index: ip = (dc-dt)/dc ×100 ip = inhibition percentage, dc = mycelium diameter in the control plate, and dt = in the eos-treated plate. javadpour et al. postharvest control of aspergillus niger in mangos 391 in vivo experiments mangos cv. halily were harvested from minab (lat. 27°07’51’’ n and long. 57°05’13’’ e) at the maturity stage of development. fruit surface was before disinfected with 2% sodium hypochlorite for 3 min, and then artificial inoculations were done by puncturing fruit surface (4 mm deep and 2 mm wide for each inoculation point) with a sterile needle on two sides of each fruit with 40 μl of a conidial suspension containing 106 ufc/ml of a. niger that it has been sprayed above each wound. after one-day of incubation at 25°c to allow conidia germination into fruit tissue, fruits were treated with 500 and 1000 μl/l eo of each plant in comparison to 0.5 and 1.0 mg/l mancozed. after the treatment (curative), all fruit trials, including the control (fruits were inoculated with conidial suspension without the treatment with essential oils), were placed into boxes and kept at 25°c for 1, 2, and 3 weeks. decay percentage of fruit was calculated as the number of decayed fruit/ total number of fruit at each replication* 100 (elanany et al., 2009). physical-chemical analysis firmness of each fruit was measured at two points of the equatorial region by using a texture analyzer with a 5 mm probe (lurton, taiwan) with units expressed in kg/cm2. surface color was measured on each fruit at two opposite sides using a chromameter (cr 400, minolta) which provided cie l*, a*, and b* values. l* is color lightness (0= black and 100= white). the a* scale shows in the maximum the red (+a*) and in the minimum the green color (a*) while the b* ranged from yellow (+b*) to blue (b*). the content of ascorbic acid (aa) expressed as mg/100 g fruit weight was determined as described by molla et al. (2011). aliquots of 10 ml of each sample was homogenized in 100 ml of extraction buffer containing 3% metaphosphoric acid. aliquots of 10 ml of homogenate was titrated against standard dye 2,6-diclorophenol indophenols to a faint pink color. the method proposed by lichtenthaler (1987) was u s e d t o d e t e r m i n e t h e t o t a l c h l o r o p h y l l a n d carotenoids content of fruit. the ‘total soluble solids’ (tss) content was determined at 20°c using a digital refractometer, and expressed as °brix. the ph of fruit juice was measured using a jenway 3320 ph meter calibrated by ph 4 and 7 buffer solutions. the ‘titratable acidity’ (ta) was determined by titration of 5 ml extract with 0.1 mol l−1 sodium hydroxide at ph 8.1 and expressed as percent citric acid (molla et al., 2011). weight loss, fruit firmness, surface color c h a n g e , c o n t e n t o f a a , t o t a l c h l o r o p h y l l , a n d carotenoids, and tss, ta and ph were determined after 1, 2, and 3 weeks of storage at 25°c. these characteristics was done with 3 replicates (3 large fruits for 1 replicate). statistical analysis t h e e x p e r i m e n t w a s c o n d u c t e d i n a randomized factorial designed whit essential oils treatment and storage time as the two factors. data were submitted to one-way analysis of variance (anova) using sas version 16.0 and means were separated by the duncan test at p<0.05 (n=3). 3. results and discussion aspergillus niger mycelia inhibition in vitro experiments showed that mycelia growth of a. niger was significantly suppressed (p<0.05) when treated with the different concentrations of each eo (fig. 1). the fungal growth was totally inhibited (ip= 100%) by all the concentrations of t. vulgaris eo and with 1500 μl/l a. persica eo. on the other hand, s. mirzayanii eo showed lower effect (ip= 72%) than other eos when tested at 1000 μl/l concentration. o u r f i n d i n g s a r e i n a g r e e m e n t t o t h e o n e s described by kohiyama et al. (2015) who reported that t. vulgaris eo was able to control the growth of a. flavus. similar observations on the prevention of fig. 1 inhibitory effect of the thymus vulgaris, salvia mirzayanii, artemisia persica and rosmarinus officinalis eos tested at 1000, 1200 and 1500 μl/l on mycelia growth of aspergillus niger cultures incubated at 25°c for 10 days. bars indicate the sd of the mean. different letters indicate significant differences in mean values (p<0.05). adv. hort. sci., 2018 32(3): 389-398 392 different pathogenic fungi by using eos have been reported in the previous studies, such as those conducted by tripathi and dubey (2004) and pawar and thaker (2006). boubaker et al. (2016) reported the antifungal activity of four thymus species eos against penicillium digitatum, penicillium italicum and geotrichum citriaurantii. pawar and thaker (2006) s h o w e d t h a t c i n n a m o m u m z e y l a n i c u m , c i n n a m o m u m z e y l a n i c u m , c i n n a m o m u m c a s s i a , cymbopogon citratus and syzygium aromaticum were the best plant sources for eos extraction showing a noticeable inhibitory effect against a. niger. it was also reported that the mycelial growth of a. niger was inhibited by application of 2.5 and 3.0 μg/ml of citrus sinensis oil (sweet orange) in potato d e x t r o s e b r o t h a n d a g a r w a t e r m e d i u m , respectively (sharma and tripathi, 2008). modifications on fungal structures induced by the eos afore-quoted might be due to interactions of their components (carvacrol, thymol, eugenol, vanillin and etc.) with cell wall synthesis, which could affect fungal growth and its morphology (rasooli et al., 2006; rao et al., 2010). some researchers have stated that some phenolic compounds present in the eos could affect the plasma membrane and the cellular organelles, such as mitochondria of the fungi by decreasing the lipid and saturated fatty acid levels and increasing the unsaturated fatty acids, resulting in the leakage of ca2+, mg2+ and k+ (sharma and tripathi, 2008). in addition, the existence of the hydroxyl groups and the aromatic nucleus could be a other important factor for the eos antimicrobial activity (numpaque et al., 2011). fruit decay suppression as shown in figure 2, the decay percentage of fruits increased with the storage time, variable from 1 to 3 weeks of incubation. the percentage of decay significantly decreased (p<0.05) with increasing of the concentration of t. vulgaris and a. persica eos after 3 weeks of storage. after one week, no significant differences were observed between control and treated fruits (data not shown). after two weeks, significant differences were found among treatments with r. officinalis eo at 500 μl/l and t. vulgaris at 1000 μl/l. at the end of experiment, after two weeks, the maximum level of decay was related to the control fruits (70%), and the minimum one was attributed to r. officinalis (500 μl/l) and a. persica (1000 μl/l) eos, reaching 12% and 13.3% decay, respectively. t h e s e d a t a a g r e e w i t h t h o s e o b t a i n e d b y ramezanian et al. (2016) who showed the possibility of using z. multiflora and t. vulgaris eos to control postharvest citrus alternaria decay (black rot). in addition, elshafie et al. (2015) reported that o. vulgare eo can control the brown rot of peach. jhalegar et al. (2015) addressed their study on the influence of lemon grass, eucalyptus, clove and neem eos against p. digitatum and p. italicum in ‘kinnow’ mandarin. these authors showed that the decay rot during storage was less in the treated fruits than in the control ones. duamkhanmanee (2008) reported that 4000 ppm lemon grass eo could control anthracnose by c. gloeosporioides decay of mangos. phenolic compounds such as carvacrol and thymol (rao et al., 2010) contained in eos have a lipophilic molecular structure, therefore it interfer with membrane-catalyzed enzymes and cell wall, causing the cell death of microbes (shirzad et al., 2011). many researchers believe that the type and the amount of phenolic compounds present in the oil can determine the antifungal activity of the eos (tripathi and dukey, 2004). weight loss the weight loss of fruit was increased strongly during the early weeks, but this increase was gradual throughout the storage period (fig. 3). after two weeks of storage, the highest and lowest weight loss was observed in the control samples and those treated with 1000 μl/l s. mirzayanii (12.7% and 9.8% respectively). during the storage, the main mango weight loss (13.2%) was found in the control fruits, while the lowest one (10.8%) was observed in r. officinalis treated fruits at 500 μl/l concentrations. the mechanism of eos for reducing physiological fig. 2 suppressive effect of the thymus vulgaris, salvia mirzayanii, artemisia persica and rosmarinus officinalis eos tested at 500 and 1000 μl/l on mangos decay after 10 and 15 days of storage compared to ‘mancozed’ (0.5 and 1.0 mg/l). bars indicate the sd of the mean. different letters indicate significant differences in mean values (p <0.05). javadpour et al. postharvest control of aspergillus niger in mangos 393 cell wall components. breakdown and the enzymatic degradation of insoluble protopectins into more simple soluble pectin can be associated with softening (willats et al., 2001). ramezanian et al. (2016) found that the eos reduced the activity of polygalacturo n a s e a n d g a l a c t o s i d a s e , w h i c h a r e s o f t e n i n g enzymes in the cell wall components, and maintained orange fruit firmness through the storage. the results obtained in the present study agree with those of maqbool and alderson (2010), who showed that by the application of lemongrass oil (0.05%) and cinnamon oil (0.4%), the firmness of banana and p a p a y a f r u i t s w a s m a i n t a i n e d d u r i n g s t o r a g e . however, tzortzakis (2007) reported that eucalyptus and cinnamon eos had no effect on the tomato and strawberry firmness. surface color change the results related to the changes in the fruit color (in terms of l*, a* and b*) of the treated mango showed that the lightness of the fruits peel was decreased throughout the storage time (table 1). the fruits treated with 1000 μl/l r. officinalis eo retained higher l* over other treatments and control samples, after three weeks of storage. the highest and lowest l* was found in r. officinalis and a. persica at 1000 μl/l concentration, respectively. the results showed that a* was significantly decreased during the storage. however, the fruits treated with eos maintained a higher a* than did the control ones. at the end of the storage, the lowest a* (7.46) and the highest a*(11.73) were found in the control and r. officinalis treated fruits at 1000 μl/l concentraloss in weight might be related to the reduction of ethylene production and the respiration rate. also, eos cover the peel of fruit, creating the water barrier between the fruit and the environment, thereby reducing water exchange (morillon et al., 2002). this agrees with previous studies showing the efficacy of eos in reducing the weight loss of cherries and grapes (serrano et al., 2005). similarly, du plooy et al. (2009) reported that the use of mentha spicata and l i p p i a s c a b e r r i m a e o s r e d u c e d w e i g h t l o s s i n ‘valencia’ oranges. fruit firmness a continuous decline in mangos firmness was observed throughout storage (fig. 4). however, the fruits treated with eos showed higher firmness than the control ones. in each stage of storage, no significant difference was identified in the firmness of fruits, at different concentrations of eos. after 3 weeks of storage, the firmness of control fruits was around 3.06 kg/cm2, while the treated fruits were significantly firmer (p<0.05). in this stage, fruits treated with mancozeb showed no significant difference, as compared with those treated with 500 μl/l t. vulgaris eo. however, r. officinalis in both concentrations showed the highest firmness, as compared with other treatments. firmness, as one of the fruits properties, is a complex sensory attribute that also includes crispiness and juiciness; it is important in determining the acceptability of horticultural crops. it has been accepted that the loss of fruit firmness throughout the storage is mainly due to the depolymerization of fig. 3 e f f e c t o f t h e t h y m u s v u l g a r i s , s a l v i a m i r z a y a n i i , artemisia persica and rosmarinus officinalis eos tested at 500 and 1000 μl/l on mangos weight loss after 1, 2, and 3 weeks of storage compared to ‘mancozed’ (0.5 and 1.0 mg/l). bars indicate the sd of the mean. different letters indicate significant differences in mean values (p <0.05). fig. 4 e f f e c t o f t h e t h y m u s v u l g a r i s , s a l v i a m i r z a y a n i i , artemisia persica and rosmarinus officinalis eos tested at 500 and 1000 μl/l on mangos firmness after 1, 2, and 3 weeks of incubation compared to ‘mancozed’ (0.5 and 1.0 mg/l). bars indicate the sd of the mean. different letters indicate significant differences in mean values (p <0.05). 394 adv. hort. sci., 2018 32(3): 389-398 tion, respectively. the results also showed that b* was increased during the storage time, but the trend in fruits treated by eos was slower than that in the control. at the end of storage, s. mirzayanii and r. officinalis, at 500 μl/l concentrations, showed the minimum b* value (40.7 and 40.6), respectively. however, control and mancozeb samples showed the maximum b* value (56.6 and 55.9), respectively. the results obtained in the present study showed that eos treatment could have better litheness with lower a* and b*, as compared to mancozed and control groups. ramezanian et al. (2016) showed the effect of the zataria multiflora and t. vulgaris eos on the black rot of ‘washington navel’ orange fruit. they found that the best color was related to zataria at 300 μl/l and thyme eos at 400 μl/l concentrations. in agreement with our findings, marjanlo et al. (2009) showed the effect of cumin eo on the postharvest quality of strawberries, finding that the essential oil treated fruits maintained a higher l* during storage in comparison with the controls. aa content ascorbic acid (vitamin c) content was gradually decreased during storage; however, its strength was lower in the treated samples (table 2). different concentrations of the eos significantly maintained ascorbic acid content, as compared to the control. overall, the most (14 mg/100 g/1) and the least (9 mg/100 table 1 effect of the thymus vulgaris, salvia mirzayanii, artemisia persica and rosmarinus officinalis eos tested at 500 and 1000 μl/l on mangos color change ± sd after 1, 2, and 3 weeks of storage compared to ‘mancozed’ (0.5 and 1.0 mg/l) in each character, different letters indicate significant differences in mean values (p<0.05). testing index treatment (concentration) storage time week 1 week 2 week 3 l* control 63.3±0.43 a 47.5±0.56 n 46.2±0.56 n mancozeb 0.5 mg/l 64.9±0.5 a 59.8±0.48 c-g 54.1±0.59 j-m mancozeb 1 mg/l 55.3±.23 f-l 55.9±0.75 f-l 56.7±0.76 f-l thymus vulgaris 500 μl/l 62.1±0.33 a-d 54.8±0.46 i-m 51.4±0.36 mn thymus vulgaris 1000 μl/l 60.6±0.54 b-f 57.8±0.49 e-j 51.8±0.66 mn salvia mirzayanii 500 μl/l 54.6±0.32 i-m 51.9±0.58 l-n 56.1±0.54 f-k salvia mirzayanii 1000 μl/l 59.9±0.33 c-g 58.4±0.44 d-g 49.7±0.38 mn artemisa persica 500 μl/l 59.7±0.43 c-g 56.9±0.58 f-l 48.7±0.65 n artemisa persica 1000 μl/l 57.9±0.23 e-j 58.8±0.75 d-g 41.4±0.65 o rosmarinus officinalis 500 μl/l 59.8±0.19 c-g 60.7±0.66 a-d 54.1±0.39 j-m rosmarinus officinalis 1000 μl/l 62.2±0.25 a-d 65.3±0.76 a 59.5±0.53 c-g a* control 16.4±0.87 12.6±0.87 g-i 7.4±0.87 l mancozeb 0.5 mg/l 17.2±0.87 15.8±0.88 a-e 8.1±0.98 l mancozeb 1 mg/l 16.8±0.99 16.1±0.98 a-d 8.6±0.99 kl thymus vulgaris 500 μl/l 16.4±0.67 16.1±0.76 b-f 8.6±0.76 kl thymus vulgaris 1000 μl/l 17.2±0.89 15.3±0.87 a-c 11.2±0.85 ij salvia mirzayanii 500 μl/l 17.1±0.78 16.7±0.88 c-f 11.1±0.76 ij salvia mirzayanii 1000 μl/l 17.6±0.77 15±0.68 e-g 8.4±0.87 kl artemisa persica 500 μl/l 17.1±0.77 14.2±0.87 f-g 10.8±0.97 ij artemisa persica 1000 μl/l 17.4±0.69 13.5±0.88 f-h 11.6±0.97 h-j rosmarinus officinalis 500 μl/l 16.8±0.90 15.8±0.98 a-e 11.7±0.98 ij rosmarinus officinalis 1000 μl/l 14.6±1.02 16.3±0.96 a-d 10.1±1.03 jk b* cntrol 25.5±0.78 ij 34.7±1.02 e 56.6±2.2 a mancozeb 0.5 mg/l 20.7±1.03 l-n 30±1.03 fg 55.9±3.2 a mancozeb 1 mg/l 21.5±0.85 l-n 32.9±1.07 ef 46.7±2.9 bc thymus vulgaris 500 μl/l 20.8±0.87 mn 25.1±0.84 h-j 46.7±3.8 bc thymus vulgaris 1000 μl/l 20.6±0.78 mn 30.6±1.06 fg 49.4±3.5 b salvia mirzayanii 500 μl/l 17.0±0.78 ab 30.7±0.94 fg 40.6±2.6 d salvia mirzayanii 1000 μl/l 23.8±1.03 i-l 27.3±0.99 gh 48.3±3.5 bc artemisa persica 500 μl/l 19.4±0.86 n 26.6±0.95 hi 47.8±3.6 bc artemisa persica 1000 μl/l 20.8±0.98 l-n 30.8±1.04 fg 47.6±3.2 bc rosmarinus officinalis 500 μl/l 20.7±1.03 l-n 24.4±0.90 i-k 40.7±2.8 d rosmarinus officinalis 1000 μl/l 22.8±0.99 j-m 23.4±1.05 i-l 45.5±3.5 c 395 javadpour et al. postharvest control of aspergillus niger in mangos g/1) amount of ascorbic acid content was detected in the fruits treated with s. mirzayanii at the concentration of 500 μl/l and control after three weeks of storage, respectively. in general, fruits treated with mancozeb showed lower ascorbic acid content in comparison with those treated with eos throughout the storage. in agreement with our findings, geransayeh et al. (2012) showed that the vitamin c content of grapes was decreased significantly during the storage; however, a higher vitamin c amount was observed in the samples treated with t. vulgaris eo. our results were nevertheless in contrast with those of marjanlo (2009) who did not detect any significant difference in the amount of ascorbic acid in strawberry fruits treated by the essential oils. carotenoids and total chlorophyll content analysis of the variance of carotenoids content revealed a significant difference (p< 0.05) between t r e a t m e n t s ( t a b l e 2 ) . t h e c o n c e n t r a t i o n o f carotenoids content was low at the initial time of storage and then significantly increased during storage. at the end of the process, control fruits showed the highest content of carotenoids (1.94 mg 100 g-1). r. officinalis and t. vulgaris, tested at 500 μl/l showing 1.09 and 1.15 (mg 100 g-1) as carotenoids content, respectively, were the lowest one, as compared with other treatments. the total chlorophyll content was gradually decreased to a lower concentration in all table 2 effect of the thymus vulgaris, salvia mirzayanii, artemisia persica and rosmarinus officinalis eos tested at 500 and 1000 μl/l on the ascorbic acid, carotenoids and total chlorophyll content ± sd in mangos after 1, 2, and 3 weeks of storage compared to ‘mancozed’ (0.5 and 1.0 mg/l) in each character, different letters indicate significant differences in mean values (p<0.05). active metabolite treatment (concentration) storage time (week) week 1 week 2 week 3 ascorbic acid control 12.3±3 cd 11±2 cd 9.5±4 d mancozeb 0.5 mg/l 13.3±4 c 12.6±3 cd 11±3 cd mancozeb 1 mg/l 13.5±4 c 12.3±3 cd 11±2 cd thymus vulgaris 500 μl/l 14.8±7 bc 13.6±4 c 12±4 cd thymus vulgaris 1000 μl/l 14.5±4 bc 13.7±4 c 12±5 cd salvia mirzayanii 500 μl/l 17.4±4 a 16.4±4 b 14±5 bc salvia mirzayanii 1000 μl/l 13.3±6 c 12.9±3 cd 12±4 cd artemisa persica 500 μl/l 15.9±7 bc 14.8±2 bc 12±5 cd artemisa persica 1000 μl/l 13.4±4 c 12.6±5 cd 11±5 cd rosmarinus officinalis 500μl/l 17.5±4 a 17±5 b 13±3 c rosmarinus officinalis 1000 μl/l 15.8±3 bc 14.5±4 bc 11±c 2 d carotenoids control 0.85±0.03kn 1.64±0.06 b 1.94±0.03 a mancozeb 0.5 mg/l 0.69±0.04 m-o 1.57±0.03 b 1.43±0.03 b-e mancozeb 1 mg/l 0.74±0.03 m-o 1.52±0.04 bc 1.48±0.08 b-d thymus vulgaris 500 μl/l 0.74±0.07 m-o 1.12±0.05 g-j 1.15±0.03 f-i thymus vulgaris 1000 μl/l 0.55±0.03 o 1.28±0.08 c-g 1.91±0.06 a salvia mirzayanii 500 μl/l 0.69±0.08 m-o 1.26±0.05 d-h 1.39±0.07 b-f salvia mirzayanii 1000 μl/l 0.7±0.06 m-o 1.1±0.06 g-k 1.53±0.03 bc artemisa persica 500 μl/l 0.56±0.05 o 0.85±0.04 l-n 1.31±0.04 c-g artemisa persica 1000 μl/l 0.6±0.06 o 0.87±0.08 j-m 1.22±0.03 e-h rosmarinus officinalis 500 μl/l 0.52±0.04 o 0.93±0.05 i-m 1.09±0.04 g-l rosmarinus officinalis 1000 μl/l 0.5±0.07 o 1.01±0.03 h-l 1.16±0.05 f-i total chlorophyll control 2.72±0.2 f 1.19±0.02 gh 0.03±0.002 i mancozeb 0.5 mg/l 3.98±0.4 e 1.31±0.02 g 0.53±0.03 h mancozeb 1 mg/l 3.44±0.2 f 1.72±0.02 g 0.31±0.02 h thymus vulgaris 500 μl/l 4.86±0.5 de 2.5±0. 6 f 0.11±0.01 h thymus vulgaris 1000 μl/l 4.33±0.2 de 2.66±0. 5 f 0.11±0.02 h salvia mirzayanii 500 μl/l 5.43±0.3 d 3.55±0.2 f 0.16±0.01 h salvia mirzayanii 1000 μl/l 7.62±0.2 bc 3.47±0.2 f 0.15±0.02 h artemisa persica 500 μl/l 9.61±0.2 a 2.58±0.7 f 0.16±0.03 h artemisa persica 1000 μl/l 8.38±0.3 b 2.35±0.5 f 0.22±0.02 h rosmarinus officinalis 500 μl/l 8.69±0.2 ab 3.27±0.8 f 0.16±0.04 h rosmarinus officinalis 1000 μl/l 8.19±0.4 b 3.3±0.6 f 0.32±0.5 h 396 adv. hort. sci., 2018 32(3): 389-398 treatments throughout the storage (table 2); however, the highest reduction was observed in the control fruits. at the end of storage, the minimum chlorophyll content (0.03) was recorded in the control, while the highest was found in mancozeb with 0.5 mg/l concentration. tss, ta and ph a gradual increase in tss percentages was determined in all treatments (table 3). generally, the fruits treated with eos had lower tss percentages than the control fruits throughout the storage. however, the treated fruits did not show any significant difference in tss, as compared with the controls. these results were in agreement with those in other studies (marjanlo et al., 2009). nevertheless, they are in discordance with rabiei et al. (2011), who reported that thyme eos treatment had a significant effect on the ph of apples. as shown in table 3, ta values were also gradually decreased during storage. at the end of storage, the maximum ta values (0.27%) were observed in a. persica (1000 μl/l), while the minimum (0.10%) was in the control fruits. among the eos treatments, r. officinalis (1000 μl/l) resulted in the lowest acidity (0.17%), this was followed by t. vulgaris (0.16%) with 1000 μl/l concentration during storage; however, no significant difference was observed between the treatments. these results are in agreement with those reported by maqbool and alderson (2010), who showed that the table 3 effect of the thymus vulgaris, salvia mirzayanii, artemisia persica and rosmarinus officinalis eos tested at 500 and 1000 μl/l on total soluble solids (tss), ph, and titratable acidity (ta) ± sd in mangos after 1, 2, and 3 weeks of incubation compared to ‘mancozed’ (0.5 and 1.0 mg/l) in each character, different letters indicate significant differences in mean values (p<0.05). quality parameter treatment storage time (week) week 1 week 2 week 3 tss control 8.9±3 c 9.6±3 b 9.9±2 a mancozeb 0.5 mg/l 8.06±3 cd 9.23±5 b 9.5±3 b mancozeb 1 mg/l 8.33±4 c 9.13±5 bc 9.5±2 b thymus vulgaris 500 μl/l 8.96±5 c 9.63±4 b 9.7±2 ab thymus vulgaris 1000 μl/l 8.83±4 c 9.76±4 ab 9.8±4 ab salvia mirzayanii 500 μl/l 8.7±4 c 9.26±2 b 9.3±3 b salvia mirzayanii 1000 μl/l 8.33±3 c 9.26±5 b 9.6±2 b artemisa persica 500 μl/l 8.66±3 c 9.3±3 b 9.6±3 b artemisa persica 1000 μl/l 8.1±5 cd 9.53±5 b 9.7±4 b rosmarinus officinalis 500 μl/l 7.5±3 d 9±4 bc 9.2±2 b rosmarinus officinalis 1000 μl/l 8.66±4 c 9.53±5 b 9.5±3 b ph control 1.15±0.03 cd 2.89±0.2 c 4.75±1.4 a mancozeb 0.5 mg/l 1.06±0.02 cd 2.38±0.4 c 4.3± 2 ab mancozeb 1 mg/l 1.09±0.02 cd 2.59±0.3 c 4.3± 2 ab thymus vulgaris 500 μl/l 1.06±0.02 cd 2.51±0.2 c 4.18± 2. 2 ab thymus vulgaris 1000 μl/l 1.26±0.02 cd 2.54±0.3 c 4.31±1.8 ab salvia mirzayanii 500 μl/l 0.99±0.02 d 2.74±0.2 c 4.24±1.9 ab salvia mirzayanii 1000 μl/l 1.12±0.02 cd 2.26±0.2 c 4.42±2 ab artemisa persica 500 μl/l 1.03±0.02 cd 2.45±0.3 c 4.48±3 ab artemisa persica 1000 μl/l 1.08±0.02 cd 2.17±0.2 c 4.47±1.4 ab rosmarinus officinalis 500 μl/l 1.02±0.02 cd 1.76±0.1 cd 4.36±1.2 ab rosmarinus officinalis 1000 μl/l 1.07±0.02 cd 2.52±0. 5 c 4.56±2.1 ab ta control 0.208±0.03 c 0.196±0.01 c 0.10±0.009 d mancozeb 0.5 mg/l 0.254±0.02 bc 0.255±0.01 bc 0.23±0.02 bc mancozeb 1 mg/l 0.27±0.02 bc 0.234±0.02 bc 0.23±0.02 bc thymus vulgaris 500 μl/l 0.328±0.02 b 0.26±0.03 bc 0.23±0.02 bc thymus vulgaris 1000 μl/l 0.308±0.01 b 0.262±0.02 bc 0.16±0.01 cd salvia mirzayanii 500 μl/l 0.384±0.02 b 0.267±0.03 bc 0.25±0.02 bc salvia mirzayanii 1000 μl/l 0.352±0.03 b 0.299±0.03 bc 0.24±0.02 bc artemisa persica 500 μl/l 0.288±0.02 bc 0.277±0.02 bc 0.23±0.02 bc artemisa persica 1000 μl/l 0.405±0.02 b 0.307±0.01 b 0.27±0.02 bc rosmarinus officinalis 500 μl/l 0.471±0.02 a 0.302±0.02b 0.26±0.02 bc rosmarinus officinalis 1000 μl/l 0.328±0.01 b 0.261±0.01 bc 0.17±0.01 cd javadpour et al. postharvest control of aspergillus niger in mangos 397 maximum reduction of the ta values was observed in the control fruits of bananas and papayas. data related to the changes in the ph of fruits during storage revealed a significant increase in all treatments (table 3). in each time, a higher ph value was found in the control groups. these results were in line with t h o s e r e p o r t e d b y d u p l o o y e t a l . ( 2 0 0 9 ) a n d tzortzakis (2007), showing no significant differences between the ph of control and treated fruits 4. conclusions the present study proves that the t. vulgaris, a. persica, r. officinalis and s. mirzayanii eos could be employed under postharvest condition to control a pathogenic isolate of a. niger on mango fruits. the effectiveness of these eos was more than mancozeb. so that, the eos here tested could be used as a natural fungicide to control an isolate of a. niger during postharvest mangos. however, further studies are needed to fully understand the antimicrobial mechanisms incited by these eos on a wide range of a. niger isolates, and evaluate their commercial implementation in order to increase the storage lifetime and quality of this marketable commodity. acknowledgements this study as research project was financially supported by hormozgan university of medical sciences, bandar abbas, iran. authors are thankful for providing necessary facilities for carrying out this work. references abdolahi a., hassani a., ghosta y., bernousi i., meshkatalsadat m.h., 2010 study of the potential use of essential oils for decay control and quality preservation of tabarzeh table grape. j. plant prot. res., 50: 45-52. boubaker h., karim h., el hamdaoui a., msanda f., leach d., bombarda i., vanloot p., abbad a., boudyach e.h., aoumar a.a.b., 2016 chemical characterization and antifungal activities of four thymus species essential oils against postharvest fungal pathogens of citrus. ind. crops prod. 86: 95-101. centeno s., calvo m.a., adelantado c., figueroa s., 2010 antifungal activity of extracts of rosmarinus officinalis and thymus vulgaris against aspergillus flavus and a. ochraceus. pak. j. biol. sci., 13(9): 452455. de sousa l.l., de andrade s.c.a., athayde a.j.a.a., de oliveira c.e.v., de sales c.v., madruga m.s., de souza e.l., 2013 efficacy of origanum vulgare l. and rosmarinus officinalis l. essential oil in combination to c o n t r o l p o s t h a r v e s t p a t h o g e n i c a s p e r g i l l i a n d autochthonous mycoflora in vitis labrusca l. 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(ed.) fungicides. intech, rijeka, croatia, pp. 538. willats w.g.t., mccartney l., mackie w., knox j.p., 2001 pectin: cell biology and prospects for functional analysis. plant molecular biology, 47: 9-27. impaginato 193 adv. hort. sci., 2018 32(2): 193-203 doi: 10.13128/ahs-21304 yield and yield components of coriander under different sowing dates and seed rates in tropical environment k.t. kassu, h.h. dawit, a.y. wubengeda, a.t. almaz, m.t. asrat ethiopian institute of agricultural research, ethiopia. key words: biomass yield, coriander, fruit yield, seed rate, sowing date. abstract: coriander makes use of favorable environmental conditions when it is sown at optimum time and rate. however, this information is very limited in the southeastern mid-highlands of ethiopia. field experiments were, therefore, conducted between 2011 and 2014 at three different research stations to determine optimum sowing dates and rates. the experiment had split plot design in randomized complete block with three replications, in which sowing dates and seed rates were the main and sub-plot treatments, respectively. the four sowing date treatments were june 20, july 10, july 30 and august 20 while the four seed rate treatments were 30, 40, 50 and 60 kg ha-1. coriander sowed in the third decade of july at arsi robe and from the first to the third decades of july at kulumsa and sagure gave the highest fruit and biomass yields. earlier sowing in the second decade of june, and delayed sowing in the second decade of august brought fruit yield reductions of 37-66 and 37% at arsi robe, 27-45 and 58-66% at kulumsa, and 24-40 and 26% at sagure, respectively. however, coriander did not respond to seed rates. owing to the enhanced yields of coriander, intermediate cultivation at a seed rate of 30 kg ha-1 was found optimum. 1. introduction coriander (coriandrum sativum l.), which belongs to the family of umbelliferae (apiaceae) is one of the most important annual spice and medicinal herb. it is grown in ethiopia and throughout the world for its seeds as well as leaves and has immense uses (diederichsen, 1996; hedburg and hedburg, 2003; parthasarathy et al., 2008; nowak and szemplinski, 2014). coriander originated from the mediterranean and western asian regions (burdock and carabin, 2009). along with central asia and near east countries, vavilov (1992) mentioned ethiopia in the lists of centers of origin for coriander. ivanova and stoletova (1990) also reported that india, northern africa, central asia and ethiopia are centers of formation and cradles for different types of coriander. there is a longstanding tradition of cultivation of coriander in ethiopia (diederichsen, 1996; geremew et al., 2014). (*) corresponding author: kasstad96@yahoo.com citation: kassu k.t., dawit h.h., wubengeda a.y., almaz a.t., asrat m.t., 2018 yield and yield components of coriander under different sowing dates and seed rates in tropical environment. adv. hort. sci., 32(2): 193-203 copyright: © 2018 kassu k.t., dawit h.h., wubengeda a.y., almaz a.t., asrat m.t. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 10 october 2017 accepted for publication 12 january 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(2): 193-203 194 the immense uses of coriander depend on the choice of fruits or green herbs, which are linked to their chemical compositions. the most important c o n s t i t u e n t s a r e t h e e s s e n t i a l a n d f a t t y o i l s (diederichsen, 1996). coriander has got significant importance as a spice in culinary, food, beverage, medicine, perfumery, pharmaceuticals and sanitary i n d u s t r i e s ( j a n s e n , 1 9 8 1 ; d i e d e r i c h s e n , 1 9 9 6 ; delaquis et al., 2002; kubo et al., 2004). on the other hand, its green foliage is used in vegetables owing to its richness in vitamins and other minerals (singh et al., 2005). in ethiopia, coriander is widely used for domestic culinary. the seeds are used for flavoring the powder of hot red pepper locally called “berbere” and used for numerous meat and vegetarian dishes, leavened flat ethiopian bread locally called “injera”, cakes and bread. the leaves are added as an aromatic herb to tea and stew locally called “wot” (jansen, 1981; geremew et al., 2014). coriander is also a good melliferous plant since it produces a considerable quantity of nectar and thereby attracts many different insects for pollination. studies indicated that one hectare of coriander allows honeybees to collect about 500 kg of honey (diederichesen, 1996). the residues left after extraction of the essential oils are used as best ruminant feed since they still contain as nearly the same digestible fat and protein content as the whole fruits (diederichesen, 1996). the success of coriander production is influenced by genetic, weather and agronomic factors (nowak and szemplinski, 2014). the maximum fruit and essential oil yields are attained only when an appropriate combination of these factors are provided for the plant (rangappa et al., 1997; gil et al., 2002). coriander is among the tropical crops and generally sown in winter season if the objective is seed for production (sharangi and roychowdhury, 2014). as a temperature-sensitive crop, it generally requires a relatively cool, comparatively dry and frost-free weather during its early stage for good vegetative growth and relatively warm temperature during flowering and reproductive stage for high yields and good quality (peter, 2004; kalra, 2008; sharangi and roychowdhury, 2014). the ideal temperature for germ i n a t i o n a n d g r o w t h o f c o r i a n d e r i s 2 0 2 5 ° c (singhania et al., 2006). coriander exploits the environment most favorably when it is sown at optimum time (kuri et al., 2015) since sowing date significantly affects the photoperiodic response of plants and determines yields and qualities (rasam et al., 2007). time of sowing controls the crop phenological development along with efficient conversion of biomass into economic yield (khichar and niwas, 2006). earliness in sowing leads to untimely flowering; however, it may also pose susceptibility to the damage of extreme cold and frost. on the other hand, delay in sowing hampers growth, yield and quality of the crop due to deficiency of soil moisture at latter stages (sharangi and roychowdhury, 2014; rashed and darwesh, 2015). determination of optimum seed rate is also a basic element for successful coriander production (rasam et al., 2007). many agronomic studies conducted in the world revealed that seed rate had a highly significant effect on the productivity and quality of coriander (diederichsen, 1996; kumar et al., 2007; ghobadi and ghobadi, 2010). both low and high seed rates resulted in reduced yield and oil concentrations. rapid life cycle of coriander allows it to fit into different growing seasons, making it possible to grow the crop under a wide range of conditions (lopez et al., 2007). cultivation of coriander in ethiopia; however, is limited to the mid to highlands (1500-2500 m a.s.l.), where sufficient soil moisture can be provided from rainfall. it can also be cultivated in the lowlands if the rainfall is sufficiently supplemented by irrigation (jansen, 1981; geremew et al., 2014). although coriander has got diverse uses, economic importance and one of the several plant species for which ethiopia is known as a center of origin and diversity (jansen, 1981; diederichsen, 1996), it is one of the most neglected or under-utilized aromatic and spice crop (beemnet and getinet, 2010). the wealth o f c o r i a n d e r i s n o t y e t e x p l o i t e d i n e t h i o p i a . compared to other crops, there is no or very limited information available on the agronomic packages. this study was, therefore, carried out to determine optimum sowing date and seed rate for increased yield of coriander in the southeastern mid-highlands of ethiopia. 2. materials and methods description of the study sites the experiment was conducted at three locations, namely arsi robe, kulumsa and sagure in the southeastern mid-highlands of ethiopia. the sites are representatives of the region, where coriander cultivation can potentially be carried out, and optimum kassaye et al. response of coriander to sowing dates and seed rates 195 sowing dates and seed rate studied. it was conducted for two seasons in 2011 and 2012 at arsi robe, and 2011 and 2014 at kulumsa and sagure. due to infestation by unknown disease, the crop could not perform well in 2012 and 2013 at kulumsa and sagure, and harvesting could not be done. arsi robe, sagure and kulumsa are located from 8.4 to 8.6 n and 40.1 to 40.4 e, 8.01 to 8.15 n and 39.2 to 39.3 e and from 7.77 to 8.03 n and 38.94 to 39.31 e, respectively. the altitudes of the locations vary from 2200 m a.s.l. at kulumsa to about 2500 m a.s.l. at arsi robe and sagure. the dominant soil type of the three locations is characterized as vertisol (iuss working group wrb, 2014). climate long-term mean annual rainfall at arsi robe, kulumsa and sagure were 937, 812 and 653 mm, respectively. hence, arsi robe and sagure had the highest and lowest, respectively rainfall with intermediate values at kulumsa (fig. 1). 56, 55 and 67% of the annual rainfall concentrated in the months of july and august at arsi robe; june, july and august at sagure and july, august and september at kulumsa. the major crop production activities are conducted between june to november; therefore, the rainfall amount and distribution during these months have significant influence on the yield and yield attributes. the highest rainfall was recorded in august at all study sites (fig. 1). from august, the rainfall amount and distribution reduce sharply and reach the lowest i n t h e m o n t h o f d e c e m b e r a t a l l l o c a t i o n s . november, december, january and february are dry months with the lowest records of rainfall amounts. long-term mean maximum temperature records of arsi robe, kulumsa and sagure were 22.3, 23.2 and 22.5°c, respectively. the corresponding values for mean minimum temperatures were 8.3, 10.5 and 8.8°c, respectively (fig. 2). february was the hottest month at arsi robe and sagure while the corresponding month at kulumsa was march. december was the coldest month at all locations with the lowest records of 4.9, 8.0 and 5.1°c at arsi robe, kulumsa and sagure, respectively. experimental set-up and procedure the experiment had split plot design in randomized complete block with three replications, in which the sowing dates and seed rates were the main and sub-plot treatments, respectively. the four sowing date treatments were june 20, july 10, july 30 and august 20 whereas the four seed rate treatments were 30, 40, 50 and 60 kg ha-1. the sowing dates were set to choose the optimum time by allowing coriander to make maximum benefit from the suitable environmental parameters, especially rainfall and temperature for its successful establishment, survival and performance. on the hand, the seed rates were set by making reference to the existing recommendation of 40 kg ha-1. the seedbed preparation started in early april and totally plowed four times prior to planting. all experimental plots at each location and season were planted with coriander (cv. keteba). the seedbeds were prepared in ridge and furrow, and seeds were drilled on raised beds by hand at 0.30m spacing between rows on the aforementioned sowing dates for all fig. 1 total mean monthly rainfall at arsi robe, kulumsa and sagure. fig. 2 mean annual maximum (a) and minimum (b) temperatures at arsi robe, kulumsa and sagure. adv. hort. sci., 2018 32(2): 193-203 196 sites in plot sizes of 1.8 m by 6 m. the spacing between plots and replications were 0.5 m and 1 m, respectively. the recommended phosphorus (20 kg p ha-1) and nitrogen (18 kg n ha-1) nutrients were uniformly applied to all plots close to the seed rows as basal dose at the time of sowing from di-ammonium phosphate (20-18 p-n). weeds were controlled by manual cleaning. data collection twenty-five randomly selected plant samples were manually cut at the ground level from the inner four rows by excluding the outer two to avoid any border effect, air-dried, the moisture content adjusted to constant level and used for measuring above ground total biomass. the harvest index was calculated by dividing the dry mass of seeds collected from the 25 plant samples and threshed manually by the dry mass of biomass, and multiplying the ratio by 100. for the measurement of fruit yield, the whole crop was harvested from a net plot area of 6 m2 (1.2 m by 5 m), subjected to air-drying and threshed manually. the fruits were detached from the biomass, cleaned and weighed. the seed moisture content was determined by placing samples from each plot in an oven at 105°c for 24 hours. the above ground total biomass and seed mass from each plot were then adjusted to 0 g kg-1 moisture content (dry weight) and expressed in kg ha-1 for statistical analysis purpose. plant height data were taken from each plot at physiological maturity from ten plant samples. data analysis all yield and yield components data were combined across sites and seasons and subjected to analysis of variance using the general linear model procedure (proc glm) of sas statistical package version 9.2 (sas institute, 2002). least significance difference (lsd) tests were employed to evaluate the means of the main and interaction effects of the treatments for each parameters measured (determined). mean separation for the interaction effects were conducted using minitab®18 statistical package (minitab inc.). when p<0.05, means values of treatments were declared as significantly different. 3. results the analysis of variance over two seasons indicated that sowing date and season as well as their interaction had very significant (p<0.001) effect on most of the traits measured at all locations (tables 1-3). sources of variation yield and yield components of coriander plant height (cm) harvest index (%) fruit yield (kg ha-1) biomass yield (kg ha-1) rep ns ns ns ns sowing date (sd) *** *** *** *** error (a) 45.64 11.12 19566.89 317296 year (y) *** ns *** *** seed rate (sr) *** ns ns ns sd x sr ns ns * ns y x sd *** *** *** *** y x sr ns ns ns ns y x sd x sr ns ns ns ns error (b) 32.67 13.5 16423.2 202979 cv 6.63 10.02 13.21 16.75 table 1 effects of sowing dates and rates by year, and their interaction on yield and yield components of coriander at arsi robe in 2011 and 2012 *** and ns means significant at p<0.001 and not significant at p<0.05, respectively. table 2 effects of sowing dates and rates by year, and their interaction on yield and yield components of coriander at kulumsa in 2011 and 2014 *** and ns means significant at p<0.001 and not significant at p<0.05, respectively. sources of variation yield and yield components of coriander plant height (cm) harvest index (%) fruit yield (kg ha-1) biomass yield (kg ha-1) rep ns ns * ns sowing date (sd) *** *** *** *** error (a) 5.86 1.83 14609.46 870629 year (y) *** *** *** ns seed rate (sr) * ns ns ns sd x sr ns *** *** ns y x sd *** *** *** ns y x sr *** *** ns ns y x sd x sr * *** ns ns error (b) 7.09 8.84 46135.74 2447414 cv 3.02 10.06 13.2 25.05 table 3 effects of sowing dates and rates by year, and their interaction on yield and yield components of coriander at sagure in 2011 and 2014 *** and ns means significant at p<0.001 and not significant at p<0.05, respectively. sources of variation yield and yield components of coriander plant height (cm) harvest index (%) fruit yield (kg ha-1) biomass yield (kg ha-1) rep ns ns ns ns sowing date (sd) *** *** *** *** error (a) 11.04 4.24 295392.79 2299583 year (y) *** *** *** ns seed rate (sr) *** ns ns ns sd x sr *** *** *** *** y x sd *** *** *** *** y x sr ns *** ns *** y x sd x sr * *** ns * error (b) 19.36 11.21 69124.93 928593 cv 4.93 10.71 18.52 20.75 kassaye et al. response of coriander to sowing dates and seed rates 197 the effects of seed rate on fruit and biomass yields were not significant; however, its interaction with sowing date and season brought significant improvement on some of the variables measured. results further showed that the interaction effects among sowing date, seed rate and season were not significant on most of the yield and yield components. the amount of variance associated with the sowing date x season interaction was the most important for this study. effect of sowing date sowing dates and their interaction with season significantly (p<0.001) affected the fruit and biomass yields of coriander at all locations except for the biomass yield at kulumsa. the highest fruit yields of coriander were attained from coriander sowed on july 30 in 2012 and 2014 at arsi robe and kulumsa, respectively; and july 30 and july 10 in 2014 and 2011 at sagure, respectively (tables 4-6). the coriander sown on july 30 and june 20 in 2012 and 2011, respectively at ari robe and july 10 in 2014 at sagure gave the highest biomass yields. fruit yields of 2713, 2028 and 2006 kg ha-1 were obtained at kulumsa in 2014, sagure in 2014 and arsi robe in 2012, respectively from coriander sowed on july 30. july 10 sown coriander at sagure gave a fruit yield of 1928 kg ha-1, which was statistically equivalent to the july 30 sown coriander. similarly, biomass yields of 4732 and 4573 kg ha-1 were harvested at arsi robe in 2012 and 2011 from july 30 and june 20 sown coriander, respectively. the highest biomass yield at sagure, 7217 kg ha-1, was found from the coriander sowed on july 10 in 2011. the result further revealed that early (june 20) and late (august 20) sown coriander produced inferior fruit and biomass yields compared to the intermediate sowing dates. the effects of sowing date and its interaction with season on the harvest index were also very significant (p < 0.001) at all locations (table 1-3). the highest harvest indexes were obtained from the august 20 sown coriander at arsi robe and kulumsa in both years (tables 4-6). the values of harvest index from the august 20 sown coriander at arsi robe were 42.7 and 40.9% in 2012 and 2011, respectively. the corresponding values from the same sowing date at kulumsa were 42.1 and 41% in 2014 and 2011, table 4 influences of sowing date and season on yield and yield components of coriander at arsi robe in 2011 and 2012 sowing date year plant height (cm) harvest index (%) fruit yield (kg ha-1) biomass yield (kg ha-1) 2011 2012 2011 2012 2011 2012 2011 2012 june 20 72 de 112 a 34 cd 28 e 239 f 1272 b 721 e 4573 a july 10 79 c 108 a 34.8 c 31.1 d 502 e 1147 c 1446 d 3702 b july 30 68 e 96 b 38.6 b 42.9 a 683 d 2028 a 1809 d 4732 a august 20 76 cd 80 c 40.9 ab 42.7 a 696 d 1193 bc 1736 d 2828 c table 6 influences of sowing date and season on yield and yield components of coriander at sagure in 2011 and 2014 table 5 influences of sowing date and season on yield and yield components of coriander at kulumsa in 2011 and 2014 sowing date year plant height (cm) harvest index (%) fruit yield (kg ha-1) biomass yield (kg ha-1) 2011 2014 2011 2014 2011 2014 2011 2014 june 20 106 a 105 a 18.9 e 20.5 de 1366 c 1493 c 7841 ab 8013 ab july 10 100 b 101 b 29.2 c 27.5 c 1835 b 1991 b 6326 c 7206 bc july 30 88 c 105 a 22 d 35 b 1876 b 2713 a 8559 a 7859 ab august 20 48 d 50 d 41 a 42.1 a 793 d 934 d 1942 d 2220 d sowing date year plant height (cm) harvest index (%) fruit yield (kg ha-1) biomass yield (kg ha-1) 2011 2014 2011 2014 2011 2014 2011 2014 june 20 96 c 108 a 33.4 b 26.5 e 1523 b 1162 c 4638 cd 4507 cd july 10 96 c 103 b 28.2 cde 29.1 cd 2006 a 1435 b 7217 a 4947 bc july 30 78 d 102 b 40.4 a 35 b 1557 b 1928 a 3846 de 5495 b august 20 64 e 66 e 30.2 c 27.4 de 888 d 845 d 3204 e 3320 e adv. hort. sci., 2018 32(2): 193-203 198 respectively. statistically equivalent harvest index (42.9%) was also provided at arsi robe from the coriander sowed on july 30 in 2012. the highest harvest index at sagure (40.4%) was obtained from the july 30 sown coriander in 2011. the influences of sowing date and its interaction on plant height was also very significant (p <0.001). generally, early and late sown coriander resulted in the tallest and shortest plant heights, respectively, which were consistent over years and locations. early sown coriander resulted in the tallest plant at arsi robe in 2012 (112 cm), kulumsa in 2011 (106 cm) and 2014 (105 cm), and sagure in 2014 (108 cm). the tallest plant heights at kulumsa in 2014 (105 cm) and arsi robe in 2012 (108 cm) were also attained from coriander sowed on july 30 and july 10, respectively. delay in sowing of coriander (august 20) brought the shortest plant height at kulumsa (48 and 50 cm in 2011 and 2014, respectively) and sagure (64 and 66 cm in 2011 and 2014, respectively). temporal variabilities significantly affected both yield and yield components of coriander at all locations (tables 1-3). most of the variables measured at arsi robe, kulumsa and sagure gained better advantage owing to sowing of coriander in 2012 than 2011, 2014 than 2011 and 2011 than 2014, respectively implied the influences of seasonal variabilities on yield and yield attributes. the effects of temporal variations on yield and yield attributes were more pronounced at arsi robe than kulumsa and sagure areas, which could be justified by the magnitudes of differences in the fruit yields recorded during the study period. the variances in the highest fruit yields between 2012 (2028 kg ha-1) and 2011 (696 kg ha-1) at arsi robe, 2014 (2713 kg ha-1) and 2011 (1876 kg ha-1) at kulumsa, and 2011 (2006 kg ha-1) and 2014 (1928 kg ha-1) at sagure were 1331, 837 and 78 kg ha1, respectively (tables 4-6). this implied that the magnitude of the variabilities associated with the fruit yields between the two years were so large at arsi robe compared to kulumsa and sagure. spatial variabilities were also accountable for large disparities in yield and yield components of coriander among the testing locations. fruit and biomass yields of coriander were superior at kulumsa compared to sagure and arsi robe. mean fruit yields of coriander combined over season at arsi robe, kulumsa and sagure were 970, 1625 and 1418 kg ha1, respectively. the corresponding values for biomass yields were 2693, 6246 and 4647 kg ha-1, respectively (tables 4-6). compared to arsi robe, kulumsa and sagure produced 68 and 46% more fruit yields and 132 and 73% more biomass yields of coriander, respectively. effect of seed rate seed rate did not bring significant effect on most of the yield and yield components of coriander measured at all locations (tables 1-3), which were consistent over years and locations. however, its interaction with sowing date and year had significant effect on some of the yield and yield attributes of coriander at all locations (tables 1-3). the fruit yields obtained from the july 30 sown coriander at a seed rate of 30 kg ha-1 at arsi robe and any one of the four seed rates at kulumsa were found to be statistically superior over all other possible sowing date x seed rate interactions. this implied that the lowest seed rate, 30 kg ha-1, could be sufficient for optimum yield of coriander in the study areas. sowing of coriander on july 30 at a seed rate of 30 kg ha-1 gave fruit yield of 1526 kg ha-1. similarly, coriander sowed on july 30 at seed rates of 30, 40, 50 and 60 kg ha-1 at kulumsa gave fruit yields of 2239, 2306, 2427 and 2204 kg ha-1, respectively, which were statistically equivalent to each other but significantly different from the other treatments (table 7). the seed rate x sowing date interactions at sagure were not significant for the fruit yields of coriander. seed rate interaction with year brought significant effect on the fruit and biomass yields of coriander at sagure only (tables 1-3). except for the biomass yield at sagure, the sowing date x seed rate x year interactable 7 influence of sowing date x seed rate interactions on the fruit yield of coriander at arsi robe, kulumsa and sagure from 2011 to 2014 sowing date seed rate (kg ha-1) arsi robe kulumsa sagure 30 40 50 60 30 40 50 60 30 40 50 60 june 20 907 de 746 f 695 f 674 f 1378 de 1293 e 1506 cde 1541 cd 1508 bcd 1272 def 1456 cde 1133 efg july 10 781 ef 909 de 822 def 786 ef 2176 b 2086 b 1702 c 1688 c 1846 ab 1708 abc 1677 abc 1650 abc july 30 1526 a 1356 b 1355 b 1184 c 2239 ab 2306 ab 2427 a 2204 ab 1544 bcd 1955 a 1683 abc 1787 abc august 20 936 d 958 d 958 d 926 de 969 f 834 f 850 f 802 f 783 h 942 fgh 917 gh 826 gh kassaye et al. response of coriander to sowing dates and seed rates 199 tions were also not significant at all locations for most of the attributes measured (tables 1-3). 4. discussion and conclusions the highest fruit and biomass yields from the july 30 sown coriander at arsi robe and kulumsa, and july 10 to 30 sown coriander at sagure attributed to the fulfillment of optimum soil moisture and thermal conditions from vegetative to reproductive stages (figs. 3-5). rainfall and its expected consequent soil moisture had significant impact on the performance of coriander at all locations. coriander seeded to the sowing dates within or after the highest precedent rainfall events provided the highest fruit and biomass yields. the third decade of july in 2011 and 2012 at arsi robe, and the first decade of july in 2011 and 2014 at sagure fell within the highest precedent rainfall amounts and linked with the highest fruit and biomass yields (figs. 3-5). the highest amount of precedent rainfall could lead to the retention of optimum amount of water in the soil, which soak the seeds, soften its cover and assist embryonic stem to be emerged very easily. compared to the other three sowing dates, the third sowing date (july 30) at arsi robe and the second sowing date (july 10) at sagure in both years were linked with the highest rainfall events of the previous 10 days, and the highest fruit and biomass yields. the highest rainfall events of the previous 10 days at kulumsa occurred in the second decade of august. though the performance of crop at its initial stages was greater, it faced acute moisture stress at its latter development stage and resulted in inferior fruit and biomass yields. compared to the remaining two sowing dates, the highest rainfall event at kulumsa was recorded in the third decade of july, which was associated with superior fruit and biomass yields of coriander. katar et al. (2016) reported increase in the fruit yield of coriander with increase in precedent rainfall. the variations in the responses of coriander to sowing time were also accounted for temperature. the superior yield and yield attributes of coriander were found to be linked to the sowing dates, when their maximum temperatures were the lowest. the maximum temperature records of the periods were the lowest in the third decade of july at arsi robe, kulumsa and sagure (figs. 3-5), which were associated with the highest fruit and biomass yields. the minimum temperatures at each location were not too low enough to affect the germination of seeds; rather it was modest for coriander. for successful germination, coriander requires low temperature since it favors germination by promoting the breakdown of reserve proteins in seeds to particular amino acids, which are necessary for growth of embryo (robinson, 1954; guha et al., 2014). temperature above optimum value hampers germination of seeds o w i n g t o t h e e n c o u r a g e d a c t i v i t i e s o f s e v e r a l m i c r o o r g a n i s m s s u c h a s b a c t e r i a a n d f u n g i . activation of microorganisms, in turn, adversely affects the embryo and endosperm of coriander seeds (naeem et al., 2002; ali et al., 2015). generally, winter crops like coriander are vulnerable to high temperature particularly during reproductive stages (kalra, 2008). earlier sowing (june 20) resulted in the fruit yield reductions of 37 and 66% in 2012 and 2011, respectively at arsi robe, 45 and 27% in 2014 and 2011, respectively at kulumsa, and 24 and 40% in 2011 and 2014, respectively at sagure. the corresponding declines in biomass yield were 60% in 2011 at arsi robe, and 36 and 18% in 2011 and 2014, respectively at sagure (tables 4-6). those reductions in yields could be because of the storage of excess soil moisture in the root zone during seeding and the adverse effect of intensive rainfall particularly in august on the leaves and flower of coriander. the relatively older leaves and flowers of coriander sown earlier (june 20 and july 10) at arsi robe and kulumsa, and (june 20) at sagure fell due to heavy rainfall in august (figs. 3-5). the premature shed of leaves and flowers reduced the performance of photosynthesis, fruit formation and ultimately the yield of coriander. the july 30 and august 20 sown coriander were not adversely affected due to the intensive rain fell in august as their leaves were still so young enough to recover soon, and the flowers were not yet blossomed. delayed sowing (august 20) of coriander also produced inferior fruit and biomass yields. with delay in sowing to august 20, fruit yield reductions of 37% in 2012 at arsi robe, 66 and 58% in 2014 and 2011, respectively at kulumsa, and 26% in 2011 at sagure were recorded. similarly, delayed sowing resulted in biomass yield reductions of 40% in 2012 at arsi robe, 77 and 72% in 2011 and 2014 at kulumsa, and 26% in 2011 at sagure (tables 4-6). the inferior yields of coriander attributed to the acute scarcities of rainfall in november at all locations (figs. 3-5). owing to delayed sowing and its consequential soil moisture stress, coriander had inadequate time to complete its vegetative growth since it entered to the reproducadv. hort. sci., 2018 32(2): 193-203 200 tive phase at quicker rates and generally the whole crop-growing period was shortened (carrubba et al., 2 0 0 6 ; s h a r a n g i a n d r o y c h o w d h u r y , 2 0 1 4 ) . consequently, inferior development of shoots and reduced yield attributes occurred (tables 4-6), which were the coriander’s response to the acute shortage of rainfall and high temperatures that encountered at latter stages in the growing season (carrubba et al., 2006; nowak and szemplinski, 2014). shortage of rainfall and its resulting soil moisture stress during the growing season brought physiological disorders such as a reduction in transpiration and photosynthesis (sarker et al., 2005; carrubba et al., 2006). besides, reduced vegetative as well as reproductive growth of coriander and their consequent decrease in seed yield could be accounted for the above optimum temperatures. the optimum temperature might have been surpassed for the late sown coriander, and adversely influenced its physiological processes including photosynthesis and respiration (sharma et al., 2003). the influences of precipitation and air temperatures on yield and yield components of coriander w e r e r e p o r t e d i n t h e w o r k s o f n o w a k a n d szemplinski (2014). bhadkariya et al. (2007) found 30.6 and 76.4% declines in the fruit yield of coriander owing to delay in sowing from march 30 to april 14 and april 29, respectively. moosavi et al. (2012) reported that with delay in sowing, coriander fruit and biomass yields reduced by 76.4 and 74.7%, respectively. moniruzzaman et al. (2015), carrubba et al. (2006) and zheljazkov et al. (2008) also reported that productivity declined as sowing postponed to latest dates. though earlier sowing resulted in the tallest plant heights, it could not increase the yields of coriander. the inferior yields attributed to logging of the crop, shed of leaves and flowers during the cropping season in response to heavy rain fell particularly in august. this result is in agreement with pan et al. (2003), carrubba et. al. (2006), bhadkariya et al. (2007), ghobadi and gobadi (2010), and moosavi et al. (2012) who reported that earlier sowing of coriander led to the highest plant height. delay in sowing resulted in stunted growth and finally led to reduced yields of coriander. the significant decrease in plant height because of delay in sowing could be correlated with higher temperatures that the plants experienced at their latter stages. this incidence shortened their growing period and assimilate-building process owing to earlier maturity. thus, plants could not maintain adequate opportunity for conducting photosynthesis; hence, their height and branch-bearing capacity could be decreased (moosavi et al., 2012). sharangi and roychowdhury (2014) found that with delay in sowing from october to december, plant height of coriander decreased significantly. moosavi fig. 3 ten-day values of rainfall, maximum and minimum daily air temperature records in arsi robe from june to october in 2011 and 2012 cropping seasons. fig. 4 ten-day values of rainfall, maximum and minimum daily a i r t e m p e r a t u r e r e c o r d s i n k u l u m s a f r o m j u n e t o october in 2011 and 2014 cropping seasons. fig. 5 ten-day values of rainfall, maximum and minimum daily air temperature records in sagure from june to october in 2011 and 2014 cropping seasons. kassaye et al. response of coriander to sowing dates and seed rates 201 (2012) also reported significant decrease in plant height with delay in sowing from march 30 to april 29. the major factors that attributed to the variations in yields of coriander among locations and seasons were related to climate particularly rainfall and soil. in their studies, rashed and darwesh (2015) found that microclimate significantly affected sowing dates implied the need for determination optimum sitespecific sowing date. rainfall had contrasting effect at arsi robe and kulumsa in 2011; it was excess at arsi robe, but optimum at kulumsa. compared to 2011, the rainfall amount and distribution in 2012 at arsi robe and 2014 at kulumsa were optimum; hence, fruit and biomass yields obtained could be superior. these results corroborate the findings of carrubba et al. (2006), who reported a highly significant dependence of coriander yields on precipitation during its growing period with linear correlation coefficient of 0.93. nowak and szemplinski (2014) also reported the significant influence of temporal variations on coriander yield and yield attributes. the rainfall amount and its influence need to be interpreted in relation to the soil types of the study areas. compared to arsi robe and sagure, where the soils are heavy vertisols, the soil type of kulumsa is relatively light vertisol. hence, the occurrence of water logging at kulumsa particularly during the months with heavy rainfalls was lower as compared to arsi robe and sagure. coriander gave the highest fruit and biomass yields in areas, where rainfall was modest and the soil was light vertisol. during the cropping season, rainfall was lower at kulumsa than sagure and arsi robe, and sagure was lower than arsi robe. for example, cumulative rainfall from the first decade of june to the end of the third decade of august at arsi robe, kulumsa and sagure were 508, 282 and 302 mm, respectively indicating rainfall at kulumsa was robust for the development of coriander. this further implied that rainfall amount had strong effect on fruit and biomass yields of coriander at the studied sites. as rainfall amount increased, fruit and biomass yields of coriander decreased; h e n c e , f r u i t a n d b i o m a s s y i e l d o b t a i n e d f r o m kulumsa was superior followed by sagure. among the tested seed rates, the lowest, 30 kg ha-1, has been found optimum because under lower seed rate, plants tended to compensate the reduced sowing density by producing new branches and result in optimum fruit and biomass yields (diederichsen, 1996). okut and yidirum (2005) reported that seed rate had no effect on harvest index of coriander while ghobadi and ghobadi (2010) observed no significant effect on 1000 fruit weights of coriander. the superior yield and yield attributes in response to the seed rate of 30 kg ha-1 signified the need for investigation under further reduced rates. diederichsen (1996) reported that based on the weight of 1000 fruits, 3 to 20 kg ha-1 fruits of coriander could be sufficient for optimum yield. the results of the current study indicated that sowing date significantly influenced the yield and yield attributes of coriander. however, the effects of seed rate and its interaction with sowing date were not significant on most of the variables measured. the optimum sowing dates that gave the highest fruit and biomass yields of coriander fall on third decade of july at arsi robe, and from first-third decades of july at kulumsa and sagure. earliness and delay in sowing resulted in inferior yields. though coriander did not respond to seed rate in this study, the lowest rate, 30 kg ha-1, was found sufficient to give optimum yield. however, further low seed rate studies need to be conducted. considering the demand of low input, fruit and biomass yields obtained throughout experimental periods, cultivation of coriander was satisfactory provided the crop was sown at appropriate sowi n g t i m e . o w i n g t o i t s e c o n o m i c b e n e f i t s , s o i l improvement, nutritional qualities and reproductive roles, wider cultivation of coriander in the southeastern mid-highlands of ethiopia and other areas with similar agro ecologies is encouraged. acknowledgements t h e f u n d i n g a n d l o g i s t i c s p r o v i s i o n o f t h e ethiopian institute of agricultural research for the e x e c u t i o n o f t h i s r e s e a r c h a c t i v i t y i s s i n c e r e l y acknowledged. the authors greatly acknowledge all members of land and water resources research team of kulumsa agricultural research center for their field and laboratory assistances. we would also like to thank the anonymous reviewers, whose comments and suggestions significantly improved this manuscript. references ali h., ayub g., elahi e., shahab m., ahmed s., ahmed 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caryophyllus l.) flower quality a.n. niyokuri 1, 2 (*), s. nyalala 2, m. mwangi 2 1 department of crop sciences, college of agriculture, animal sciences and veterinary medicine (cavm), university of rwanda, p.o. box 210 musanze, rwanda. 2 department of crops, horticulture and soils, faculty of agriculture, egerton university, p.o. box 536-20115, egerton, kenya. key words: bioslurry, carnation, flower quality, plant biostimulant, residual effect. abstract: a greenhouse experiment was conducted in finlays, lemotit flower farm in kenya to determine the residual effect of bioslurry and an amino acids plant biostimulant on carnation flower quality. a second flush of carnation plants growing in previous experimental plots was used. this experiment was laid out in a split plot design with three replications. four levels of bioslurry: 0, 0.125, 0.25 and 0.5 l m-2 were applied in the main plots while four levels of plant biostimulant: 0, 2.0, 2.5 and 3.0 l ha-1 were used in the sub-plots. results showed that there was no significant residual effect of bioslurry on studied parameters. residual effects of plant biostimulant applied at 2.0, 2.5 and 3.0 l ha-1 resulted in a significant increase in carnation flower stalk length by 1.08 to 1.72 cm compared to control. however, there was negligible reduction of the flower stalk diameter (0.1 mm) and no significant residual effect of plant biostimulant on flower head size. moreover, there were no residual interactive effects of bioslurry and plant biostimulant on studied parameters. these results suggest that plant biostimulant can be used to improve the flower stem length in the subsequent flush of carnation plants supplied with a full dose of inorganic fertilizers. 1. introduction carnation (dianthus caryophyllus l.) is a popular cut-flower throughout the world (roychowdhury and tah, 2011). it is preferred to other cut flowers in several exporting countries as it lasts longer after being cut, has a wide range of attractive forms and colours, has the ability to withstand long distance transportation and significant ability to rehydrate after continuous shipping (salunkhe et al., 1990; kanwar and kumar, 2009; (*) corresponding author: nnari26@gmail.com citation: niyokuri a.n., nyalala s., mwangi m., 2018 residual effects of bioslurry and amino acids plant biostimulant on carnation (dianthus caryophyllus l.) flower quality. adv. hort. sci., 32(1): 137-142 copyright: © 2018 niyokuri a.n., nyalala s., mwangi m. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 7 august 2017 accepted for publication 23 october 2017 ahs advances in horticultural science short note adv. hort. sci., 2018 32(1): 137-142 138 renukaradya et al., 2011). although it is indigenous to the mediterranean region, carnation can be grown in almost every climate in glasshouses, plastic houses, and shade nets as well as in open field (aydinsakir et al., 2011). it was fourth among the top ten imported cut flower to t h e n e t h e r l a n d s a f t e r r o s e s , s t j o h n ’ s w o r t (hypericum spp.) and gypsophila with a turnover of €18 million (floraholland, 2017). it was also one of the main cut flowers exported by kenya in 2012 (cbi, 2013) and among the leading cut flowers locally used in flower arrangements and in value addition of flowers, in form of bouquets. in 2014, carnations contributed 4% of the domestic value of floriculture in kenya (hcd, 2015). the quality of carnations is currently affected by many problems such as calyx splitting and short stem length for some varieties. to meet the required quality parameters such as stem length and girth, flowers size and number, farmers resort to heavy application of inorganic fertilizers and synthetic plant growth regulators. although this results in increased production and quality, it adversely affects soil productivity and the environment. this is because pesticides, phosphorus and nitrate used in carnation production represent the major agricultural pollutants that threaten the environment (nardi et al., 2016). with the increasing relevance of social and environmental standards, current research is focusing on developing alternative systems in crop production through development of unconventional and nonpollutant solutions. currently, there are limited organic alternatives to meet plant nutrients. moreover, organic alternatives for plant growth regulators are lacking in many crops and limited in other crops such as carnations. use of compost derived from plants and/or animal wastes as soil amendment or fertilizer additive has been reported as an alternative in the production of several ornamental plants. moreover, bioslurry, the residual manure generated through anaerobic decomposition of various organic materials is conside r e d a q u a l i t y o r g a n i c f e r t i l i z e r ( i s l a m , 2 0 0 6 ) . similarly, the use of biostimulants in sustainable agriculture has been growing particularly for their capacity of enhancing nutrition efficiency and stress response (du jardin, 2012). biostimulants can be obtained from different organic materials and include complex organic materials, humic substances, beneficial chemical elements, peptides and amino acids (protein hydrolysates), seaweed extracts, inorganic salts, chitin and chitosan derivatives, antitranspirants, amino acids and other n-containing substances (du jardin, 2015; nardi et al., 2016). many studies (karki, 2001; islam, 2006; shahbaz, 2011; jeptoo et al., 2013; shahariar et al., 2013) reported yield increases and quality improvement on many crops such as okra (hibiscus esculentus l.), maize (zea mays), cabbage (brassica oleracea var. capitata) and carrot (daucus carota) following bioslurry application. studies by nahed et al. (2009 a, b), paradiković et al. (2011) and mondal et al. (2015) reported that plant biostimulants could be successfully used in the production of ornamental and other horticultural crops such as antirrhinum majus, gladiolus (gladiolus grandflorum l.) sweet yellow pepper (capsicum annuum l.) and eustoma grandiflorum. h o w e v e r , m a n y s t u d i e s o n l y e v a l u a t e d t h e bioslurry and biostimulant direct effects, whereas there are only a few studies which focused on their residual effects on the quality and yield of many crops including carnations. this study was therefore aiming at evaluating residual effects of bioslurry and plant biostimulant on flower quality in carnation plants which previously received applications of bioslurry and plant biostimulant. 2. materials and methods experiment location the study was conducted in a greenhouse at lemotit flower farm of finlays horticulture kenya ltd. situated in kenya at latitude 0o 22’ south and longitude 35o 18’ east, from march to september 2015. experimental design and treatments application carnation ‘walker’ plants planted on soil media at a density of 36 plants per m2 were used. these plants had received drench applications of bioslurry and plant biostimulant, four times at bi-weekly intervals after pinching (three weeks after transplanting) during the period september 2014 to february 2015. the experimental design was a split-plot design with three replications. the main plot measured 5.5 x 1 m (5.5 m2) while the sub-plot was 1 x 1 m (1 m2). buffer zone of 0.5 m and 1 m separated inter-plots and individual main blocks respectively. cow dung bioslurry was applied in main plots at the rate of 0.125, 0.25, 0.5 l m-2 and control diluted in one litre of water prior to application. rates of plant biostimulant used were 2.0, 2.5 and 3.0 l ha-1 and control thoroughly mixed with water at the rate of 5000 l ha-1 and they niyokuri et al. residual effects of bioslurry and amino-acids on carnation flower quality 139 were applied to the sub-plots during the period of september 2014 to february 2015. bioslurry used had at wet basis a ph of 7.44, 0.23% of n, 4.58 ppm of p, 89.3 ppm of k, 4.31 ppm of ca, 19.91 ppm of mg and density of 1.0195 kg l-1. the plant biostimulant used was hicure®, an amino acids based plant biostimulant. this plant biostimulant contains a balanced mixture of free amino acids (with higher proline and glycine contents) and peptides (hydrolysed protein) of natural origin. it is composed of amino acids and peptides (62.5%), total nitrogen (10.9%) and organic carbon (29.4%). after harvest in february 2015, carnation plants were allowed to grow for subsequent season in order to study the residual effects of bioslurry and plant biostimulant. maintenance practices all treatments benefited from a weekly application of mineral fertilizers through fertigation using: 3.06 g n, 3.51 g p205, 5.19 g k20, 1.71 g ca and 0.74 g mg, plus trace elements per square metre. routine crop management practices included irrigation, supporting, weeding, training, disbudding and pest management. harvesting was done at the paint brush stage when petals started to elongate outside the calyx. data collection and analysis data were collected from 10 tagged sample plants per sub-plot on three flower quality parameters namely the length of flower stalk, diameter of flower stalk and flower head size (diameter and head length). the length of flower stalk was measured in centimetres from the point just below the bud to the point of origin of branch on the main stem at harvest; diameter of flower stem was measured in millimetres using digital vernier callipers. the flower head length was recorded in millimetres from the point just below the calyx to the upper point of the flower while the flower head diameter was recorded in millimetres at harvesting from each harvested cut flower at paint brush stage using digital vernier calliper. all data were subjected to analysis of variance (anova) using genstat 14th edition. separation of means was performed using the tukey’s honest significant difference (hsd) test at p≤0.05. 3. results there was no significant residual effect of bioslurry on measured parameters (table 1). however, a significant residual effect of plant biostimulant on plants which had received any level of plant biostimulant was observed on flower stalk length and flower stalk diameter (table 2). carnations plants that had received 2.0, 2.5 and 3.0 l of plant biostimulant had significantly longer flower stalks and significantly thinner flower stalks compared to the control (table 2). the application of different rates of plant biostimulant did not show a significant residual effect on flower head size (diameter and head height) as presented in table 2. the interaction between different levels of bioslurry and those levels of plant biostimulant had no significant residual effects on measured parameters (table 3). 4. discussion and conclusions results of this study showed a significant increase of flower stalk length as a result of the residual effect of plant biostimulant. this increase of flower stalk length may be as a result of enhancement of macro nutrient uptake by plant biostimulant (calvo et al., 2014; rose et al., 2014) which rapidly improves the growth compared to treatments without plant biostimulant. the other probable reason would be the direct uptake of amino acids which are immediately used by carnation plants for their growth and develtable 1 residual effect of bioslurry on carnation flower quality bioslurry levels (l m-2) flower stalk length (cm) flower stalk diameter (mm) flower head diameter (mm) flower head length (mm) 0 81.01 5.52 21.77 40.58 0.125 81.18 5.56 22.04 40.44 0.25 81.02 5.55 21.86 40.56 0.5 81.15 5.55 21.81 40.48 table 2 residual effect of plant biostimulant on carnation flower quality levels of plant biostimulant (l ha-1) flower stalk length (cm) flower stalk diameter (mm) flower head diameter (mm) flower head length (mm) 0 80.12 b* 5.628 a 21.97 40.53 2 81.20 a 5.512 b 21.83 40.47 2.5 81.20 a 5.515 b 21.74 40.58 3 81.84 a 5.528 b 21.93 40.48 * means in the same column with the same letter are not significantly different at p≤0.05 using tukey’s hsd test. adv. hort. sci., 2018 32(1): 137-142 140 opment (calvo et al., 2014). there are strong evidences that the increase in flower stalk length may be attributed to residual auxins and gibberellins like activities of the plant biostimulant (brown and saa, 2015). in fact, both auxins and gibberellins are important in plant cell division and elongation (ertani et al., 2009; calvo et al., 2014 and colla et al., 2014). results of this study are in agreement with previous findings by ertani et al. (2009) and colla et al. (2014). colla et al. (2014) reported an increase in coleoptile elongation rate when compared to the control, in a dose-dependent fashion, comparable with the effects of indole-3-acetic acid following the treatment of maize with the protein hydrolysate. the same study provided additional evidences of a gibberellin-like activity of protein hydrolysate when application of plant-derived protein hydrolysate “trainer” at all doses significantly increased the shoot length of the gibberellins deficient dwarf pea plants by an average value of 33% in comparison with the control treatment. similar results previously reported by ertani et al. (2009) showed that the treatment of lettuce with both protein hydrolysate based fertilizers resulted in an increase in the epicotyl length comparable with the effects of exogenous gibberellic acid. the occurrence of this residual effect was perhaps the result of previous down-regulation mechanisms which affected the effect of plant biostimulant in the previous season. ammonium, which was used as a source of nitrogen, has been reported to down-regulate amino acids (henry and jefferies, 2003 cited by gioseffi et al., 2012; thornton and robinson, 2005 cited by gioseffi et al., 2012). the observed reduction in flower stalk diameter is suspected to be a gibberellin-like activity which promoted the flower stalk length (ertani et al., 2009; colla et al., 2014) at the expense of flower stalk diameter. the absence of residual effect of plant biostimulant on flower head size was possibly due to constant supply of nutrients through fertigation. the flower head size is usually a result of carbohydrates stored for subsequent growth and reproductive processes (islam et al., 2010). although there are evidences that biostimulants can enhance macro nutrient uptake (calvo et al., 2014; rose et al., 2014), it is possible that the residual effect of plant biostimulant was limited to stimulating the elongation of flower stalk. hence, the residual effect may have contributed much during early stages when carnation plants started re-growing after the harvest by improving growth and nutrients assimilation as previously revealed by colla et al. (2014). bioslurry did not show any significant residual effect on the flower stalk length, flower stalk diameter and the flower head size. generally, nutrients in cow dung slurry and poultry manure slurry are released in higher amounts compared to their original state (haque et al., 2015). this is because bioslurry, with its narrower c:n than farmyard manure, shows better results on soil nutrient availability at early stages of its application while, farm yard manure affects the nutrient uptake to the plant in more consistent manner because its mineralization occurs at later stages (muhmood et al., 2014). however, results of our study are not in agreement with shahzad et al. (2015) who reported that the application of bioslurry and composted poultry manure as a bio-fertilizer improves soil organic matter contents and availability of soil nutrients (n, p and k) to the subsequent crop, resulting in increased crop productivity and reducing the cost of fertilizer to subsequent crop. in fact, the residual effect of bioslurry may depend on its initial content, characteristics and the quantity applied and for these reasons, the residual effect of bioslurry on carnation grown in the following season was very limited. based on results of this study, we can conclude that the application of plant biostimulant on carnations plants supplied with a full dose of inorganic fertable 3 residual effects of the interaction of bioslurry and plant biostimulant on carnation flower quality bioslurry levels (l m-2) level of plant biostimulant (l ha-1) flower stalk length (cm) flower stalk diameter (mm) flower head diameter (mm) flower head length (mm) 0 0 79.94 5.57 21.81 40.61 2 81.07 5.48 21.74 40.57 2.5 80.83 5.51 21.71 40.45 3 82.21 5.51 21.8 40.70 0.125 0 79.75 5.65 22.13 40.55 2 81.30 5.53 21.81 40.46 2.5 81.68 5.51 21.79 40.67 3 81.97 5.56 22.44 40.08 0.25 0 80.28 5.67 22.06 40.49 2 81.14 5.52 21.95 40.50 2.5 80.85 5.50 21.76 40.60 3 81.82 5.51 21.69 40.64 0.5 l 0 80.52 5.61 21.88 40.47 2 81.29 5.52 21.84 40.35 2.5 81.44 5.55 21.7 40.58 3 81.35 5.53 21.81 40.41 niyokuri et al. residual effects of bioslurry and amino-acids on carnation flower quality 141 tilizers may have residual effect on the subsequent production flush. however, the residual effect of bioslurry on carnations grown in the same conditions may depend on its nutrient content and physicochemical characteristics. further works would be necessary to study the application of both products under lower rates of inorganic fertilizers and extend over many productions flushes to find out the extent of their residual effects. acknowledgements t h e a u t h o r s a r e t h a n k f u l t o j a m e s f i n l a y s company for providing the research plant materials and allowing us to conduct experiments in their lemotit farm. references aydinsakir k., tuzel i.h., buyuktas d., 2011 the effects of different irrigation levels on flowering and flower quality of carnation (dianthus caryophyllus l.) irrigated by drip irrigation. afr. j. biotechnol., 10(66): 14826-14835. brown p., saa s., 2015 biostimulants in agriculture. front. plant sci., 6: 671. calvo p., nelson l., kloepper j.w., 2014 agricultural uses of plant biostimulants. plant soil, 383: 3-41. cbi, 2013 cbi tradewatch cut flowers and foliage. survey report. cbi, ministry of foreign affairs, www.cbi.eu. colla g., rouphael y., canaguier r., svecova e., cardarelli m., 2014 biostimulant action of a plantderived protein hydrolysate produced through enzymatic hydrolysis. front. plant sci., 5: 448. du jardin p., 2012 the science of plant biostimulants. a bibliographical analysis. final report “ad hoc study report on bio-stimulants products”, pp. 1-37. du jardin p., 2015 plant biostimulants: definition, concept, main categories and regulation. sci. hort., 196: 3-14. ertani a., cavani l., pizzeghello d., brandellero e., a l t i s s i m o a . , c i a v a t t a c . , n a r d i s . , 2 0 0 9 biostimulant activity of two protein hydrolyzates in the growth and nitrogen metabolism of maize seedlings. j. plant nutr. soil sci., 172: 237-244. f l o r a h o l l a n d , 2 0 1 7 f a c t s a n d f i g u r e s 2 0 1 6 . https://www.royalfloraholland.com/en. gioseffi e., de neergaard a., schjoerring j.k., 2012 interactions between uptake of amino acids and inorganic nitrogen in wheat plants. biogeosciences, 9: 1509-1518. haque m.a, jahiruddin m., rahman m.m., saleque m.a., 2015 phosphorus mineralization of bioslurry and other manures in soil. j. environ. waste manag., 2(2): 79-83. hcd, 2015 horticulture. validated report 2014. horticulture crops directorate, pp. 1-68. islam m.r., rahman s.m.e., rahman m., oh d.h., ra c.s., 2010 the effects of biogas slurry on the production and quality of maize fodder. turk. j. agric. for., 34: 91-99. islam m.s., 2006 use of bioslurry as organic fertilizer in bangladesh agriculture. international workshop on the use of bioslurry, domestic biogas programmes, 2728 september, bangkok, thailand. jeptoo a., aguyoh j.n., saidi m., 2013 improving carrot yield and quality through the use of bio-slurry manure. sustainable agric. res., 2(1): 164-172. kanwar j.k., kumar s., 2009 influence of growth regulators and explants on shoot regeneration in carnation. hort. sci. 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effects of different tillage systems, bioslurry and poultry manure on soil properties and subsequent wheat productivity under humid subtropical conditions of pakistan. intl. j. biosci., 6 (11): 99-108. impaginato 169 adv. hort. sci., 2018 32(2): 169-175 doi: 10.13128/ahs-22018 application of calcium to decrease yellow sap contamination at different positions of garcinia mangostana l. y. tanari 1 (*), d. efendi 1, 2, r. poerwanto 1, 2, d. sopandie1, k. suketi 1 1 department of agronomy and horticulture, faculty of agriculture, bogor agricultural university, 16144 bogor, west java, indonesia. 2 center for tropical horticulture studies, kampus ipb baranangsiang, jln. raya pajajaran, 16144 bogor, west java, indonesia. key words: ca pectate, sector, shaded, transpiration, well-exposed. abstract: the present research aimed at studying the effects of ca application, through soil fertilization, on yellow sap contamination based on the position of the fruits on the canopy of the tree. the tree was divided into 6 sectors based on the differences in light exposure i.e. sector 1, 2, and 3 for shaded fruit positions and sector 4, 5, and 6 for well-exposed (to light) fruit positions. the present study used a randomized complete block design (rcbd), consisting of 2 treatments i.e. 0 kg ca/tree and 4.8 kg ca/tree. the results revealed that ca treatment lead to an increase in ca-pectate content in pericarp. in addition, the exposed fruit position allegedly increase the absorption of ca-pectate to the fruit. thus, it is important to both apply ca on the soil and ensure that the fruit, in the canopy, gets enough light to decrease the occurrence of yellow sap contamination. the well-exposed position of the fruit, in the 4.8 kg ca/tree treatment during anthesis, had increased the ca-pectate content of the pericarp which, in turn, resulted in a decrease in yellow sap contamination in segment, aryl, and rind of the mangosteen fruit. 1. introduction yellow sap is a sap which is naturally produced in each organ of the mangosteen, excepts the root. indeed, it constitutes the main constraint in the indonesian mangosteen agribusiness industry due to the fact that it causes the fruit flesh (aryl) to have a bitter taste and a less attractive look (osman and milan, 2006). statistical data (2015) revealed that only 14.8% of the total indonesian mangosteen production is exported as a consequence of high percentage of yellow sap contamination. yellow sap is found in the yellow sap duct which is surrounded by typical epithelium cells (dorly et al., 2008). it will contaminate the surface of the fruit (or aryl) if the epithelial cells of the secretory duct break as a result of cadeficiency. the break of the epithelial cells is connected with the extreme changes in groundwater during the developmental process of the fruit (pechkeo et al., 2007), and the differences in growth rate (*) corresponding author: yulindatanari@yahoo.co.id citation: tanari y., efendi d., poerwanto r.. sopandie d., suketi k., 2018 application of calcium to decrease yellow sap contamination at different positions of gancinia mangostana l. adv. hort. sci., 32(2):169-175 copyright: © 2018 tanari y., efendi d., poerwanto r., sopandie d., suketi k. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 10 october 2017 accepted for publication 12 january 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(2): 169-175 170 between the seed and the aryl with the fruit pericarp during the growing phase of the fruit (poerwanto et al., 2010). according to previous research, the break of yellow sap duct is related to the concentration of ca. indeed, ca content of the pericarp of mangosteen contaminated fruit (by yellow sap) is lower compared t o t h a t o f a n o r m a l f r u i t ( p o o v a r a d o m , 2 0 0 9 ; kurniadinata et al., 2016). according to marshner (2012), ca structurally functions to strengthen the cell wall, plant tissues, and the stability of the membrane. mortazavi et al. (2016) reported that ca can minimize cell membrane injury. the increased effect of ca application can be explained by its role in cell membrane structure. meanwhile, seligmann et al. (2009) stated that ca in plant plays an important role regarding the strength of the mechanical tissue and the determination of the fruit quality. however, ca is an immobile nutrient that could not be translocated from plant tissues. thus, developing leaves and fruits fully depend on the transmission of ca in the transpiration stream of the xylem. according to qiang and ling (2005), transpiration is the main factor that promotes ca movements and a low transpiration rate will result in a low addition of ca2+ to aerial organs. mangosteen is a small or medium height tree with a straight, symmetrically branched to form a conical and very tight canopy which leads to both a low light intensity and temperature in the shaded internal part compared to the well light-exposed parts (sector). the described architecture is believed to influence the microclimate of various parts of the plant canopy, causing differences in transpiration rate, which in turn, is believed to cause differences in ca absorption rate to the fruit. crisosto et al. (1995) report the greater the light interception by an individual fruit and its surrounding leaves the better its quality. fruit that developed in the more shaded inner canopy p o s i t i o n s h a v e a g r e a t e r i n c i d e n c e o f i n t e r n a l breakdown than fruit from the high light, outer canopy positions. erez and flore (1986) reported that f r u i t e x p o s e d t o l i g h t e x p e r i e n c e d a q u a l i t y improvement by pigmentation of peach that is also assumed to relate to the sink strength of the fruit. research, on the relationship between both ca and the fruit position in the canopy and the occurrence of yellow sap, become capital in determining the sector with a high contamination potential. the present research was aimed to determinate the effects of ca on yellow sap contamination based on the position of the fruit in the tree canopy 2. materials and methods time and place the present research was conducted in tandolala village, poso district, central sulawesi, from october 2015 to april 2016. tandolala village is situated at an altitude of 508 mdpl with a ph (soil) of 4.8, and a rainfall of 192.8 mm/month. the observations on yellow sap contamination were carried out in the laboratory of natural science at the university of sintuwu maroso poso. analyses of total ca and pericarp capectate contents were performed in the laboratory of soil chemistry and fertility, bogor agricultural university. materials ten productive plants, at an average 40 years old (with a planting distance ranging from of 4x4 m to 6x6 m), an average height of 16 meters and a canopy diameter of 4 m, were used in the present study. dolomite [camg(co3)2], containing 30% ca, was used as ca source. the canopy of the tree was divided into 6 sectors as follows: sector 1 inner bottom, sector 2 outer bottom, sector 3 inner middle, sector 4 outer middle, sector 5 inner top, and sector 6 outer top. the tree was divided into sectors based on the modifications of setiawan et al. (2012) techniques (fig. 1). parameters, such as light intensity, temperature, and humidity, were measured on weeks 4, 8, and 12, on each sector by placing the measuring tool (thermohydrometer) in each sector. afterwards, the light intensity was recorded as displayed on the measuring fig. 1 fruit positions in the canopy of the tree. adapted from setiawan et al., 2012. s= sector. tanari et al. calcium application against yellow sap contamination in mangosteen 171 tool. light intensity, temperature and humidity averages for each sector are presented in table 1. experimental design a randomized complete block design (rcbd), consisting of 2 treatments i.e. 0 kg ca/tree and 4.8 kg ca/tree (equivalent to 16 kg dolomite/tree), was used as experimental design in the present study. fertilization with ca was carried out during anthesis by sowing in the path that was already made around the mangosteen tree (below the crown of the plant) and recovering it with soil. based on the light intensity measurement results in table 1, the observations on yellow sap contamination were carried out on both shaded and wellexposed positions. a fruit was considered shaded if the perceived light intensity is low. meanwhile, it was categorized as well-exposed if the average received light intensity is high. shaded fruit positions were identified in sectors 1, 2 and 3, with an average light intensity of 481 lux, while well exposed fruit positions were identified in sectors 4, 5 and 6 with an average light intensity of 1229 lux. the total sample from each sector was 12 fruits (72 fruits per tree). thus, a total of 720 mangosteen fruits were used as samples. the transpiration rates, of the leaves, were measured at both shaded and well-exposed positions. the results of the above measurements will be used to estimate the transpiration rates of the fruits in various positions. the transpiration rate was measured by means of a lux meter. measurements harvesting was carried out 16 weeks post-anthesis (wpa). the observations on the percentage of yellow sap (pys) were carried out in order to determine the percentage of contaminated fruit in the group of fruits that was observed. a fruit was considered contaminated although the yellow sap, that pollutes both the aryl and the rind just one spot (small patch). the percentages of contaminated aryl (pca), contaminated rind (pcr), and contaminated segment (pcs) was calculated by means of the following equations: pca = (total yellow sap contaminated aryl /total fruit sample) x 100 pcr = (total yellow sap contaminated rind/total fruit sample) x 100 pcs = (total yellow sap contaminated segment/total fruit segment sample) x 100 yellow sap contamination score shows the severity level of a fruit contaminated by yellow sap and ranges from 1 (very well) to 5 (very bad). observations on aryl fruit and yellow sap contaminated rind scores referred to kurniadinata et al. (2016 ) method as presented in table 2 and 3. ca pectate analyses referred to an analysis method developed by setyaningrum et al. (2011) which uses dry fruit sample. the samples were ground into partitable 1 light intensity, temperature, humidity, and transpiration rate at different fruit positions (exposed and shaded) score description 1 very good, clean white aryl, no yellow sap between aryl and rind, and fruit vessels as well 2 good, 1-2 yellow sap stains (small patch) on one end of the aryl, but does not make the fruit bitter 3 good enough, the presence of some yellow sap stains (patch) on one end of the aryl or between the segments and the littering aryl 4 bad, presence of yellow sap stains/blobs at the end of the segments, between the segments or the fruit vessels, making the fruit bitter 5 very bad, the presence of large yellow sap stains/blobs at the end of the segments, between the segments or at the fruit vessels, making the fruit bitter with a clear colored aryl table 2 yellow sap contamination score on aryl score 1= very good/without contamination, up to score 5= very bad /high contamination score. table 3 yellow sap contamination score on rind score 1= very good/without contamination, up to score 5= very bad /high contamination score. score description 1 very good, flawless rinds with no visible yellow sap. 2 good, flawless rinds with 1-5 yellow sap stains (small patch) which dry without affecting the color of the fruit 3 good enough, flawless rinds with 6-10 yellow sap drops which dry and do not affect the color of the fruit 4 bad, flawed rinds due to medium/large yellow sap clumps, there are 1-2 yellowing streams 5 very bad, flawed rinds with more than one large yellow sap clumps with lots of yellowing streams on the rind of the fruit and a dull fruit color fruit positions light intensity (lux) temperature (oc) humidity (%) transpiration rate (hpa/s) sector 1 414.44 26.54 70.98 shaded 0.03 sector 2 536.00 26.80 70.46 sector 3 492.76 26.08 71.54 sector 4 1581.51 27.04 69.77 well-exposed 0.06 sector 5 944.60 26.74 72.04 sector 6 1163.11 27.26 70.97 adv. hort. sci., 2018 32(2): 169-175 172 cles, added with ion free water and shacked for 2 hours. afterward, the solution was centrifuged for 15 minutes at a speed of 3000 rpm. the supernatant was then filtered to collect the pellet to which a 1 mol l-1 of nacl was added, shacked for 2 hours and centrifuged for 15 minutes. the extraction result was analyzed atomic absorption spectrophotometer (aas) in order to obtain data on ca pectate. statistical analysis pca, pcr, pcs, total ca contents, and ca-pectate were analyzed using sas 9.1.3 program, which was followed by duncan’s post-hoc comparison (at a significance level of 5%) test. meanwhile, data on fruit score, yellow sap contaminated aryl were analyzed by mean of a kruskal wallis test, which was followed by dunn test. 3. results the application of ca had an effect on the decrease in yellow sap contamination in mangosteen (table 4). the lowest percentage of yellow sap contaminated segment was observed in the 4.8 kg ca/tree (exposed fruit position), which significantly differed to those of other treatments. the percentage of yellow sap contaminated segment showed significant decline of 81% with application of 4.8 ca/tree on exposed position compared to treatment with 0 kg ca/tree on shaded position, 71% compared to treatment with 0 kg ca/tree on exposed position and 73% compared to 4.8 kg ca/tree on shaded position. the percentage of yellow sap contaminated aryl, in the 4.8 kg ca/tree treatment (exposed fruit position), showed an average percentage of 14.2%, which was not different to that of the 0 kg ca/tree treatment (23.9%). no differences were observed among treatments application of 4.8 kg ca/tree in terms of yellow sap contaminated rind percentage. the percentage of yellow sap contaminated rind was considerably high with treatment of 0 kg ca/tree, which was 79.9% on shaded position and 73.9% on exposed position. while the application of 4.8 kg ca/tree showed that yellow sap contamination was still considerably high at 61.3% on shaded position and 53.5% on shaded position. although the numbers were still high, there was a decline of 33% with application of 4.8 ca/tree compared to without ca application on shaded position. according to martias et al. (2012), percentage of yellow sap contaminated that was higher than 50% was considered very high. table 5 presents the yellow sap contamination scores of both aryl and rind. the best yellow sap contaminated aryl score was observed at the wellexposed fruit position with the application of 4.8 kg ca/tree, and significantly differed to other treatments. meanwhile, the yellow sap contaminated rind score in the 4.8 kg ca/tree treatment revealed similar results for both shaded and well-exposed fruit positions (table 5). accumulation of ca in fruit pericarp was a representation of adsorbed soil ca by plant. total ca content of the fruit pericarp did not significantly differ among treatments, but was significantly different to that of the ca-pectate contents. the ca-pectate contable 4 percentage yellow sap contamination in fruit segment, aryl and on rind (16 wpa) numbers followed by different letters within the same column showed significant differences in dmrt test (α= 5%). shaded (sector 1, 2 and 3) with a light intensity of 481 lux, exposed (sector 4, 5 and 6) with a light intensity of 1229 lux. table 5 score of yellow sap contamination in aryl and on rind (16 wpa) data were analyzed based on kruskal wallis test. numbers followed by different letters within the same column showed significant differences based on dunn test (1%). score 1: very good; score 2= good; score 3= good enough; score 4= bad; score 5= very bad. treatments fruits contaminated by yellow sap (%) pericarp ca content (ppm) segment aryl rind total pectate 0 kg ca/tree shaded 21.3 a 45.7 a 79.9 a 1088.9 552.02 b exposed 13.6 a 23.9 b 73.9 ab 2100.0 528.87 b 4.8 kg ca/tree shaded 15.0 a 34.9 a 61.3 bc 1500.0 576.40 b exposed 4.0 b 14.2 b 53.5 c 1566.7 754.60 a f-test * * ns * treatments yellow sap contaminated aryl and rind score in 1-5 aryl rind 0 kg ca/tree shaded 1.99 a 2.38 a exposed 1.67 c 2.17 b 4.8 kg ca/tree shaded 1.77 b 1.90 c exposed 1.23 d 1.74 c dunn-test * * tanari et al. calcium application against yellow sap contamination in mangosteen 173 tent was higher in the ca treated fruit (well-exposed position) (table 6). the percentage of yellow sap contaminated rind and aryl was reduced because of increased ca-pectate in fruit pericarp, proving that ca-pectate had a role in strengthening cell wall epithelium which compile the yellow sap duct making the cell stronger and kept yellow sap from leaking and contaminate the aryl and rind. leaves transpiration rate measurement, on week 4, 8, and 12, resulted in average transpiration rates of 0.03 hpa/s (shaded position) and 0.06 hpa/s (well exposed position) (table 1). this data supported the measurement result of light and temperature on table 1, that the higher the temperature and light intensity, the higher the transpiration rate. 4. discussion and conclusions ca is an immobile nutrient, for its absorption rate follows the transpiration pathway in the xylem. thus, a deficiency in ca often occurs in fruits that do not transpire as much as the leaves. in the present study, the transpiration rate of the fruits was estimated by observing parameters such as light intensity, temperature and humidity in each sector, and also the transpiration rates of leaves at both the exposed and shaded positions. data shown in table 4 showed that the percentage of yellow sap contamination on the rind was higher than aryl and segment. yellow sap contamination was caused by the same factor which was the lack of ca on epithelium cell of yellow duct sap, but triggered by different things. according to dorly et al. (2008), yellow sap contamination on the aryl was caused by turgor and mechanical pressure, that was the pressure of aryl and seed growth outward during fruit enlargement. while contamination on the rind was caused by turgor pressure of pericarp cell, or by insect, fungal, or bacterial attack. fruits that were exposed to a high light intensity (table 1) and treated with 4.8 kg ca/tree had highest ca-pectate content (table 6) which was a conseq u e n c e o f t h e h i g h l i g h t i n t e n s i t y r e c e i v e d . marschner (2012) stated that part of the pectin, in the leaves, is in the form of ca-pectate in a high light intensity condition. ca strengthens the main plant cell wall in a crosslinking with the pectin. formation of calcium pectate due to the binding of calcium with pectins has been found beneficial to increase the strength of cell wall and middle lamella (carpita and mccann, 2000). the high ca-pectate content is believed to be a result of light intensity not only on the leaves but also on the fruit that possibly increases photosynthesis rate and transpiration rate, as demonstrated by the results of the present research. such fruits (well-exposed, treated with ca, and showing highest ca-pectate), showed also lowest yellow sap contamination levels. a high ratio of ca-pectate over total ca content (48% of the total ca) (table 6) could decrease yellow sap contamination through the strengthening of the epithelial cell walls in the yellow sap duct. a research, conducted by setyaningrum et al. (2011), revealed that the capectate content represented 20% of the total ca, which tend to reduce yellow sap contamination in mangosteen. the improvement of fruits quality (low yellow sap contamination) is related to high light intensity of fruits exposition. physiologically, light has a direct influence by photosynthesis and indirect influence throught plant’s growth and development as the results of direct metabolic responses (fitter and hay, 1991). the improvement of fruits quality is caused by t h e d i s t r i b u t i o n o f p h o t o s y n t h a t e t o t h e f r u i t exposed to light. the high temperature and light intensity in the exposed tree canopy resulted in higher transpiration rate at exposed positions compared to the shaded ones (table 1). similar results were observed by caleb et al. (2013) who demonstrated that temperature and humidity have significant effects on the transpiration rate. the high leaves transpiration rate at the exposed position is believed to be a consequence of higher ca translocation from the root to parts of the leaf (including part of the exposed fruit), for the ca is translocated along with water during the transpiration process. the high transpiration rate led to a high translocation of ca from the root to the fruit, since ca is translocated along with water during numbers followed by different letters within the same column showed significant differences in dmrt test (α= 5%). shaded (sector 1, 2 and 3) with a light intensity of 481 lux, exposed (sector 4, 5 and 6) with a light intensity of 1229 lux. table 6 calcium total and ca-pectate content in pericarp and percentage ratio of ca-pectate/total ca at 16 wpa treatment ca content in pericarp (%) percentage pectate/totaltotal pectate 0 kg ca/tree shaded 0.11 0.06 b 50.5 exposed 0.21 0.05 b 23.8 4.8 kg ca/tree shaded 0.15 0.06 b 40.0 exposed 0.17 0.08 a 47.1 f-test ns * ns adv. hort. sci., 2018 32(2): 169-175 174 the transpiration process. hansen (1980) stated that ca is transported in the fruit by means of water distribution through the xylem. gilliham et al. (2011) demonstrated that the mechanism of ca uptake is through the apoplast of the root along with the mass flow which follows the apoplast or the symplast pathway to the xylem. according to qiang and ling (2005) and white and broadley (2003), the transport through the apoplastic pathway mainly depends on the transpiration, while the symplast pathway is selective in controlling ca2+ to the xylem which depends on ca2+ requirement at the canopy. if there is a deficiency in ca supply of the plant, the plant may experience damages at the cell level. thus, the supplementation of ca is required on soil with low ca content. according to martias et al. (2012), yellow sap contamination could be directly prevented by the ca availability in the soil. amor and leo (2006) stated that the ca concentration of the plant significantly decrease due to a low supply. in addition, hocking et al. (2016) demonstrated that low supply and transport of ca would result in a ca deficiency, leading to damages of both membrane and cell walls of the fruit. the results revealed that ca treatment lead to an increase in ca-pectate content in pericarp. in addition, the exposed fruit position allegedly increase the absorption of ca-pectate to the fruit. thus, it is important to both apply ca on the soil and ensure that the fruit, in the canopy, gets enough light to decrease the occurrence of yellow sap contamination. the well-exposed position of the fruit, in the 4.8 kg ca/tree treatment during anthesis, had increased the ca-pectate content of the pericarp which, in turn, resulted in a decrease in yellow sap contamination in segment, aryl, and rind of the mangosteen fruit. acknowledgements the authors would like to thank the ministry of research, technology and higher education for funding and supporting the present research through doctoral dissertation program 2017. references amor f.m., leo f.m., 2006 differential effect of transpiration and ca supply on growth and caconcentration of tomato plants. sci. hort., 111: 17-23. caleb o.j., mahajan p.v., opara u.l., 2013 impact of temperature and relative humidity on the transpiration rate of pomegranate arils. journal of food, 11(3): 199-207. carpita n., mccann m., 2000 the cell wall, pp. 52-108. in: buchanan b.b., g. wilhelm, and r.l. jones, (eds.) biochemistry and molecular biology of plants. john wiley & son, chichester, uk, pp. 1280. central bureau of statistics, 2015 production of fruits in indonesia 2015. jakarta indonesia (in indonesian). crisosto c.h., mitchell f.g., johnson s., 1995 f a c t o r s i n f r e s h m a r k e t s t o n e f r u i t q u a l i t y . postharvest news and information, 6(2): 17-21. dorly s., tjitrosemito s., poerwanto r., juliarni, 2008 secretory duct structure and phytochemistry coumpounds of yellow latex in mangosteen fruit. hayati j. of biosciences, 15: 99-104. erez a., flore j.a., 1986 the quantitative effect of solar radiation on ‘redhaven’ peach fruit skin color. hortscience, 21: 1424-1426. fitter ah., hay r.k.m., 1991 environmental physiology of plants. gadjah mada university press, yogyakarta, pp. 421. (in indonesian). gilliham m., dayod m., hocking b.j, xu b., conn s.j., kaiser b.n., leigh r.a. tyerman s.d., 2011 calcium delivery and storage in plant leaves: exploring the link with water flow. j. exp. bot., 62(7): 2233-2250. hansen p., 1980 crop load and nutrient translocation, pp. 201-212. in: atkinson d., j.e. jackson, and r.o. sharples (eds.) mineral nutrition of fruit trees. butterworths, london, uk, pp. 452. hocking b., tyerman s.d., burton r.a. gilliham m., 2016 fruit calcium: transport and physiology. plant sci., 7(569): 1-17. kurniadinata o.f., depari s.o.s., poerwanto r., efendi d., wachjar a., 2016 solving yellow sap contamination problem in mangosteen (garcinia mangostana) with ca2+ application based on fruit growth stage. communications in biometry and crop science, 11(2): 105-113. marschner h., 2012 mineral nutrition of higher plants. academic press, usa, pp. 672. martias, poerwanto r., anwar s., hidayat r., 2012 relation between soil nutrient availability with yellow sap occurrence in mangosteen fruit. j. hort., 2 (22): 111-118. 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( i n indonesian). poovaradom s., 2009 growth and nutrient uptake into mangosteen (garcinia mangostana l.) fruit. the proceeding of international plant nutrition colloquium xvi, pp. 1-6. qiang y.h., ling y.l., 2005 uptake and transport of calcium in plants. j. of plant physiology and molecular biology. 31 (3): 227-234. seligmann r, wengrowicz u., tirosh d., yermiyahu u., tal a.b., schwartz a.b., 2009 calcium translocation and whole plant transpiration: spatial and temporal measurements using radio-strontium as tracer. proceeding of international plant nutrition colloquium xvi, pp. 1-8. setiawan e., poerwanto r., fukuda f., kubota n., 2012 meteorological conditions of mangosteen orchard in west java, indonesia and seasonal changes in c-n ratio of their leaves as affected by sector (position in canopy) and tree age. scientific report of the faculty of agriculture okayama university, japan. 10: 39-47. setyaningrum y.i., dorly, hamim, 2011 morphophysiology response and yellow latex reduce on external calcium application on mangosteen fruit ( g a r c i n i a m a n g o s t a n a l . ) . – p r o c e e d i n g o f t h e national seminar of indonesian horticulture, pp 89237. (in indonesian). white p.j., broadley m.r., 2003 calcium in plants. ann bot., 92 (4): 487-511. impaginato 311 adv. hort. sci., 2017 31(4): 311-317 doi: 10.13128/ahs-21031 investigation on rooting ability of twenty olive cultivars from southern italy c. cirillo 1, r. russo 1, f. famiani 2, c. di vaio 1 (*) 1 università degli studi di napoli federico ii, dipartimento di agraria, via università, 100, 80055 portici (na), italy. 2 università degli studi di perugia, dipartimento di scienze agrarie alimentari ed ambientali, 06121 perugia, italy. key words: cuttings, germplasm, naa, nad, olea europaea l., propagation. abstract: the effects of two different types of auxins (660 ppm alpha-naphthaleneacetic acid naa in liquid solution or 750 ppm alpha-naphthaleneacetamide nad dispersed in a talcum powder) and cuttings from three different portions of the shoots (basal, middle and apical) on the rooting ability of twenty autochthonous olive cultivars were investigated in two growing seasons (spring and autumn). the results showed that the autochthonous olive cultivars of the campania region are characterized by a wide variability in the potential rhizogenic ability. the two periods of cutting collection (march and september) significantly affected the rooting aptitude of the cultivars, indicating that in some cultivars the cuttings collected in autumn had a higher rooting rate than those collected in spring. the effects of naa and nad on rooting strongly depended on interaction with the cultivar, time of collection (autumn or spring) and type of cuttings (basal, medium or apical). among the twenty cultivars tested, we found only eight cultivars with a satisfactory rooting ability after hormonal applications (ortolana, racioppella, tenacella, tonda, biancolilla, carpellese, cornia and pisciottana). in general, the apical and the median portions of the shoots gave the best rooting results. 1. introduction among the vegetative propagation methods of olive cultivars (olea europaea l.) the use of semi-hardwood cuttings is the most common, since rootings are easy to prepare and the requirement in special equipment is negligible and cheap (cimato, 1999; ismaili et al., 2011). this is why in the mediterranean basin olive is mainly propagated by cuttings, a propagation method that relies on the ability of the cuttings to form adventitious roots (fabbri et al., 2004). while some cultivars are easily propagated by this technique, others are difficult-to-root and this poses a challenge for their preservation and commercialization (hartmann and kester, 1975; hartmann et al., 1990; carfi et al., 1994; porfírio et al., 2016). rooting aptitude of the different olive cuttings depends on both intrinsic and extrinsic factors (wiesman and lavee, 1994; hechmi et al., (*) corresponding author: divaio@unina.it citation: cirillo c., russo r., famiani f., di vaio c., 2017 investigation on rooting ability of twenty olive cultivars from southern italy. adv. hort. sci., 31(4): 311-317 copyright: © 2017 cirillo c., russo r., famiani f., di vaio c. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 30 july 2017 accepted for publication 26 october 2017 ahs advances in horticultural science adv. hort. sci., 2017 31(4): 311-317 312 2013), such as the genotype (avidan and lavee, 1978; fabbri et al., 2004; chiancone et al., 2011), the age of the mother tree, the timing of cutting collection as well as the type of cuttings (fontanazza and jacoboni, 1975; del rio et al., 1991; khabou and trigui, 1999). taking this background into consideration, the main aim of this study was to assess the rooting ability of olive cuttings obtained from twenty autochthonous olive cultivars of the campania region, by using different combinations of auxin treatments (naa and nad), times of collection of cuttings (spring and autumn) and portions of shoots to prepare the cuttings (basal, middle or apical). the cultivars considered in this study have been previously evaluated in terms of vegetative-productive characteristics and oil quality, and some of them present agronomical behaviors and quality of the oils which make them interesting for the use in new olive orchards, also for the production of oils with a strong yet greatly diverse typicality (di vaio et al., 2013). this makes very important to know their intrinsic ability to root and the best combination of factors (hormones, time of cutting collection and portion of shoots to use) to utilize in order to obtain the best rooting results from each cultivar. 2. materials and methods experimental site and plant material the trial was carried out in 2014/2015 at the experimental station “improsta” of the campania region, located in battipaglia (40°37′ 00’’ n lat, 15°03′ 23’’ e long, 72 m above sea level) sele plain (salerno, south italy). the average annual rainfall and the average minimum and maximum temperature of the area were 988 mm, 10.9°c and 21.0°c, respectively. the experimental station hosts a germplasm collection orchard, established in 2001, which includes all the main autochthonous olive cultivars of the region. trees were trained at central leader and spaced 6 × 3 m. among the available germplasm, 20 cultivars were selected for their economic importance and potential to be used in new orchards considering their good agronomical characteristics (productivity and resist a n c e t o b i o t i c a n d a b i o t i c s t r e s s e s ) a n d h i g h quality/typicality of the oils, since some of them also allow the production of certified oils under the protected designation of origin. the cultivars were grouped according to their origin (main province of cultivation). therefore, the selected olive cultivars were four from the province of avellino (‘ogliarola campana’, ‘ravece’, ‘ritonnella’ and ‘ruveia’), five from the province of benevento (‘femminella’, ‘ortice’, ‘ortolana’, ‘pampagliosa’ and ‘racioppella’), f o u r f r o m t h e p r o v i n c e o f c a s e r t a ( ‘ a s p r i n i a ’ , ‘caiazzana’, ‘tonda’ and ‘tenacella’) and seven from the province of salerno (‘biancolilla’, ‘carpellese’, ‘cornia’, ‘oliva bianca’, ‘pisciottana’, ‘rotondella’ and ‘salella’) (di vaio et al., 2013). cutting collection and preparation the cuttings were collectioned in the autumn (end of september) and in the next spring (second decade of march). for each cultivar, three trees were selected by following homogeneity criteria for developmental stage and productivity. the semi-hardwood cuttings were obtained in autumn (a) and spring (s) from 30 cm long one-year shoots/season. each shoot was divided in three different portions (basal, middle and apical) (180 cuttings/cultivar/season). cuttings were 10 cm long and presented 4 nodes and 2 pairs of terminal leaves. rhizogenic treatments for each combination of cultivar, collection time, portion of shoot and hormonal treatment three groups of ten cuttings each were used. the trial was conducted under mist system in cold greenhouse conditions. two different commercial formulations of auxins, alpha-naphthaleneacetic acid (naa) in liquid formulation (hydroalcoholic solution at 30% alcohol) at the concentration of 660 ppm (t1) and alphanaphthaleneacetamide (nad) at the concentration of 7 5 0 p p m ( t 2 ) i n t a l c u m p o w d e r f o r m u l a t i o n (0.075/100 w.w.) were compared to untreated control (t0). these concentrations were used considering the good results obtained with naa 500-1000 ppm in other cultivars (denaxa et al., 2011). the t1 treatment was performed by dipping 2 cm basal part of cuttings in the hydroalcoholic solution for 5 seconds, whereas the t2 treatment by dipping 2 cm basal part of cuttings in distilled water first and then in the powder. the t0 (control) was obtained by dipping 2 cm basal part of cuttings in distilled water for 5 seconds. cuttings were then placed in perlite filled rooting benches provided with basal heating (substrate temperature 22-24°c) and with mist system to get periodically wet the cuttings avoiding their dehydration (air humidity about 90-95%). rooting sampling and score semi-hardwood cuttings were evaluated 70 days after the rooting treatments and each cutting was cirillo et al. rooting ability of twenty olive cultivars from southern italy 313 scored for the rooting rate, the number of roots/cutting (primary and secondary roots), and the length of roots/cutting (the length of the different roots of each cutting was summed). the percentage of rooting was calculated as the number of rooted cuttings with respect to the total number of cuttings per treatment. statistical analysis all data were statistically analyzed by three-way analysis of variance (anova) using the spss 13 software package (spss 13.0 for windows; spss inc., chicago, il). whenever the two-way interaction was significant, a one-way anova was performed. to separate treatment means for each measured parameter, duncan’s multiple range test was performed at a significance level of p≤0.05. 3. results and discussion adventitious rooting process in cuttings is still to be unraveled under the genetic point of view, however, as referred in studies on italian and international olive cultivars (fontanazza and baldoni, 1989; chiancone et al., 2011), the capability of cuttings of forming adventitious roots is mainly affected by the cultivar. among the wide number of local and worldwide grown cultivars, the main part of them displays a poor aptitude for rooting of cuttings, whereas few cultivars are known to be well rooting cultivars. a dataset of the international olive council (ioc, 2005) reported the results of a screening on rooting rate of 426 cultivars: 59 cultivars showed an average rooting rate of 1.5%, 213 cultivars reached an average rate of 21.3%, whereas the rooting rate recorded in 86 cultivars was approximately 54% and only 68 (=16% of the total) showed a rate of rooting higher than 70%. in the present study, rooting rate of the cuttings was significantly affected by the cultivar (c), by the interaction between the cultivar and the time of sampling (c × ts) and by the interaction between the hormonal treatment and the time of sampling (t × ts) (table 1). the highest percentage of rooted cuttings, over the rooting treatment and the time of sampling, was recorded in cv. ortolana (66%), followed by cv. racioppella (54.6%) and cv. biancolilla (51%) (fig. 1). on the contrary, cv. ogliarola campana, cv. ortice and cv. salella showed the lowest rooting rate (14.9%, 15.8% and 11.2%, respectively). timing of cutting collection, according to the literature, is the second pivotal element, following the cultivar, to be concerned about, since very relevant differences in the success of rooting process may depend on this factor (hartmann and loreti, 1965). thus, the definition of the most suitable season for cutting collection from mother plants has been a critical item for researchers dealing with fruit trees species, although the fine tuning of protocols ready to use in the nurseries is still lacking, especially if single cultivars are considered. the c × ts interaction highlighted that the cultivars considered in this study responded differentially to the time of cutting sampling (fig. 2). for instance, among the three cultivars that showed the highest rooting rate, cv. ortolana and cv. raciopella reached higher percentages of rooting when the cuttings were collected during spring season, whereas the rooting rate of cv. biancolilla was significantly increased by using the cuttings collected in autumn (fig. 2). on the contrary, the cultivars that showed the lowest rooting rate (cvs. ogliarola campana, ortice and salella) did not ns= non significant; *, **; ***= significant at p≤ 0.05, ≤ 0.01, 0.001, respectively. fig. 1 average rooting rate (mean ± standard error) of semihardwood cuttings of the olive cultivars of the campania region. bars with the same colour correspond to cultivars of the same province. abbreviations of the names of the cultivars: ogl = ogliarola campana, rav = ravece, rit = ritonnella, ruv = ruveia, fem = femminella, orti = ortice, orto = ortolana, pam = pampagliosa, rac = racioppella, asp = asprinia, cai = caiazzana, ten = t e n a c e l l a , t o n = t o n d a , b i a = b i a n c o l i l l a , c a r = carpellese, cor = cornia, ob = oliva bianca, pis = pisciottana, rot = rotondella, sal = salella. means followed by different letters are significantly different for p ≤ 0.001. source of variance rooting rate (%) cultivar (c) *** hormonal treatment (t) ns time of sampling (ts) ns c × t ns c × ts *** t × ts * c × t × ts ns table 1 analysis of variance for cultivars, hormonal rooting treatments, time of sampling and their interactions on rooting rate of olive cuttings adv. hort. sci., 2017 31(4): 311-317 314 perform differently comparing the two times of sampling (fig. 2). the overall influence of hormonal treatments (naa or nad) and time of sampling (t × ts) on cutting rooting rate indicated that the effect of treatments was significantly different on the cuttings collected in spring (fig. 3). in particular, the lowest rooting rate was observed on t0 cuttings collected in spring (fig. 3). o n t h e b a s e o f c u t t i n g r o o t i n g r a t e , t h e international olive council classified the cultivars in four ranks: 0-5%; 5-40%; 40-70% and 70-100% (ioc, 2005). according to this classification, the twenty cultivars tested in the present experiment resulted to be distributed in three different groups: twelve between 5 and 40% (‘ogliarola campana’, ‘ravece’, ‘ritonnella’, ‘ruveia’, ‘femminella’, ‘ortice’, ‘pampagliosa’, ‘asprinia’, ‘caiazzana’, ‘oliva bianca’, ‘rotondella’, ‘salella’); seven between 40 and 70% (‘racioppella’, ‘tenacella’, ‘tonda’, ‘biancolilla’, ‘carpellese’, ‘cornia’ a n d ‘ p i s c i o t t a n a ’ ) ; o n e b e t w e e n 7 0 a n d 1 0 0 % (‘ortolana’). as far as root number and length are concerned, the effects of cultivar, hormonal treatments and portion of the shoot and their first level interactions were all significant (table 2). the twenty cultivars of olive exhibited a high variability in the number and length of roots produced both in autumn and spring experiments. the root number and length were increased by the rhizogenic treatments. generally, both the liquid and powdery formulations used for the rooting stimulation of the cuttings were effective in improving rooting emission and growth. with regard to the number of roots, our findings indicated that the liquid treatment with 660 ppm naa was the most effective, showing, with respect to the control, an average increase of about 266% in cuttings from the basal, median and apical portions of the shoots. the same treatment was particularly effective in stimulating the length growth of the roots, with an average increase of 236% compared to the control. furthermore, the root number and length of cuttings collected in autumn and spring were significantly affected by the interaction between the cultivar (c) and hormonal rooting treatment (t) (table 2). for instance, the average root number in autumn cuttings was 2.7 roots per cutting, and the application of t h e r h i z o g e n i c t r e a t m e n t t 2 , c o m p a r e d t o t 0 , induced a significant increase of the number of roots in nine cultivars: ortolana (+245%), cornia (+169%), tonda (+160%), ruveia (+165%), tenacella (+192%), asprinia (+312%), biancolilla (+320%), pisciottana fig. 3 average rooting rate (mean ± standard error) of semih a r d w o o d c u t t i n g s a s a f f e c t e d b y t h e i n t e r a c t i o n between the hormonal rhizogenic treatments (t0= control no hormonal treatments; t1= naa treatment; t2= nad treatment) and the time of sampling. a= autumn; s= spring. means followed by different letters are significantly different for p ≤ 0.05. fig. 2 average rooting rate (mean ± standard error) of semih a r d w o o d c u t t i n g s a s a f f e c t e d b y t h e i n t e r a c t i o n between the cultivar and the time of sampling. a= autumn; s= spring. abbreviations of the names of the cultivars: ogl = ogliarola campana, rav = ravece, rit = ritonnella, ruv = ruveia, fem = femminella, orti = ortice, orto = ortolana, pam = pampagliosa, rac = racioppella, asp = asprinia, cai = caiazzana, ten = t e n a c e l l a , t o n = t o n d a , b i a = b i a n c o l i l l a , c a r = carpellese, cor = cornia, ob = oliva bianca, pis = pisciottana, rot = rotondella, sal = salella. means followed by different letters are significantly different for p≤0.001. source of variance root number root length (cm/cutting) autumn spring autumn spring cultivar (c) *** *** *** *** hormonal treatment (t) *** *** *** *** portion (p) *** ** *** *** c × t ** *** *** ** c × p * *** * *** t × p * ns ns * c × t × p ns *** ns * ns= non significant; *, **; ***= significant at p≤ 0.05, ≤ 0.01, 0.001, respectively. table 2 analysis of variance for cultivars, hormonal rooting treatments, shoot portion and their interactions on number and length of roots of olive cuttings obtained in autumn or spring cirillo et al. rooting ability of twenty olive cultivars from southern italy 315 (+610 %) and carpellese (+236 %) (fig. 4a). the average root length in autumn cuttings was 4.2 cm per cutting, and, similarly to the root number, it was also increased by the rhizogenic treatment t2 in ten cultivars: ortolana, ravece, cornia, tonda, r u v e i a , t e n a c e l l a , a s p r i n i a , b i a n c o l i l l a a n d pisciottana (fig. 4 b). a similar pattern was observed in the spring cuttings (fig. 5 a, b), even though the fig. 4 effects of the interaction of the cultivars and the hormonal rhizogenic treatment (t0= control no hormonal treatments; t1= naa treatment; t2= nad treatment) on root number/cutting (a) and root length/cutting (b) of semi-hardwood cuttings collected in autumn. the dotted line shows the average value of all c u l t i v a r s u n d e r o b s e r v a t i o n . abbreviations of the names of the cultivars: ogl = ogliarola campana, rav = ravece, rit = ritonnella, ruv = ruveia, fem = f e m m i n e l l a , o r t i = o r t i c e , o r t o = o r t o l a n a , p a m = pampagliosa, rac = racioppella, asp = asprinia, cai = caiazzana, ten = tenacella, ton = tonda, b i a = b i a n c o l i l l a , c a r = carpellese, cor = cornia, ob = oliva bianca, pis = pisciottana, rot = rotondella, sal = salella. fig. 5 effects of the interaction of the cultivars and the hormonal rhizogenic treatment (t0= control no hormonal treatments; t1= naa treatment; t2= nad treatment) on root number/cutting (a) and root length/cutting (b) of semi-hardwood cuttings collected in spring. the dotted line shows the average value of all cultivars under observation. abbreviations of the names of the cultivars: ogl = ogliarola campana, rav = ravece, rit = ritonnella, ruv = ruveia, fem = femminella, orti = ortice, o r t o = o r t o l a n a , p a m = pampagliosa, rac = raciopp e l l a , a s p = a s p r i n i a , c a i = caiazzana, ten = tenacella, ton = tonda, bia = biancolilla, car = carpellese, cor = cornia, o b = o l i v a b i a n c a , p i s = pisciottana, rot = rotondella, sal = salella. adv. hort. sci., 2017 31(4): 311-317 316 values of both the average number and length of roots were higher than those recorded in autumn. the root number of autumn cuttings was also affected by the interaction between the hormonal treatment (t) and the shoot portion (p). indeed, the number of roots per cutting increased from the bottom (b), to the middle (m), and then to the apical (a) portion of collected shoots with the highest value recorded in the apical cuttings treated with t1 (4.5 roots/cutting) followed by t2 treated apical and middle cuttings (3.9 and 3.4 roots/cutting, respectively) (fig. 6 a), whereas the lowest number of roots was observed in the basal cuttings of untreated control (t0) and t1 (1.5 roots/cuttings) (fig. 6 a). with respect to the control and t1, a significant increase of the root number was observed in the basal cutting treated with t2 (2.8 roots/cuttings). the capability of olive cuttings on forming adventitious roots is known to be related to the portion (basal, middle or apical) of the shoots, even though different cultivars can respond differentially. a general hypothesis has been formulated on the higher aptitude for rooting of subapical cuttings that seems to be related to a possible h i g h e r c o n t e n t i n a u x i n i c c o m p o u n d s d r i v e n basipetally from the young leaves of cuttings (fabbri et al., 2004). on the other hand, only the root length of spring cuttings was significantly affected by the interaction between the hormonal treatment (t) and the shoot portion (p) (table 2, fig. 6 b), with the highest values (11.4 cm) recorded in the apical cuttings treated with naa in liquid formulation (t1) and in basal, middle and apical cuttings (10.3, 10.9, and 12.2 cm, respectively) treated with nad in powdery formulation (t2). 4. conclusions this study highlighted that the olive cultivars belonging to autochthonous germplasm of the campania region are characterized by a wide variability in the potential rhizogenic activity. overall the two sampling periods of cutting collection (march and september) significantly affected the rooting aptitude of several cultivars, indicating that in some cultivars the cuttings collected in autumn may have a higher rooting rate than the spring collected ones. moreover, it was possible to evaluate the rhizogenic aptitude of the cultivars belonging to the olive campanian germplasm under the influence of rhizogenic treatments. the results showed that the effects of naa and nad on rooting strongly depended on interaction with the cultivar, time of collection (autumn or spring) and type of cuttings (basal, medium or apical). in general, the apical and the median portions of shoots were confirmed to be the most suitable for improving the rooting of cuttings. references avidan b., lavee s., 1978 physiological aspects of the rooting ability of olive cultivars. acta horticulturae, 79: 93-101. carfi c.h., ben hadj salah h., msallem m., haddar t., hellali r., 1994 effet des doses d’aib et des dates de prélévement sur la rhizogenése des boutures de 6 variétés d’olivier (olea europaea l.) “chetoui”, “meski”, “picholine”, “besbessi”, “chemlali”, “arbequina”. revue inat, vol. 9, no. 1-2. chiancone b., macaluso l., germanà m.a., 2011 prove sulla radicazione di talee di cultivar siciliane di olea europaea l. acta italus hortus, 1: 370-375. cimato a., 1999 l’élevage des plants d’olivier en pèpinière. innovations technologiques et leur application en oléiculture et oléotechnie. séminaire international, firenze (italia). del rio c., rallo r., caballero j.m., 1991 effect of carbohydrate content on the seasonal rooting of vegetative and reproductive cuttings of olive. j. hortic. sci., fig. 6 effects of the interaction of the hormonal rhizogenic treatment (t0= control no hormonal treatments; t1= naa treatment; t2= nad treatment) and shoot portion used to make the cuttings (b= basal; m= medium; a= apical) on root number/cutting (a) of semi-hardwood cuttings collected in autumn and on root length/cutting (b) of semi-hardwood cuttings collected in spring. means followed by different letters are significantly different for p≤0.05 (root number) and p≤0.05 (root length). cirillo et al. rooting ability of twenty olive cultivars from southern italy 317 66(3): 301-309. denaxa n.k., vemmos s.n., roussos p.a., kostelenos g., 2011 the effect of iba, naa and carbohydrates on rooting capacity of leafy cuttings in three olive cultivars (olea europaea l.). acta horticulturae, 924: 101-109. di vaio c., nocerino s., paduano a., sacchi r., 2013 characterization and evaluation of olive germplasm in southern italy. j. sci. food agric., 93(10): 2458-2462. fabbri a., bartolini g., lombardi m., kailis s.g., 2004 olive propagation manual, pp. 2-7. in: fabbri a., g. bartolini, m. lombardi, and s.g. kailis. olive propagation manual. csiro publishing, landlinks press, collingwood vic, australia, pp. 145. fontanazza g., baldoni l., 1989 innovazioni tecnologiche in olivicoltura. olivicoltura: innovazioni tecnologiche e valutazione dei risultati economici in alcune r e a l t à a z i e n d a l i . f o r m e z , a r c h i v i o d e i c o r s i d i formazione, pp. 41-54. fontanazza g., jacoboni n., 1975 indagine sugli effetti della defogliazione e della degemmazione di talee di olivo sulla rizogenesi in camera di nebulizzazione. frutticoltura, 9: 25-30. hartmann h.t., kester d.e., 1975 plant propagation: principles and practices. prentice-hall inc., englewood cliffs, nj, usa, pp. 662. hartmann h.t., kester d.e., davies f.t., 1990 plant propagation principles and practices. prentice-hall inc., englewood cliffs, nj, usa, pp. 547. hartmann h.t., loreti f., 1965 seasonal variation in rooting leafy olive cuttings under mist. proc. amer. soc. hort. sci., 87: 194-198. hechmi m., khaled m., abed s., el-hassen a., faiez r., m’hamed a., 2013 performance of olive cuttings (olea europaea l.) of different cultivars growing in the agro-climatic conditions of al-jouf (saudi arabia). am. j. plant physiol., 8(1): 41-49. ioc, 2005 olive nursery production and plant production techniques. international olive council http://www. internationaloliveoil.org/projects/paginas/sectionb.htm. ismaili h., ianni g., dervishi a., 2011 study of main factors influencing olive propagation. j. int. environ. appl. & sci., 6(4): 623-629. khabou w., trigui a., 1999 optimisation of the hardwood cutting as a method of olive tree multiplication. acta horticulturae, 474: 55-58. porfírio s., sonon r., gomes da silva m.d.r., peixe a., cabrita m.j., azadi p., 2016 quantification of free auxins in semi-hardwood plant cuttings and microshoots by dispersive liquid-liquid microextraction/ microwave derivatization and gc/ms analysis. anal. methods, 8: 6089-6098. wiesman z., lavee s., 1994 vegetative growth retardation, improved rooting and variability of olive cuttings in response to application of growth retardants. plant growth regul., 14: 83-90. impaginato 421 adv. hort. sci., 2018 32(3): 421-431 doi: 10.13128/ahs-23361 biochemical, physiological changes and antioxidant responses of cut gladiolus flower ‘white prosperity’ induced by nitric oxide h. kazemzadeh-beneh 1 (*), d. samsampour 1, s. zarbakhsh 2 1 department of horticulture science, faculty of agriculture and natural resources, university of hormozgan, bandar abbas, iran. 2 department of horticulture science, plant breeding and biotechnology, faculty of agriculture, shiraz university, shiraz, iran. key words: anthocyanin, catalase, cut gladiolus flower, enzymatic antioxidant system, nitric oxide (no), sodium nitroprusside (snp) abstract: sodium nitroprusside (snp), as nitric oxide (no) donor, has been considered by postharvest researchers as one of the best option for slowing the processes controlling senescence in cut flowers. here, we investigate the role of no on postharvest physiology and vase life of the gladiolus grandiflorus cv. white prosperity. vase life markedly extended by snp at 150 μm from 3 day to 7.33 day and thus those inducer effects were doseand time-dependent. snp at 125 μm interdependent on vase life time period was observed to be the optimal dose for improving of relative fresh weight (rfw), peroxidase (pod), and total monomeric anthocyanin (tma) in cut flowers. supplementing vase solution with snp indicated significant increase in water uptake of cut flowers and consequently protected to decline in rfw due to alleviate water losses stress. snp was maintained the level of total soluble protein, lipid peroxidation, and pod, whereas it enhanced the level of catalase (cat) and tma in flower petals. summary of our results revealed that snp exogenous prolongs vase life via maintaining protein degrade, scavenging free radical in term of anthocyanin and enzymes antioxidant, decreasing polyphenol oxidase, inhibiting lipid peroxidation, and improving membrane stability in ‘white prosperity’ cut flowers. 1. introduction floriculture is an emerging and fast expanding globalized market and subsequently studies on postharvest handling of cut flowers occupy a fundamental position (gul and tahir, 2013). therefore, the postharvest longevity of flowers have a vital importance in evaluating the value of the each horticulture plant. this aspect can be particularly hold good with cut flowers and it is a necessity for extended handling and transportation periods. cut flowers are greatly perishable, and consequently they have short vase life and also are exposed to early senescence processing, which restricts efficient marketing of economically significant ornamental plants (*) corresponding author: kazemzadehhashem@yahoo.com citation: kazemzadeh-beneh h., samsampour d., zarbakhsh s., 2018 biochemical, physiological changes and antioxidant responses of cut gladiolus flower ‘white prosperity’ induced by nitric oxide. adv. hort. sci., 32(3): 421-431 copyright: © 2018 kazemzadeh-beneh h., samsampour d., zarbakhsh s. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 6 june 2018 accepted for publication 16 september 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(3): 421-431 422 (nasibi et al., 2014). however, postharvest senescence is a major restriction to the marketing of many species of cut flowers and so much appreciable efforts have been dedicated to developing postharvest treatments to extend the marketing period or increasing postharvest longevity (vajari and nalousi, 2013). sodium nitroprusside (snp), as donor nitric oxide (no, is one of the postharvest treatments which recently using from it for improving postharvest life of horticulture crops has exceptionally increased. postharvest application of snp has been shown to be effective in extending the postharvest life of a range of flowers, fruits and vegetables when applied as a short term fumigation treatment at low concentrations (wills et al., 2000). no is a short-lived bioactive molecule, which is considered to function as prooxidant as well as antioxidant in plants. no molecule is now documented as an important signaling molecule and reported to be involved in various key physiological processes such as plant defense mechanism, abiotic stress resistance, germination, stimulate antioxidant compounds, decrease lipid peroxidation, growth and development of plants etc. (zhao et al., 2004). furthermore, it was also revealed that plant response to such stress or like drought, high or low temperature, salinity, heavy metals and oxidative stress derived from reactive oxygen species (ros), is moderate by no (mandal and gupta, 2014 ). no is recognized as a biological messenger in plants and it has been proved that no is effective for increase the vase life of cut flowers because it can be may play role as anit-ethylene synthesized from wounded or non-wounded organ (abasi, 2014). liao et al. (2009) reported that no may act as an antagonist of ethylene in cut rose flowers senescence. optimum snp levels could postponement the climacteric phase of many tropical fruits and elongate the post-harvest shelf life of a wide range of horticultural crops by inhibiting ripening and senescence (singh et al., 2013). gladiolus is one of the four famous cut flowers in the world (bai et al., 2009). gladiolus cut flowers have extremely used to decorate graves and celebrate major life events in iran. likewise, the longevity of cut flowers is one of the main challenges of florists today. first data concerning about the effect of snp on differential activity of antioxidants and expression of sags (senescence associated genes) in relation to vase life of gladiolus cut flowers (gladiolus grandiflora cv. snow princess) has been reported by dwivedi et al. (2016). finding of their study suggested that the application of snp increases vase life by increasing the scavenging mechanism of reactive oxygen species (ros) in terms of antioxidants activity, membrane stability and down-regulation of ggcyp1 gene expression in gladiolus cut flowers. under condition in plants subjected to snp, not only the responses of various genotypes or cultivars to snp may be multiresponse, but also the responses rely on dose-and cultivar-dependent, physiological growth state, and environmental factors status. the same trend has been stated by naing et al. (2017) who found that snp promoted the vase life of the cut gerbera flowers via a delay in the time to stem bending; however, all three gerbera cultivars responded to snp and the effects were found to be doseand cultivar-dependent. in the previous study, it has been demonstrated that the snp dose that was best for one cultivar was not suitable for another; thus, variation in the o p t i m a l d o s e o f s n p a m o n g c u l t i v a r s f o r t h e enhancement of their vase life could result from differences in their genetic background (naing et al., 2017). hence, whether snp participates in improving of cut flowers of white prosperity cultivar has not been yet reconnoitered. however, the purpose of the present study was to evaluate the effect induced by nitric oxide donor namely, snp, on the enzymatic antioxidant activity, biochemical and physiological processes of cut gladiolus (gladiolus grandiflorus cv. white prosperity) flowers in order to extend their vase life and postharvest shelf-life. 2. materials and methods plant material and snp treatments cut flowers used in the experiment were g. grandiflorus cv. white prosperity. cut gladiolus flowers were obtained from a commercial grower presented in mahallat city, as famous central commercial production of ornamental plant, in iran at normal harvest maturity and transferred immediately to laboratory of the postharvest physiology and technology r e s e a r c h , f a c u l t y o f a g r i c u l t u r e a n d n a t u r a l resources, hormozgan university at jun, 2017 and the experiments were established on the same day. flowers stems ends were recut under tap water to eliminate air emboli, to inhibit vascular blockage, and to trim to a uniform length of 70 cm. stock solutions of snp (enzo life sciences) were prepared following the manufacturer’s instructions. uniform cut flowers kazemzadeh-beneh et al. postharvest physiology of cut gladiolus flower “white prosperity” 423 were placed in holding solutions, that containing of snp, na2 [fe (cn) 5 no]. 2h2o (sigma-aldrich), as no donor (0, 25, 50, 75, 100, 125 and 150 μm) plus 3% sucrose as carbohydrate supplement. for control set, flowers were dipped in distilled water plus 3% sucrose. finally, the flowers stems were placed in 500 ml bottles with 250 ml of each mentioned solutions containing different concentrations of the snp solutions + 3% sucrose and they were maintained at a temperature of 23±3°c, 60±5% relative humidity and under a 12 h photoperiod using cool-white fluorescent lamps (24 μmol m-2 s-1 irradiance) during experimental period. there were three bottles (21 flowers) per treatment and the experiment was done seven treatments. to escape from photodegradation of snp (release of a nitrosyl ligand and a cyanide ion), the bottles were shielded with black nylons. snp treatment was applied as a continuous treatment and flower stems were kept in solutions till the end of vase life. vase life and water uptake the vase life was determined based on wilting of more than one-third of the petals of flower and vase life termination of each floret was considered as soon as the first symptom of wilting was observed. indeed, it was defined as the number of days in vase life required for one-third of the florets of each spike to lose its ornamental value (lost turgor and wilted). water uptake was measured by periodically weighting the vase of a control bottle without cut flowers and bottles containing flowers. finally, vase water u p t a k e w a s d e t e r m i n e d u s i n g t h e f o r m u l a (rezvanypour and osfoori, 2011): water uptake (ml day-1 g-1 fresh weight) = (st-1-st)/wt where st= solution weight (g) at = days 1, 4, 8 and st-1= solution weight (g) on the preceding day, and wt = fresh weight of the cut flower (g) on t days. number of opened, unopened florets and relative fresh weight o n e a c h s p i k e , t h e n u m b e r o f o p e n e d a n d unopened florets was recorded from the beginning of the experiment to until 20 days after snp treatments. the fresh weight was measured every 4 days and relative fresh weight (rfw) of cut flowers was calculated by the following equation: rfw (%) = (wt/w t=1 ) × 100 where wt = weight of cut flowers (g) at t = days 1, 4, 8 and wt=1 = the initial fresh weight of the same cut flower (g) on day 1 (rezvanypour and osfoori, 2011). antioxidant enzyme assays antioxidant enzyme activities were determined in the third floret from the base of spike at three time points (days 1, 4, and 8). the 100 mg of floret tissue from controls and snp treatments were removed, were homogenized with mortar and pestle in 1 ml 50 mm epps buffer (ph 7.8) containing 0.2 mm edta and 2% pvp, and were ice-covered for the analysis of antioxidant activity. the homogenates were centrifuged at 4°c for 20 min at 12 000×g and the obtaining supernatants were used to evaluate of antioxidant enzyme activities. catalase (cat) activity was assayed as described by chance and mahly (1995) as follows: the assay reaction mixture of cat contained 50 mm phosphate buffer (ph 7.8), 15 mm h2o2, and crude enzyme. the decomposition of h2o2 was followed at 240 nm (e = 39.4 mm-1 cm-1). absorbance values were quantified using standard curve generated from known concentrations of h2o2. for the measurement of peroxidase (pod) activity, the reaction mixture contained 50 mm phosphate buffer (ph 7.8), 13 mm guaiacol, 5 mm h2o2 and enzyme. the reaction was started by adding 300 μl of h2o2 (0.03%). the pod activity was determined by the increase in absorbance at 470 nm due to guaiacol oxidation (e = 26.6 mm-1 cm-1) (chance and mahly, 1995). the polyphenol oxidase (ppo) activity was assayed in 2.8 ml of reaction mixture comprised 2.5 ml of 50 mm potassium phosphate buffer (ph 7.8), 0.3 ml substrate containing 0.2 ml pyrogallol and 0.1 ml crude enzyme (kar and mishra, 1976). the reaction mixture was mixed and the ppo activity was determined in absorbance at 420 nm (6.2 mm-1 cm-1). it’s to be remembered that the blank cuvette consisted of 3.0 ml potassium phosphate buffer (ph 7.8). the results of antioxidant enzymes activitie was expressed as units (u) per mg fw. anthocyanin content assay petals were cut from controls and snp treatments of at three time points (days 1, 4, and 8) and were frozen for the analysis of total monomeric anthocyanin (tma). tma content in petals extract was determined by the ph-differential method based on two buffer system described previously by giusti and wrolstad (2005). to measure the absorbance at ph 1.0 and 4.5, the samples were diluted 2 times with ph 1.0 potassium chloride buffer (0.025 m) and ph 4.5 sodium acetate buffer (0.4 m), respectively. therefore, the tma content analyses of prepared mixtures were performed following the methods of giusti and wrolstad (2005). adv. hort. sci., 2018 32(3): 421-431 424 lipid peroxidation the level of lipid peroxidation in petals tissue was measured by determination of malondialdehyde (mda), which is recognized to be breakdown products of lipid peroxidation, at the end of time points (days 8). the mda content was determined with the thiobarbituric acid (tba) reaction. temporarily, 0.2 g of sample tissue was homogenized in 5 ml 0.1% tca. the homogenate was centrifuged at 10000 g for 5 min. 4 ml of 20% tca containing 0.5% tba were added to 1 ml aliquot of the obtained supernatant. the mixture was heated at 95 ºc for 15 min and cooled immediately on ice. the absorbance was measured at 532 nm by a spectrophotometer. the value for the non-specific absorption at 600 nm was subtracted from the above value. the level of lipid peroxidation was expressed as mmol of mda formed using an extinction coefficient of 155 mmol-1 cm-1 (heath and packer, 1968). total soluble proteins total soluble proteins (tsp) content of petals at the three time point (days 1, 4, and 8) was determined according to the method of bradford (1976) using bovine serum albumin as standard. statistical analysis the experiment was carried out in completely randomized design (crd) with three replications. three flowers stems were used for each replication and thus, the experiment was done with seven treatments and three replication per treatment. the nonnormalize date of the total soluble protein, pod enzyme activity and rfw of cut flowers were normalized with kurtosis and skewness test; so, their transformed date used for analyzing. data were statistically analyzed using analysis of variance (anova) in sas software (version 9.4, sas institute inc., cary, nc, usa). correlations among the evaluated parameters were analyzed using pearson’s correlations (p<0.05 and p<0.01). mean comparisons to identify significant differences between treatments were performed using least significant difference (lsd) at the p<0.01 or 0.01 level of probability. 3. results vase life, rfw and water uptake application of snp markedly enhanced the time to vase life for white prosperity cultivar (p < 0.01). results showed that the bottle solution containing snp + sucrose, significantly increased the vase life of cut flowers compared to the control solution (distilled water), as maximum vase life with higher concentration of snp treatments was verified near the end of storage (table 1). however, it positive impacts on increasing vase life was dose-dependent: 150 and 125 μm were displayed to be the best concentration for vase life (7.33 and 5.66 days) of ‘white prosperity’, respectively, whereas the other concentrations lower than the 125 μm did not markedly influenced vase life as compared to control (p<0.01). generally, based on the results of vase life, ‘white prosperity’ exhibited a longer vase life (4.33 days) when exposed to 150 μm no as compared to control (3 days). the prolonged vase life in snp-treated cut flowers were approximately associated with increasing in floral opening of cut flower by 150 μm treatment (table 1). a d i r e c t s i g n i f i c a n t r e l a t i o n s h i p w a s d e t e c t e d between snp and floral opening (%); however, increasing in snp concentration resulted in increasing in floral opening percentage. statistically, the floral abscission and un-opened flower did not affected by snp treatments as compared to control (p<0.01). values followed by the same letter within a column indicate they are not significantly different (p< 0.01) by least significant difference (lsd). table 1 effect of different concentrations of sodium nitroprusside (as nitric oxide donor) on vase life, flower opening and floral abscission in cut gladiolus flowers (gladiolus grandiflorus cv. white prosperity) treatments vase life (days) full-opened flower (%) un-opened flower (%) floral abscission (%) sodium nitroprusside 0 µm 3.0 c 58.60 b 19.21 a 24.39 a sodium nitroprusside 25µm 3.0 c 60.33 ab 15.51 a 22.99 a sodium nitroprusside 50 µm 3.33 c 69.83 ab 14.83 a 18.02 a sodium nitroprusside 75 µm 4.0 bc 66.92 ab 15.87 a 17.19 a sodium nitroprusside 100 µm 3.33 c 72.38 ab 10.52 a 19.72 a sodium nitroprusside 125 µm 5.66 ab 74.81 ab 6.38 a 18.79 a sodium nitroprusside 150 µm 7.33 a 80.25 a 6.52 a 13.21 a kazemzadeh-beneh et al. postharvest physiology of cut gladiolus flower “white prosperity” 425 significantly increased water uptake (12.33±0.56 and 12.69±1.51 ml day-1 fw) compared to the control (9.16±0.81 ml day-1 fw) resulted in 34.60% and 38.53% increase in vase solution uptake on day 1, respectively; however, they were also conserved the same manner on day 4 or day 8. in contrast, the low concentration did not sufficiently play protective role to inhibit water losses on 4 days, which was also detected that the 25 and 50 μm accelerated water losses, even faster than controls, especially on 8 days. lipid peroxidation the data, belong to mda concentration of flowers petals representing the level of lipid peroxidation is revealed in figure 2a. measurement of mda demonstrated that vase solutions containing 125 and 150 μm snp significantly decreased mda production in comparison to control (p<0.01). overall, it was predictable that white prosperity without treatment (control) showed higher mda concentration than the snp treatments. results found inversely correlation between lipid peroxidation and higher snp concentration. at any specified level of snp concentration, the production of lipid peroxidation product was lesser in treatments at the end experiment (8 days) comparison to control. thus, the vase solutions having as shown in figure 1a, rfw (data normalized; 4.6 is equal to 100%) in cut gladiolus flowers of control gradually was declined during the vase life period, while the decline in rfw was no observed by snp treatments throughout the vase life. it is notable that the rfw of the snp treatments solutions except to 125 μm at three point time did not significantly difference with those in control solutions at initial point time (day 1). so, the presence of snp in vase solutions displayed a protective role to the inhibition of rfw decline in vase life period, even when the vase l i f e o f c u t f l o w e r s e n d e d . o n l y s n p w i t h concentration 125 μm (5.60±0.08) in vase solution prolonged the rfw 22.55% higher than other concentrations on day 4 or day 8 in comparison to those in controls at initial time (fig. 1a). senescence is a process characterized by water loss and desiccation of plant tissues. during vase life period, water uptake gradually was declined in both some of the snp treatments (25, 50, and 75 μm) and control cut flowers (fig. 1b). generally, the water uptake with snp concentration 100, 125, 150 μm were higher than those under control condition, respectively (p<0.01). at the initial point time (day 1) of vase life, the white prosperity showed a rapid response to high snp concentrations for promoting water uptake; however, the water loss was not observed during its vase life period. the vase solutions containing snp at concentration 125, 150 μm fig. 1 the effects of different concentrations of sodium nitroprusside (as nitric oxide donor) on physiological changes of gladiolus grandiflorus cv. white prosperity cut flowers during vase life. data are means of three replications. vertical bars indicate standard deviation. fig. 2 effect of different concentrations of sodium nitroprusside (as nitric oxide donor) on malondialdahyde (mda), as an indicator of lipid peroxidation, total soluble protein in gladiolus grandiflorus cv. white prosperity cut flowers during vase life. vertical bars with the same letters did not show significantly different using lsd method at p<0.01 significant level. 426 adv. hort. sci., 2018 32(3): 421-431 snp at concentration 125, 150 μm significantly declined the lipid peroxidation of cell membrane (0.463±0.04 and 0.61±0.04 mmol g-1 fw) compared to the control (1.55±0.07 mmol g-1 fw) resulted in 70.13% and 60.65% decline in product induction of lipid peroxidation, mda, on days 8 prior to senescence appearance in cut flowers. total soluble proteins the chemical analysis for tsp of the flower petals exhibited that snp significantly increased the tsp during vase life period in comparison with control (p<0.01) (fig. 2b). the protein degradation of flower petals in control was higher than snp treatments; however, the total soluble protein gradually was declined in control across days. thus, not only snp lead to help to the inhibition of protein degradation in flowers petals on day 4 or day 8, but also it caused in delaying the senescence of gladiolus flowers. so, all of the snp treatments except to 150 μm displayed a protective or maintain role for protein degradation in cut flowers. overall, only increase in tsp was observed with 150 μm snp and also was recorded highest tsp for its on day 8; however, the protein degradation did not occurred by 150 μm snp treatment during vase life period. furthermore, the prolong vase life of white prosperity flowers can be strongly associated with increasing in tsp and inhibiting from its degradation in flower petals during vase life. enzymatic antioxidant and non-enzymatic antioxidant activities anova analysis with mean comparison showed that antioxidant enzymes and non-enzymatic antioxidant activities in flower petals differed significantly between control and treatments in white prosperity (p<0.01). as expected, the ppo activity (u/mg fw) was continually increased in control during vase life, which this tendency was also approximately found for 25 μm (fig. 3a). the low concentration from 25 to 75 μm did not sufficiently decrease the ppo activity in flower petals comparison to control (p<0.01). so, the decrease in ppo activity was observed by 100, 125, and 150 μm treatments on day 4 or day 8, respectively; however, increasing snp concentration in vase solution resulted in markedly decreasing ppo activity in comparison to controls at initial time of vase life (p<0.01). it can be predictable that the positive effect of snp on maintaining or decreasing ppo activity was high dose-dependent. it is now well recognized that high ppo activity accelerate to senescence and to induce browning in plant tissues. generally, the high concentrations of snp to white prosperity cut flowers, check the activity of ppo enzyme, lead to help in delaying the senescence of gladiolus flower via preventing the ppo activity compared to control (p<0.01). as shown in figure 3b, the pod activity (u/mg fw) significantly decreased in control flowers throughout vase life, while the cat activity (u/mg fw) in control flowers displayed a constant tendency at all of the 3 point time of vase life (fig. 3c) comparison to snp treatments(p<0.01). it is appears that all of the treatments except to 125 μm significantly played a protective role to conserve the decrease of pod activity during vase life (p<0.01). the highest pod activity obtained by 125 μm on 8 days, according to lsd test at p<0.01. however, the positive effect of snp on pod activity was doseand time-dependent: 125 μm was observed to be the optimal concentration for pod activity on day 4. thus, in white prosperity, low concentrations did not adequately increase pod activity, which was also found for concentrations higher than the optimal levels (fig. 3b). in concerning about cat activity, the positive relation was detected between cat activity and snp treatments; however, increasing in snp concentration resulted in increasing cat activity, especially on day 4 or day 8, compared to control (p<0.01) (fig. 3c). the results of the present study indicated that with more addition snp concentration by 100 to 150 μm into vase solution was lead to positively increase in cat activity at each of three time points during vase life. the results of lsd test (p<0.01) indicated that tma degradation was gradually happened in control flowers during vase life (fig. 3d). the snp treatments not only significantly prevented from the tma degradation but also they were greatly enhanced the tma p r o d u c t i o n o v e r 8 d a y s , c o m p a r e d t o c o n t r o l (p<0.01). at the during vase life, the low concentrations of snp demonstrated a protective role to inhibit tma degradation in flower petals in comparison to control (p<0.01). the furthest increase in the tma production was archived for 125 μm (0.273 ± 0.037 mg l-1), and 150 μm (0.193±0.015 mg l-1) with a significant difference compared to control, respectively. hence, improved tma in flower petals likely to pod activity was doseand time-dependent: 125 μm was observed to be the optimal concentration for tma content. thus, in white prosperity, low concentrations did not adequately increase tma content, which was also detected for concentrations higher than the optimal levels on day 4 or day 8. 427 kazemzadeh-beneh et al. postharvest physiology of cut gladiolus flower “white prosperity” pearson correlation analysis reveals interactions between physiological, biochemical and antioxidant system related traits in order to arrange for an overview of the associations between physiological, biochemical traits, and antioxidant system activity, the pearson correlation test used for analyzing and thus was investigated all of the significant associations, as presented in table 2. from this analysis 23 positive and 11 negative significant correlations was achieved. among them, some correlations were expected, such as the positive and negative correlations observed between antioxidant system activity, for example, cat activity and tma content (r= 0.89, p<0.01), and ppo activity and tma content (r= -0.95, p<0.01) on days 8, respectively. with regard to physiological traits, the results of paired linear correlation indicated that rfw was positively correlated with cat (r= 0.85, p<0.05 on days 4), and pod activity (r= 0.86, p<0.05 on days 4 and r= 0.81, p<0.05 on days 8), and tma content (r= 0.79, p<0.05 on days 8), while was negatively correlated with ppo activity on day 4 (r= -0.80, p<0.05) and day 8 (r= -0.87, p<0.05) of the white prosperity vase life. also, the pearson correlation of water uptake with cat activity (r= 0.80, p<0.05 on days 1; r= 0.82, p<0.05 on days 4; r= 0.86, p<0.05 on days 8) and with tma content (r= 0.81, p<0.05 on days 8) was positive significant, whereas displayed a negative significant with ppo activity (r= -0.79, p<0.05 on days 4 and r= -0.85, p<0.05 on days 8) (table 2). however, suggesting that the snp treatment is a key inhibitor to water loss and an inducer to antioxidant system for delaying the senescence of gladiolus flowers during vase life, especially on 4 and days 8. total soluble protein had a positive correlation with tma content and a negative correlation with ppo activity. tma indicated a positive correlation with water uptake, rfw, total soluble protein, and cat activity and a negative correlation with ppo and pod activity. cat activity was positively correlated with physiological traits, tma and negatively correlated with ppo and pod activity. however, according to the results of pearson correlation, suggesting that snp might be an important protective or inducer involved in the physiological, biochemical process and antioxidant system in white prosperity vase life that can be alleviate to water loss, rfw, and to browning process, which lead to early senescence appearance. 4. discussion and conclusions the postharvest longevity of cut flower has a critical importance in determining the value of crop. recently, snp, a no donor known to be a signal molecule involved in biotic and abiotic stress tolerance, has been increasingly used to extend the vase life of fig. 3 evaluating the effects of different concentrations of sodium nitroprusside (as nitric oxide donor) on enzymatic and non-enzymatic antioxidant system changes during vase life period of gladiolus grandiflorus cut flowers. vertical bars indicate standard deviation. 428 adv. hort. sci., 2018 32(3): 421-431 cut flowers, such as rose, gladiolus, and carnation (naing et al., 2017). first data concerning about application exogenous snp to improve vase life of g. grandiflora cv. snow princess cut flower has been reported by dwivedi et al. (2016). it is generally accepted that different genotypes or cultivars might indicate different physiological or biochemical responses to exogenous snp, which is the effects induced by it may be rely on doseand cultivardependent. some published evidences supports no acting as a negative regulator during leaf senescence, but also there is opposite result in this regard; no enhances flower abscission and senescence in cut racemes of lupinus havardii wats (sankhla et al., 2003; guo and crawford, 2005). thus, the properly effects of snp on enhancing physiological and biochemical processes for one cultivar, may not be suitable for another, which is due to differences in their genetic background. this aspect has also been confirmed by naing et al. (2017), who found that snp dose that was best for one cultivar of gerbera cut flower was not suitable for another; thus, variation in the optimal dose of snp among cultivars for the enhancement of their vase life could result from diff e r e n c e s i n t h e i r g e n e t i c b a c k g r o u n d . h e n c e , whether snp participate in improving of cut flowers of white prosperity cultivar has not been yet reconnoitered. therefore, in the current study, we investigated the role of snp in the enhancement of physiological, biochemical responses, and antioxidant activity to extend vase life of gladiolus grandiflorus cv. white prosperity cut flower. cut flower senescence is linked to a sequence of highly regulated physiological and biochemical processes such as degradation of proteins, dna content, peroxidation lipids and membrane leakage, degradation of macromolecules, cellular decompartmentalization, floral abscission, color change, leaf yellowing, and weight loss (buchananwollaston et al., 2003; nasibi et al., 2014). in this study, results of our findings revealed that the physio l o gi c a l , b i o c h em i c a l , a n d a n t i o x i d a n t a c t i vi t y induced by snp in white prosperity cultivar were more different than those induced in snow princess cultivar, a previous study by dwivedi et al. (2016), which it may be due to differences in their genetic background. hence, in present study, snp was significantly promoted the vase life of ‘white prosperity’ cut flowers through help to delay the senescence appearance and desiccation on tissue or organ level; however, it effects were discovered to be doseand time-dependent. vase life positively associated with table 2 pearson correlation between physiological and biochemical characteristics of gladiolus grandiflorus cv. white prosperity cut flowers affected by sodium nitroprusside during vase life period wu= water uptake; rfw= relative fresh weight; tsp= total soluble protein; ppo= polyphenol oxidase activity; pod= peroxidase activity; cat= catalase activity; tma= total monomeric anthocyanin, the 1, 2, and 3 representing vase life time for each variable on day 1, day 4, and day 8. ns, *, ** non-significant, correlation is significant at the 0.05 and the 0.01level, respectively. rfw1 has no computed because at least one of the variables was constant. traits wu1 wu2 wu3 rfw2 rfw3 tsp1 tsp2 tsp3 ppo1 ppo2 ppo3 pod1 pod2 pod3 cat1 cat2 cat3 tma1 tma2 tma3 wu1 1 0.932 ** 0.919 ** 0.499 ns 0.595 ns -0.499 ns 0.261 ns 0.408 ns 0.288 ns -0.584 ns -0.713 ns -0.741 ns 0.152 ns 0.152 ns 0.803 * 0.746 ns 0.927 ** 0.421 ns 0.655 ns 0.732 ns wu2 1 0.849 * 0.698 ns 0.753 ns -0.383 ns 0.517 ns 0.616 ns 0.48 ns -0.579 ns -0.801* -0.666 ns 0.347 ns 0.347 ns 0.752 ns 0.829 * 0.981 ** 0.434 ns 0.752 ns 0.854 * wu3 1 0.627 ns 0.722 ns -0.388 ns 0.430 ns 0.553 ns 0.40 ns -0.791* -0.850* -0.787* 0.271 ns 0.271 ns 0.780 * 0.871 * 0.862 * 0.25 ns 0.697 ns 0.811 * rfw2 1 0.988 ** 0.208 ns 0.747 ns 0.687 ns 0.802 * -0.575 ns -0.809* -0.295 ns 0.866 * 0.866 * 0.296 ns 0.854 * 0.656 ns 0.233 ns 0.534 ns 0.745 ns rfw3 1 0.100 ns 0.713 ns 0.677 ns 0.774 * -0.663 ns -0.872* -0.406 0.817 * 0.817 * 0.352 ns 0.892 ** 0.724 ns 0.311 ns 0.565 ns 0.794 * tsp1 1 -0.009 ns -0.208 ns 0.071 ns 0.36 ns 0.342 ns 0.615 ns 0.585 ns 0.585 ns -0.514 ns -0.215 ns -0.436 ns -0.333 ns -0.319 ns -0.405 ns tsp2 1 0.971 ** 0.558 ns -0.64 ns -0.75 ns -0.432 ns 0.445 ns 0.445 ns 0.405 ns 0.685 ns 0.543 ns -0.062 ns 0.771 * 0.820 * tsp3 1 0.526 ns -0.693 ns -0799* -0.561 ns 0.299 ns 0.299 ns 0.591 ns 0.751 ns 0.642 ns -0.073 ns 0.851 * 0.889 ** ppo1 1 -0.272 ns -0.615 ns 0.056 ns 0.747 ns 0.747 ns 0.16 ns 0.775 * 0.365 ns 0.068 ns 0.154 ns 0.507 ns ppo2 1 0.897** 0.868* -0.21 ns -0.21 ns -0.486 ns -0.7 ns -0.682 ns -0.286 ns -0.734 ns -0.847* ppo3 1 0.739 ns -0.45 ns -0.45 ns -0.556 ns -0.910** -0.832* -0.36 ns -0.742 ns -0.956** pod1 1 0.152 ns 0.152 ns -0.688 ns -0.532 ns -0.786* -0.363 ns -0.817* -0.791* pod2 1 1.000** -0.14 ns 0.548 ns 0.269 ns 0.185 ns 0.086 ns 0.318 ns pod3 1 -0.14 ns 0.548 ns 0.269 ns 0.185 ns 0.086 ns 0.318 ns cat1 1 0.661 ns 0.749 ns -0.124 ns 0.779 * 0.682 ns cat2 1 0.786* 0.125 ns 0.646 ns 0.862 * cat3 1 0.482 ns 0.823 * 0.894 ** tma1 1 0.084 ns 0.3 ns tma2 1 0.887 ** tma3 1 kazemzadeh-beneh et al. postharvest physiology of cut gladiolus flower “white prosperity” 429 rfw, water uptake, tsp content, tma, enzyme antioxidant activity and lipid peroxidation. at the start of vase life, there was a noticeably increase and then a constant tendency in water uptake of white prosperity cut flowers during their vase life, which suggested that snp might have a protective role in cut flowers against water losses stress (fig. 1b). the rapid increase in initial water uptake was dose-and time-dependent, while increase in rfw was more dose-dependent (fig. 1a). the 125 μm concentration was observed to be the optimal concentration for increasing in rfw. the rfw increase obtained in white prosperity is in isagreement with results pronounced by dwivedi et al. (2016) in snow princess. the inhibition or improvement in rfw across days under no condition is probbly attributed to the excessive potential of water uptake, leading to a stability or promote in cell turgidity pressure, which restricts burning from reserved carbohydrates in respiration and limits fresh weight reduction. an association of improved water uptake and inhibited fresh weight reduction has been reported by vajari and nalousi (2013) in carnation and naing et al. (2017) in gerbera cut flower. overall, the ‘white prosperity’ in snp (150 μm) had prolonged vase life over control, which was strongly associated with increased water uptake and improved rfw. the damage to the plant cell’s biomembrane liable to senescence process, decrease in the ratio of unsaturated fatty acids, change mobility of the cell membrane, and generate free radicals are resulted in an increase in the concentration of malondialdahyde (mda), which is an indicator of lipid peroxidation and of injury to the plant cell membrane (chen, 2009). so, the higher membrane stability plays a key role in inhibiting leakage of electrolytes, sugars, pigment, solute leakage, and also lipid peroxidation as well as in delay senescence during gladiolus cut flowers postharvest (ezhilmathi et al., 2007; ghadakchiasl et al., 2017). our results showed that the change in membrane stability and lipid peroxidation occurrence resulting from the mda production were alleviated by snp concentrations (150, 125 μm) on day 7 after treatment and therefore protected and reduced white prosperity mda production in cell membrane (fig. 2a). these results are in agreement with those reported earlier by mansouri (2012), who suggested that snp prolonged the vase life of chrysanthemum flowers, which was accompanied by decreasing in the electrolyte leakage, levels of mda and lipid peroxidation. indeed, the role of no in prevention of lipid peroxidation is related to the ability of no to react with lipid alcoxyl (lo•) and lipid peroxyl (loo•) radicals and stop the chain of peroxidation in a direct fashion (beligni and lamatina, 1999). the role of snp in reducing membrane lipid peroxidation has previously been stated by liao et al. (2012) and dwivedi et al. (2016). many researchers have been shown that protein degradation and also shortage protein due to consumption it instead of soluble carbohydrate during senescence process for respiration in petals are the most important causes for shortening cut flowers vase life (rezvanypour and osfoori, 2011). in addition, snp significantly maintained the tsp degradation in cut flowers petals, while in absence snp increased the tsp degradation to a greater rate than snp treatments on day 4 and day 8 (fig. 2b). the tsp measured in vase solution supplemented by 150 μm was distinctly higher than those placed in controls on day 8. the proteins are the basic components of all cell activities, their reduction degrades enzymes and causes higher production of free radicals, as well as reducing protein synthesis (saed-moucheshi et al., 2014). therefore, it was clear that the proteins degradation during vase life significantly inhibited by snp supplements in the vase life of ‘white prosperity’. the increase or protect of tsp degradation by snp application in strawberry (ghadachiasl et al., 2017) and in peanuts (verma et al., 2010) has also been claimed. earlier studies have been confirmed that snp may either be directly scavenging ros and thus decreasing lipid peroxidation, or it may be modulating the activity of antioxidant system (beligni and lamatina, 1999; saed-moucheshi et al., 2014). various studies have demonstrated that the vase life of cut flowers is modulated by antioxidant enzymes and nonenzymatic antioxidant activities (vajari and nalousi, 2013). thus, supplemented vase solutions with snp stimulated a higher enzymatic or non-enzymatic antioxidant activity in flower petals during vase life period. the ppo catalyzes the browning reaction and results in the formation of quinine, which is subsequently polymerized to varying degree leading to production of brown pigments (dubravina et al., 2005). the ppo activity greatly was reduced by snp, while the pod and cat activity greatly promoted by snp during progress senescence (fig. 3). the results were in accordance with the findings of ghadakchiasl et al. (2017) and dwivedi et al. (2016). indeed, the no synthesized by snp in tissue plant acts signaling adv. hort. sci., 2018 32(3): 421-431 430 molecule to enhance the enzymatic antioxidant activity such as sod and cat and ultimately protects proteins degradation as well as lipid peroxidation against free radicals. so, pod and cat high activity induced by snp showed a negatively correlation with ppo activity and thus they blocked ppo activity during vase life (table 2). approximately, high and markedly negative between ppo and more traits examined in current study were also detected in pearson correlation analysis. furthermore, tma degradation in flower petals protected by snp, while tma degradation increasable induced in flower petals without presence snp during vase life period. however, positive effects induced by snp in both pod activity and tma were doseand time-dependent, therefore, the 125 μm was selected as an optimal concentration for tma and pod activity (fig. 3b, d). antioxidant compounds such as vitamin c, glutathione, and anthocyanin plays vital role, as non-enzymatic system, in protecting cell against destructive chemical compounds such as free radicals and reactive oxygen species (ros) that are constantly produced by the cell metabolism and their concentration increases under stress conditions (kazemzadeh et al., 2015). however, snp increased tam content at any time and level of snp concentration in comparison to controls. the high and significantly positive correlation between tma anthocyanin with cat activity and total soluble protein was obtained by pearson analysis (table 2). with progress senescence during vase life, tma probably scavenged free radicals due to oxidative stress, consequently, inhibited more deterioration of membrane and protein degradation in flower petals. in conclusion, vase life period in the g. grandiflorus cv. white prosperity cut flower is likely to be associated with many parameters, particularly fresh weight content, water uptake, enzymatic or nonenzymatic antioxidant activities, membrane stability and lipid peroxidation. furthermore, it was found that positive effects induced by snp on vase life distinctly were doseand time-dependent and were also genetic back ground cultivar-dependent in comparative responses between white prosperity with snow princess, which has previously been reported by dwivedi et al. 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species, urban agriculture. abstract: urbanization and poverty have brought to worse life conditions in towns of many developing countries, including difficult availability of food, especially fresh. urban agriculture and horticulture can contribute to the availability of fresh foods, officinal and medicinal plants, but the little availability of irrigation and surface to destine for cropping suggest the convenience of little water consuming species, with little needs of soil fertility and that can be eaten entirely. opuntia fiscus-indica (l.) mill. corresponds at all these requirements, and it is a very promising strategic species that can be eaten completely (green parts, fruits and even flowers), it has good nutritional values and also interesting medical properties. a trial has been done to compare the initial productivity of cladodes multiplied in pots, car tires and open field. our results suggest that the prickly pear can be cropped better in large exhausted tires than in small plots also saving money for the materials. 1. introduction urbanization and poverty cause food insufficiency in many tropical and subtropical countries and this problem is enhanced in towns and urban peripheries where land plots to crop are few and small and there is irregular availability of foods in local markets. a few examples: accra (ghana) is a growing town that loses about 2.600 hectares of farm land every year to buildings and consequently reduces agricultural land availability nearby and into the town. the peripheral agriculture in hanoi (vietnam) produces at least 150.000 tons of fruit and vegetables per year. in cuba urban and peripheral agriculture gives around 60 percent of horticultural produce consumption in large towns. still in cuba due to the constraints caused by trade embargo, a growing percentage of the agricultural production is provided by urban agriculture: in 2002 more than 14.000 ha of urban yards produced 3.100.000 tons of food, and 90% of the habana fresh produce comes from local urban farms. in 2003 more (*) corresponding author: andrea.pardini@unifi.it citation: pardini a., massolino m., grassi c., 2017 opuntia ficus-indica (l.) mill. growing in soil and containers for urban agriculture in developing areas. adv. hort. sci., 31(4): xxx-xxx copyright: © 2017 pardini a., massolino m., grassi c. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 17 may 2017 accepted for publication 22 september 2017 ahs advances in horticultural science adv. hort. sci., 2017 31(4): xxx-xxx 192 than 200.000 cubans worked in the expanding urban agriculture sector (cuban ministry of agriculture, 2013). in kinshasa (democratic republic of the congo), urban agriculture is producing an estimated 75.000 to 85.000 tons of vegetables per year that is 65% of the town supply. agriculture is being pushed far from many towns, with increasing costs of food transport, packing and conservation enhanced by the bad conditions of rural roads, and heavy losses in transit. in these areas, growing fruit and vegetables in and around cities increases the supply of fresh produce and improves the economic access to food for poor people (fao 2015) that spend 60-80% of their income on food. urban families may actually grow crops or raise small animals, and so produce some of their own food (cohen and garret, 2009). moreover many foods available in towns and peripheries of d e v e l o p i n g c o u n t r i e s a r e i n t r o d u c e d w i t h i n t e r n a t i o n a l a i d a n d c o n s e q u e n t l y t h e y a r e a constraint to food sovereignty. opuntia ficus-indica is a wild plant and a cultivated crop, whose value is largely underestimated. its green parts are commonly used in environmental protection against soil erosion and as forage (mulas and mulas, 2004), the biomass is used for biogas (rosato, 2014), industrial sectors use prickly pear fruits and leaves as raw materials for cosmetics, drinks and food additives (saenz et al., 2013). its flowers are useful to honey bees, but all its above ground parts are also good meals in form of fruits, fresh and cooked salads, soups, refreshing beverages, flour for breads and pastas. fruits and cladodes are very nutritional (saenz et al., 2013; rodriguez et al., 1996), and their use a human functional food dates back to pre-columbian times (ramirez et al., 2010). especially young cladodes (nopalitos in latin america) conserve water that is very useful in hot dry areas for both humans and livestock, around 9% crude protein, low fats (just around 1%), several minerals especially potassium (220 mg per 100 grams), calcium (16-33 mg), phosphorus (13-28 mg), and vitamins especially c vitamin (ascorbic acid 40 mg per 100 grams), with the best nutritive values being in the young cladodes (retamal et al., 1987). the seeds are commonly considered a waste of the food industry however the extraction of oil is under study for applications in food, pharmaceutical and cosmetic industries (de wit et al., 2017). calories content vary within 25 and 50 kcal per 100 grams dry which is comparable to the most common fruits, moreover it has important pharmaceutical effects including antioxidants and also anti-cancerogenic effects (livrea and tesoriere, 2006). for all this uses the prickly pear has to be considered a multipurpose species quite useful also in many agroforestry systems. opuntia ficus-indica is a species of the cactaceae with cam metabolism that favors water conservation, even if young cladodes have a c 3 metabolic pathway (mulas and mulas, 2004) that causes water recall from lower-older cladodes during the day reducing water availability for the whole plant on benefit of the growth of younger parts (wang et al., 1997). opuntia ficus-indica is a wild plant native of mexican deserts, with very little needs of soil fertility and capable of growth even without any management. few parasites treat this plant. all this makes this crop possible in most tropical-subtropical areas with enhanced hot and dry season, with poor soils and little management. it can be cropped also in permaculture and with this techniques it becomes an additional strategic plant useful as disasters relief crop beside short cycle cereals, beans and vegetables, after people displacement due to earthquakes (see in haiti capital town), hurricanes (as common in caribbean islands like cuba), or after war situations (like recently happened in somaliland, iraq, libya) when the whole national or regional agro-food system has to be restarted. one of the problems related to prickly pear food harvesting and processing is related to the spines or glochids (hichy hair) that can be removed by immersion in water and mechanical treatment or fire burning. however there are spineless varieties (var. inermis) commonly used for forage, and also varieties almost without glochids that can be handled safely and make easy harvesting and processing, even if the absence of defensive parts make the plants more sensible to animal predation. due to the good nutritional values, the little water and soil needs and the easy management, prickly pears can be advised as a strategic crop for urban and peripheral areas with difficult climate or poor soil, as a green fence and also cropped in pots and exhausted tires as common in many towns of tropical developing countries. the productivity of prickly pear has been studied for field production or in greenhouse but still oriented to later in field transplanting. researches have been done about how cladode size, their position at planting can influence initial growth and production (bakali et al., 2016) and has been suggested that pardini et al. opuntia ficus-indica growing in soil and containers for urban agriculture 193 horizontal planting is the best technique in arid regions, and that cladode size influence early dry matter production. the same authors proved that cladode orientation to the sun, and the planting depth had not significant effects on early production. however singh and vijai singh (2003) suggest that planting vertical is generally the best technique together with using cladodes 12 months old, and planting in spring. the effects of planting cladode p a r t s i n s t e a d o f w h o l e h a s b e e n s t u d i e d b y stambouli-essasi et al. (2015), that suggest the possibility to use just parts in order to save planting material. concerning plant densities in open field, ruiz-espinosa et al. (2008), suggest plantations of 60.000 plants per hectare. concerning different clones, several authors have got diversified results in d i f f e r e n t e n v i r o n m e n t s , t h u s s u g g e s t i n g a relationship clone-environment (flores, 1992; floreshernandez et al., 2004; ruiz-espinosa et al., 2008). although there are few data available on opuntia cultivation in containers, these are not intended for food production. our research is oriented to the production of a strategic food plant and has compared the productivity of prickly pear cladodes grown in soil (as the case of green fencing) or in pots (the case of urban agriculture in balconies) and in exhausted tires (as common in backyards, small plots or balconies of urban peripheries of many developing countries). 2. materials and methods the trial was done on a spineless cultivar of opuntia ficus-indica (l.) mill. without glochids imported from cuba in the winter 2011, “milpa alta” that is the most cultivated in mexico (gallegosvasquez and mondragon-jacobo, 2011), it is an erected cultivar with elliptic cladodes, spineless and practically without glochids, with yellow fruits. the introduced parts have been multiplied for four years in order to have the necessary number of green parts, and then planted for the trial in central italy (florence hills, 250 m asl, exposed to south) on the first of may 2015 and then again on may 2016. the location has 750 mm average annual rainfall with 159 during the 90 summer days, 14.5c° average annual temperature, in the period 1971-2000 there was an average of 62 days with maximum temperatures above 30c°. the soils of the trial location are mainly clayey (45% clay). the initial plantation has been done using only young cladodes produced by mother plants the summer before (august 2014), planted after winter on 1st may 2015, thus 8 months old. a second plantation was done in 2016 after winter on 1st may, starting from cladodes produced by mother plants in the summer before (august 2015), thus also these 8 months old. all the cladodes used for first and second planting had similar size, 5-7 cm width and 20-25 cm length in order to have very similar conditions of all plants at the beginning. each cladode was planted in a pot or in a tire or straight in the soil. in extensive soil they were planted in three single lines at distances of 50 cm on the line, simulating a soil area available similar to what they have in plots or tires. pots were 20 cm large x 30 cm deep that is a size quite common in local canned food tins that are used as flower pots, exhausted tires were borrowed all the same size 70 cm diameter (45 inner) x 23 cm width that is a 4wd type very common in developing countries. one pot has been got from each tire. tires were put on cemented area to avoid rooting in external soil. the tires were brought back to the giver after the end of the trial for ecological elimination by existing regulations. pots and tires were filled with the same soil were cladodes were also planted straight. pots and tires were disposed in three lines alternated with the three lines of plants planted in the soil, and mixed in a randomized block design. treatments were not irrigated, in the attempt to reproduce conditions of absence or minimal management, common in strategic crops in harsh areas including home yards, balconies and peripheral areas where the water available serves mainly for human needs. thus we got three treatments: 1) extensive soil, 2) pots, 3) tires. each treatment was implemented with 8 initial cladodes (8 repetitions), for a total of 24 plants per year. measurements were done at the end of the growing season (30 september 2015 and 30 september 2016 for all treatments) on: total number of new cladodes originated from each planted (counting); fresh weight of new cladodes (harvesting all new cladodes and weighing fresh with scale), the fresh weight has been considered because this food is eaten fresh (like pineapple or papaya, as examples); dry weight (after fresh weighting, all new cladodes were split in two halves to favor dehydration and oven dried for 5 days at 80°c, then weighted again to calculate the percentage of dry matter). adv. hort. sci., 2017 31(4): xxx-xxx 194 fruits cannot be produced in the trial location during one growth season because it is too short. statistical analysis was done with lsd at p= 0.05. 3. results and discussion number of new cladodes the number of new cladodes (table 1) was the highest in extensive soil (6 in the average of the two years) and the least in pots (2.5 in the average). obviously roots explore a larger area of extensive soil than in pots or tires and consequently have higher water and nutrients availability, moreover soil temperature in pots and tires (that are black) rises more than in the soil and this in turn causes higher evaporation and reduces water availability. this somehow contrasts with the ability of a container to conserve water better than extensive soil, but this doesn’t happen in full summer when high temperatures cause strong evaporation. tires are larger than pots and probably have given intermediate yields because there is more soil than in pots, small plots have brought the plants to stressed conditions very rapidly whilst tires have provided better conditions for some longer time. the higher number of cladodes in the second year (5.0 in the average of treatments, in comparison to 3.7 of the first year) can be due to warmer temperatures that persisted up to the end of the summer 2016 whilst several cool and some rainy days happened in the year before. fresh weight of new cladodes the fresh weight (table 2) was higher in the extensive soil (778 grams in the average of two years) than in tires (508.5 grams) and in pots (only 172 grams). the higher yield in 2016 (554.7 grams in the average of all treatments) than in 2015 (417.7) is probably due to warmer summer temperatures of 2016 in comparison to 2015. dry weight of new cladodes and percentage of dry matter the percentage of dry matter (table 3) was higher in 2016 (14% in the average of all treatments) than in 2015 (12%) probably because the warmer temperatures of 2016 favored evaporation whilst the cooler days and some rains increased water content in 2015. the total dry matter yield was similarly higher in 2016 (75.4 grams in the average of all treatments) than in the year before (50.9 grams). the highest yield was got in 2016 in extensive soil (94.4 grams in the average of the two years, with a maximum of 114 grams in 2016) and the lowest was got in pots (24,3 grams in the average of the two years, with a minimum of 19.5 grams in 2015). 4. conclusions most households in towns, especially in developing countries, have not soil available and must rely on small and cheap containers for gardening, under this point of view, tires not only are cheaper then pots and they are easily available in many tropical towns, but they also provide better growing conditions for opuntia ficus-indica than commercial pots. of course extensive soil is a better condition for table 1 number of new cladodes in the three treatments in the two years of trial and average table 2 fresh weight (grams) of new cladodes in the three treatments in the two years of trial and average different letters show significantly different values at p=0.05. table 3 dry weight (dw, grams) and dry matter percentage (dm, %) in new cladodes in the three trial treatments in the two years of trial and average different letters show significantly different values at p=0.05. treatment number of new cladodes 2015 2016 average extensive soil 5 a 7 a 6.0 pot 2 c 3 c 2.5 tire 4 b 5 b 4.5 average 3.7 5.0 4.3 treatment fresh weight of new cladodes (grams) 2015 2016 average extensive soil 679 a 877 a 778.0 pot 150 c 194 c 172.0 tire 424 b 593 b 508.5 average not irrigated 417.7 554.7 482.2 treatment dry weight of new cladodes 2015 2016 average dw(g) dm(%) dw(g) dm(%) dw(g) dm(%) extensive soil 74.7 a 11 b 114.0 a 13 b 94.4 12.0 pot 19.5 c 13 a 29.1 c 15 a 24.3 14.0 tire 50.9 b 12 ab 83.0 b 14 ab 67.0 13.0 average not irrigated 48.4 12.0 75.4 14.0 61.9 13.0 pardini et al. opuntia ficus-indica growing in soil and containers for urban agriculture 195 growing prickly pears than into containers, at least referring to water needs and taking into account that in extensive soil problems can rise from weeds competition more than in pots or tires. the production in our trial in italy has not been much because of the short warm season (4 months, with only three having summer temperatures) and because we planted cladodes not yet rooted. we can reasonably consider that production would be much higher in the tropics and subtropics where temperatures are good all the year through, using, already rooted plants. the production could be increased much if some waste water can be used in the household for irrigation. the yield of new cladodes can integrate the diet and be a useful supply for a small family during the worst part of a dry season of 3-4 months, can be also a source of herbal medicine extremely useful to families. although the limiting climatic conditions of our trial, the results maintains their value of comparison within treatments also for tropical countries. a couple of issues to be investigated are whether food production in tires is economically more convenient than transforming them into handmade items such as sandals (it is a common practice), and check food quality for eventual absorption of toxic contaminants from exhausted tires into edible plants. finally, a next trial on the use of prickly pear as a strategic plant, could be to start growing cladodes in tires or pots and when grown use some of these to plant green fences. the use of prickly pear for green fences can also be advised in order to shift the common use of planting the poisonous euphorbia spp. (usually euphorbia trigona) that has been much diffused in many and large 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doi: 10.13128/ahs-20832 solar radiation levels modify the growth traits and bromatological composition of cichorium intybus f. schwerz 1 (*), j. sgarbossa 2, t. olivoto 3, e.f. elli 4, a.c.m. aguiar 2, b.o. caron 2, d. schmidt 2 1 luiz de queiroz college of agriculture, university of são paulo (esalqusp), department of plant science, piracicaba, sp, brazil. 2 federal university of santa maria, frederico westphalen campus, departament of agronomic and environmental sciences, frederico westphalen, rs, brazil. 3 federal university of santa maria, department of plant science, santa maria, rs, brazil. 4 luiz de queiroz college of agriculture, university of são paulo (esalqusp), department of biosystems engineering, piracicaba, sp, brazil. key words: chicory, growth rate, radiation use efficiency, shading screens. abstract: shading greenhouse may be an effective method to achieve a suitable environment for crop growth and to enhance crop yield and quality in places or seasons where there is high light intensity. therefore, solar radiation levels may modify the biomass accumulation and bromatological composition. different solar radiation levels (100%, 70% and 50% of available solar radiation) were simulated in order to determine crop responses to these factors in chicory (cichorium intybus l. var. foliosum). a hydroponic experiment was conducted in an experimental greenhouse in the city of frederico westphalen, rio grande do sul, brazil. plants grown in lower solar radiation levels are more efficient in converting solar radiation into dry matter, had a higher lipid content, increased chlorophyll indices a, b and total, as well as reduced leaf thickness, acid detergent fiber, cellulose, and lignin content, presenting more attractive bromatological features for commercial production. in this study it was demonstrated that the use of shading screens is an effective method to attenuate the solar radiation, this is especially relevant in places or seasons where there is high light intensity, which contribute to achieve better characteristics of the chicory produced. 1. introduction growth rate, dry matter production and radiation use efficiency are considered important variables when analyzing yield-limiting factors and their interactions in different production environments. dry matter accumulation under non-limiting conditions is directly related to the amount (*) corresponding author: felipe_schwerz@hotmail.com citation: schwerz f., sgarbossa j., olivoto t., elli e.f., aguiar a.c.m., caron b.o., schmidt d., 2017 solar radiation leves modify the growth traits and bromatological composition of cichorium intybus. adv. hort. sci., 31(4): 257265 copyright: © 2017 schwerz f., sgarbossa j., olivoto t., elli e.f., aguiar a.c.m., caron b.o., schmidt d. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 17 december 2016 accepted for publication 29 june 2017 ahs advances in horticultural science adv. hort. sci., 2017 31(4): 257-265 258 of intercepted photosynthetically active radiation (caron et al., 2012). therefore, variation in total biomass due to limiting biotic and abiotic factors may be attributed to changes in the solar radiation levels. changes in the solar radiation level of a given environment may affect the photosynthetic apparatus of plants. lower solar radiation level reduces the photosynthetic rate and hence plant growth. when radiation levels is above the light saturation point, the reaction center of the psii is inactivated and frequently damaged, constituing a phenomenon called photoinhibition (taiz et al., 2017). due it, studies have identified a reduction of quantum efficiency, photosynthetic rate, and possible damage to the p h o t o s y n t h e t i c a p p a r a t u s ( z h a n g e t a l . , 2 0 0 8 ; murchie et al., 2009) beyond changes on the yield, bromatological and nutritional aspects, when plants are exposed both to suboptimal or super optimal radiation levels (dario et al., 2015; bianculli et al., 2016). achieving an appropriate environment in greenhouses in subtropical regions has become one of the main challenges for chicory producers, due to a large amount of solar radiation transmitted into the greenhouse and then converted into sensible and latent heat. thus, greenhouse cooling methods and their impact present a considerable problem that requires a solution (abdel-ghany and al-helal, 2010). a strategy used in greenhouses in order to provide an appropriate environment for plant growth and to increase crop yield is the shading screens (sethi and sharma, 2007; ganguly and ghosh, 2011). shading is one of the most inexpensive ways to reduce heat accumulation and to modify the greenhouse environment (sethi and sharma, 2007; holcman and sentelhas, 2012), besides promoting higher rates of diffuse radiation. solar radiation use efficiency (ɛb) of crops is determined by the slope of the linear regression between produced dry matter and photosynthetic active radiation (par) intercepted by the leaves (monteith, 1965, 1972, 1977; van heerden et al., 2010). reduced solar radiation availability within the greenhouse environment may influence various traits of chicory, especially the leaf area index, the thickness of leaves and chlorophyll content, because cultivation in shaded environments often results in thinner leaves (wherley et al., 2005; liu et al., 2016). these leaves contain a greater total chlorophyll content per unit of fresh weight when compared to leaves grown under full sunlight. chlorophyll content of each leaf, per unit area, may be smaller in environments with reduced incident solar radiation (wild and wolf, 1980; taiz et al., 2017). research showing the change of yield, and bromatological and nutritional traits as a function of incident solar radiation level, both as suboptimal and optimal levels were shown by bianculli et al. (2016), dario et al. (2015), and kataria and guruprasad (2015). to the authors’ knowledge, this study is the first to investigate the impact of different solar radiation levels on plant growth, radiation use efficiency and biomass partitioning in chicory. information that reveals the impact of solar radiation levels on chicory as well as the bromatological composition are relevant in order to improve some management in a greenhouse environment. in addressing this lack of information, the following hypotheses were investigated: (i) solar radiation use efficiency is reduced in the plants grown under high solar radiation level; and (ii) shading screens provide an appropriate environment for plant growth, resulting in plants with better bromatological composition. these hypotheses justify the following aims: (i) to determine the influence of different solar radiation level on plant traits; and (ii) to evaluate the bromatological compositon of chicory plants under different solar radiation levels. 2. materials and methods study area the study was conducted in an experimental greenhouse in the city of frederico westphalen, rio grande do sul, brazil. a hydroponic experiment was performed between the months of march 2016 to june 2016. the geographical location of the experiment was 27°23’ s, 53°25’ w, 490 m asl. according to the köppen climate classification, the climate is cfa, i.e., humid subtropical with mean annual temperatures of 19.1°c, and varying maximum and minimum temperatures of 38 and 0°c, respectively (alvares et al., 2013). management of hydroponic system chicory (chicory intybus l. var. foliosum) seeds were inserted into phenolic foam board on 25 march 2016. seedlings were transplanted into a system called “seedling tray” on 3 april 2016, when they reached two to three true leaves; seedling tray had 40 mm-hydroponic channels (3-cm deep, spaced by 7 cm between channels and 10 cm between plants in the channels), with 3% declivity. seedlings remained schwerz et al. solar radiation levels modify the growth traits of cichorium intybus 259 in this system until they reach a developmental level of five true leaves. after the seedling tray stage, the final transplant to the growth tray was performed on 11 april 2016. each final growth tray was formed by 11 hydroponic channels (6-m long, 0.10-m wide and 0.05-m deep), subjected to a 4% declivity. the spacing was 0.20 m between plants in the channels and 0.20 m between channels. thus, three hydroponic benches had 33 channels, with 242 plants per bench, 726 plants throughout the experiment composed the system. a 1-hp pump coupled to a fiberglass tank with a capacity of 1000 liters powered the hydroponic system. the nutrient solution used was prepared with 4 0 0 m g l 1 o f a c o m m e r c i a l h i d r o g o o d ® f e r t (hidrogood modern horticulture, brazil); these values were considered to be a full dose. nutrient solution was pumped inside the hydroponic channels and was collected at the end of each channel by a system of closed system gutters. the nutrient solution used w a s p r e p a r e d w i t h 4 0 0 m g l 1 o f a c o m m e r c i a l hidrogood® fert (hidrogood modern horticulture, brazil); these values were considered to be a full dose. irrigation was performed in an on-and-off system in periods of 15 minutes throughout the day (6 am 7 pm.); and 15 minutes each two hours during the night (7 pm 6 am). potential of hydrogen (ph) and electric conductivity of the nutritive solution were assessed daily with a digital ph meter (ph009ia model), and with a conductivity meter (az8301 model), respectively. the ph of the nutrient solution was kept at 6.0 (±0.5) using sulfuric acid (10% concentration of h2so4) or very low sodium hydroxide (2% of naoh). nutrients were replaced when the electrical conductivity of the nutritive solution reached 50% of its initial concentration. solar radiation level and experimental design the experimental design was a randomized complete block, arranged in a factorial arrangement (solar radiation level x evaluation periods) with four replications. different solar radiation level (srl) were simulated using black polyethylene meshes, fixed 1.0 m above the hydroponic benches. the following treatments were applied: 100% of available srl (without mesh over the plants), 70% of available srl (30% transmissivity mesh), and 50% of the available srl (50% transmissivity mesh). each simulated radiation level treatment composed a different hydroponic bench and the meteorological conditions in all treatment were the same, differing only the solar radiation levels in each hydroponic bench. for the growth traits analysis, the chicory plants were collected from the central hydroponic channels of each treatment, beginning seven days after the transfer to the final growth tray. the evaluations were performed weekly until the average fresh mass of experimental plants hit 250 g (harvest point). destructive evaluation consisted of two whole plants in each replication, totaling eight plants per treatment in each period. in laboratory the sectioning plants was performed, including the preparation of leaf discs in order to determine the leaf area and dry matter partitioning. the total dry matter (tdm) of the plants was determined from the sum components (root, stem and leaves). each component was gathered and placed into pre-identified individual paper sacks. the sacks were then kept in a forced circulation oven at 60°c until a consistent mass was obtained. the samples were later weighed on a precision balance in order to obtain the dry mass of each component, which together resulted in the tdm. growth rates and bromatological analysis in each evaluation period, the following variables were determined using the average values of dry matter (dm) and leaf area index (lai): specific leaf area (sla), leaf area ratio (lar), leaf weight ratio (lwr), absolute growth rate (agr), relative growth rate (rgr) and net assimilation rate (nar). for more details about the used metrics of evaluation and the determination of these variables, see thornley (1976) and gardner et al. (1985). the chlorophyll index a, b, total and a/b ratio were determined with a cfl 1030 chlorophyll meter. we selected, in the last evaluation period, fully expanded leaves from the upper third of 10 plants in each replication. all collected dry matter samples were properly prepared and subjected to bromatological analysis; the following traits were determined: ash (ash, % of dm), lipids (lip, % of dm), crude protein (cp, % of dm), neutral detergent fiber (ndf, % of dm), acid detergent fiber (adf, % of dm), lignin (ln, % of dm), hemicellulose (hc, % of dm), cellulose (cel, % of dm) and soluble carbohydrates in neutral detergent (scnd, % of dm). for the ash content determination, the method aoac 923.03 (1995) was used, which consider a temperature of 550°c. lipid content was quantified according to the method proposed by bligh and dyer (1959). the values of ndf, adf and ln were determined, followed by the calculations for estimating hc, cel and csnd as proposed by senger et al. (2008), and the crude protein (n x 6.25) was determined by micro-kjeldahl method (method 960.52) of aoac (1995). adv. hort. sci., 2017 31(4): 257-265 260 radiation use efficiency production of dry matter was based on the model proposed by monteith (1977), where dry matter production was calculated from intercepted photosynthetically active radiation (ipar) multiplied by the use efficiency (εb). the εb was calculated by the ratio between the average production of accumulated tdm and the ipar involved in the production of biomass according to the following expression: dm= b*ipar where dm = dry matter, in g; = conversion efficiency of in biomass produced, in g mj–1 and = intercepted photosynthetically active radiation, in mj m–2. estimation of accumulated photosynthetically active radiation was determined according to the expression proposed by varlet-grancher et al. (1989): *ipar=0.95*(inpar)*(1-e (-k*lai)) where ipar = intercepted photosynthetically active radiation, in mj m-2; inpar = incident photosynthetically active radiation, in mj m-2; k = light extinction coefficient. the k value calculated in the current study for the chicory plants was 0.13. lai = leaf area index. the light extinction coefficient (k) was calculated using the following equation: k= in(rn/rt) lai where k= light extinction coefficient, rn= solar radiation measured under the plant canopy (mj m-2); rt= radiation above the plant canopy (mj m-2); lai= leaf area index. leaf area index was calculated by the following expression: lai= la/ua where lai = leaf area index; la = leaf area, in cm2; ua = useful area per plant, in cm2. the fraction of photosynthetically active radiation was considered 47% of the incident solar global radiation found in rio grande do sul (assis and mendez, 1989). the estimation of accumulated photosynthetically active radiation was based on monteith (1977) and varlet-grancher et al. (1989). the par transmissivity of greenhouse cover was calculated based on an assessment using a quantum sensor, 50 cm height from the ground level, measuring 40 random points inside and outside of the greenhouse weekly during the trial period. to calculate transmissivity, the following equation was used: t = (100*pari)/paro where t= transmissivity, in %; pari= photosynthetically active radiation inside of the greenhouse; paro= photosynthetically active radiation outside of the greenhouse. with the transmissivity data of greenhouse and polyethylene meshes, pari was estimated for each treatment, according to the following expressions: par100%=pari par70%=pari*0.7 par50%=pari*0.5 where photosynthetically active radiation inside of the greenhouse; environment with 70% of photosynthetically active radiation available and environment with 50% of photosynthetically active radiation available. the values of incident global solar radiation during the study were obtained with the automatic climatological station of the national institute of meteorology, located at 300 m from the study site (27°39’ s and 53°43’ w). chicory growth and nutritional composition variables were statistically analyzed with the software sas 9.0 (sas institute 2002). data were initially examined for homogeneity of variance and then subjected to analysis of variance. tukey test (p>0.05) was used to compare the difference between the treatments. 3. results radiation use efficiency in chicory the radiation use efficiency and photosynthetically active radiation accumulated values in the different solar radiation level during the conduct of the study are shown in figure 1. it was observed that the fig. 1 radiation use efficiency (εb) in g mj-1 and photosynthetically active radiation levels (par) accumulated over cichorium intybus cycle. adm = accumulated dry matter, pariac = photosynthetically active radiation intercepted accumulated. schwerz et al. solar radiation levels modify the growth traits of cichorium intybus 261 plants growing under reduced radiation availability (50% and 70%) presented respectively 51.6% and 40.1% higher radiation use efficiency than those growing under 100% of solar radiation level. plants grown in low levels of photosynthetic active radiation were more efficient in converting the radiant energy into accumulated dry matter. growth rates and leaf traits the absolute growth rate values were greater in the higher level of solar radiation from 42 dat up to the crop harvest (fig. 2a). the relative growth rate showed changes depending on the age of the plants, but for the solar radiation levels was observed difference only at 14 and 21 dat (fig. 2b). in relation to the net assimilation rate was not observed changes in the values in function of the solar radiation level, however, the largest net assimilation rate values were observed at 21 dat (fig. 2c). the attenuation of 50% and 70% of solar radiation reduced the leaf area index of the plants by 51.8% and 19.8% on average when compared with the plants growing under 100% of solar radiation level from 42 dat up to the crop harvest (fig. 3a). for the specific leaf area, significant differences can be observed from transplanting up to 21 dat, where the plants under lower radiation levels showed the higher specific leaf area (fig. 3b). in relation to the leaf area ratio, it was identified higher value only at 7 dat for the plants under 100% of solar radiation level (fig. 3c). additionally, for the leaf weight ratio was not verified difference in the different shading levels (fig. 3d). plants grown in lower radiation levels showed higher chlorophyll index a, b, and total, which were 19.4, 36.4 and 25.3% higher than those observed for the 100% of solar radiation level, respectively. fig. 2 absolute growth rate (agr), relative growth rate (rgr) and net assimilation rate (nar) in cichorium intybus grown under different solar radiation levels. lowercase letters denote differences within each day after transplanting by tukey test (p<0.05). bars represent average values ± se (n=8). fig. 3 leaf area index (lai), specific leaf area (sla), leaf area ratio (lar) and leaf weight ratio (rlw) in cichorium intybus grown under different global solar radiation levels. lowercase letters denote differences within each day after transplanting by tukey test (p<0.05). bars represent average values ± se (n=8). adv. hort. sci., 2017 31(4): 257-265 262 however, plants without light restriction showed higher chlorophyll-a/b ratio (fig. 4). biomass partitioning and bromatological composition the pattern of dry matter accumulation in the leaves, roots and stem of the chicory plants was similar for all solar radiation levels; however, there was observed an increase in the proportion of leaves during the crop cycle, with a decrease in the percentage of roots and stem of the plants. this pattern of partitioning was observed for all solar radiation levels (fig. 5). reduced levels of solar radiation increased the lipids and ash contents and decreased the neutral detergent fiber, acid detergent fiber, lignin, hemicellulose and cellulose values that characterized less rigid leaves. the crude protein content and soluble carbohydrates in neutral detergent were not affected by shading levels (fig. 6). in overall, the bromatological traits of chicory plants were affected by the solar radiation levels. 4. discussion and conclusions solar radiation levels modify the radiation use efficiency and growth rates the results showed that growing conditions have a striking effect on the radiation use efficiency in relation to its capacity to convert solar radiation into dry biomass. the efficiency of plants to convert solar energy into biomass was higher in low solar radiation fig. 4 chlorophyll-a, chlorophyll-b, and total chlorophyll index and chlorophyll-a/b ratio in cichorium intybus grown under different solar radiation levels. lowercase letters denote differences between solar global radiation levels by tukey test (p<0.05). bars represent average values ± se (n=8). fig. 5 biomass partitioning (%) in each days after transplanting (dat) among leaves, roots and stem in cichorium intybus grown under different solar radiation levels. bars represent average values ± se (n=8). fig. 6 ash percentage (ash), lipids (lip), crude protein (cp), neutral detergent fiber (ndf), acid detergent fiber (adf), lignin (ln), hemicellulose (hm), cellulose (cel), soluble carbohydrates in neutral detergent (csnd) in cichorium intybus leaves growing in different solar radiat i o n l e v e l s . l o w e r c a s e l e t t e r s d e n o t e d i f f e r e n c e s between solar global radiation levels by tukey test (p<0.05). bars represent average values ± se (n=8). schwerz et al. solar radiation levels modify the growth traits of cichorium intybus 263 level. the higher efficiency of chicory plants was not enough to compensate the limitations of solar radiation. this is justified by the higher leaf area index observed under high levels of radiation. the highest leaf area index observed for plants growing under 100% of solar radiation may be related to the higher rate of photosynthesis, since there were no limitations on the availability of solar radiation. according to taiz et al. (2017), sun leaves increase co2 assimilation having more availability of rubisco, and can dissipate excess of light energy due to a large pool of components in the xanthophyll cycle. in this sense, it is important to emphasize that plants with higher conversion efficiency are not always the ones that result in higher yield. this affirmation was confirmed in this study, where the chicory plants grown under reduced solar radiation levels were more efficient into convert solar radiation into biomass, however, showed lower leaf area index. this response can be explained due the morphological changes occurred in function of the shading conditions. the shading effect provided by the solar radiation level of 50% significantly increased the specific leaf area of the plants mainly up to 14 days after transplantation. the decrease in leaf thickness of plants grown at higher levels of shading maybe linked to the fact that plant prefers to spend photoassimilated for the expansion of the leaf area (cooper and qualls, 1967; carvalho et al., 2006; gondim et al., 2008; lenhard et al., 2013), thus ensuring itself a greater possibility of intercepting solar radiation. in chicory, the attenuation of solar radiation above the plants with the use of shading screens resulted in an alteration in leaf thickness which was responsible for changes in the chlorophyll content and bromatological composition (sims and pearcy, 1992; terashima et al., 2001; oguchi et al., 2003). this plant response is correlated, where a reduction in the leaf thickness can imply in changes in the chlorophyll content and bromatological composition depending on the intensity of leaf thickness alteration in function of the level of shading. the morphological changes were considered positive, as the leaves of these plants were more tender. this characteristic may be considered in the market commercialization due to a better acceptability of the consumer’s for products with greater bromatological features. reduced solar radiation levels improve morphological and bromatological traits of chicory the current study confirmed the difference in the bromatological quality of chicory plants grown in different solar radiation level. in overall, a higher lipids content and lesser fiber, lignin, hemicellulose and cellulose components were observed under reduced solar radiation levels. this result reveals an important bromatological change, which confers more quality to the chicory produced. the reduction of lipid content in chicory plants under high solar radiation levels may be associated with the occurrence of light saturation and photoinhibition. according to taiz et al. (2017), the light saturation in the leaves of most species occurs between 500 and 1000 µmol m−2 s−1, whereas the full solar radiation can provide around 2000 µmol m−2 s−1. excessive light causes damage to chloroplasts, one of the locations of fatty acids synthesis (halliwell, 1987; mittler, 2002; gill and tuteja, 2010). the higher content of lipids in chicory plants produced under reduced solar radiation level is an important finding that can help people health, once lipids deliver energy and essential fatty acids, being essential for fat-soluble vitamin absorption, and may c o n t a i n n a t u r a l a n t i o x i d a n t s ( l i n d l e y , 1 9 9 8 ) . considering the consumer’s point of view, the higher content of lipids and reduction of fiber, lighin, hemicellulose and cellulose components are favorable, since the leaves will present better characteristics for consumption. such effects produced by the attenuation of solar radiation can be explained due to the fact that shading reduces the availability of assimilates used for the development of secondary cell walls (kephart and buxton, 1993). additionally, this fact is relevant because the lignin and cellulose components are important substances against abiotic stress, including uv-b radiation (rozema et al., 1997). in phalaenopsis orchids, e.g., an increase in the lignin content in the leaves and roots of the plants where observed when the light intensity was increased, being this result associated with the induction of pal, cad and pod activities (copur and tozluoglu, 2008). regarding the morphological and physiological changes, the highest content of chlorophyll-a, b and total observed for the plants grown under 50% of solar radiation level is related to the need to increase the utilization of the available radiation for the plants. the current study confirmed the ability of plants to compensate the low level of radiation by increasing the number of photosynthetic pigments, which resulted in a higher radiation use efficiency by plants (fig. 1). higher chlorophyll content in plants grown under adv. hort. sci., 2017 31(4): 257-265 264 low solar radiation level is consistent with the findings of minotta and pinzauti (1996), cardillo and bernal (2006), and hazrati et al. (2016). a lower chlorophyll a/b ratio in the lowest light intensity treatment is due to chlorophyll-b being degraded more slowly in shaded environments (lee et al., 2000). information generated in the current study is relevant, as it provides valuable information to vegetable’s producers, assisting in the strategy to minimize the effect of the high solar radiation inside of the greenhouses. we proved the attenuation of solar radiation is an effective method to improve morphological and bromatological aspects of chicory. by using this simple method, both researchers and farmers might have a competitive advantage in planning their cropping systems, mainly in sites or seasons where the high light intensity is a limiting factor for chicory cropping. chicory plants grown in lower solar radiation levels are more efficient in converting solar radiation into dry matter, have a higher lipid content, chlorophyll index a, b and total, reduced leaf thickness, acid detergent fiber, cellulose, and lignin content, which confirm the hypothesis tested. in this study it was demonstrated that the use of shading screens is an effective method to attenuate the solar radiation; this is especially relevant in places or seasons where there is high light intensity, which contributes to achieve better characteristics of the cichory produced. acknowledgements the authors are grateful to the national council for scientific and technological development (cnpqbrazil), for the productivity scholarship of the coauthor braulio otomar caron and for their financial support for the authors felipe schwerz and tiago olivoto. references a b d e l g h a n y a . m . , a l h e l a l i . m . , 2 0 1 0 characterization of solar radiation transmission through plastic shading nets. j. sol. energy mater. sol. cells., 94: 1371-1378. alvares c.a., stape j.l., sentelhas p.c., moraes gonçalves j.l., sparovek g., 2013 köppen’s climate classification map for brazil. meteorol. z., 22: 711728. aoac, 1995 official 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identification of strawberry phenolics with antioxidant and human cancer cell antiproliferative properties. j. agric. food chem., 56: 670-675. impaginato 185 adv. hort. sci., 2018 32(2): 185-191 doi: 10.13128/ahs-21864 effects of nano-silver pulsing, calcium sulfate and gibberellin on an antioxidant molecule and vase life of cut gerbera flowers s.-s. shafiee-masouleh department of genetics and breeding, ornamental plants research c e n t e r ( o p r c ) , h o r t i c u l t u r a l s c i e n c e s r e s e a r c h i n s t i t u t e ( h s r i ) , agricultural research education and extension (areeo), mahalatt, iran. key words: antioxidant, flavonoid, ga4+7, gerbera jamesonii. abbreviations: nano-silver= ns; deionized water= di; calcium sulfate= cs; gibberellin4+7 = ga; anthocyanin leakage= al; total soluble solids= tss. abstract: the aim of this study was to evaluate interactions between ns coupled with cs and ga on flavonoid, cell membrane behavior and extending the vase life of cut gerbera. pulse treatments of flowers were conducted in ns at concentrations of 0 (di), 3 or 9 mg/l for 24 h. then, flowers were treated with preservative solutions containing calcium sulfate (0, 10 or 20 mm) and ga4+7 (0 or 20 mg/l), plus 1.5% sucrose in all preservative solutions. pulse treatments with 3 or 9 mg ns/l and holding in solution containing 20 mm cs compared to the control treatment (holding in the solution of sucrose following pulse treatment in di) significantly extended vase life by 8 days. according to the antioxidant role of flavonoids, and lower amounts of flavonoid in the flowers that pretreated with ns, therefore, it may be said that ns prevented from microbial attack. 1. introduction gerberas (gerbera jamesonii) are well-known flowers for the variety of their colors, and are popular in the world flower trade (liu et al., 2009 a; solgi et al., 2009). however, often the growers and florists suffer further loss from short vase life of gerberas. for example, the mean of vase life in some cultivars of gerbera (‘bayadere’ and ‘sunway’) are reported only between 6 to 10 days when water tap is used (shabanian et al., 2018). gerberas are ethylene insensitive, but bacterial plugging of the xylem is a main cause of early and rapid senescence in their cut flowers (liu et al., 2009 a). the decrease of water uptake and consequently the increase of the ratio between transpiration and water uptake (i.e., high value of (*) corresponding author: shafiee.masouleh@areeo.ac.ir citation: shafiee-masouleh s.-s., 2018 effects of nanosilver pulsing, calcium sulfate and gibberellin on an antioxidant molecule and vase life of cut gerbera flowers. adv. hort. sci., 32(2): 185-191 copyright: © 2018 shafiee-masouleh s.-s. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 10 october 2017 accepted for publication 12 january 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(2): 185-191 186 water balance) will be caused as a result of xylem obstruction, and will end the cut flower vase life. nano-silver (ns) pulse and continuous treatments for cut flowers are newly used and are known as novel agents of anti-microbial (liu et al., 2009 a; solgi et al., 2009; lü et al., 2010). as a novel antiseptic, ns is used in the medical industry, silver embedded fabrics, water purification and vegetable disinfection. due to their high surface area to volume ratio, among other unique chemical and physical properties, ns formulations provide full contact with microorganisms and are highly effective as germicides. ns particles can connect the cell membranes and penetrate into bacteria. then, ns can disrupt the respiration and cell division and cause the cell death. ns releases silver ions (ag+) within bacterial cells, silver ions have bactericidal activity (liu et al., 2009 a; solgi et al., 2009; nair et al., 2010; sharon et al., 2010; lü et al., 2010; liu et al., 2012). naghsh (2010) described the inhibited meiosis in aspergillus niger due to ns activity. alavi and dehpour (2010) reported that the nano-silver solution is effective on greenhouse cucumber downy mildew disease. liu et al. (2009 a) and lü et al. (2010) observed that ns pulse treatments extended vase life of the cut gerbera and the rose flowers. also, solgi et al. (2009) reported that ns continuous treatments inhibited the growth of bacteria in the solution and xylem vessels and increased vase life of cut gerbera flowers. calcium increase postharvest longevity of fresh cut flowers (gerasopoulos and chebli, 1999; de c a p d e v i l l e e t a l . , 2 0 0 5 ; s o s a n a n , 2 0 0 7 ) . t h i s increased postharvest longevity may be due to a delay of physiological events related to senescence, such as a decrease in water uptake, increased water transpiration loss, decreased fresh weight, stem bending (sosa nan, 2007). since the level of soluble carbohydrates will be maintained by the treatment of gibberellin (ga) (ranwala and miller, 2000; whitman et al., 2001; hatamzadeh et al., 2010), therefore, ga can have a positive effect on the water balance. furthermore, sucrose in the preservative solutions maintains water balance, in addition to act as a food source (solgi et al., 2009). the objective of this research was to evaluate the interactions of calcium sulfate and gibberellin continuous treatments by ns pulse treatments on flavonoid as an antioxidant component, and vase life of cut gerberas. flavonoids have antioxidant effects and can be effective on the vase life. antioxidant molecules can be efficient systems to protect cells against pathogen and water deficit-induced oxidative stress, and this prevents the senescence and cell death (shabanian et al., 2018). 2. materials and methods plant material cut gerbera (gerbera jamesonii cv. pink elegance) flowers that were grown in standard hydroponic greenhouse conditions were purchased from a flower and plant growing company (pakdasht, tehran, iran). flowers were harvested by pulling the stems off in the plants when 2-3 rows of stamens of the bisexual disc florets were mature (gerasopoulos and chebli, 1999; solgi et al., 2009) in the morning. stem bottom of harvested flowers was put in the flower capsule containing deionized water (di). flowers were packed and transported within 8 h to the laboratory. in the laboratory, stems were re-cut to a length of 45 cm into the di to remove air emboli (liu et al., 2009 a; solgi et al., 2009). flowers were re-cut 2-3 times, when it was necessary. the flowers were placed in a controlled environment room at 20±2°c with 60±10% r.h. and 12 µmol/m.s. light intensity (cool white fluorescent lamps; 12 h/day). experimental design and treatments solutions of pulse treatment were prepared in two concentrations of ns (3 or 9 mg/ l), and di was used as a control treatment. flowers were treated f o r 2 4 h w i t h t h e t w o c o n c e n t r a t i o n s o f n s (nanonasb-pars company, iran) or di. each pulse treatment contained 18 flowers. following pulse treatment, the flowers were individually kept into 1000 ml glass vases containing 500 ml of fresh solutions (as continuous treatment) that were prepared at second day of the experiment and were not renewed. in the continuous treatments, three concentrations (0, 10 or 20 mm) of calcium sulfate, cs, (caso42h2o, merck company), and two concentrations (0 or 20 mg/ l) of ga4+7 (serva company, usa) were used. in the all continuous treatments, sucrose 1.5% was used. there were three replications and three samples per treatment in a completely randomized design as factorial experiment. each sample was one flower per bottle. data were analyzed using three-way analysis of variance (proc glm), and the means shafiee-masuleh antioxidant molecule and vase life of cut gerbera flowers 187 were compared by tukey’s test (hsd) at p≤0.05 using sas (9.1) statistical software. correlation coefficients between vase life and cell conditions and flavonoid were conducted by spss (version 11.5). regression analysis (path analysis) was taken to determine the major factors that affect vase life (dependent variable). the independent explanatory variables were anthocyanin leakage, tissue ph, tss and flavonoid. the software used for path analysis was spss/pc+ “stepwise” (version 11.5). vase life vase life was recorded from harvest time by the time the flowers showed symptoms of petal wilting or curling, stem bending (≥90°) or breaking, therefore, the flowers were visited daily. total flavonoid assay in termination of the vase life, to extract total flavonoid, 20 ml of acidic methanol (1% hcl) was added to 0.2 g fresh weight of petals, and the mixture was stirred for 48 h in the dark. the extract was used to measure total flavonoid content immediately (chang et al., 2002). total flavonoid content was measured by aluminum chloride colorimetric assay (chang et al., 2002; kumar et al., 2008). an amount of 200 µl of plant extract was added to 600 µl of methanol, 40 µl alcl3 (10%), 40 µl of potassium acetate (1 m) and was made to 2000 µl by distilled water. the solution was vigorously mixed and after keeping at room temperature in the dark for 30 min, the absorbance was measured against reagent blank at 510 nm with a spectrophotometer (t80+ uv/vis spectrometer, pg instruments ltd). the calibration curve of standard solutions of catechin (5-40 µg/ ml of 1% hcl in methanol) was drew (y= 0.0003x + 0.1654, r2= 0.9989). total flavonoid content of flower was expressed as mg catechin equivalent per 100 g of fresh weight. anthocyanin leakage to evaluate the effects of treatments on the cell membrane structure, anthocyanin leakage was measured to observe the stability of plasma membrane. at tenth day of vase life, 0.5 g of petals was sliced to pieces of 1×1 cm, these pieces were washed in di water two times and within a period of 2 h. then 10 ml of di water was added to samples. after 12 h in 25°c, the absorbance was recorded with a spectrophotometer (t80+ uv/vis spectrometer, pg instruments ltd) at 525 nm (poovaiah, 1979). tissue ph measurement tissue ph was measured according to the method of hill (1999) to consider the treatment influences on the conditions of cell reactions. in termination of vase life, 2 g of petals was crushed in liquid n2, and then was placed at -80°c for 48 h. the frozen tissues were removed from -80°c, thawed at 20°c, then were frozen in liquid n2 again and placed at -80°c for a further 36 h. after thawing at 20°c again, 25 ml distilled water was added to tissues in the test tube, then were frozen at -20°c for 24 h. the ph of filtered fluid was recorded after thawing with a ph meter. total soluble solid total soluble solid (tss) of petal juice was measured with a refractometer (ceti, belgium) as °brix (roein et al., 2009). 3. results and discussion interactions between ns and cs significantly extended the vase life (fig. 1). ns pulse treatments and then holding in preservative solutions (10 or 20 mm cs) extended the vase life compared to di pulsing and preservative solution containing sucrose (control treatment) (fig. 1). however, no significant (p<0.05) difference was found among various concentrations of ns and cs to extend the vase life. also, interactions between cs and ga4+7 had significant effect on the extension of the vase life (p<0.05) (fig. 2). the longest vase life was found in the pulse treatment with 3 mg ns/l and preservative solution containing 20 mm cs. it was 7.5 days (i.e., 62.5%) added to vase life compared to the control (fig. 1). gerbera is insensitive to ethylene (liu et al., 2009 a); therefig. 1 interactions between nano-silver (0, 3 or 9 mg/l) and calcium sulfate concentrations (= 0 mm; = 10 mm; ▲= 20 mm) on the vase life (days) in cut gerbera flowers. the means with same letters have no significant differences (msanova = 6.27; type 1 error, hsd0.05), n=3. adv. hort. sci., 2018 32(2): 185-191 188 fore, ns effects for extending the vase life of gerbera are not related to anti-ethylene effects of ns. therefore, it must be explained that the basic role of ns is to prevent from bacterial plugging of the xylem (liu et al., 2009 a, b; solgi et al., 2009; chaloupka et al., 2010; lü et al., 2010); then increasing water uptake and calcium. according to gerasopoulos and chebli (1999), post-calcium uptake prevents appearing the symptoms of the end of vase life in gerbera [wilting, petal curling, stem bending (in this cultivar was not observed) and breaking (in this cultivar was observed partially)]. the longest vase life was obtained in preservative solutions containing 10 mm cs without ga4+7 [approximately 19 days, i.e., 32.3% more than treatment with 20 mg/l ga, without cs, which had significant (p<0.05) differences with solutions containing 20 mg l-1 ga4+7 without cs] (fig. 2). gibberellin can enhance hydrolization of starch to glucose, and during enzymatic process sucrose will be produced. more production of sucrose causes strength of cell walls. having more sugar in tissues preserves them of early disruption and increases their longevity (halevy and mayak, 1981). also, whitman et al. (2001) determined that ga4+7 sprayed t o l i l i u m l o n g i f l o r u m h a d a p o s i t i v e e f f e c t t o decrease foliar chlorosis and increased vase life contrary to our results; moreover, it decreased the effect of cs. there were significant interactions (p<0.05) between ga, cs and ns on total flavonoid at the end of vase life (table 1). the highest flavonoid content table 1 the effect of nano-silver pulsing and continuous treatments by calcium sulfate and gibberellin on the biochemistry factors of cut gerbera flowers fig. 2 interactions between calcium sulfate (0, 10 or 20 mm) and ga4+7 concentrations (= 0 mg ga4+7/l; = 20 mg ga4+7/l) on the vase life (days) in cut gerbera flowers. the means with same letters have no significant differences (msanova = 1.41; type 1 error, hsd0.05), n=3. treatments flavonoid (mg equivalent catechin per 100 g f.w.) anthocyanin leakage (absorbance in 525 nm) tissue ph tss (° brix)pulse treatment continuous treatment ns (mg/ l) csz (mm) ga (mg/ l) 0 0 0 3933.3 bcd 0.165 a 4.98 abcd 11.2 a 20 2350.0 d 0.160 abc 5.15 a 10.8 ab 10 0 4516.7 abcd 0.161 abc 4.94 bcd 11.1 a 20 3877.8 bcd 0.161 abc 4.98 abcd 11.3 a 20 0 8183.3 ab 0.160 abc 4.91 bcd 11.2 a 20 8933.3 a 0.157 bc 4.94 bcd 9.9 ab 3 0 0 5711.1 abcd 0.159 abc 4.98 abcd 9.4 ab 20 2988.9 cd 0.163 ab 5.01 abc 10.0 ab 10 0 5655.6 abcd 0.155 c 4.88 bcd 9.0 ab 20 3433.3 bcd 0.157 bc 4.83 cd 8.9 ab 20 0 7544.4 abc 0.156 bc 4.86 cd 8.8 ab 20 5377.8 abcd 0.155 c 4.83 cd 7.9 b 9 0 0 2933.3 cd 0.156 bc 4.96 abcd 9.8 ab 20 3100.0 cd 0.157 bc 5.05 ab 10.4 ab 10 0 2766.7 cd 0.155 c 4.85 cd 8.3 ab 20 5322.2 abcd 0.156 bc 4.89 bcd 8.5 ab 20 0 6488.9 abcd 0.156 bc 4.82 d 9.5 ab 20 3711.1 bcd 0.157 bc 4.86 cd 8.9 ab anova (mean of square) ns 815234.8 ns 0.0001 ** 0.04 ** 18.97 ** cs 50446502.1 ** 0.0001 ** 0.12 ** 4.47 * ga 12438400.2 * 0.00000002 ns 0.02 * 0.74 ns ns x cs 6853137.9 y 0.00002 * 0.0008 y 1.56 y ns x ga 696546.46 y 0.00002 * 0.01 y 0.47 y cs x ga 2485082.3 y 0.000003 y 0.01 y 1.52 y ns x cs x ga 5213477.41 y 0.00001 y 0.0009 y 0.1 y the means with different letters are significant (hsd0.05). n= 3.** = significant at p≤0.01; * = significant at p≤0.05; ns = not significant. y= type i error. shafiee-masuleh antioxidant molecule and vase life of cut gerbera flowers 189 was measured in the petals of flowers that were treated with di and kept in the solution containing 20 mm cs and 20 mg/l ga. whereas treated flowers with di and kept in the solution containing 20 mg/l ga showed the lowest total flavonoid. the antioxidant role of flavonoids was revealed for the flowers that were not pulsed by ns, because, the microbial attack might be a signal to synthesize the flavonoids (khatiwora et al., 2010). when calcium (meyer et al., 1973) and gibberellin (ranwala and miller, 2000; hatamzadeh et al., 2010) were made available, they activated the reducing of nitrate to produce phenylalanin, and to form simple carbohydrates, respectively. therefore, the pathway of flavonoid synthesis was completed and total flavonoid was increased. phenylalanie transforms into 4-coumaroyl-coa in the phenylpropanoid pathway, and finally enters the flavonoid synthesis pathway (falcone ferreyra et al., 2012). the most significant interactions (p<0.05) were observed between ga, cs and ns on the anthocyanin leakage. in the flowers which were not treated with ns and/or cs, anthocyanin leakage was highest (table 1). the stability of cell membrane will have been decreased by factors as senescence, microbial (attacking by micro-organism) or no microbial (deficit of calcium) diseases. the measurement of anthocyanin leakage at half of vase life could be a gauge to evaluate the stability of cell membrane. the accumulation of calcium in middle lamella of cell wall increases the stability of cell membrane and decreases anthocyanin leakage (nikbakht et al., 2008). ns prevents microbial attack and decreases senescence and keeps stability (liu et al., 2009 a; solgi et al., 2009; lü et al., 2010). the most tissue ph was recorded at pre-treatment with di and keeping in solution containing 20 mg ga/l, and the least tissue ph was at pulsing with 9 mg ns/l and keeping in calcium sulfate solution (20 m m ) . g e n e r a l l y , s i g n i f i c a n t d i f f e r e n c e s w e r e observed between treatments (p<0.05) (table 1). schmitzer et al. (2010) explained that increasing the cell sap ph causes the developing flowers from the bud to senescence stage. the most tss was measured in the flowers of control treatment, and the least tss was recorded in the flowers which were pulsed with 3 mg ns/l and kept in solution containing 20 mm cs and 20 mg ga/l. significant differences (p<0.05) were observed between two mentioned treatments. however, no one has the significant differences with other treatments. gebremedhin et al. (2013) interpreted that tss will be increased by more water uptaking to provide the required substrate for respiration. the analysis of correlation coefficients (table 2) shows the negative significant correlation (p≤0.01) between vase life and anthocyanin leakage and tss. furthermore, there is positive significant correlation between anthocyanin leakage and tss (table 2). for the last parameter, the coefficient of multiple determinations (r2) was 0.479 in linear model for the vase life (table 3). this coefficient gives the proportion of the total variation in the dependent variable (vase parameters vase life al tissue ph tss flavonoid vase life 1 al -0.656 ** 1 tissue ph -0.353 0.412 1 tss -0.692 ** 0.757 ** 0.375 1 flavonoid -0.338 0.253 -0.373 0.206 1 vase life linear model variable b se b standard β t significance constant 32.265 4.04 7.987 0 tss -1.586 0.414 -0.692 -3.832 0.001 multiple r 0.692 r2 0.479 adjusted r2 0.446 standard error 1.82 anova sum of squares df mean squares f significance regression 48.834 1 48.834 14.688 0.001 residual 53.197 16 3.325 total 102.031 17 ** significant in p<0.01 (two-tailed correlations), n=18. table 3 vase life of gerbera flowers regressed (stepwise regression) against anthocyanin leakage, tissue ph, tss and flavonoid table 2 correlation coefficients between vase life (days), anthocyanin leakage (al), tissue ph, total soluble solids (tss) and flavonoid (mg equivalent catechin per 100 g f.w.), n=3 adv. hort. sci., 2018 32(2): 185-191 190 life) explained by the predictors included in the model. thus, from among four independent variables, total soluble solids explained 47.9% of the observed total variation in the vase life, and other independent variable (anthocyanin leakage, tissue ph and flavonoid) had a lesser role in the vase life. furthermore, the test statistic in linear model s h o w e d t h a t c o e f f i c i e n t o f t s s n e g a t i v e l y a n d significantly (p≤0.01) influenced vase life (table 3, fig 3). 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longiflorum. sci. hort., 89: 143-145. impaginato 23 adv. hort. sci., 2019 33(1): 23-31 doi: 10.13128/ahs-23015 biochemical characterization of artichoke (cynara cardunculus var. scolymus l.) spring genotypes from marche and abruzzo regions (central italy) a. galieni, 1 (*) f. stagnari 2, m. pisante 2, c. platani 1, n. ficcadenti 1 1 centro di ricerca orticoltura e florovivaismo, crea, via salaria, 1, 63077 monsampolo del tronto (ap), italy. 2 facoltà di bioscienze e tecnologie agroalimentari e ambientali, università degli studi di teramo, via r. balzarini, 1, 64100 teramo, italy. key words: antiradical activity, artichoke genotypes, capitula quality traits, total phenolic content. abstract: ten artichoke genotypes from marche and abruzzo regions [ascolano (as), castorano (cs), clone monsampolo, jesino (je), mazzaferrata (mz), montelupone a, montelupone b, urbisaglia1 (ub_1), urbisaglia2 and violetto tardivo di pesaro] were characterized for their quality traits and peculiar enduse attitudes, in comparison with the reference romanesco clone c3 (cl_c3). total polyphenols content (tpc), total flavonoid content (tfc) and antiradical activity were assessed in the receptacle and external bracts of both main and first order capitula. cl_c3 showed high tpc and tfc values in the receptacle of the main flower heads (7.4 mg gallic acid equivalents, gae, g-1 dry weight, dw, and 3.6 mg rutin equivalents, rue, g-1 dw, respectively), confirming its attitude for fresh consumption. je and ub_1 showed great and stable (among main and first order capitula) head quality, highlighting their potential for breeding programs to enhance the content of functional compounds. conversely, mz and as could be appreciable for processing or pharmaceutical applications, being characterized by great tpc (external bracts, first order capitula: 2.8 and 2.7 mg gae g-1 dw, respectively) and tfc (external bracts, first order capitula: 2.1 and 2.7 mg gae g-1 dw, respectively) values in the waste parts. high correlations between tpc and tfc with antiradical activity were also observed. our results suggest the possibility to promote the utilization in genetic breeding programs of the autochthonous artichoke populations, according to their peculiar characteristics, including also their biochemical composition. 1. introduction globe artichoke [cynara cardunculus l. var. scolymus (l.) fiori] belongs to the family of asteraceae (compositae) and it is an herbaceous perennial crop mainly cultivated in the mediterranean basin (about 65% of world production) followed by americas and china (sihem et al., 2015, lombardo et al., 2017). in italy it plays an important role in the agro-food (*) corresponding author: angelica.galieni@crea.gov.it citation: galieni a., stagnari f., pisante m., platani c., ficcadenti n., 2019 biochemical characterization of artichoke (cynara cardunculus var. scolymus l.) spring genotypes from marche and abruzzo regions (central italy). adv. hort. sci., 33(1): 23-31 copyright: © 2019 galieni a., stagnari f., pisante m., platani c., ficcadenti n. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 9 april 2018 accepted for publication 12 december 2018 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(1): 23-31 24 chain with over 43.8 kha and approximately 366 kt of floral heads produced (fao, 2016). the italian gene pool of globe artichoke includes hundreds of varieties and ecotypes, grouped into four main types i.e. “catanesi”, “romaneschi”, “spinosi” and “violetti”. according to the harvesting period, they are classified as early or late distinct clonal varietal groups, with the former having a typical autumn-winter cycle in the southern regions, while late including spring genotypes mainly grown in central regions (ciancolini et al., 2013 a, b). the varietal constitution of artichoke is restricted to clonal selection carried out within local populations propagated by agamic way which represent a patrimony of agrobiodiversity and a biological, cultural and economic heritage (ficcadenti et al., 2013). however, several cases of homonymy and synonymy (different varieties are called with the same name in the first case, while the same variety comes call with different names in the second) as well as unsatisfactory, uniformity and identity of accessions occur (ficcadenti et al., 2013). traditional agricultural and food production must be safeguarded to avoid processes of globalization and homologation and, consequently, identification, collection, characterization and conservation of agrobiodiversity as well as development of genetic improvement strategies, particularly linked to nutritional and organoleptic traits, have been undertaken in these years (mauromicale and ierna, 2000; ficcadenti et al., 2010; ciancolini et al., 2012). recently, a renewed and growing interest for artichoke cultivation has been observed worldwide mainly due to its potential uses as functional food: its large immature inflorescences, called capitula or heads, represent a rich source of bioactive compounds including polyphenols with a strong antiradical activity (schütz et al., 2004) inulin, fibres and minerals (lattanzio et al., 2009; lombardo et al., 2010; pandino et al., 2011). furthermore, the utilization of by-products of artichoke processing (i.e. external bracts) involves animal feedstuff (megías et al., 2002) or extraction of functional molecules (larossa et al., 2002). it emerges that the types and amount of bioactive substances and their activity (i.e. polyphenols content and the related antioxidant activity) could be used to characterize and select specific genotypes. nowadays, only few studies have investigated on polyphenols content, discriminating among the different head parts of artichoke (see for example f r a t i a n n i e t a l . , 2 0 0 7 ; l o m b a r d o e t a l . , 2 0 1 0 ; soumaya et al. 2013; sihem et al., 2015); besides, the simultaneous determinations of total polyphenols and flavonoids content with radical scavenging capability, have not been considered at all. consequently, in the present work we aimed at investigating such important biological properties in eleven spring accessions of artichoke collected in central italy (marche and abruzzo regions). the primary objectives were to obtain a preliminary: (i) genotype’s characterization of the selected central-italy artichoke accessions from a biochemical point of view; (ii) evaluation of the suitability of the selected artichoke genotypes for fresh consumption or by-products production. 2. materials and methods plant material, management practices and head sampling the study was carried out in 2014 at the experimental field of the research centre for vegetable and ornamental crops, council for agricultural research and economics (crea-of), located in monsampolo del tronto (ap) (latitude 42°52’59.1” n, longitude 13°48’01.9” e), in the coastal area of the marche region (central italy) a typical area for globe artichoke cultivation. t e n a r t i c h o k e a c c e s s i o n s f r o m m a r c h e a n d abruzzo regions, named as “clone monsampolo” (cl_msp), “ascolano” (as), “castorano” (cs), “jesino” (je), “mazzaferrata” (mz), “montelupone a” (ml_a), “montelupone b” (ml_b), “urbisaglia1” (ub_1), “urbisaglia2” (ub_2) and “violetto tardivo di pesaro” (vt_ps), were collected on the base of their peculiar sensory features and were compared with the reference genotype “romanesco clone c3” (cl_c3), characterized by high market standards of the flower heads (purple with green shades, round shape, regular size and thick consistency). the selected globe artichoke genotypes differ for their biological and morphological profiles, as briefly synthetized in table 1. plant material (shoots, named “carducci”) was transplanted in august 2011 in rows spaced 1.00 m apart with row spacing of 1.20 m; each plot (artichoke genotype) consisted of thirty plants. the fertilization program, typical of the area, consisted in: 150 kg ha-1 of n, 80 kg ha-1 of phosphorus pentoxide (p2o5) and 100 kg ha-1 of potassium oxide (k2o), respectively. the experimental field was kept weedfree by mechanical weed control and no pest control was needed. galieni et al. biochemical differences among artichoke genotypes 25 at the marketing stage, six capitula per artichoke genotype were harvested, without floral stem, in two subsequently times (10th april and 10th may, considered as early and mid-spring), allowing to compare both main (first sampling data) and first order (second sampling data) capitula. each flower head was separated into ‘external bracts (∼15 bracts)’ (waste part) and ‘receptacle’ (edible fraction), freeze-dried, homogenized and stored at -20°c until biochemical characterization. chemical analysis the extraction of polyphenols and flavonoids were carried out as described by gouveia and castilho (2012 a). the folin-ciocalteu reagent method was used to evaluate the total polyphenols content (tpc) of the external bracts and receptacle following the method of gouveia and castilho (2011). plant extracts were dissolved in methanol (10 mg ml-1); aliquots of 50 µl were added to 1.25 ml of folin-ciocalteu (dilution, 1:10) and 1.0 ml of a 7.5% na2co3 solution. solutions were maintained at room temperature for 30 min and the tpc was determined at 765 nm using a beckman du640b spectrophotometer (beckman coulter, brea, california, usa). gallic acid standard solutions were used to calibrate the method, so results were expressed as mg gallic acid equivalents (gae) per g-1 dry weight (dw). total flavonoids content (tfc) was calculated following the procedure described by gouveia and castilho (2012 a) and estimated as rutin equivalents (rue), i.e. expressed as mg rue g-1 dw. methanolic solutions (500 µl of sample solution) of the plant extracts (2.5 mg ml-1) were mixed with 1.5 ml of methanol, 2.8 ml of water, 100 µl of potassium acetate (1 m) and 100 µl of aluminium chloride (10% in methanol). the absorbance of reaction mixture was read after 30 min at room temperature and at 415 nm using a beckman du640b spectrophotometer. the radical scavenging activity of the extracts was determined using the stable radicals: (i) 2,2’-azinob i s ( 3 e t h y l b e n z o t h i a z o l i n e 6 s u l p h o n i c a c i d ) teac/abts assay (abts) (re et al., 1999), modified as described by gouveia and castilho (2012 a); and (ii) 2,2-diphenyl-1-picrylhydrazyl dpph assay (gouveia and castilho, 2012 b). in each assay, trolox was employed as reference standard and results were expressed as µmol trolox equivalent (te) g-1 dw. reagents and solvents were purchased from sigma chemicals co. (st. louis, mo). all reagents were of analytical grade. statistical analysis in order to test (f-test) the effect of genotype on all the investigated variables, a one-way analysis of variance (anova) was performed. the experiment was conducted following a complete randomized design and each sampled capitula represented a single repetition. when significant differences were detected, the means were compared based on the standard error of the difference (sed) between means, with significance being assigned using the least significant difference (lsd) value at the 5% (p<0.05) level of significance. before the anova, the d a t a w e r e a n a l y z e d t o t e s t f o r n o r m a l i t y a n d homoschedasticity assumptions, through graphical methods. to interpret and summarize the association between treatments (artichoke genotypes: cl_c3, cl_msp, as, cs, je, mz, ml_a, ml_b, ub_1, ub_2 and vt_ps) and variables (tpc, tfc, abts, dpph) the principal component analysis (pca) was applied. the pca was performed separately for main and first order capitula; each principal component (pc) was table 1 head characteristics of the eleven selected genotypes of globe artichoke genotype acronym colour of outer bracts colour of inner bracts bracts "clone c3" cl_c3 green with purple shades yellow spineless "clone monsampolo" cl_msp green yellowish-green spineless "ascolano" as purple with green shades yellow-greenish with purple shades spineless "castorano" cs purple with light green shades yellow-purple spineless (but mucronate) "jesino" je purple with green shades yellow purple spineless "mazzaferrata" mz purple with green shades yellow-greenish spineless "montelupone a" ml_a purple with green shades yellow-purple spineless "montelupone b" ml_b purple with green shades yellow-purple spineless (but mucronate) "urbisaglia 1" ub_1 purple yellow-purple spineless "urbisaglia 2" ub_2 purple yellow-purple spineless "violetto tardivo ps" vt_ps purple yellow-purple spine adv. hort. sci., 2019 33(1): 23-31 26 calculated as a linear combination of the standardized original variables by using the eigenvectors of the correlation matrix. the results were visually explored in a two-dimensional pca correlation biplot: standardized pc1 and pc2 scores were plotted as symbols, while the correlations between pcs and standardized variables (factor loadings) were plotted as vectors. statistical analyses were performed with the r software (r core team, 2017). 3. results and discussion the tpc and tfc in the receptacle and external bracts of main and first order capitula of the eleven artichoke genotypes, are reported in table 2. tpc ranged from 1.5 to 9.2 mg gae g-1 dw, while tfc ranged from 1.8 to 4.1 mg rue g-1 dw, matching with the literature data or, in some circumstances, resulting slightly higher (lombardo et al., 2010; pandino et al., 2011; gouveia and castilho, 2012 a; pandino et al., 2012 a; sihem et al., 2015; dabbou et al., 2017; marques et al., 2017; petropoulos et al., 2017). both traits were significantly (p<0.05) influenced by genotype: differences are related to both head part (receptacle or external bracts) and their location on plant architecture (main or first order capitula) (table 2). clear trends were observed: je gave the highest tpc content (5.1 mg gae g-1 dw, averaged over head parts and harvest time), followed by ub_1, ub_2 and vt_ps (5.0, 5.0 and 4.3 mg gae g-1 dw on average, respectively), while cl_msp resulted as one of the worst genotypes in terms of polyphenols concentratable 2 total polyphenols content [tpc, mg gallic acid equivalents (gae) g-1 dry weight (dw)] and total flavonoids content [tfc, mg rutin equivalents (rue) g-1 dw] in the receptacle and in the external bracts of different artichoke genotypes genotype§ main capitula first order capitula tpc (mg gae g-1 dw) tfc (mg rue g-1 dw) tpc (mg gae g-1 dw) tfc (mg rue g-1 dw) receptacle cl_c3 7.4 ± 0.11 3.6 ± 0.23 3.8 ± 0.57 2.5 ± 0.38 cl_msp 3.6 ± 0.28 1.9 ± 0.16 3.1 ± 0.09 1.9 ± 0.12 as 5.7 ± 0.42 2.4 ± 0.21 3.9 ± 0.55 2.2 ± 0.19 cs 6.3 ± 0.91 2.5 ± 0.30 2.6 ± 0.03 1.9 ± 0.08 je 8.2 ± 1.09 3.3 ± 0.44 5.4 ± 0.47 3.0 ± 0.34 mz 4.9 ± 0.73 2.8 ± 0.17 6.8 ± 1.35 3.5 ± 0.61 ml_a 5.8 ± 1.34 4.1 ± 1.01 2.1 ± 0.19 1.8 ± 0.19 ml_b 4.3 ± 0.22 2.3 ± 0.31 4.0 ± 0.64 3.1 ± 0.66 ub_1 8.0 ± 0.58 3.4 ± 0.36 4.7 ± 0.66 2.9 ± 0.69 ub_2 9.2 ± 1.55 3.9 ± 0.74 4.4 ± 0.15 2.6 ± 0.15 vt_ps 6.8 ± 0.45 3.5 ± 0.42 4.2 ± 0.45 3.2 ± 0.45 f-test ** * ** * sed 1.2 0.7 0.9 0.6 external bracts cl_c3 3.2 ± 0.12 2.6 ± 0.07 2.1 ± 0.06 2.3 ± 0.18 cl_msp 2.2 ± 0.15 2.0 ± 0.00 1.7 ± 0.11 2.0 ± 0.10 as 3.0 ± 0.19 2.4 ± 0.08 2.7 ± 0.42 2.7 ± 0.30 cs 2.9 ± 0.28 2.1 ± 0.17 2.1 ± 0.04 2.6 ± 0.20 je 3.9 ± 0.53 2.5 ± 0.18 3.0 ± 0.46 2.9 ± 0.56 mz 2.4 ± 0.14 2.5 ± 0.08 2.8 ± 0.16 2.1 ± 0.04 ml_a 2.1 ± 0.25 2.4 ± 0.25 1.5 ± 0.08 2.3 ± 0.02 ml_b 2.1 ± 0.16 2.0 ± 0.07 2.2 ± 0.28 2.3 ± 0.28 ub_1 4.8 ± 0.30 2.9 ± 0.17 2.4 ± 0.03 2.3 ± 0.22 ub_2 4.1 ± 0.61 2.7 ± 0.25 2.2 ± 0.19 2.3 ± 0.02 vt_ps 4.0 ± 0.11 3.2 ± 0.05 2.2 ± 0.05 3.0 ± 0.21 f-test ** ** ** ns sed 0.4 0.2 0.3 data refer to both main and first order capitula (two different harvest times, at early and mid-spring 2014). means ± standard errors of n=6 independent replicates are reported. * p<0.05; ** p<0.01; *** p<0.001; ns = not significant. sed, standard error of differences between means. § the list of the used acronomys is reported in table 1. galieni et al. biochemical differences among artichoke genotypes 27 tion in artichoke heads (on average 2.7 mg gae g-1 dw) together with ml_a and ml_b (on average 2.9 and 3.2 mg gae g-1 dw, respectively) (table 2). these results were quite confirmed by tfc data (table 2), indicating those genotypes’ suitable for fresh consumption rather than food processing. lower antioxidant compounds (i.e. polyphenols) is, indeed, considered a qualitative trait required by industry, thanks to the scarce propensity to enzymatic browning phen o m e n a a f t e r c u t t i n g a n d s t o r a g e o p e r a t i o n s (lattanzio et al., 1994; lombardo et al., 2010). the reference genotype (cl_c3) confirmed its high value for fresh consumption, registering higher tpc and tfc, only in the combination early-spring harvest (main capitula)/receptacle (table 2), mostly appreciated by consumers and with the highest commercial value. as previously observed (fratianni et al., 2007; lombardo et al., 2010; pandino et al., 2011; pandino et al., 2012 b; pandino et al., 2013 a; sihem et al., 2015), polyphenols were not uniformly distributed in the different floral head parts (fig. 1): regardless of the harvest time, higher tpc values were observed in the receptacle (5.2 mg gae g-1 dw, averaged over genotypes) while lower in the external bracts (2.7 mg gae g-1 dw, averaged over genotypes). besides, no differences emerged in terms of tfc (2.5 vs. 2.8 mg gae g-1 dw in external bracts and receptacle respectively, averaged over genotypes). the different amount of antioxidant compounds in the various head parts is of interest to identify genotypes rich in these molecules in the by-products (external bracts) and hence interesting for the industrial processes (i.e. animal feedstuff, fiber production, recovery of functional ingredients) (femenia et al., 1998; larossa et al., 2002; megías et al., 2002; lattanzio et al., 2009). nonetheless, we observed some differences in terms of relative tpc among artichoke accessions, with particular regards in terms of relative tfc (fig. 1): the genotypes cl_msp, as and cs were characterized by the highest relative tfc in the waste products (fig. 1), suggesting useful utilization for the by-products processes, with external bracts representing a potential innovative source for flavonoid extraction. lastly, tpc values lowered in both receptacle and external bracts shifting from main to first order capitula (fig. 2a); this trend was confirmed by all the accessions with the exception of mz (fig. 2a), which gave the highest tpc values in the first order flower heads (table 2), so maintaining a high content of functional compounds during the growing cycle. a similar behavior was recorded for some of the selected artichoke accessions in terms of tfc (see for example cl_msp, as and ml_b) (fig. 2b). this was probably attributable to the environmental conditions recorded during the harvest season. despite the solar radiation levels show the stronger effect on polyphenols accumulation in the artichoke’ receptacle (pandino et al., 2013 b), in our study the lower temperatures observed in april (-10% on average with respect to may considering the mean air temperatures recorded during the first 10 days of each month) could have affected tpc and tfc, as previously observed in other crops (klimov et al., 2008; hykkerud et al., 2018). fig. 1 relative proportion (as percentage) of total polyphenols content [tpc, gallic acid equivalents (gae) g-1 dry weight (dw)] and total flavonoids content [tfc, mg rutin equivalents (rue) g-1 dw] in the receptacle and external bracts averaged over both main and first order capitula of different globe artichoke genotypes (see table 1 for the list of acronyms). data represent means ± standard errors, n=12 independent replicates. fig. 2 variations normalized to main capitula values (dashed line) of (a) total polyphenols content [tpc, mg gallic acid equivalents (gae) g-1 dry weight (dw)] and (b) total flavonoids content [tfc, mg rutin equivalents (rue) g-1 dw] in the receptacle and external bracts of different globe a r t i c h o k e g e n o t y p e s ( s e e t a b l e 1 f o r t h e l i s t o f acronyms). data represent means ± standard errors, n=6 independent replicates. 28 adv. hort. sci., 2019 33(1): 23-31 radical scavenging activity (abts and dpph assays) registered differences among thesis similar to those observed for tpc and tfc data (table 3). abts values ranged from 21.2 to 146.8 µmol te g-1 dw and dpph ranged from 12.8 to 204.3 µmol te g-1 dw, showing the same order of activity previously found in other antioxidant capacity assays on artichoke (gouveia and castilho, 2012 b; rouphael et al., 2017). the highest activity was concentrated in the receptacle (85.7 and 103.8 µmol te g-1 dw for abts and dpph, respectively vs. 41.9 and 38.3 µmol te g-1 dw for abts and dpph, respectively in the external bracts) regardless of genotype and harvesting time (table 3) (sihem et al., 2015). also for these biochemical traits, je, ub_1 and ub_2 ranged at the first positions while cl_msp as the genotype with the lowest abts and dpph values (table 3). moreover, we found significant (p<0.001) linear relationships between these variables, confirming previous results (alghazeer et al., 2012; lombardo et al., 2013). follows the higher measured pearson’s correlation coefficients: tpc vs. abts, r=0.84; tpc vs. dpph, r=0.91; tfc vs. abts, r=0.76; tfc vs. dpph, r=0.81. indeed, phenolic compounds are known to have the ability to block the chain reaction of reactive oxygen and nitrogen species through different pathways involving (i) direct reaction with free radicals, (ii) sequester metal ions able to spread the chain reaction, (iii) synergic action with other antioxidants (khasawneh et al., 2014). the relationships between tpc, tfc, abts and dpph, classified based on the analysed capitula parts table 3 radical scavenging activity (µmol trolox equivalents (te) g-1 dw) obtained from two different assays: trolox equivalent antioxidant capacity with 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (abts) and 2,2-diphenyl-1-picrylhydrazyl (dpph) in the receptacle and in the bracts of different artichoke genotypes data refer to both main and first order capitula (two different harvest times, at early and mid-spring 2014). means ± standard errors of n=6 independent replicates are reported. * p<0.05; ** p<0.01; *** p<0.001; ns = not significant. sed, standard error of differences between means. § the list of the used acronomys is reported in table 1. genotype§ main capitula first order capitula abts (µmol te g-1 dw) dpph (µmol te g-1 dw) abts (µmol te g-1 dw) dpph (µmol te g-1 dw) receptacle cl_c3 146.8 ± 6.91 165.5 ± 7.75 61.1 ± 14.29 61.3 ± 26.77 cl_msp 71.3 ± 8.85 47.3 ± 12.82 42.3 ± 7.39 31.7 ± 2.95 as 87.2 ± 14.10 109.9 ± 14.97 73.4 ± 7.23 78.2 ± 14.87 cs 97.4 ± 28.90 127.2 ± 31.56 34.4 ± 5.83 33.5 ± 7.20 je 119.5 ± 19.14 185.6 ± 34.58 76.0 ± 12.66 56.9 ± 11.52 mz 86.6 ± 19.63 108.4 ± 28.80 95.0 ± 12.93 178.2 ± 42.51 ml_a 104.2 ± 32.54 144.8 ± 57.25 31.4 ± 3.08 26.2 ± 9.39 ml_b 68.2 ± 8.77 80.0 ± 12.90 82.3 ± 19.76 47.2 ± 12.23 ub_1 140.3 ± 16.58 171.5 ± 19.28 67.5 ± 12.89 111.0 ± 26.09 ub_2 128.2 ± 27.06 204.3 ± 49.60 95.2 ± 5.22 91.7 ± 9.76 vt_ps 107.4 ± 7.40 138.2 ± 12.15 70.2 ± 16.40 84.3 ± 22.80 f-test ns * ** ** sed 42.4 16.7 28.5 external bracts cl_c3 57.3 ± 5.56 42.9 ± 4.49 37.6 ± 8.53 18.2 ± 2.77 cl_msp 27.6 ± 0.77 12.8 ± 3.78 21.2 ± 3.39 16.1 ± 2.57 as 45.5 ± 4.93 37.0 ± 4.82 40.8 ± 3.63 42.6 ± 10.25 cs 34.1 ± 8.87 38.1 ± 6.55 32.1 ± 2.04 24.2 ± 3.38 je 64.7 ± 12.85 80.5 ± 13.40 54.4 ± 10.96 53.7 ± 16.83 mz 27.5 ± 2.79 21.7 ± 5.90 48.1 ± 5.17 34.0 ± 8.95 ml_a 31.9 ± 6.81 36.6 ± 1.91 27.4 ± 3.67 17.9 ± 5.71 ml_b 27.5 ± 1.78 27.2 ± 5.04 31.5 ± 10.47 20.9 ± 7.52 ub_1 80.9 ± 8.72 93.9 ± 10.09 35.8 ± 4.19 38.4 ± 2.52 ub_2 58.8 ± 16.82 71.4 ±20.49 39.1 ± 6.21 19.1 ± 6.07 vt_ps 57.6 ± 2.45 59.4 ± 4.90 39.2 ± 6.46 36.7 ± 3.85 f-test ** ** ns ** sed 11.5 12.7 10.8 galieni et al. biochemical differences among artichoke genotypes 29 (i.e. receptacle tpc_rec, tfc_rec, abts_rec and dpph_rec and external bracts tpc_bra, tfc_bra, abts_bra and dpph_bra), and the eleven artichoke accessions, were summarized by pca. the results, on the basis of harvest time, are graphically displayed in two correlation bi-plots (figs. 3a and 3b, respectively); in table 4 are reported the factor loadings, the eigenvalues and the percentage of the explained variance. in early-spring harvesting time (i.e. referring to main capitula data), the first and second principal components explained 90.2% of the total data variability (80.7 and 9.5% for pc1 and pc2, respectively) (table 4). the variables were grouped into two distinct clusters, separated by pc2: in the upper right quadrant, we found all the chemical data related to the external bracts samples (tpc_bra, tfc_bra, abts_bra and dpph_bra) while in the bottom right s e c t i o n , t h o s e r e l a t e d t o t h e r e c e p t a c l e o n e s (tpc_rec, tfc_rec, abts_rec and dpph_rec); all the variables reached high pc1 scores (scores from 0.952 to 0.788 for tpc_rec and tfc_rec, respectively) (table 4). regarding genotypes, cl_c3, ub_2, je, vt_ps and ub_1 clustered separately on the right along pc1 and were positively associated with all the investigated variables; conversely, cl_msp exhibited the highest negative pc1 score (-1.533) followed by ml_b, mz, cs, as and ml_a (fig. 3a). with respect to the mid-spring harvesting time (i.e. referring to first order capitula data), the eigenvalues for pc1 and pc2 were 5.28 and 1.71, respectively, thus capturing 87.4% of the total data variability (table 4). again, variables were clearly separated by pc2 while genotypes by pc1 (fig. 3b). in particular, all the variables reached high pc1 scores with the exception of tfc_bra, which was mainly correlated with pc2 (scores: 0.328 and 0.886 for pc1 and pc2, respectively; table 4). je and mz showed the higher pc1 scores (1.302 and 1.568, respectively) despite they performed very differently with respect to pc2 (scores: 1.668 and -1.679 for je and mz, respectively). as a consequence, je was related to higher tpc, abts and dpph values in the waste fractions (external bracts) while mz in the edible parts (receptacle). pca proved to be a useful tool to summarize the biochemical characteristics of the different investifig. 3 two dimensional principal component analysis (pca) correlation bi-plot [main capitula/first harvest time (a) and first order capitula/second harvest time (b)]: symbols show the standardized scores on pc1 (x-axis) and pc2 (yaxis) for the eleven artichoke genotypes (see table 1 for the list of acronyms); vectors coordinates represent the correlations between standardized variables [total polyphenols content in receptacle and external bracts (tpc_rec and tpc_bra, respectively), total flavonoids content in receptacle and external bracts (tfc_rec and tfc_bra, respectively), radical scavenging activity obtained from two different assays in the receptacle and external bracts (abts_rec and abts_bra, respectively; dpph_rec and dpph_bra, respectively)] and pcs. table 4 principal component analysis (pca): factor loadings, eigenvalues and percentage of the explained variance variables main capitula first order capitula pc1 pc2 pc1 pc2 tpc_core 0.952 -0.093 0.941 -0.288 tfc_core 0.788 -0.561 0.853 -0.242 dpph_core 0.927 -0.286 0.777 -0.477 abts_core 0.916 -0.163 0.831 -0.356 tpc_bratee 0.903 0.418 0.926 0.228 tfc_bratee 0.823 0.084 0.328 0.886 dpph_bratee 0.933 0.220 0.762 0.572 abts_bratee 0.930 0.315 0.912 0.226 eigenvalue 6.455 0.761 5.284 1.711 explained variance (%) 80.692 9.515 66.056 21.382 adv. hort. sci., 2019 33(1): 23-31 30 gated artichoke genotypes, and clear conclusions could be obtained, confirming the results in terms of single investigated biochemical parameters. in particular, the reference genotype cl_c3 and the accessions ub_1, ub_2, je and vt_ps confirmed higher tpc, tfc and, consequently, antiradical activity in the main capitula, highlighting their important attitude for fresh consumption. this greater head quality was maintained during all the growing season (i.e. as quality traits of the first order capitula) only for ub_1, je and vt_ps. other genotypes, such as as and, principally, mz, were clearly characterized by higher bioactive compounds in the first order flower heads and by smaller capitula. these accessions could represent a promising potential as germplasm for future breeding programs to select elite cultivars, characterized by: (i) higher and stable quality traits suitable for fresh consumption (i.e. je); (ii) high concentrations of biochemical compounds, especially in the waste products, to be used for processing or pharmaceutical applications although further investigations on smaller and waste flower heads are needed. 4. conclusions in conclusion, our results confirm that the capitula of globe artichoke could be considered a functional food thanks to its relevant content of bioactive compounds accumulated in both receptacle and external bracts. the properties of the external bracts could be usefully exploited for other end-use purposes, although they are still edible fractions (fratianni et al., 2007; pandino et al., 2011). a great and appreciable variation among genotypes in terms of chemical composition and nutritional value exists. such biodiversity of the accessions of abruzzo and marche regions should be exploited and utilized, taking into account the 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catalase, cellulose, lignin, membrane stability index, superoxide dismutase. abstract: gerbera flower belongs to the composite family and is one of the top five cut flowers in the world in terms of production and consumption, which has a great economic value in the international flowering industry. this study was designed to evaluate whether calcium pre-harvest application, provided through 0, 0.5, 1 and 1.5% of calcium chloride (cacl2) and calcium nitrate (cano3), could extend the day of stem bending of gerbera cut flower. in the present study, we used two gerbera cultivars ‘intense’ and ‘rosaline’ as resistant and sensitive to stem bending, respectively. for evaluation of associated traits with stem bending, the produced flowers were kept in a vase solution containing 200 mg/l hydroxyquinoline with temperature conditions of 20°c. the results showed that day of stem bending of flowers extended 9.62 and 10.37 days by application of 1% cacl2 for ‘rosaline’ and ‘intense’ respectively. all treatments were effective in the increasing relative water content of flower due to increase water uptake. the results also revealed that the calcium pretreatment delayed flowers senescence and increased antioxidant enzyme activity. application of calcium resulted in an increase in membrane stability index in the cut flowers of both cultivars, providing evidence for delay of senescence in calcium-treated cut flowers. also, results showed that calcium application significantly increased lignin, cellulose and hemicellulose content of both cultivars. the maximum and the minimum lignin and cellulose content were observed in resistant and sensitive cultivars, respectively. in general, pre-harvest application of calcium (especially 1% cacl2) with increasing of antioxidant enzyme activity and stem lignification led to decreasing of stem bending disorder in both cultivars. 1. introduction gerbera is one of the most important ornamental plants in the world (*) corresponding author: m.hassanpour150@gmail.com citation: aghdam m., hassanpour asil m., ghasemnezhad m., mousavi mirkalaei s.a.a., 2019 effects of pre-harvest applications of different source of calcium on the cell wall fractions and stem bending disorder of gerbera (gerbera jamesonii l.) cultivar flowers adv. hort. sci., 33(1): 57-65 copyright: © 2019 aghdam m., hassanpour asil m., ghasemnezhad m., mousavi mirkalaei s.a.a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 9 march 2018 accepted for publication 4 october 2018 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(1): 57-65 58 used widely as cut flower or potted flower (çelikel and reid, 2002; perik et al., 2014). gerbera, followed by rose, chrysanthemum, tulip, and lily, occupies fifth position in top ten cut flower in world in terms of its production and consumption (nair et al., 2006; perik et al., 2014). the longevity or postharvest life of gerbera flowers depends on the extent of stem bending and the lack of stem strength (naing et al., 2017; nazarideljou and azizi, 2015). in this regard, although flowers of some gerbera cultivars are apparently not wilted or faded, their stems have drooped due to their inappropriate stem strength; and based on flower quality and grading, flowers with stem bending more than 30° are not worth maintaining (çelikel and reid, 2002; perik et al., 2014). the lack of mechanical protection and cell wall lignification (especially secondary cell wall) as well as lowering stem strength are of more likely reasons for emerging stem bending in gerbera (perik et al., 2012). in addition to its remarkable effect on stem hardening and growing gerbera uprightly, lignin plays an utmost role in promoting water flow inside xylem vessels (vanholme et al., 2010). also, there is a negative relationship between the rate of stem lignification and the extent of stem bending (perik et al., 2012). accordingly, increasing stem lignification directly influences stem strength and thereby reducing stem bending of gerbera cut flowers (nazarideljou and azizi, 2015). in past years, many attempts have been made to increase postharvest life and quality of cut flowers through different techniques. in cut flowers industry, inappropriate condition and inadequate nutrition often lead to a reduction in the quality and quantity of produced gerbera flowers; and regarding these issues can economically improve gerbera flowers production (khangoli, 2001). calcium is an essential element affecting on plant growth and development; and its accumulation in plants facilities pectin polymers’ linkage so as to improve mechanical strength of stem in line with reducing stem bending and extending flowers vase life (gerasopoulus and chebli, 1999; helper, 2005; li et al., 2012). the role of intra and intercellular calcium on cell metabolism change is attributed to its effect on the membrane structure and function and cell walls (ferguson and drobak, 1988). the results of previous studies showed that calcium, due to its role in the synthesis of pectin located in cell wall of xylem vessels, facilitates water translocation in stem and inhibits stem bending (van ieperen and van gelder, 2006). also, applying calcium as pre or postharvest treatments caused to i n c r e a s e t h e v a s e l i f e o f m a n y c u t f l o w e r s (gerasopoulos and chebli, 1999; hepler, 2005; li et al., 2012; geshnizjany et al., 2014). in spite of existing such documents, the effects of calcium on reducing stem bending of gerbera cut flowers are in needs o f m o r e i n v e s t i g a t i o n s . t h e r e f o r e , i n p r e s e n t research, the effect of different calcium concentrations in forms of chloride and calcium nitrate on stem bending and other related parameters in two gerbera cultivars were scrutinized. 2. materials and methods this research was conducted using a factorial experiment in completely randomized design (crd) with four replicates and fourteen plots in each replicate and five pots in each plot (totally 280 pots) in local commercial greenhouse (persian gulf, tehran, iran). based on the sensitivity to stem bending, two cultivars of gerbera, namely ‘intense’ as a tolerant cultivar and ‘rosaline’ as a sensitive cultivar to stem bending (nazarideljou and azizi, 2015), were used in this experiment. gerbera seedlings were cultivated in pots with 18 cm diameter filled with coco peat and perlite (1:1) maintained in greenhouse with 2025/15-18°c (day/night), 70±5% relative humidity and 14 h with a light intensity of 40 µmol-2 m-2 s-1 (naing et al., 2017). before receiving the treatments, the seedlings were feeded based on a nutritional formula used by most commercial greenhouses in iran (table 1). elements content (mmol/l) macro-elements nitrogen 10 phosphorous 1.8 potassium 5.5 magnesium 2 sulphate 3 micro-elements iron 40 magnesium 5 zinc 5 copper 1 molybdenum 1 boron 30 table 1 nutrient elements used in gerbera plant fertilization during excremental period aghdam et al. stem bending disorder of gerbera 59 after planting seedlings in pots and undergoing their perfect establishment, experimental seedlings received calcium chloride and calcium nitrate treatments at three levels (0.5%, 1%, and 1.5%) in form of foliage spray (mehran et al., 2007) while controls received distilled water. the flowers were harvested when two to three rows of ray floret got color but the stamens still did not open. after preparing of flowers stem in the length of 40 cm, they were placed in the erlenmeyer containing 500 cc distilled water and hydroxiqinolin sulphate (hqs) in concentration 200 mg/l. then, they were transferred into the ambient temperature and a light with intensity of 2 8 µ m o l 2 m 2 s 1 a n d 6 0 % r e l a t i v e h u m i d i t y (gerasopoulos and chebli, 1999). the treatments were measured on 9th of experiment. stem bending was determined by assessing the accompanying change in the position of the flower head as described previously (perik et al., 2012). ten excised petals per each flower were weighed (fresh weight, fw) and placed in distilled water in the dark for 6 hours to allow them to reach full turgidity and, hence, to determine their turgid weight (tw). these samples were then dried at 70°c for 24 h and their dry weight (dw) was recorded. finally, relative water content was calculated using the fw-dw/tw-dw (abdolmaleki et al., 2015). membrane stability index (msi) was recorded using an electrical conductivity meter based on ezhilmathi et al. (2007). petal samples were rinsed and immersed in 10 ml of distilled water. the samples were incubated at room temperature for 60 min with shaking (150 rpm). the electrical conductivity of the solution (ec1) was read after shaking using a conductivity meter. samples were incubated in 95°c water bath for 20 min the second reading (ec2) was taken after the solutions had cooled to room temperature. msi was calculated as [1-(ec1/ec2)] ×100. petal samples (0.2 g) was homogenized with 2 ml of 50mm phosphate buffer (ph=7) containing 1 mm edta, 1mm phenylmethylsulfonyl fluoride (pmsf) and 2% polyvinylpyrrolidone (pvp) (w/v) in ice waterbath and then centrifuged at 12000 g for 20 min at 4°c. the supernatant was collected and used for enzyme assay with a uv-visible spectrophotometer (cary 100, varian, usa). t h e a c t i v i t i e s o f e n z y m e s w e r e d e t e r m i n e d according to standard methods previously reported for cat (ec1.11.1.6) (aebi, 1984) and sod (ec 1.15.1.1) (giannopolitis and ries, 1977). enzyme activities were expressed as enzyme units.mg-1 protein. protein content was determined according to the method of bradford (1976). the cell wall materials of the proximal end of flower stem were fractioned following the method of li et al. (2012). briefly, the stems were ground into fine powder in liquid nitrogen and extracted with 95% alcohol, then washed twice with boiling alcohol and methyl alcohol: chloroform (1:1, v/v), respectively. finally, the cell wall residues were dried over night at 50°c and used for analysis. hemi-cellulose content was determined by the phenol colorimetric assay (dubois et al., 1956) and the cellulose content was m e a s u r e d b y t h e a n t h r o n e c o l o r i m e t r i c a s s a y (updegraff, 1969). lignin content was determined following the method of müse et al. (1997). all measurements were reported as µg/mg dw. stem calcium concentration was determined by atomic absorption spectrophotometer (abdolmaleki et al., 2015). the data were analyzed using sas software and the comparing means was carried out using duncan multiple range test at 5% probability and the drawing figures were carried out using excel 2013 software. 3. results and discussion stem bending the results of variance analysis showed that the main effects of treatment and cultivar (p<0.01) and their interaction effects (p<0.05) were significant on stem bending (table 2). all calcium treatments, except 0.5% calcium nitrate in ‘intense’, significantly increased the days until onset of stem bending. also, calcium chloride had better effect on stem bending in ‘rosaline’. stem bending is the most important factor affectdependent variable cultivar (c) treatment (t) c × t stem bending ** ** * relative water content ns ** ns membrane stability index ** ** * catalase activity ** ** ** superoxid dusmutase activity * ** * lignin content ** ** * cellulose content ** ** ** hemicellulose content ns ** * calcium content ** ** ns table 2 analysis of variance (anova) for cultivar, calcium treatment and their interactions effects on evaluated dependent variables in gerbera cut flower ** and * represent significance at the 1 and 5% probability levels, respectively, and ns represents non-significance at p<0.05. adv. hort. sci., 2019 33(1): 57-65 60 ing the quality of cut flowers, flower vase life, and flower loss during harvesting in gerbera. genetic, nutrition, storage temperature, and the lack of water balance in xylem vessels are of the most important factors facilitate stem bending (van meeteren, 1978; perik et al., 2012; 2014). reduction in rigidity of the stems carrying flower is associated with the reduction in the formation of lignin compounds. in this regard, it has been stated that the higher-vase life cultivars of chrysanthemum contain more lignin compared to lower-vase life ones (lv et al., 2011). it has been described that tolerance to stem bending is contributed to the presence of a sufficient amount of sclerenchyma and lignin in the stems of flowers (perik et al., 2012). so, each factor being able to increase the stem rigidity also reduce stem bending. calcium ions maintain cell wall rigidity as they bind to pectin molecules, thereby increasing cell wall stiffness. the positive effects of calcium chloride on the reduction of stem bending in gerbera were proved by gerasopoulos and chebli (1999). also, perik et al. (2014) reported that calcium treatments decreased stem bending in gerbera cultivars likely due to enhancing stem rigidity. it seems that calcium i n h i b i t s v a s c u l a r b l o c k a g e a n d e s c a l a t e s s t e m strength in line with reduction in stem bending and consequently increasing flower vase life (fig. 1). relative water content the results showed that just treatment had a significant effect on relative water content (rwc). the effects of cultivar and its interaction with treatment were not statistically significant (table 2). compared to controls, all applied calcium treatments effectively fig. 2 the main effects of calcium treatments on petals relative water content of two gerbera cultivars. bars represent standard error of four replicates. values with the different letters are significantly different according to duncan’s multiple range test at p<0.05. fig. 1 the effects of calcium treatments on day of stem bending of two gerbera cultivars. bars represent standard error of four replicates. values with the different letters are significantly different according to duncan’s multiple range test at p<0.05. increased rwc and among all applied treatments, 1% calcium chloride had the better effect on rwc while 1.5% calcium nitrate gained the lowest effects on rwc (fig. 2). the results of this research are in agreements with those obtained by cortes et al. (2011) who reported that calcium treatments increased the relative fresh weight of cut flowers. in gerbera, applying calcium treatments extended flowers vase life by enhancing water uptake by stem and maintaining flower water content (geshnizjany et al., 2014). also, it has been stated that the flower fresh weight and solution a b s o r p t i o n d e c r e a s e d w h e r e a s f l o w e r w i l t i n g increased, these mainly occur due to water loss by different organs of flowers (borochov and woodson, 1989; urban et al., 2002). in other words, when the stem vessels are blocked, flower spent the absorbed water through transpiration and subsequently reduced rwc (urban et al., 2002; van meeteren, 1978). in results, upholding a proper water uptake by cut flowers is of crucial issues in order for extending t h e v a s e l i f e o f f l o w e r s ( u r b a n e t a l . , 2 0 0 2 ) . whenever the rate of transpiration exceeds more than that of water uptake, water deficiency occurs and leads to flower wilting and reduction in flower weight (van meeteren, 1978). in current research, the significant effect of calcium treatments on i n c r e a s i n g t h e p e t a l r w c o f c u t f l o w e r s w a s observed, it can be resulted from a reduction in stem blockage owing to increasing lignin biosynthesis. membrane stability index the results of variance analysis revealed that the main effects of cultivar and treatment as well as their interaction cultivar × treatment on msi was significant (table 2). also the mean comparison of the aghdam et al. stem bending disorder of gerbera 61 interaction effect revealed that calcium treatments used in both cultivars significantly increased msi. it was found a significant different among calcium treatments of both forms on msi. the highest rate of msi was observed in ‘intense’ cultivar by 1% calcium chloride which increased as much as 13% to controls, but the highest rate of msi was found in ‘rosaline’ c u l t i v a r b y a p p l y i n g 1 % c a l c i u m n i t r a t e w h i c h increased as much as 15% to controls (fig. 3). in general, the senescence in plants is an oxidative and controlled process including biological, physiological, hormonal, and structural changes destroying macro molecules such as protein, nucleic acids, and lipids (iqbal et al., 2017). the lack of balance between generating reactive oxygen species (ros) and cleaning ros by plant defensive systems paves the way for inducing oxidative stresses which inflect damage on d i f f e r e n t p a r t s o f c e l l s a n d d e s t r o y t h e m . t h e destruction of cell membrane is one of the processes which intensifies in the presence of different ros, r e s u l t e d i n i n c r e a s i n g c e l l e l e c t r o l y t e l e a k a g e (abdolmaleki et al., 2015). in our research, pretreatment of different sources of calcium increased msi in petals of gerbera cut flowers. calcium is supposed to alleviate the adversary effect of ethylene on initiating cell senescence through inhibiting cell membrane destruction and consequently to extend the vase life of cut flowers (de capdeville et al., 2005). also, the structural and functional roles of calcium on maintaining cell membrane and cell walls have been proved (ferguson and drobak, 1988). in other words, according to the results of our experiment, calcium treatments were found to enhance antioxidant enzyme activities resulted in decreasing oxidative stress and preserving cell membrane structure. our results are in agreement with those of abdolmaleki et al. (2015) who reported that applying calcium preharvest treatment retarded damage to cell membrane and hence increased vase life of rose cut flowers. it appears that calcium treatments directly increase cell wall stability and indirectly influence enzyme processes in order to increase the content of msi in gerbera cut flowers. cat and sod activity the results showed that the interaction effects of cultivar × treatment was significant in terms of catalase (cat) and superoxide dismutase (sod) (table 2). compared to controls, all applied treatments significantly increased cat in both cultivars and the effects of the treatments in both cultivars were the same (fig. 4a). also, calcium nitrate and calcium chloride significantly improved sod activities on both cultivars. the highest sod activity was observed in ‘intense’ cultivar by using 1.5% calcium nitrate, whereas in ‘rosaline’ cultivar it was found by applying 1.5% calcium chloride (fig. 4b). the process of senescence in cut flowers is usually accompanied with a modulation in the antioxidant enzymes’ activities (borochov and woodson, 1989). fig. 3 the effects of calcium treatments on petals membrane stability index of two gerbera cultivars. bars represent standard error of four replicates. values with the different letters are significantly different according to duncan’s multiple range test at p<0.05. fig. 4 the effects of calcium treatments on catalase (cat) (a) and superoxide dismutase (sod) (b) activities of gerbera petals. bars represent standard error of four replicates. values with the different letters are significantly different according to duncan’s multiple range test at p<0.05. 62 adv. hort. sci., 2019 33(1): 57-65 the results of different studies showed that increasing antioxidant enzyme activity extended flower longevity because these antioxidants retarded the process of senescence (ezhilmathi et al., 2007). it has been demonstrated that calcium treatments, through changing in antioxidants activities, delayed flower senescence and maintained chlorophyll and protein contents in gladiola flower during storage (sairam et al., 2011). in this respect, zhao et al. (2006) showed that calcium chloride enhanced the activities of cat and sod in the cut flowers of rosa hybrida and dendrobium phalaenopsis. in current research, both forms of calcium increased cat and sod activities in gerbera flowers (fig. 4). it is obvious that improving antioxidant activities prevents cell membrane from breakdown and maintains membrane integrity. calcium may serve as a secondary messenger in a pathway to stimulate the synthesis of antioxidant enzymes (jiang and zhang, 2003). with respect to what mentioned above, calcium may indirectly stimulate the antioxidant activities and consequently reduce the oxidative stresses through lessening the peroxidation activity and maintaining the membrane integrity, or it may directly keep the membrane integrity and cell wall and delay the process of senescence in cut flowers. lignin, cellulose, and hemicellulose the results showed that the interaction effect of c u l t i v a r × t r e a t m e n t w a s s i g n i f i c a n t o n l i g n i n (p<0.05), cellulose (p<0.01), and hemicellulose (p<0.05) (table 2). comparing to ‘rosaline’, more lignin and cellulose were observed in ‘intense’, but the rate of hemicellulose in both cultivars was not significantly different (fig. 5). application of both forms of calcium at all their concentrations significantly improved the rates of lignin and hemicellulose in both cultivars (fig. 5a and 5b). the highest rate of lignin was found in ‘intense’ by 0.5% calcium nitrate while it was obtained in ‘rosaline’ by 1.5% calcium chloride (fig. 5a). on the other way, all calcium treatments increased cellulose in ‘intense’ in comparison to control, but just 1% and 1.5% calcium chloride and 0 . 5 % o f c a l c i u m n i t r a t e i n c r e a s e d c e l l u l o s e i n ‘rosaline’ as comparing to controls (fig. 5b). one of probable factors on providing stem bending in cut flowers is contributed to the lack of sufficient lignin formation in cell wall, especially in the secondary wall besides its effect on stem rigidity, causes to take up water continuously by flowers (vanholme et al., 2010). peroxidase (pod) and phenylalanine ammonia lyase (pal) are of key enzymes involving in the pathway of lignin biosynthesis; their roles in stem strength and delaying stem b e n d i n g i n g e r b e r a f l o w e r s h a v e b e e n p r o v e d (nazarideljou and azizi, 2015). it has been reported that the cut flowers prepared from the long-lasting cultivars contain more pod and pal compared to short -lasting cultivars (lv et al., 2011; nazarideljou and azizi, 2015). in the present research, ‘intense’ showed a higher tolerance to stem bending than ‘rosaline’ because of containing more lignin and cellulose. the results of this research also revealed that calcium treatments significantly increased lignin, cellulose, and hemicellulose contents in gerbera. perik fig. 5 the effects of calcium treatments on lignin (a), cellulose (b), and hemicellulose (c) of gerbera petals. bars represent standard error of four replicates. values with the different letters are significantly different according to duncan’s multiple range test at p<0.05. aghdam et al. stem bending disorder of gerbera 63 et al. (2014), by investigating the effects of different treatments on stem rigidity, figured out that using calcium chloride lessened stem bending in gerbera flowers through increasing stem strength resulted from lignin formation. in this regard, li et al. (2012) reported that applying calcium chloride at pre-harvest stage significantly enhanced the rate of pectin, lignin, cellulose, and hemicellulose in the cut flowers of herbaceous peony. also, it has been stated that using some materials such as ethylene and salicylic acid, through affecting on the some enzymes like pal involving in lignin biosynthesis, had a direct influence on stem bending of flower (ferrante et al., 2007). also, naing et al. (2017) reported that applying sodium nitroprusside augmented the gene expression of lignin biosynthesis; and likewise pal extended flower vase life and diminished stem bending of gerbera flowers. calcium content the interaction effect of cultivar × treatment was not significant in terms of calcium, but the main effects of treatment and cultivar were significant (table 2). the rate of calcium in ‘intense’ was observed higher than of that in ‘rosaline’ (fig. 6a). in other words, all calcium treatments significantly increased the amount of calcium in comparison to controls. there was a significant difference among all treatments and 0.5% calcium nitrate and calcium chloride had the lowest effect on calcium content of the stem cut flower (fig. 6b). calcium is an essential element effecting on the plants’ growth and development processes in a way that its accumulation in them facilitates creating the connections among pectin polymers; and accordingly it escalates the mechanical strength of stem and lignin production. eventually, they lead to reducing s t e m b e n d i n g a n d i n c r e a s i n g f l o w e r l o n g e v i t y (gerasopoulos and chebli, 1999; li et al., 2012). the internal and inter cellular roles of calcium are contributed to its part in plants leading to the changes on cell metabolism as well as its effect on the struct u r e a n d f u n c t i o n o f c e l l w a l l a n d m e m b r a n e (ferguson and drobak, 1988). the results showed that application of calcium, due to its role in synthesis of the pectin located in cell wall of xylem vessels, improved water translocation in stem and prevented cut flowers from stem bending (van ieperen and van gelder, 2006). in reality, a reduction in the resistance towards water conductivity in plants is one of calcium duties for extending vase life of cut flowers (cortes et al., 2011). calcium improves the flower longevity and this may delay many issues such as physiological phenomena related to senescence, reduction in water uptake, and losing more water from flower through transpiration, and thereby reducing flower fresh weight and stem bending (gerasopoulos and chebli, 1999; de capdeville et al., 2005; sosanan, 2007). in current research, applying calcium increased calcium concentration in stem of cut flowers. in a similar way, using different forms of c a l c i u m a t g r o w t h s t a g e o f h e r b a c e o u s p e o n y enhanced the internal calcium, lignin formation, and consequently the rigidity of the stems carrying flowers (li et al., 2012). nikbakht et al. (2008) reported that calcium accumulation in the stem of gerbera delayed stem bending. therefore, it seems that an increase in the content of calcium inside stems is associated with the reduction of stem bending and extending flower longevity. 4. conclusions in general, the results showed that applying calcium in different forms of nitrate and chloride reduced the stem bending in gerbera cultivars. both forms of calcium had a positive effect on gerbera cultivars, but mostly calcium chloride had better effect on stem fig. 6 the main effects of cultivar (a) and calcium (b) treatments on calcium content of two gerbera flower stem. bars represent standard error of four replicates. values with the different letters are significantly different according to duncan’s multiple range test at p<0.05. adv. hort. sci., 2019 33(1): 57-65 64 bending in comparison to calcium nitrate; and its 1% concentration compared to other its concentrations gained the best results. the positive effect of calcium 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(in japan). impaginato 3 adv. hort. sci., 2019 33(1): 3-11 doi: 10.13128/ahs-22935 the effect of cutting type, leaf area, leaf number, putrescine and indole-3butyric acid on the rooting of ficus cuttings (ficus elastica roxb. ex hornem.) m. ghasemi ghehsareh 1 (*), m. kosh-khui 2 1 d e p a r t m e n t o f h o r t i c u l t u r a l s c i e n c e s , c o l l e g e o f a g r i c u l t u r e , shahrekord university, shahrekord, iran. 2 department of horticultural sciences, college of agriculture, shiraz university, shiraz, iran. key words: auxin, cutting, plant propagation, polyamine, rubber fig. abstract: in order to study the importance of lateral or apical buds and also the possibility of replacing the role of bud and leaf with putrescine (put) and auxin, three experiments were conducted using leaf-bud cuttings with intact leaf blade (full blade) or halved-blade, and terminal cuttings having 1 to 4 leaves of ficus elastica. treatments included iba (0, 2000 mg/l) and put (0, 1000, 2000, 4000 mg/l). comparing the lateral cuttings with intact or halved-blade showed that the rooting of the cuttings with intact leaf blade was better. the longest root length and the best rooting index were observed in cuttings treated with 1000 mg/l put + 2000 mg/l iba. the highest root number resulted from the iba treatment. in cuttings with halved-leaf blade, put along with iba improved the indices of rooting in comparison with the control. the rooting of leaf-bud cutting was better in comparison with apical cuttings and the application of put with iba increased rooting indices in both types of cuttings and there was no significant difference between the different concentrations of put. results showed that terminal cuttings with three and four leaves had the longest, heaviest and most abundant roots, and that the rooting index resulted in the highest value. the rooting of leaf-bud cutting with intact leaf blade was better than that of one-leaf apical cutting. in general, the experiment showed that in the one-leaf terminal cutting, the apical bud has a negative effect on rooting and the increase in the leaf area or the application of put with auxin improves rooting. 1. introduction rooting of cuttings is a method of vegetative propagation and is one of the most important methods of clonal propagation for many plants. stimulating the formation of adventitious roots in stem cuttings with the application of auxin is well-known (altman, 1972; bolat, 1995). however, other factors do also play a role and may be restrictive under certain cir(*) corresponding author: mghasemi1352@gmail.com citation: ghasemi ghehsareh m., kosh-khui m., 2019 the effect of cutting type, leaf area, leaf number, putrescine and indole-3-butyric acid on the rooting of ficus cuttings (ficus elastica roxb. ex hornem.). adv. hort. sci., 33(1): 3-11 copyright: © 2019 ghasemi ghehsareh m., kosh-khui m. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 23 march 2018 accepted for publication 7 august 2018 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(1): 3-11 4 cumstances. inhibitors (hess, 1969), rooting co-factors (hess, 1969; heaser and hess, 1972), auxin antagonists (heaser and hess, 1972; batten and goodwin, 1981) and nutrients (nanda et al., 1971; heaser and hess, 1972) are specified to have a role in regulating the process of rooting. it has been found that cuttings without buds do not produce roots even when they are treated with auxin. this suggests that another factor other than auxin which is likely to be produced in the bud is essential for root formation (hartmann et al., 2011). it has also been specified that the presence of leaves on cuttings has a stimulatory effect on rooting (reuveni and raviv, 1980). the stimulatory effect of the leaves on rooting in stem cuttings has been well-specified in an experiment on avocado (reuveni and raviv, 1980). the cuttings of hard rooting cultivars soon lost their leaves under the mist system, while the leaves remained on rooted cuttings for a long time (9 months). after five weeks, it was observed that the amount of starch found in the planting bed of easy-rooting avocado cuttings was five times more than that of the beginning of the experiment. in hibiscus, the maintenance of leaves on cuttings increased rooting (van overbeek et al., 1946). carbohydrates that are transmitted from the leaves are important for root development. but the effects of improving leaves and bud on rooting are likely to be related to other factors (hartmann et al., 2011). it has been reported that carbohydrates and nitrogen affect the onset of adventitious roots. using tomato cuttings, researchers explained the interactions between carbohydrates and nitrogen in regulating root and shoot growth and adventitious root formation (kraus and kraybill, 1918; starring, 1923; reid, 1924 a, b). cuttings from plants grown under conditions of high carbohydrates availability (i.e., high light intensity) relative to nitrogen availability produced roots that grew vigorously (reid, 1924 a, b; schrader, 1924). van overbeek et al. (1946) reported that the rooting stimulation effect of leaves in red hibiscus cuttings could be replaced with sucrose and nitrogen. in the study of the effect of lateral and apical buds on rooting of avocados under in vitro conditions, it was reported that the apical buds had the highest rooting percentage, root number and root length, but lateral buds had no significant effect on these indices (zulfiqar et al., 2009). the effect of polyamines on the rooting of woody plants varies. put, spermidine (spd), and spermine (spm), along with iba, improved the rooting of hazel cuttings (rey et al., 1994). on the contrary, higher l e v e l s o f e n d o g e n o u s p u t , s p d a n d s p m w e r e observed at maturity stage (difficult to root) compared to the juvenile stage (easy to root) (ballester et al., 1999). the rooting of micro cuttings of olives was increased with the use of polyamines and naa, but did not increase in almonds, pistachios, chestnut, jujuba, apricots and walnuts (reuveni and raviv, 1 9 8 0 ) . i n v i t r o s t u d i e s s h o w e d a n i n c r e a s e i n polyamine levels, especially put, with root formation by culturing leaf explants of the datura innoxia (chriqui et al., 1986) and passiflora alta-caerulea (desai and mehta, 1985). investigating the role of polyamines during adventitious root formation with the evaluation of the de-bladed petiole rooting in juvenile stage (easy-to-root) and maturity stage (difficult-to-root) in hedera helix showed that auxin stimulated the root formation at the juvenile stage (easy to root), but did not have a positive effect on the mature phase. adding put, spd and spm with or without naa, did not affect the rooting in juvenile or m a t u r e p h a s e . b u t a s i g n i f i c a n t i n c r e a s e w a s observed in endogenous levels of put and spd in cuttings treated with auxin (geneve and kester, 1991). thus, the polyamines may have the role of a secondary messenger for rooting (hartmann et al., 2011). ghasemi ghehsareh and khosh-khui (2016) showed that put along with auxin improved rooting and increased the quality of roots in ficus leaf-bud cuttings. according to previous studies regarding the positive role of buds, leaves, glucose, nitrogen, auxins and polyamines in rooting of cuttings, this experiment was conducted to examine the importance of lateral and apical buds, as well as the possibility of replacing the role of buds and leaves with put and auxin. for this purpose, three separate experiments were carried out using leaf-bud cuttings with intact leaf blade or leaf blade trimmed to 50%, and terminal cuttings of the rubber fig (ficus elastica roxb. ex hornem.) having 1 to 4 leaves with iba and put treatments. 2. materials and methods experiment conditions the experiments were carried out using ficus cutt i n g s i n t h e g r e e n h o u s e o f t h e d e p a r t m e n t o f horticultural sciences of shiraz university. the medium containing washed sand was equipped with a bottom heat system at a temperature of about 22°c. ghasemi ghehsareh and kosh-khui rooting of ficus elastica cuttings 5 an intermittent mist system was employed to provide moisture, and the misting was carried out daily at 10, 13 and 16 hours, and each session lasting 1 minute. the greenhouse temperature was between 21 to 25°c during daytime and 15 to 18°c at night. first experiment effect of leaf size, auxin and put. healthy and uniform leaf-bud cuttings included leaf blade, petiole and a part of the stem with a length of 3 to 4 cm with lateral buds from the mid-section of the annual stems were taken in january. in half of the cuttings, the leaf blade was halved, and the rest remained intact. the cuttings were kept in a benomyl fungicide solution with a concentration of 2000 ppm for five minutes and then were washed with distilled water. after removing the surface moisture, the cuttings were treated with put [zero (distilled water), 1000, 2000, and 4000 mg/l)], indole-3-butyric acid (iba) (zero and 2000 mg/l, the best concentrations resulti n g f r o m t h e p r e v i o u s e x p e r i m e n t ) ( g h a s e m i ghehsareh and khosh-khui, 2016) and their combinations by dipping the stems in the solutions for 10 seconds and then planted in the bed. second experiment effect of leaf-bud or terminal cutting, auxin and put. in this experiment, leaf-bud and terminal cuttings with one leaf were used. growth regulators were used and other conditions were similar to the first experiment. third experiment effect of leaf-bud or terminal cutting, leaf area and leaf number. in this experiment, the rooting of different cuttings including leaf-bud cuttings with halved blade, leaf-bud cuttings with intact blade and terminal cuttings with 1, 2, 3 and 4 leaves were compared. all cuttings were treated with iba at a concentration of 2000 mg/l. other conditions were similar to the first experiment. in all three experiments, after 2 months, the cuttings were removed from the bed and after isolating the medium form the roots, root fresh weight, root length (length of the longest root), root number were measured. to determine the quality of the roots, rooting index was calculated. for this purpose the cuttings were visually grouped into five groups including cuttings with heavy, medium and weak rooting, rootless but alive cuttings and dead cuttings. for each group, a coefficient was considered as the weight of rooting. number 5 was for heavy rooting, 4 for medium rooting and 3 for weak rooting. a coefficient value of 2 was used for rootless but alive cuttings, and a coefficient value of 1 was used for dead cuttings (criley, 2011; ghasemi ghehsareh and khosh-khui, 2016). statistical design and data analysis the first and second experiments were conducted as factorial in a completely randomized design with three replications. each replication included 10 cuttings. the third experiment was conducted in a completely randomized design with three replications, each with 6 cuttings. statistical analysis of the data was performed using sas 9.13 software and the mean values were compared using lsd (p≤0.05). 3. results first experiment the results of analysis of data variance showed that the treatments had a significant effect on the root length, root number, root fresh weight and root quality. maximum root length (8.9 cm) was caused by iba 2000 mg/l + put 1000 mg/l treatment in fullblade cuttings, but did not show any significant difference with iba 2000 mg/l + put 2000 or 4000 mg/l on full-blade cuttings. furthermore, no significant difference was observed between the mentioned results and the result of treatment with iba 2000 mg/l + put 1000 or 2000 mg/l on halved blade leaves. the lowest root length was observed in cuttings with h a l v e d l e a f b l a d e , a n d g e n e r a l l y , r o o t l e n g t h decreased with a decrease in leaf area. in terms of the number of main roots, the highest number (13.3) was obtained by iba 2000 mg/l on fullblade, and the lowest number of leaves was obtained with halved blade cuttings, without growth regulators. the application of put did not significantly affect the number of main roots. the highest root fresh weight was observed by the application of put 4000 mg/l with iba treatment in full-blade cuttings, which did not have significant difference with other concentrations in full leaves and put 4000 mg/l with auxin in cuttings with halved blade leaves. in fact, by increasing put concentration, the root fresh weight increased in both types of cuttings and the amount of increase was more in cuttings with halved-blade leaves (table 1). in terms of root quality, the highest rooting index (4.7) was observed by treatment with put (1000 mg/l) + iba (2000 mg/l) in full-blade cuttings. the highest rooting index in halved-blade cuttings (3.8) adv. hort. sci., 2019 33(1): 3-11 6 was observed by put 1000 mg/l + iba treatment. the lowest rooting index (2.3) was observed in control treatment with halved-blade cuttings. in general, cuttings with leaf blade trimmed to 50% had roots with lower quality compared to cuttings with full leaf. with increasing the put concentration, the quality of the roots first increased and then decreased (table 2). second experiment the results showed that the interaction of cutting types (leaf-bud or terminal cutting) with growth regulators had a significant effect on rooting characteristics. the terminal cuttings were not rooted without the application of growth regulators, but rooting improved with the application of rooting growth regulators. the longest root length (9.7 cm) was found in leaf-bud cuttings, and there was not a significant difference between different treatments in this cutting. in the terminal cuttings, the use of put + auxin in comparison with the control treatment and auxin alone significantly increased the root length, but there were no significant differences between different concentrations of put. the highest main root number (14.8) was observed in the leaf-bud cutting treated with put 4000 mg/l + iba 2000 mg/l. the application of iba as well as increased put concentration increased the number of roots. in the terminal cuttings, the highest number of roots (6.4) was observed by treatment with 2000 mg/l of put along with iba, which did not differ significantly in different concentrations (table 3). the highest root fresh weight was observed in leaf-bud cuttings. the highest fresh weight (2.9 g) was obtained by using auxin alone, and by put at 2000 mg/ml + iba, which did not differ significantly with other concentrations of put. in the terminal cuttings, the application of put and its increased concentration increased the fresh weight of the root. the best root quality (4.0) was observed in the leaf-bud cuttings treated with put (2000 mg/l) +iba. in the terminal cuttings, the application and increase of the concentration of put improved the quality of the roots as compared with other treatments (table 4). table 2 effect of leaf size, auxin and put on root quality (rooting index) of ficus leaf bud cutting table 1 effect of leaf size, auxin and put on root length, root number and root fresh weight of ficus leaf bud cutting * means with similar letters (lowercase letters for whole means and capital letters for means of rows and columns) are not significant at 5% level of probability using lsd. leaf size treatments mean control aux2000 aux2000+put1000 aux2000+put2000 aux2000+put4000 root length intact leaf blade 3.6 c ∗ 6.5 ab 8.917 a 8.417 ab 6.917 ab 6.87 a halved-blade 0.833 d 5.833 bc 6.75 ab 8.333 ab 5.917 bc 5.5333 b mean 2.2167 c 6.1667 b 7.8333 ab 8.375 a 6.416 7 b root number intact leaf blade 1.333 e 13.333 a 8.167 b-d 8.5 bc 10.333 b 8.3333 a halved-blade 0.5 e 7.167 cd 5.833 d 6.0 d 6.667 cd 5.2333 b mean 0.9167 c 10.25 a 7.0 b 7.25 b 8.5 ab root fresh weight intact leaf blade 0.475 d 1.6533 a 1.5233 ab 1.7533 a 1.93 a 1.467 a halved-blade 0.4833 d 0.7033 cd 0.83 b-d 0.9067 b-d 1.2167 a-c 0.828 b mean 0.4792 b 1.1783 a 1.1767 a 1.33 a 1.5733 a leaf size treatments high (×5) medium (×4) low (×3) alive (×2) sum of weights rooting index intact leaf blade control 0 0 20 10 80 2.7 aux2000 13 13 4 0 129 4.3 aux2000+put1000 20 10 0 0 140 4.7 aux2000+put2000 20 10 0 0 140 4.7 aux2000+put4000 20 5 5 0 135 4.5 halved-blade control 0 0 10 20 70 2.3 aux2000 0 17 13 0 107 3.6 aux2000+put1000 0 25 5 0 115 3.8 aux2000+put2000 5 10 15 0 110 3.7 aux2000+put4000 5 7 18 0 107 3.6 ghasemi ghehsareh and kosh-khui rooting of ficus elastica cuttings 7 third experiment by comparing the rooting of leaf-bud cuttings having either full or halved-leaf blade, and terminal cuttings having 1, 2, 3 or 4 leaves, it was observed that by increasing the leaf area, the rooting was also increased. accordingly, the longest roots were obtained in the terminal cuttings with 4 leaves (13.1 cm), followed by terminal cuttings with 3 leaves (9.75 cm). the shortest roots (1.47 cm) were obtained in leaf-bud cuttings with halved-blade. terminal cuttings with 4 leaves produced the highest main root number (6.4) which did not have a significant difference with two and three leaf cuttings. the highest root fresh weight (1.72 g) was observed in four leaf cuttings. the lowest root number, root fresh weight and rooting index were observed in one-leaf terminal cuttings (table 5). in terms of rooting index, the highest rate (3.6) was observed in three and four leaf cuttings (table 6). generally, the experiment showed that the best rooting was obtained in the three or four leaf terminal cuttings and it was weaker in oneleaf terminal cuttings compared to other cuttings. table 4 effect of leaf bud or terminal cutting, auxin and put on root quality (rooting index) of ficus cutting table 3 effect of leaf bud or terminal cutting, auxin and put on root indices of ficus cutting * means with similar letters (lowercase letters for whole means and capital letters for means of rows and columns) are not significant at 5% level of probability using lsd. * means with similar letters (lowercase letters for whole means and capital letters for means of rows and columns) are not significant at 5% level of probability using lsd. table 5 effect of cutting type, leaf area and leaf number on root indices of ficus elastica leaf area/cutting type root length root no. root fresh weight half leaf/leaf bud 1.47 c ∗ 3.83 bc 0.49 cd full leaf/leaf bud 2.25 c 4.6 ab 0.83 bc 1 leaf/terminal 1.92 c 2.33 c 0.22 d 2 leaf/terminal 6.92 b 3.33 ab 0.68 bc 3 leaf/terminal 9.75 a 4.25 ab 0.93 b 4 leaf/terminal 13.0 a 6.42 a 1.72 a cutting type treatments mean control aux2000 aux2000+put1000 aux2000+put2000 aux2000+put4000 root length terminal 0.000 c ∗ 0.900 c 4.200 b 4.300 b 4.170 b 2.7140 b leaf bud 9.718 a 8.518 a 8.532 a 9.582 a 8.750 a 9.0200 a mean 4.8590 bc 4.7090 c 6.3660 ab 6.9410 a 6.4600 a root number terminal 0.000 c 1.200 c 4.000 b 6.400 b 5.600 b 3.4400 b leaf bud 4.167 b 10.833 a 11.667 a 13.333 a 14.833 a 10.9666 a mean 2.083 c 6.017 b 7.833 ab 9.867 a 10.217 a root fresh weight terminal 0.0000 c 0.2300 c 0.9460 b 1.3120 b 1.4790 b 0.7934 b leaf bud 1.2000 b 2.9000 a 2.4550 a 2.9032 a 2.4318 a 2.3780 a mean 0.6000 b 1.5650 a 1.7005 a 2.1076 a 1.9554 a cutting type treatments high (×5) medium (×4) low (×3) alive (x2) sum of weights rooting index terminal 0 0 0 0 30 60 2.0 aux2000 2 3 4 21 76 2.5 aux2000+put1000 1 2 24 3 91 3.0 aux2000+put2000 3 7 18 2 101 3.4 aux2000+put4000 2 6 19 3 97 3.2 leaf bud 0 0 2 24 4 88 2.9 aux2000 1 18 11 0 110 3.7 aux2000+put1000 2 22 6 0 116 3.9 aux2000+put2000 6 17 7 0 119 4.0 aux2000+put4000 5 14 11 0 114 3.8 8 adv. hort. sci., 2019 33(1): 3-11 4. discussion and conclusions first experiment our test results showed that the application of put along with auxin increased the main root length and root fresh weight, which is consistent with the results of other researchers (cristofori et al., 2010; birlanga et al., 2015; ghasemi ghehsareh and khoshkhui, 2016). the studies of root formation in the mung bean showed that in the rooting phase stimul a t e d w i t h a u x i n , t h e e n d o g e n o u s p o l y a m i n e s increase (friedman et al., 1982; jarvis et al., 1983). desai and mehta (1985) and chriqui et al. (1986) studied the rooting of leaf explants in vitro which showed that at the time of root formation the amount of polyamines, especially put, increases. t h e y s h o w e d t h a t t h e a c t i v i t y o f o r n i t h i n e decarboxylase as a key enzyme in put biosynthesis increased in auxin-treated explants, but the use of polyamines in an auxin-free environment did not l e a d t o r o o t i n g . t h e i n c r e a s e i n e n d o g e n o u s polyamines during root formation by in-vitro auxin is reported to stimulate the formation of callus, leaf discs and de-bladed petiole explants (malfatti et al., 1983; desai and mehta, 1985; chriqui et al., 1986; tiburcio et al., 1989; geneve and kester, 1991). this increase in put in the formation and extension of the roots shows that put is a biochemical marker for root differentiation (tiburcio et al., 1989). researchers have shown that put, spm and spd inhibit root formation (friedman et al., 1982; jarvis et al., 1983; palavan-ünsal, 1987). also, schwartz et al. (1986) reported that the activities of ornithine decarboxylase (odc) and arginine de-carboxylase (adc) enzymes of put biosynthesis pathway increase during lateral root formation in zea mays. the biosynthesis of the polyamines is associated with cell division in the organogenesis process. in most cases, polyamines have not been able to stimulate root formation in the absence of auxin (birlanga et al., 2015), and numerous studies have shown that the simultaneous application of auxins and polyamines increases rooting (sankhla and upadhyaya, 1988; birlanga et al., 2015). biondi et al. (1990) showed that the polyamines do not directly influence the induction of adventitious root in the prunus avium micro-cuttings, but do play a role in the later stages of root development and elongation. however, put improved the quality of roots in our experiment. in this experiment, the decrease in leaf area caused a decrease in rooting, which is consistent with akinyele (2010) results in bachholzia coriaria. it was reported that leaf size had a very significant effect on rooting, and that the rooting of full-leaf cuttings was better than half-leaf ones. our results showed that the highest number of main roots was obtained by auxin treatments alone and the combination of put along with auxin reduced the number of main roots, but increased root length a n d w e i g h t . o n t h e o t h e r h a n d , r o o t i n g i n d e x increased in treatments containing both auxin and putrescine in full-blade cuttings. it seems that put, along with auxin, increases root weight by increasing the number of root branches and also the root length, and in comparison with using auxin alone, it reduced the number of thick roots, thereby improving the quality of the roots. based on our results, the simultaneous application of auxin and put has been shown to improve rooting, and to some extent it has been able to compensate for the decrease in leaf a r e a i n c u t t i n g s w i t h h a l v e d b l a d e s . t h i s w a s observed in a manner that no significant difference existed among the indices of root length and root weight, even when comparing cuttings with full and half blades treated with put 2000 or 4000 mg/l. this suggests that perhaps a part of the positive role of the leaf blade in rooting is related to polyamines and their derivatives. second experiment results of comparisons between one-leaf terminal and leaf-bud cuttings showed that rooting rate in terminal cuttings was zero without using growth regulatable 6 effect of cutting type, leaf area and leaf number on root quality (rooting index) of ficus elastica leaf area/cutting type high (×5) medium (×4) low (×3) alive (×2) sum of weights rooting index half leaf /leaf bud 0 5 8 5 54 3 full leaf/leaf bud 2 6 5 5 59 3.44 1 leaf/terminal 0 5 4 8 48 2.89 2 leaf/terminal 0 9 6 3 60 3.33 3 leaf/terminal 2 9 5 2 65 3.61 4 leaf/terminal 3 12 1 0 66 3.67 ghasemi ghehsareh and kosh-khui rooting of ficus elastica cuttings 9 tors and treatment of cuttings with put and iba significantly increased rooting in comparison to the use of auxin alone, thereby confirming the results of the first experiment and indicating that part of the positive role of leaf in rooting is related to put. on the other hand, the rooting conditions of the leaf-bud cuttings is better than that of the terminal cuttings, which indicates that the apical buds have had a negative effect on rooting. the results of this experiment are consistent with previous reports by al-zebari and al-brifkany (2015), where it was observed that the rooting of citron (citrus medica) cuttings was mostly successful in cuttings obtained from the middle of the stem and the least rate of rooting was in the apical cuttings. this indicates that the apical bud is not only a strong source of auxin, compared to the lateral bud (thimann and skoog, 1933; thimann et al., 1934), but also a strong consumer which cannot play a positive role in rooting without the presence of a leaf. concerning the inhibitory effect of the apical buds on the growth of lateral buds, it has been reported that the apical bud is a strong consumer of auxin and sucrose. in this regard, thimann (1937) suggested that the optimal auxin concentration for stem elongation is more than the concentration necessary for the development of lateral buds. there is also evidence that the apical bud is a strong consumer which limits access to sugars for lateral buds (taiz et al., 2015). this strong sink effect of the apical bud can also have a negative effect on rooting. a study on in vitro rooting of avocado showed that the apical buds had a better rooting than the lateral buds (zulfiqar et al., 2009). on the other hand, rooting is a highly critical and energy-demanding process that is influenced by the complex interaction between sucrose and hormonal levels (birlanga et al., 2015). roberts and fuchigami (1973) studied the effect of seasonal changes on auxin and rooting of the douglas-fir stem cuttings. they showed that at the time of removing cuttings from the beds, rooted cuttings showed a certain activity in buds, but most of the cuttings with very active buds had not produced roots. lanphear and meahl (1963) observed that rooting occurred more actively at or before the bud break, but it decreased sharply with increasing bud activity. the decline was probably due to the competition for growth factors necessary for rooting. third experiment by comparing the leaf-bud cuttings with half and full blade, and terminal cuttings with 1, 2, 3 and 4 leaves, all of which were treated with iba 2000 mg/l, was observed showed that increasing the leaf area, improves rooting. however, by comparing leaf-bud cuttings and one-leaf terminal cuttings, leaf-bud cuttings had a better rooting, which confirmed the results of the second experiment, indicating that the apical bud probably acted as a strong consumer and reduced rooting. the increase in the number of leaves improved rooting so that the highest rooting was observed in terminal cuttings with 3 and 4 leaves. this suggests that, although the apical bud is a strong source of auxin, it was not able to compensate for the lack of factors necessary for rooting. here, the importance of other roles of the leaf becomes clear. the results of this experiment are consistent with reports by other researchers (leakey et al., 1982; badji et al., 1991; tchoundjeu and leakey, 1996; tchoundjeu et al., 2002; atangana et al., 2006; opuni-frimpong et al., 2008). in an experiment by leakey (leakey, 2004), stem cuttings of the khaya senegalensis showed that rooting was limited to leafy cuttings. the inability of rooting in leafless cuttings was associated with the rapid evacuation of carbohydrates in stem tissues, but their concentration in leafy cuttings increased (leakey et al., 1982). this shows that rooting depends on the formation and consumption of carbohydrates after the cuttings are separated from the mother plant (leakey and coutts, 1989). van overbeek et al. (1946) reported that rooting stimulation by the presence of leaves on red hibiscus cutting could be replaced with sucrose and organic or inorganic nitrogen. welander (1976) s h o w e d t h a t i n v i t r o r o o t i n g o f s u g a r b e e t hypocotyls was stimulated by an increase in sucrose and inorganic nitrogen in the presence of high concentrations of iaa, whereas low concentrations of auxin were ineffective. gabryszewska (2011) studied s y r i n g a v u l g a r i s u n d e r i n v i t r o c o n d i t i o n s a n d showed that increasing the amount of sucrose in the medium causes spontaneous root formation on cultivated plantlets in the presence of low levels of nitrogen salts. the planting of rosa ‘improved blaze’ shoots for the purpose of investigating the effect of sucrose and inorganic nitrogen on adventitious root formation showed that high concentrations of sucrose led to the production of higher and more elongated roots (hyndman et al., 1981). therefore, the role of the leaf in helping rooting can occur by the presence of carbohydrates through photosynthesis and also by polyamines as a source of nitrogen. in general, this study shows that the application of put along with auxin improves rooting and root quality in ficus cuttings. moreover, in terminal cutadv. hort. sci., 2019 33(1): 3-11 10 tings, due to the competition for nutritional and hormonal factors necessary for the simultaneous growth of buds and the occurrence of rooting, a certain amount of leaf area is necessary to provide these factors. the application of put along with auxin can partly satisfy this requirement in cuttings with smaller leaf areas. references akinyele a.o., 2010 effects of growth hormones, rooting media and leaf size on juvenile stem cuttings of buchholzia coriacea engler. ann. for. res., 53(2): 127133. altman a., 1972 role of auxin in root initiation in cuttings. proceedings of international plant propagation society, london, uk, 22: 284-294. al-zebari s.m.k., al-brifkany 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agronomy, universidade tecnológica federal do paraná, via do conhecimento, km 1, pato branco, paraná, brasil. 2 universidade tecnológica federal do paraná, estrada para boa esperança, km 4, dois vizinhos, paraná, brasil. key words: fruit growing, irradiation, vine, vitis sp. abstract: the aim of this study was to evaluate the use of reflective materials on the soil surface and management practices on the physicochemical and biochemical quality of the merlot grapes (vitis vinifera l.). the experimental design was of randomized blocks, in factorial 3 x 2 (“material type” x “management practice”), with 4 replications, using 4 plants by plot. the “management practice” factor was divided into two levels, with and without its realization. for “material type”, two reflective films were tested in the soil surface, it being white raffia plastics of polypropylene (reflective film 1), metallic raffia plastic (reflective film 2) and without the use of any reflective material. when they reach the harvesting point in the 2009/2010 and 2010/2011 production cycles, the physicochemical and biochemical evaluations of fruit quality were submitted; physiological characteristics of the plant and microbiological properties of soil were also evaluated. the use of reflective material on the soil surface improved the quality of the grape in some aspects, especially determining the reduction of the berry dropping and, consequently, an increase in the productivity. furthermore, it provided greater microbial activity in the soil, which may be important for grapevine. the cultural practices had no influence on the physicochemical and biochemical quality of the vine fruits, providing only a greater number of berries per bunch. 1. introduction grape is one of the most economically important fruits in the world, with brazil in 2014 occupying 78,765 hectares, while production was 1,454,183 tons (fao, 2014; camargo et al., 2011). approximately 57% of this production was used for winemaking (mello, 2011), with the rest consumed in natura or as dried fruits (conde et al., 2007). viticulture for winemaking is mainly concentrated between the 30th and 50th parallel of north latitude and between 30th and 45th of south lati(*) corresponding author: porto@alunos.utfpr.edu.br citation: porto a.h., wagner junior a., bandei̇ra cabral v., ci̇tadi̇n i., zanela j., nunes i.b., , conceição p.c., 2019 reflective materials and management practices on the physicochemical and biochemical quality of merlot grapes. adv. hort. sci., 33(1): 39-48 copyright: © 2019 porto a.h., wagner junıor a., bandeira cabral v., citadin i., zanela j., nunes i.b., conceição p.c. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 10 july 2018 accepted for publication 4 october 2018 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(1): 39-48 40 tude. the main climates that allow the cultive of vines are those of temperate, mediterranean and arid areas, in different levels (tonietto and mandelli, 2003). however, the state of paraná, brazil, presents potential for the cultivation of grapes destined for the in natura consumption and for the elaboration of juices or wines. in particular, in the southwest region of paraná there are numerous vine orchards in small rural properties even though the grapes produced in this region had sporadically shown inferior quality to what is considered as good for wine and/or juice, especially when related to soluble solids content. fruit quality is linked directly to genetic characteristics, edaphoclimatic factors and cultural practices. the management adopted in the vine orchard usually aims to promote the penetration of solar rays inside the canopy, allowing the leaves located in the lower part of the plant to increase the photosynthetic rate and increase the availability of the photo assimilates for the fruits (mota et al., 2009). the solar radiation is one of the main factors to obtain fruits with high quality, with regard to size, firmness, sugar content, coloration, anthocyanins, starch content and acidity (procton and lougheed, 1976; erez and flore, 1986; jackson, 1989). thus, the increase in the efficiency of its use is fundamental for obtaining more attractive fruits. among the management techniques performed in the pre-harvesting of the grape, which can improve the penetration of light and aeration inside the canopy, favouring the obtaining of quality fruits, are green pruning, defoliation, thinning, removal extra shoots (suckering) in the grape tree leaf (mendonça et al., 2016). however, many winegrowers are resistant to these practices, preferring the adoption of management techniques that allow them to obtain fruits with quality, without the need to increase the demand for labour and the costs in the property. one of the alternatives that can be adopted is the use of reflective materials in the soil, since they allow to increase the total light absorbed and the distribution of the same inside the canopy, improving the quality of the fruits (chavarria and santos, 2009). in ‘fuji’ apple the use of reflective materials in the soil was able to increase fruit quality as the red intensity of the epidermis and increase the size of fruits from the interior of the canopy (andris and crisosto, 1996). the use of reflective plastics, under the canopy of plants, is a very used management technique in some countries of america, europe and asia. the main objective is to reflect the sunlight to the inside of the crown, to intensify the coloration of the fruit epidermis, to improve the flavour, as well as to antecipate maturation (trevisan et al., 2006; meinhold et al., 2011). this additional light is beneficial for photosynthesis and anthocyanin production (layne et al., 2002). the principle of the use of these materials is the ability to reflect the solar radiation to the interior of the canopy and it can provide a 40% increase in the photosynthetically active radiation reflected when the soil is completely covered and, around 24%, when partially covered (green et al., 1995). the present study aimed to evaluate the use of reflective materials and management practices on plant physiology and physicochemical and biochemical quality of merlot grape (vitis vinifera l.). 2. materials and methods the experiment was conducted in a commercial vineyard in the dois vizinhos city pr, brazil, in santa lucia community (25° 51’ 08” s, 53° 06’ 15” w, 594 m of altitude), for two productive cycles (2009/2010 and 2010/2011), indicated as cycles 1 and cycles 2 in the following text. the studied vineyard was planted in 2004 with merlot (vitis vinifera l.) grafted 1103 rootstock paulsen (v. berlandieri x v. rupestris). the spacings between rows are 3.0 m and between plants are 2.0 m in an espalier system, totalling 1.666 plants per hectare. the experimental design was of randomized blocks, in multifactorial 3 x 2 (“material type” x “management practice”), with 4 replications, considering each 4 plants as a plot, the two central ones being useful, disregarding those near the border. the “management practice” factor was divided into two levels, with and without its realization. the treatments with the management practices consisted of the adoption of the shoot topping, withdrawal of tertiary branches, disbudding and defoliation. in the treatments without the management practices, only the disbudding was done. for “material type”, two reflective films were tested in the soil: polypropylene white raffia plastic (reflective film 1) and metallic raffia plastic (reflective film 2), both placed below the projection of the porto et al. reflective materials and management on quality of merlot 41 crown of the plants, in the lines and between the lines. the films were placed in the vineyard 30 days after plants broke their dormancy; as third level, none film was used. upon reaching the harvesting point, in both cycles, the fruits were harvested and taken to the l a b o r a t o r y o f p l a n t p h y s i o l o g y , ( u n i v e r s i d a d e t e c n o l ó g i c a f e d e r a l d o p a r a n á c a m p u s d o i s vizinhos) for physicochemical and biochemical evaluations. t h e eval u ated vari ab l es were ro t i n ci d en ce (bunch and berry), bunch and berries weight, number of berries and bunches, berry drop, total soluble solids content (°brix) (ss) titratable total acidity (tta) (g tartaric acid 100 ml-1), ss/tta ratio, ph and production per plant (kg). the incidence of rot was determined visually, considering fruits with damage when they presented lesions and typical characteristics of pathogen attack. the berry drop was determined by the percentage of berries detached from the general total of the bunch. the ss contents were determined by refractometry. the titratable acidity was determined by titrating 10 ml of juice in 90 ml of water diluted with 0.1 n naoh solution to ph 8.1; the results being expressed in g of tartaric acid 100 ml-1. analysis of biochemical variables of productive cycle 1 fruits included total proteins, total phenols, total and reducers sugars, phenylalanine ammonialyase enzyme (pal), flavonoids and anthocyanins, constituting a total of four samples per treatment. for total protein dosage, the pulp samples from the fruits of each treatment were macerated in a mortar with 10 ml of 0.2 m phosphate buffer (ph 7.5). then the material was centrifuged (14.000 g for 10 min at 4°c) and the supernatant collected. for the quantification of the total protein content in the samples, the bradford test (1976) was used and the spectrophotometer, model uv-sp2000-spectrum at 630 nm, with bovine serum albumin as standard. the quantification of the total phenolic compounds of the pulp of the fruits of each treatment was carried out in two stages; the first one, following the method adapted from bieleski and turner (1966) and the second one according to jennings (1991). the total soluble sugar concentrations of fruit pulp from each treatment were determined by the phenol-sulfuric method as described by dubois et al. (1956). the activity of pal was evaluated based on the difference in absorbance resulting from the conversion of phenylalanine to trans-cinnamic acid (hyodo et al., 1978). were evaluated the efficiency of water use (%), co2 assimilation rate (μmol co2 m-2 s-1), water cond u c t a n c e ( m o l h 2o m 2 s 1) , i n t r a c e l l u l a r c o 2 concentration (μmol co2 mol-1), transpiration rate (mmol h2o m-2 s-1) and foliar temperature (°c) in the productive cycle 1 and productive cycle 2. these physiological analyses of the plants were performed on three different dates (8th, 15th and 21st of january, 2010) for the productive cycle 1 and a date (14th of december, 2010) during the productive cycle 2. the gas exchange readings always started at 9:30 a.m. using an open gas measurement system equipped with a li-6400xt infrared gas analyser (irga li-cor, lincoln, nebraska usa) and an artificial source of red and blue light, performed on the fully developed and healthy middle third leaves of two plants per plot. the microclimatic conditions in the sample chamber were maintained constant during the readings, being 1100 μmol m-2 s-1 par (photosynthetically active radiation) and ambient co2 concentration (average of 383 μmol co2 mol-1). microbiological analyses of the soil were also performed according to the type of coverage adopted, by means of the quantification of the respiratory activity of the same, according to method proposed by öhlinger (1993). the analyses were carried out in the laboratory after the collection of the same, in the period of seven days after the harvest of each productive cycle to the depth of 10 cm. maximum, average and minimum temperatures of the plant body were obtained after reflection of the solar radiation for the productive cycle 1, captured by infrared images (thermographic) by a therma cam sc500 camera, always installed in the direction of the region of the plant, at 1 m of distance from the projection of the plant, discarding those of the border of each treatment. the temperat u r e w a s o b t a i n e d u s i n g t h e t h e r m a c a m 2 0 0 professional software based on the readings of 15th and 22nd of january, 2010 starting at 9:00 a.m. the results of all variables were submitted to analysis of variance and the means were compared by the tukey’s test (α = 0.05), except for the soil microbiological analyses where the duncan’s test (α = 0.05) was applied. the data expressed in percentage was transformed by sine arc √(x/100) and when expressed in numbers by √(x + 1), according to the lilliefors’ normality test. all analyses were performed b y t h e s t a t i s t i c a l s o f t w a r e s a n e s t ( z o n t a a n d machado, 1984). adv. hort. sci., 2019 33(1): 39-48 42 3. results and discussion in the course of the experiment, excessive rainfall occurred during the two productive cycles (figs. 1a and 1b). in addition to the occurrence of hail in productive cycle 1, which impaired the performance of phytosanitary treatments, creating a favourable environment for disease incidence, it being observed a m o n g t h e s e , a g r e a t e r i n c i d e n c e o f m i l d e w (plasmopara viticola), close to 25% of bunches or berries. there were no statistically significant differences for the multifactorial interactions between the type of material and the management practices in the physicochemical variables (table 1) during productive cycles 1 and 2 and biochemical (table 2) of productive cycle 1, except for the ss/att ratio of the productive cycle 2. when factors were analyzed separately, significant results were observed, except for the total weight of berries and bunch, berry drop, yield per plant and total sugars concentration for the factor “material type” of production cycle 1 (table 3) and number of berries per bunch for the factor “management practice” of cycle 1 (table 4). for berry drop of the grapes, in the productive cycle 1, it was verified that the use of the reflective film 2 (metallized raffia plastic) presented the lowest values in comparison to the reflective film 1 (white raffia plastic) and without film (table 1). however, in production cycle 2 these values were lower in relation to cycle 1. the highest values of berry drop productive cycle 1 obtained in the reflective film 1 and without film, may be related to the greater attack of fungal diseases in the berries, because the cycle had an average temperature around 25°c and a high incidence of rainfall (280 mm) near the harvest season fig. 1 data obtained from inmet’s meteorological station, from productive cycle 1 (2009/2010) (a) and productive cycle 2 (2010/2011), located at universidade tecnológica federal do paraná campus dois vizinhos pr. table 1 berry drop (%), bunch weight (g), weight of total and individual berries (g), ss (°brix), att (g of tartaric acid 100 ml-1), ph, ss/tta ratio, number of berries per bunch, number of bunches, rot berries and bunches (%), production per plant (kg) of merlot grapes submitted or not to management practices with or without the use of reflexive films on the soil, in productive cycle 1 of (2009/2010) and productive cycle 2 of (2010/2011) ns= non-significant by the f test. ** significant at 5% of probability.t1= with management practices and no reflexive film; t2= with management practices with reflexive polypropylene white raffia plastic (film 1); t3= with management practices with reflexive metallic raffia plastic (film 2); t4= without management practices and no reflexive film; t5= without management practices with reflexive polypropylene white raffia plastic (film 1) and (t6) without management practices with reflexive f metallic raffia plastic (film 2). treatment (z) berry drop bunch weight total weight of berries berry weight (g) ss att ph ss/att berries no. per bunch bunches no. berries with mildew bunch with mildew production (kg) productive cycle 1 (2009/2010) t1 12.71 ns 132.4 ns 125.70 ns 1.73 ns 16.64 ns 5.57 ns 3.44 ns 2.99 ns 72.76 ns 25.67 ns 25.24 ns 24.02 ns 3539.04 ns t2 15.29 162.65 155.17 2.43 15.74 4.71 3.47 3.34 63.62 27.69 20.44 25.57 4222.51 t3 4.59 155.96 148.5 1.98 15.89 4.84 3.53 3.28 74.87 26.71 21.96 24.92 4535.66 t4 16.76 117.29 111 1.98 16.64 5.34 3.42 3.12 56.06 23.91 25.24 22.5 2831.5 t5 11.36 148.45 139.96 2.22 15.74 4.77 3.43 3.3 62.95 30.06 20.45 27.48 4503.96 t6 4.68 157.17 150.16 2.48 15.89 4.51 3.45 3.52 60.65 29.12 21.96 25.66 4554.81 cv (%) 18.77 14.06 14.29 18.33 7.98 8.29 2.21 16.52 8.12 8.79 4.54 5.1 24.37 productive cycle 2 (2010/2011) t1 1.59 ns 250.58 ns 237.82 ns 1.75 ns 60.25 ns 4.04 ns 3.67 ns 4.14** 137.2 ns 60.25 ns 4.47 ns 15.61 ns 15321.1 ns t2 4.8 259.4 241.74 1.76 62.63 3.77 3.36 4.44 139.75 62.62 6.25 15.98 16389.38 t3 5.42 283.37 268.07 1.74 70.25 4.58 3.06 3.72 152.97 70.25 4.13 14.13 19997.24 t4 3.72 245.29 233.95 1.78 59.63 4.4 3.37 3.86 131.97 59.62 6.04 11.26 14907.88 t5 3.63 265.81 253.84 1.75 60.88 4.08 3.2 3.93 145.06 60.88 4.85 17.11 16278.66 t6 3.78 246.34 238.1 1.62 70.3 4.43 3.35 4.48 147.22 69.38 8.06 18.99 17504.40 cv (%) 27.72 18.25 17.92 6.6 25.57 10.93 2.99 11.79 16.16 25.57 49.36 26.24 30.64 porto et al. reflective materials and management on quality of merlot 43 (fig. 1), favouring the development of pathogens. although they did not present statistically significant difference in both cycles, it is assumed that the use of metallized raffia plastic as a reflective film beneath the merlot vines had provided greater heat in the area of the crown projection as compared to the use of white raffia plastic without film creating an environment with low levels of moisture, unfavourable to the attack of diseases. the same was visualized in productive cycle 2 whose incidence of fungal diseases attack was reduced, resulting in lower values of berry drop. this hypothesis can be verified by the analysis of plant surface temperatures (minimum, average and maximum) after reflection of the solar radiation, captured by infrared (thermographic). in fact, we found divergent results for the type of cover and management practice, and the use of reflective film allowed an increase of up to 3°c in the average and maximum temperatures, in relation to the non-use of reflective film (tables 3 and 4, respectively). the same significant effect was not obtained for the maximum, average and minimum temperatures of the plants during the second evaluation (table 2). treatment (z) flavonoids (mg/100 g) phenols (mg/g) total sugar (mg/g) anthocyanins (mg/100 g) pal (uabs/min/ mg prot) proteins (mg/g) surface temperature in 2nd evaluation intracellular co2 concentration min (°c) med (°c) max (°c) 1st evaluation 3rd evaluation t1 159.12 ns 1.78 ns 26.92 ns 128.08 ns 0.09 ns 0.92 ns 26.33 ns 30.23 ns 37.90 ns 255.55 ns 242.07 ns t2 153.31 1.01 28.02 119.43 0.02 1.02 25.2 29.98 38.77 251.23 250.12 t3 110.41 1.32 30.76 86.01 0.04 0.79 24.85 31.38 41.65 246.61 241.07 t4 121.79 1.56 26.82 94.87 0.04 0.45 25.92 29.6 35.75 253.19 235.12 t5 151.74 1.63 27.76 118.22 0.03 0.74 26.83 31.13 39.73 255.55 244.82 t6 137.52 1.27 28.16 107.13 0.39 0.54 23.05 31.1 38.6 246.94 242.71 cv (%) 31.83 33.38 5.67 30.82 282.43 71.27 8.13 4.76 7.99 3.1 3.99 table 2 flavonoids (mg/100 g), phenols (mg/g), total and reducing sugar (mg/g), anthocyanins (mg/100 g), pal (uabs/min/mg prot) and proteins (mg/g), minimum, average and maximum surface temperatures of the second evaluation, intracellular co2 concentration (μmol co2 mol -1) of the first and third evaluation, of merlot vine submitted or not to management practices and the use or not of reflexive films on the soil, in productive cycle 1 (2009/2010) ns= non-significant by the f test. ** significant at 5% of probability. (z) t1= with management practices and no reflexive film; t2= with management practices with reflexive polypropylene white raffia plastic (film 1); t3= with management practices with reflexive metallic raffia plastic (film 2); t4= without management practices and no reflexive film; t5= without management practices with reflexive polypropylene white raffia plastic (film 1) and (t6) without management practices with reflexive f metallic raffia plastic (film 2). table 3 berry drop (%), total weight of bunches and berries (g), production per plant (kg) and total sugar (mg/g), minimal, medium and maximum surface temperatures during the first evaluation, of merlot vines submitted or not to management practices with the use or not of reflexive films on the soil, in productive cycle 1 (2009/2010) ns= non-significant by the f test. *means followed by the same lowercase letter in the column don’t differ significantly by the level of 5% by tukey’s test. (z) reflexive polypropylene white raffia plastic (reflexive film 1); reflexive metallic raffia plastic (reflexive film 2). type of material (z) berry drop total weight of berries bunch weight production per plant total sugar surface temperatures in first evaluation min (°c) med (°c) max (°c) reflexive film 1 13.26 a* 147.57 a 155.55 a 4519.81 a 27.92 ab 20.43 ns 29.84 ab 39.98 ab reflexive film 2 4.63 b 149.33 a 156.57 a 4388.66 ab 29.46 a 18.81 30.45 a 41.55 a no film 14.68 a 118.35 b 124.84 b 3185.27 b 26.87 b 18.26 29.09 b 38.20 b cv (%) 24.62 14.29 14.06 24.37 5.67 14.35 2.75 5.83 table 4 minimum, medium and maximum surface temperatures during the first evaluation, number of berries per bunch in merlot vines submitted or not to management practices, in productive cycle 1 (2009/2010) * means followed by the same lowercase letter in the column do not differ significantly at 5% level of significance by tukey’s test. management practice surface temperature in first evaluation n° of berries per bunch min (°c) med (°c) max (°c) with management 20.75 a * 29.79 a 38.63 b 70.33 a without management 17.58 b 29.79 a 41.8 a 59.86 b cv (%) 14.35 2.75 5.83 5.67 44 adv. hort. sci., 2019 33(1): 39-48 regarding the bunch weight, total berries weight, and production per plant, the highest values were obtained when the reflective films 1 and 2 (white raffia plastic and metallized raffia plastic, respectively) were used, and when observed in the productive cycle 2 these values were even more expressive, but without statistical difference for the effect of the treatments (table 1). observing the productive cycles 1 and 2, we noticed the alternation of productivity, which may be related to the lower pluviosity in the final period of development of the fruits (fig. 1) and lower incidence of fungal diseases, providing best sanitary conditions of the vines resulting in increased production. it is believed that the use of reflective films in productive cycle 1 may have provided an increase in the use of the photosynthetically active radiation by the plant, since when it reaches the films, it is reflected, mainly in the regions below the canopy, usually more shaded, increasing the production of photo assimilates, which allowed a greater weight of bunch and berries and, consequently, greater production. this fact can also be observed for productive cycle 2, however, with no statistical difference. the influence of the radiation on the weight of the berries was also verified by cargnello (1992), kliewer and lider (1968), and todic et al. (2007), who verified that the weight of berries in plants exposed directly to the radiation was superior to those kept under shading conditions. this fact was also verified by morrison (1988), reporting that shading of the leaves undermined the weight of the berries. during fruit development, shading affects the photosynthetic rate, which consequently limits carbohydrate sources for their development (garriz et al., 1998). in part, this hypothesis can be verified by the results obtained regarding the intracellular co2 concentration during the second evaluation in productive cycle 1, since there was a significant effect for the interaction between the factors tested (table 5), demonstrating that when the management practice was performed, there was a lower value with the use of reflective film 2 (metallized raffia plastic), followed by the use of reflective film 1 (white raffia plastic) and during productive cycle 2, the results remained favourable for the use of some kind of material, however, with no statistical difference. it is assumed that these lower values in the intracellular co2 concentration with the use of the reflective films are due to the greater photosynthetic activity by the plant, quickly converting all co2 absorbed into photo assimilates, as previously attributed to the results regarding bunch weight, total of berries weight and production per plant. the same effect of the films was obtained for the contents of total sugars, with a higher mean obtained in fruits, where the plants had reflective films 1 and 2, which is also related to the greater use of the photosynthetically active radiation. in grapevine, kliewer (1980) described that the fruits located inside the canopy, therefore, with lower illumination, presented less accumulation of sugar. this fact also occurred in the fruits of those plants that did not have reflective films below the canopy, which demonstrates that the use of these is important for the greater use of photosynthetically active radiation. when submitted to the management practices the ss/att ratio of the fruits obtained higher results significantly using the reflective film 1 and without the use of reflective films. however, when they observed the non-use of management practices, the use of reflective material did not present statistical difference (table 6). the same phenomenon can be also observed concerning, the management practice. with its realizatable 6 ss/att ratio in merlot vines submitted or not to management practices with or without reflexive films on the soil, in productive cycle 2 (2010/2011) * means followed by the same lowercase letter in the column don’t differ significantly by the level of 5% by tukey’s test. (z) reflexive polypropylene white raffia plastic (reflexive film 1); reflexive metallic raffia plastic (reflexive film 2). table 5 intracellular co2 concentration (μmol co2 mol-1) during the second evaluation in merlot vines submitted or not to management practices with or without reflexive films on the soil, in productive cycle 1 (2009/2010) material type (z) ss/att ratio with management without managment reflexive film 1 4.44 aa 3.93 aa reflexive film 2 3.72 ba 4.48 aa no film 4.14 aa 3.86 aa cv (%) 11.8 material type (z) intracellular co2 with management without management reflexive film 1 266.35 aba* 259.50 aa reflexive film 2 261.19 ba 261.53 aa no film 274.13 aa 254.73 ab cv (%) 2.03 * means followed by the same lowercase letter in the column don’t differ significantly by the level of 5% by tukey’s test. (z) reflexive polypropylene white raffia plastic (reflexive film 1); reflexive metallic raffia plastic (reflexive film 2). porto et al. reflective materials and management on quality of merlot 45 tion, it is believed that there was increase in the photosynthetic rate of the plant, as results in the intracellular co2 concentration (tables 5, 7, and 8), since practices like disbudding and defoliation allow higher incidence of light to enter the plants. in part, the greater light penetration can be proven by obtaining the increase in the minimum temperature of the plants during the first evaluation, when the management practice is carried out (table 4), which is advantageous for greater metabolic activity of the plant, consequently providing increase in energy production and photo assimilates. however, when there was no management practice the plants showed a higher maximum temperature in comparison with those in which it was carried out (table 4). this demonstrates that the accomplishment of the management practice besides increasing the minimum temperature of the plant, provides lower maximum temperature, favouring the photosynthetic activity, since very high temperatures can provide stomatic closure, reducing the entrance of co2, which diminishes the photosynthetic activity in addition to reducing the absorption of water and nutrients. on the other hand, the average surface temperature of the plant presented equal means with or without the management practice (table 4). as for the other physiological variables (water use efficiency, co2 assimilation rate, water conductance, transpiration rate and leaf temperature), there was no significant interaction or for the factors individually during the production cycle 1 in the three evaluations carried out (table 7), also verified for intracellular co2 concentration in the first and third evaluation (table 2), and in the water use efficiency, co2 assimitable 7 water use efficiency (%), co2 assimilation rate (μmol co2 m-2 s-1), water conductance (mol h2o m-2 s-1), transpiration rate (mmol h2o m-2 s-1), leaf temperature (°c), intracellular co2 concentration (μmol co2 mol-1) of merlot vines submitted or not to management practices with or without reflexive films on the soil, during the first, second and third evaluations, in productive cycle 1 (2009/2010) ns= non-significant by the f test. (z) (t1) with management practices and no reflexive film; (t2) with management practices with reflexive polypropylene white raffia plastic (film 1); (t3) with management practices with reflexive metallic raffia plastic (film 2); (t4) without management practices and no reflexive film; (t5) without management practices with reflexive polypropylene white raffia plastic (film 1) and (t6) without management practices with reflexive f metallic raffia plastic (film 2). treatment (z) water use efficiency (%) co 2 assimilation rate (μmol co 2 m-2 s-1) water conductance (mol h 2 o m-2 s-1) transpiration rate (mmol h 2 o m-2 s-1) leaf temperature (°c) 1ª ev. 2ª ev. 3ª ev. 1ª ev. 2ª ev. 3ª ev. 1ª ev. 2ª ev. 3ª ev. 1ª ev. 2ª ev. 3ª ev. 1ª ev. 2ª ev. 3ª ev. t1 0.26 ns 0.25 ns 0.38 ns 12.12 ns 10.55 ns 10.9 ns 0.23 ns 0.24 ns 0.18 ns 4.76 ns 4.76 ns 2.94 ns 32.59 ns 31.58 ns 28.3 ns t2 0.27 0.27 0.38 12.57 11.93 11.8 0.26 0.25 0.23 4.69 4.69 3.41 31.82 31.16 28.38 t3 0.31 0.3 0.37 11.92 12.53 10.69 0.22 0.23 0.2 4.57 4.57 3.27 32.45 31.13 28.58 t4 0.28 0.3 0.39 12.44 11.52 10.98 0.23 0.22 0.18 4.5 4.5 2.96 31.84 31.21 28.6 t5 0.24 0.27 0.39 12.96 11.05 11.5 0.26 0.21 0.2 5.34 5.34 3.23 32.94 31.82 28.49 t6 0.26 0.3 0.37 12.9 12.62 11.78 0.23 0.25 0.17 4.34 4.34 2.8 31.78 30.93 28.02 cv (%) 15.03 9.78 10.72 7.22 9.69 10.72 10.85 15.89 20.67 11.69 13.37 13.43 2.99 2.07 1.41 table 8 intracellular co2 concentration (μmol co2 mol-1), water use efficiency (%), co2 assimilation rate (μmol co2 m-2 s-1), water conductance (mol h2o m-2 s-1), transpiration rate (mmol h2o m-2 s-1), leaf temperature (°c) of merlot vines submitted or not to management practices with or without reflexive films on the soil, in productive cycle 2 (2010/2011) treatment (z) intracellular co 2 concentration water efficiency (%) co 2 assimilation rate (μmol co 2 m-2 s-1) water conductance (mol h 2 o m-2 s-1) transpiration rate (mmol h 2 o m-2 s-1) leaf temperature (°c) t1 261.31 ns 1.43 ns 14.67 ns 0.23 ns 3.29 ** 24.44 ns t2 263.7 1.46 14.36 0.23 3.2 24.51 t3 263.09 1.46 14.09 0.22 3.36 24.32 t4 260.05 1.41 15.02 0.23 3.23 24.37 t5 263.43 1.44 14.71 0.23 3.67 24.5 t6 261.82 1.44 14.44 0.22 3.04 24.26 cv (%) 2.78 7.84 7.85 8.74 7.48 2.85 ns= non-significant by the f test. (z) (t1) with management practices and no reflexive film; (t2) with management practices with reflexive polypropylene white raffia plastic (film 1); (t3) with management practices with reflexive metallic raffia plastic (film 2); (t4) without management practices and no reflexive film; (t5) without management practices with reflexive polypropylene white raffia plastic (film 1) and (t6) without management practices with reflexive f metallic raffia plastic (film 2). adv. hort. sci., 2019 33(1): 39-48 46 lation rate, water conductance for productive cycle 2. the transpiration rate of productive cycle 2 showed significant interaction indicating that the use of reflective film 2 in conjunction with the management practice were superior to the other treatments, which possibly may have contributed to the quantity and quality of the fruits in the cycle productive 2 (table 9). it was verified that the adoption of management practices in spite of not influencing most of the variables analysed in the present study, provided a greater number of berries when carried out on the merlot vines in productive cycle 1 (table 4), demonstrating the importance of its execution for the winegrower. it is believed that the lower number of berries in the plants without management practices can be due to the attack of fungal diseases. champagnol (1984) and fregoni (1998) reported in their studies that mildew, after effective fruiting, may be one of the causes for the lower number of berries per bunch. indeed, the practices of shoot topping, suckering, withdrawal of tertiary branches and defoliation may have improved the penetration of light and air in the area of projection of the plants, not allowing the formation of favourable microclimate for fungal diseases. regarding the microbiological analysis of the soil, a statistical difference was observed for the type of reflexive film used in productive cycle 1, with the highest microbial activity in the soil when white raffia plastic and metallized raffia plastic were used. the latter did not differ statistically from the treatment without reflexive film on the soil (table 10). for production cycle 2, although the values obtained were higher, which makes it favourable for productivity, the same did not present statistical differences. it is assumed that the use of the white or metallic raffia plastic films to reflect the solar radiation allowed lower temperature in the soil, as well as reduced water loss, favouring the full metabolic activity of soil microorganisms, given the maintenance of conditions optimum temperature and humidity, decreasing problems with water stress for the plant. hungria et al. (1997) mentioned that the reduction of soil microbiota impairs the temporary fixation of nutrients, increasing their losses and resulting in soil impoverishment. in general, soils with high microbiological activity indicate low human interference with the environment and are, in principle, desirable for crops, since they contribute to a faster decomposition and re-synthesis of organic matter, to nutrient cycling, to specific biochemical transformations (nitrification, denitrification, oxidation and sulphur reduction) for biological nitrogen fixation (silveira and freitas, 2007). thus, the use of reflective material on the soil presents additional benefits to the crop and the environment, and according to the peculiarities of the area, the effects of the application of the technology on the soil microbial population can become as important or more than the benefits obtained directly on the quality of the fruits. attention must be paid to determining the most appropriate moment for the installation of the reflective film in the soil, since the influence of the radiation on the quality of the fruit varies according to the phenological stage of the crop (weston and barth, 1997; kader, 2002; meinhold et al., 2011). material type (z) transpiration rate (mmol h 2 o m-2 s-1) with practice without practice reflexive film 1 3.20 b a 3.67 a a reflexive film 2 3.36 a a 3.04 a ab no film 3.29 a a 3.23 a b cv (%) 7.48 table 9 transpiration rate (mmol h2o m-2 s-1) of merlot vines submitted or not to management practices with or without reflexive films on the soil, in productive cycle 2 (2010/2011) * means followed by the same lowercase letter in the column don’t differ significantly by the level of 5% by tukey’s test. (z) reflexive polypropylene white raffia plastic (reflexive film 1); reflexive metallic raffia plastic (reflexive film 2). table 10 microbiological respiratory activity of the soil according to the material type addopted for merlot vines, in productive cycle 1 (2009/2010) and productive cycle 2 (2010/2011) * means followed by the same lowercase letter in the column don’t differ significantly by the level of 5% by tukey’s test. (z) reflexive polypropylene white raffia plastic (reflexive film 1); reflexive metallic raffia plastic (reflexive film 2). material type (z) microbiological resiratory activity (mg of co2 kg of soil-1) productive cycle of 2009/2010 reflexive film 1(z) 45.65 a reflexive film 2 43.45 ab no film 35.75 b cv (%) 11 productive cycle of 2010/2011 reflexive film 1 100.10 ns reflexive film 2 102.3 no film 82.13 cv (%) 30.87 porto et al. reflective materials and management on quality of merlot 47 4. conclusions the use of reflective films on the soil improved the quality of merlot grapes in some aspects as reducing berry drop, consequently increasing productivity. management practices here tested didn’t have influence on the physicochemical and biochemical characteristics of the fruits, resulting only in more berries per bunch. the use of reflective films on the soil provided higher microbiological activity in the soil, which may be important for grapevines. references andris h., crisosto c., 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análise estatística para microcomputadores. sanest. ufpel, pelotas, pp. 56. impaginato 335 adv. hort. sci., 2018 32(3): 335-341 doi: 10.13128/ahs-22472 ascorbic acid content and senescence in blueberry (vaccinium corymbosum l.) during storage a. spinardi (*), i. mignani dipartimento di scienze agrarie e ambientali (disaa), università degli studi di milano, via celoria, 2, 20133 milano, italy. key words: controlled atmosphere (ca), malondialdehyde, oxidative stress, redox state and quality. abstract: blueberry consumption increases because its health properties linked to antioxidants, easy cultivation and profitability. the ability to preserve fruits in controlled atmosphere (ca) allows extending the marketing calendar. the work evaluates parameters linked to the cellular redox state of blueberry fruits, cv. brigitta, stored at 0°c at different atmosphere regimes (ca1= 10% co2, 4% o2 and ca2= 9% co2, 2% o2, compared to air as control). during storage, quality was assessed by the content of ascorbic acid (aa), antioxidant and index of fruit metabolic status, and of malondialdehyde (mda), cell membranes oxidative stress and senescence marker; soluble solids content, titratable acidity and dry matter were also determined. storage in ca increases the blueberries shelf life, particularly at the intermediate times; after 2 months there is a drastic lowering in aa levels and differences among treatments are no longer detectable. ascorbate is confirmed to be an excellent index of oxidative stress in fruit senescence processes. in control, along with the aa decrease, there is a higher mda content, in particular up to the intermediate dates. ca2 proves to be the most suitable atmosphere for delaying the senescence process. titratable acidity and soluble solids remain constant in all samples throughout storage. 1. introduction the blueberries (vaccinium corymbosum l.) industry is rapidly increasing because of its healthy properties linked to antioxidant content and its meeting the consumer expectation of healthy food (gosch, 2003; kähkönen et al., 2003). therefore the blueberry growing area is rapidly extending in many regions, taking advantage by the adaptability and ease of cultivation of this crop and its profitability. blueberry cultivation is well adapted to mountain and hill soil and climate conditions and to oriented organic or environmental friendly agricultural methods with integrated pest management, endowing the growing areas with a benefit of local production and environmental respect. moreover blueberry, as other small fruit, can be established as small-scale farms giving extra income to family businesses. (*) corresponding author: anna.spinardi@unimi.it citation: spinardi a., mignani i., 2018 ascorbic acid content and senescence in blueberry (vaccinium corymbosum l.) during storage. adv. hort. sci., 32(3): 335-341 copyright: © 2018 spinardi a., mignani i. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 8 january 2018 accepted for publication 17 may 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(3): 335-341 336 the quality of this fruit and the content of healthpromoting compounds are influenced by many factors, such as environmental conditions, genetic diversity and degree of maturity at harvest (ehlenfeld and prior, 2001; connor et al., 2002). blueberry has often been reported to be a highly perishable fruit, thus its commercial value could be strongly affected by storage conditions. its profitability may take interesting advantage by a storage and shelf life extension that can be achieved by cold storage (0-1°c) and controlled atmosphere (ca) with low oxygen (1-4 kpa o2) and high carbon dioxide (9-12 kpa co2) concentrations (krupa and tomala, 2007). after harvest, blueberry quality and product losses are mainly due to dehydration, weight loss, shrivel and fungal spoilage. to understand better the effects of long-term storage on the antioxidant components of blueberry the present study evaluates some parameters related to the cellular redox state of berries of the late cultivar brigitta, stored at 0°c in different ca regimes. 2. materials and methods full ripe ‘brigitta’ berries were harvested in the valtellina area (northern italy, lat: 46.1653333, long: 9.6461111) from 10-year-old plants. the same day of harvest, fruits were put in polyethylene punnets containing about 200 g of fruits each, labelled, weighed and randomly assigned to each of the different storage conditions. berries were stored at 0°c, 95% relative humidity in the following controlled atmosphere modes: ca1: 4 kpa o2 and 10 kpa co2; ca2: 2 kpa o2 and 9 kpa co2. the control was kept in the air (20.1 kpa o2 and 0.03 kpa co2). samples were taken for quality assessment at the following storage times: 0, 12, 33, 47, 61, 76, 94, 110, 132, 147 days. at each sampling time, 3 punnets per storage condition were removed from the storage and held at -80°c until chemical analysis and quality measurements. each parameter was determined on 3 replicates, obtaining one fruit sample from each of the 3 punnets per storage condition and time. dry matter was determined on 50 g of homogenate placed in crucibles and left in an oven at 70°c for 24 hours. a s c o r b i c a c i d w a s e x t r a c t e d i n a 6 % ( w / v ) metaphosphoric acid solution. the amount of 7.5 g of blueberries was homogenized in a mortar with 10 ml of cold extraction medium and centrifuged at 10,000 g at 4°c. the supernatant was transferred into a 25ml volumetric flask at 4°c. the pellet obtained by c e n t r i f u g a t i o n w a s w a s h e d w i t h 7 m l o f c o l d metaphosphoric acid solution and centrifuged. the supernatants were combined and brought to a final volume of 25 ml with cold 6% metaphosphoric acid. after filtration through 0.2-µm nylon filter, a 10-µl sample aliquot was injected onto an inertsil ods-3 (5 µm; 4.6 mm × 250 mm) gl science column at 20°c attached to a series 200 lc pump (perkinelmer, norwalk, ct, usa). the column was eluted with 0.02 m orthophosphoric acid at a flow rate of 0.7 ml/min and ascorbic acid was monitored at 254 nm with a uv-975 intelligent uv-vis detector (jasco model 7800, tokyo, japan). ascorbic acid was identified by the retention time and quantification was achieved according to the concentration of a corresponding external standard (sinelli et al., 2008). the determination of the thiobarbituric acid-reactive-substances (tbars) content was carried out using a 5% trichloroacetic acid extract. five grams of mesocarp were homogenized in 25 ml of 5% (w/v) trichloroacetic acid then centrifuged at 4°c at 10,000 g for 30 min. the extract was added to an aqueous solution of 15% (w/v) tca and 0.5% (w/v) 2-thiobarbituric acid. samples were mixed and heated at 95°c for 15 min in a water bath, cooled and centrifuged at 4,000 g for 15 min. samples were then analyzed in a spectrophotometer (jasco, model 7800, tokyo, j a p a n ) a t 5 3 2 , 6 0 0 , a n d 4 4 0 n m . t h e v a l u e o f a b s o r b a n c e a t 5 3 2 n m w a s p u r g e d f r o m t h e absorbance at 440 nm and at 600 nm due to sucrose and tonon-specific turbidity (cocetta et al., 2016). tbars concentration was expressed in mda equivalent (nmol/g fw) following the equation (du and bramlage, 1992): {[(a 532 a 600 ) (a 440 a 600 )(8.4/147)/157 000]}106 for the determination of the titratable acidity (ta), a 5 g sample of homogenized blueberry puree was diluted with 30 ml of distilled water. the ta was measured after 15 sec stirring by titration with 0.1 n naoh to an end-point of ph 8.3 by a compact titrator d (crison strumenti spa, carpi, italy). the acidity was expressed as meq/100 g fw. total soluble solids (tss), expressed as percent of soluble solids, were determined by a hand refractometer (atago mod., n1, tokyo, japan) on juice obtained from squeezing the berries. analysis of variance was performed by spss software, ibm spss statistics 22 (spss inc., chicago, il), using general linear model univariate analysis. sources of variation were time of storage and atmosphere regimes. significant differences between spinardi and mignani ascorbic acid content and senescence in blueberry 337 m e a n s w e r e c a l c u l a t e d b y t u k e y ’ s m e a n t e s t . differences at p≤0.05 were considered as significant. 3. results blueberry fruit cv. brigitta showed at harvest a percentage of dry matter of 12.52% (fig. 1). within the first 47 day storage period, berries maintained in air and in controlled atmospheres exhibited similar dry matter contents (13.45% in control; 12.55% and 12.14% in ca1 and ca2 stored berries, respectively). thereafter, the level of dry matter increased in control samples until end of storage and was higher than in treated samples. in berries stored in both ca regimes dry matter did not change throughout the entire storage period. at the end of the trial dm accounted for 14.52% of the weight of control fruit and for 11.69% and 11.54% of the weight of ca1 and ca2 stored fruit, respectively. the trend of total soluble solids fairly followed the parallel sample variation in dry matter content during storage (fig. 2). soluble solid content increased in control fruit during the first storage period, from 11.10% at harvest to 12.70% at 47 days storage, and during the trial it was always higher in fruit stored in air than that in berries stored in ca1 and ca2, except at 132 days of storage. during the trial, berries stored in ca showed no differences in this parameter until day 147, when a decrease was recorded. comparing total soluble solids at the end of storage period with respect to the beginning, the amounts were 5% higher in berries maintained in air and 12% and 17% lower in berries under ca1 and ca2, respectively. the titratable acidity (11.91 meq/100 g fw at harvest) remained stable in the first 94 days of storage in all the samples and no differences were detectable among treatments (fig. 3). after this sampling time, ta increased in all samples, reaching a maximum value at day 132 and either maintaining higher values at the last sampling point, in fruit under controlled atmosphere regimes (20.27 meq/100 g fw in ca1 and 19.69 meq/100 g fw in ca2 after 147 days storage), or decreasing, in control, at the end of storage to values not statistically different from those at the beginning of the trial (10.92 meq/100 g fw). fig. 1 changes in blueberry dry matter during cold storage (0°c) in different atmosphere regimes (air; ca1= 4 kpa o2 and 10 kpa co2; ca2= 2 kpa o2 and 9 kpa co2). values are mean ± se of triplicate samples. in the table, different lowercase letters indicate significant differences among values after different storage periods at each atmosphere regime and different capital letters indicate significant differences among values at different atmosphere regimes, at each sampling time (p≤0.05). fig. 2 changes in blueberry soluble solid content during cold storage (0°c) in different atmosphere regimes (air; ca1= 4 kpa o2 and 10 kpa co2; ca2= 2 kpa o2 and 9 kpa co2). values are mean ± se of triplicate samples. in the table, different lowercase letters indicate significant differences among values after different storage periods at each atmosphere regime and different capital letters indicate significant differences among values at different atmosphere regimes, at each sampling time (p≤0.05). fig. 3 changes in blueberry titratable acidity during cold storage (0°c) in different atmosphere regimes (air; ca1= 4 kpa o2 and 10 kpa co2; ca2= 2 kpa o2 and 9 kpa co2). values are mean ± se of triplicate samples. in the table, different lowercase letters indicate significant differences among values after different storage periods at each atmosphere regime and different capital letters indicate significant differences among values at different atmosphere regimes, at each sampling time (p≤0.05). adv. hort. sci., 2018 32(3): 335-341 338 ascorbic acid content (0.35 mg/100 g fw at harvest) increases after the first 12 days of storage in control fruit by 377% and in ca1 and ca2 stored samples by 218% and 239%, respectively (fig. 4). after this sampling time the levels in the control progressively declined reaching the minimum level of 0.18 mg/100 g fw, while ascorbate levels remained higher in the berries stored in both ca regimes until the day 61 (1.31 mg/100 g fw in ca1 and 1.39 mg/100 g fw in ca2). from that date the ascorbic acid content of treated fruit decreased drastically and after the sampling time at 94 days storage ascorbate content was no longer detectable in any sample. in control fruit tbars levels (197.95 nmol/g fw at harvest) decreased at the first sampling date and then increased markedly (+33%), reaching a maxim u m a f t e r 3 3 d a y s o f s t o r a g e ( f i g . 5 ) . t b a r s decreased over time, starting at 94 days of storage, from 222.10 nmol/g fw to 138.18 nmol/g fw at the end of storage. in ca stored berries, the trend of tbars content progressively decreased, but no significant differences were observed compared to harvest date, except for fruit stored in ca1 for 61 days. at the end of the trial, tbars levels reached values of 170.32 and 143.9 nmol/g fw in ca1 and ca2 stored berries, respectively. 4. discussion and conclusions dry matter content, linked to water and weight loss and to metabolic activity, is an important factor affecting blueberries storage life. as blueberries have a high surface-to-volume ratio, they are prone to water loss. the similar values showed by all the samples during the first 47 day of cold storage are in accordance with the research findings of duarte et al. (2009). they reported a markedly low (0.9%) weight loss found in ‘brigitta’ fruit at 48 d of cold storage, regardless of the gas mixture used, either air or ca. the higher percentages of dry matter determined in control starting from the sampling date after 61 days is due to the greater loss of water sustained by the berries kept in the air with respect to the samples stored in ca1 and ca2. in contrast to our data, alsmairat et al. (2011) reported an effect of ca on moisture loss, yielding a 13-fold difference between the 0% co2/21% o2 treatment (0.25% weight loss) and the 19% co2/2% o2 treatment (3.3% weight loss). the authors reported that the greater weight loss for the highest co2/low o2 treatment could be associated to physical causes, e.g. the greater flux of dry gas through the ca chambers, and secondly because high levels of co2 can stress blueberry fruit. on the other hand, in our trial a slight impact on moisture loss in control fruit may stem from similar physical causes, because the ca chambers are smaller than the storage room of control fruit and also because control storage condition could be more stressful to berries than the ca. blueberry fruit cv. brigitta at harvest showed a total soluble solids content and titratable acidity according to other research data (hancock et al., fig. 4 changes in blueberry ascorbic acid content during cold storage (0°c) in different atmosphere regimes (air; ca1= 4 kpa o2 and 10 kpa co2; ca2= 2 kpa o2 and 9 kpa co2). values are mean ± se of triplicate samples. in the table, different lowercase letters indicate significant differences among values after different storage periods at each atmosphere regime and different capital letters indicate significant differences among values at different atmosphere regimes, at each sampling time (p≤0.05). fig. 5 changes in blueberry malondialdehyde level, expressed as tbars content, during cold storage (0°c) in different atmosphere regimes (air; ca1= 4 kpa o2 and 10 kpa co2; ca2= 2 kpa o2 and 9 kpa co2). values are mean ± se of triplicate samples. in the table, different lowercase letters indicate significant differences among values after different storage periods at each atmosphere regime and different capital letters indicate significant differences among values at different atmosphere regimes, at each sampling time (p≤0.05). spinardi and mignani ascorbic acid content and senescence in blueberry 339 2008; gündüz et al., 2015). soluble solids content and titratable acidity level are important quality parameters that account for the flavor of fruit. total soluble solids of control samples increased by 13% during the first storage period of 47 days, according to chiabrando et al. (2009) who recorded changes in soluble solid content in cv. coville, with significantly higher values after 28 days of storage in air at 0°c. this increase in control samples cannot be completely explained on the basis of water loss by transpiration and could be more closely associated to the final events of the ripening process. although blueberry is expected to stop sugar accumulation once the fruit is picked (nonclimacteric behavior) and does not have starch to support soluble sugar synthesis after harvest, an increase in carbohydrate levels may be a consequence of cell wall degradation. in fact, during this storage period of 47 days no significant increase in dry matter linked to a change in m o i s t u r e c o n t e n t i s o b s e r v e d i n c o n t r o l f r u i t . thereafter, soluble solids content did not change until end of the trial. similar research findings were reported on cultivars brigitta (duarte et al., 2009), b l u e c r o p a n d i v a n h o e ( b e a u d r y e t a l . , 1 9 9 8 ) , burlington (forney et al., 2003). in ca stored fruit, soluble solids content shows similar values throughout the storage period until the last sampling time, when it decreased significantly both in ca1 andca2 stored berries. the steady values of total soluble solids shown by the samples stored in ca starting from the beginning of cold storage can be due to a stronger effect of ca on fruit metabolism compared to air-stored fruit. tritatable acidity showed no changes in all samples stored up to 94 days. this is in accordance with previous studies carried out by chiabrando et al. (2009) on ‘bluecrop’ and ‘coville’ (fruit stored for 35 days in air) and smittle and miller (1988) on rabbiteye blueberry (fruit stored for 42 days at different atmosphere regimes) which showed titratable acidity not to be affected by storage duration or atmosphere. on the other hand, after this storage period of 94 days, the parameter increased markedly at day 132 and then remained stable in ca stored fruit until end of the trial. schotsmans et al. (2007) reported a significant increase in titratable acidity during ca storage (2.5% o2, 15% co2) of rabbiteye blueberry ‘centurion’ fruit from 35 days onwards. in contrast to ca stored fruit, in control fruit titratable acidity decreased after the peak at day 132 t o l e v e l s s i m i l a r t o t h a t r e c o r d e d a t h a r v e s t . according to this trend, harb et al. (2014) found no significant differences in titratable acidity between air-stored and ca stored blueberries cv. duke after 4 weeks storage, but a 8 week storage period resulted in significant higher level of titratable acidity in ca stored fruit respect to control fruit. increased titratable acids contents of blueberries did not correspond proportionately and cannot be explained with declining moisture content of the berry, rather may be linked to the onset of senescence and to the release of additional acids associated with softening and cell wall breakdown (proctor and peng, 1989). the higher levels in titratable acidity at the end of storage in ca stored fruit compared to control could indicate that such fruit still had more reserves remaining whereas the reserves for the airstored fruit were partially depleted. ascorbic acid is an antioxidant which concur to fruit quality and it is a marker of the metabolic status of fruit. growing season, location, agricultural practices, cultivar and crop ripeness affected to varying degrees ascorbic acid levels at harvest. the levels of 1.7 mg/100 g recorded at full ripeness in control fruit of ‘brigitta’ were low compared to some other research data (10.2 mg/100 g reported by golding et al., 2014; 13.6 mg/100 g reported by kozos et al., 2014) but similar to other studies (4.8 mg/100 g reported by spinardi et al., 2009; 2.6 mg/100 g reported by sinelli et al., 2008). during the first 12 days of storage ascorbic acid levels increased markedly and reached a maximum in air-stored fruit, when the ripening process is completed. the significant increment in ascorbic acid content may rather be due to a partial disassembly of cell wall polysaccharides (davey et al., 1999; gilbert et al., 2009; cruz-rus et al., 2011) than to the activation of the ascorbic acid biosynthetic pathway. it could also be related to a more efficient ascorbic acid recycling pathway (e.g. enzymes of the ascorbateglutathione cycle), which plays an important role in the response and adaptation to the stress (stevens et al., 2008). after this storage period, ascorbic acid content in control samples progressively diminished. this steady decrease could be due to less effective recycling of ascorbic acid or the presence of factors that promote oxidation of the ascorbic acid pool such as the enzymatic activity of ascorbate peroxidase (apx) or the direct interaction with reactive oxygen species (ros). zhou et al. (2014) found a progressive increment in apx activity in blueberries until 45 days of cold storage in air and a higher level after 60 days compared to the beginning of storage. throughout 340 adv. hort. sci., 2018 32(3): 335-341 storage, they found also a steady increase in superoxide radical production rate and hydrogen peroxide content. in contrast to the decrease recorded in control fruit after the first sampling time, ascorbic acid content did not change in ca stored fruit up to 61 days, when the levels were significantly higher than in control fruit. this evidence is partially in contrast to a previous report on cv. bluecrop (harb et al., 2010) that indicate that a marked loss in ascorbic acid over the entire storage period of 6 weeks occurred under all storage conditions (ranging from 0% co2, 18% o2 to 24% co2, 2% o2) and stated that increasing co2 and/or decreasing o2 partial pressures within the storage atmosphere did not decisively change this loss. on the other hand, harb et al. (2010) found that storing fruit under low o2 combined with high co2 level (up to 18%) resulted in better preservation of ascorbic acid and that the highest co2 level (24%) was injurious and resulted in lower ascorbic acid content. the malondialdehyde (mda) content (determined as tbars level) of 198 nmol/g fw at harvest is similar to the levels reported on blueberries (zhou et al., 2014), on pears (cocetta et al., 2016) and grapes (xu et al., 2009). the levels of mda were coherent in part with the ascorbic acid levels, and reflected more the differences between the various treatments than the trend of a single treatment during the storage period. in control fruit the decreasing trend of ascorbic acid levels during storage is accompanied with a significant higher malondialdehyde content than that recorded in fruit maintained in ca2, up to 94 days of storage. malondialdehyde is considered a biochemical marker of lipid peroxidation of membranes. this data demonstrates a greater oxidative damage to the cellular components of fruit in a natural atmosphere. the drastic decrease in the ascorbic acid content in the second part of the storage period is not in fact accompanied by an increase in mda levels. this could be due to the onset of senescence processes associated to a loss of cellular integrity and compartimentalisation, as a result of the drastic lipid peroxidation of cell membranes caused by ros accumulation, and therefore not to a recovery of oxidative stress. the analysis of the tbars content shows a positive effect especially of ca2 on the fruit, in which the senescence process is delayed and there is no increase in the levels of this parameter. the present work confirms the ascorbic acid content as a parameter closely associated to the cellular metabolic state and therefore an excellent index of oxidative stress that intervenes in the processes of senescence of the fruits during storage. moreover, t h e b l u e b e r r y ‘ b r i g i t t a ’ s t o r a g e i n c o 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w.t., peng x.l., luo y.b., wang j.a., guo x., huang k. l., 2009 physiological and biochemical responses of grapefruit seed extract dip on ‘redglobe’grape. lwtfood sci. and technol., 42(2): 471-476. zhou q., ma c., cheng s., wei b., liu x., ji s., 2014 changes in antioxidative metabolism accompanying p i t t i n g d e v e l o p m e n t i n s t o r e d b l u e b e r r y f r u i t . postharvest biol. techn., 88: 88-95. impaginato 569 adv. hort. sci., 2018 32(4): 569-573 doi: 10.13128/ahs-22942 field evaluation of new kabuli chickpeas lines for the production of canned seeds p. casini dipartimento di scienze delle produzioni agroalimentari e dell’ambiente, università degli studi di firenze, piazzale delle cascine, 18, 50144 firenze, italy. key words: canned products, canned seeds, chickpea, cicer arietinum l., seed grade, tuscany. abstract: there is an increased demand for canned chickpeas worldwide, which has also resulted in increased chickpea cultivation in italy. the availability of italian chickpea varieties, suitable for industrial transformation, is limited. the objective of the present study was to evaluate the field production of new kabuli chickpea accessions and their suitability for industrial transformation. thirteen accessions, provided by the international centre for agricultural research of turkey, together with ‘blanco sinaloa’, a commercial mexican variety, were cultivated in cesa, tuscany, in 2016 and 2017. the average seed yield was 2.8 and 3.4 t ha-1, respectively, in 2016 and 2017. increased yield in 2017 was attributable to a more balanced rainfall pattern, compared to that in 2016 which induced increased vegetative growth. based on production stability over the two-year trial, the varieties flip08-69c, flip-160c and flip05-157c turned out particularly suitable for cultivation. the climatic conditions in 2017 favored an increased presence of a grade b seed classification [220-250 seeds per 100 g (caliber 8)] which is preferred by industry. most varieties showed potential regarding seed dimension classification, however, of the varieties with a good production, flip05-157c was also characterized by seed gradation stability. 1. introduction in recent years, there has been a change in the style of food consumption by italian people. this includes the growing interest in legumes as an alternative protein source to meat. the lower fat content of legumes appears to be a more appreciated characteristic. here, we are referring to “food safety”, where consumers are prepared to incur higher costs when purchasing foods for health safety and towards guaranteeing environmentally friendly production techniques (dixon and sumner, 2003; fratianni et al., 2014). the industrial uses of legumes vary, ranging from animal feed (flour (*) corresponding author: paolo.casini@unifi.it citation: casini p., 2018 field evaluation of new kabuli chickpeas lines for the production of canned seeds. adv. hort. sci., 32(4): 569-573 copyright: © 2018 casini p. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 27 march 2018 accepted for publication 4 july 2018 ahs advances in horticultural science short note adv. hort. sci., 2018 32(4): 569-573 570 and supplements) to the processing of the seeds for human consumption, especially in the form of preserved (canned) products. according to 2016 statistics, the italian market for canned vegetables is valued at approximately 400 million euros, representing an increase of +1.2% compared to the previous year (limonta, 2016). the same data showed that, in the face of production decreases in sweet corn, green beans and peas, there is an increase in beans and chickpeas. more specifically, the categories “beans” and “other legumes” (chickpeas and lentils) constitute more than 60% of the market. as regards chickpeas, with a 62% share, an increase of 10.2% was recorded. as an inevitable consequence, there was an increase in the area dedicated to chickpea cultivation in italy (palumbo, 2017), from 5.000 to almost 16.000 ha, from 2008 to 2017, respectively. this notable increase is attributable, not only to the aforementioned food trends, but also to agronomic benefits such as reducing the use of both fertilizers and pesticides. in turn, this serves to improve the structure and fertility of the soil and, above all, a more sustainable return to crop rotation with cereals (palumbo, 2017). these benefits were already sought after, as early as the beginning of 2000 (watson et al., 2017). concomitant with italian scenario for chickpea cultivation, there has been a steady increase in the world production of chickpeas from 63.4 to 77.5 million tons, from 2009 to 2015, respectively (muehlbauer and sarker, 2017). this positive trend in the chickpea market has naturally resulted in the involvement of the italian canning industry, always attentive to the needs of the market. an increasing number of industries, through their own incentive, are producing the raw material directly in italy just to meet the needs of “food safety”. in this way, through cultivation contracts, they are certain to direct cultivation towards the production of the raw material, adhering, as closely as possible to both product requirements and processing techniques for the production of canned chickpeas. the italian consumer prefers kabuli chickpeas. the characteristics of “kabuli” include a light color, preferably with a caliber of 8 (220-250 seeds per 100 g), a typical shape with rough surface, and with a thin integument that still adheres to the seeds once cooked (palumbo, 2017). the availability of italian chickpea varieties, suitable for industrial transformation, is rather limited. wi t h reg a rd t o c en t ra l i t a l y, m o re s p ec i f i c a l l y tuscany, experimental tests conducted in the ‘80s and ‘90s (casini, 1987, 1989), showed that either autumn or end of winter sowing subjected the cultivation to high risks of anthracnose [ascochyta rabiei (pass.) labr. trot.]. more recent research (radicetti et al., 2012), has highlighted that “pascià” and “principe” are the two most widespread italian varieties, suitable for industrial transformation with yields varying between 2.5 and 2.7 t ha-1. however, industry is continually looking for new varieties either for use in genetic improvement programs or for use towards better production, tolerance to diseases and technological qualities. the aim of the present study was to evaluate the field production of new accessions of kabuli chickpeas and their suitability for industrial transformation based on the preferred seed dimensions classification characteristics. 2. materials and methods t h e f i e l d e x p e r i m e n t s w e r e c a r r i e d o u t i n tuscany, central italy, in 2016 and 2017 at the “ c e n t r o p e r i l c o l l a u d o e d i l t r a s f e r i m e n t o dell’innovazione di cesa (arezzo)”, 43° 18’ n; 11° 47’ e; 242 m a.s.l. the cultivation environment was characterized of a neutral, loamy-sandy soil. the principle physical and chemical characteristics of the soil were as follows: sand 36%, loam 38%, and clay 26% respectively. the soil ph was 7.0. total n was 0.11% and p (olsen) 13 ppm. exchangeable ca, mg and k were 4123, 595 and 141 ppm, respectively. thirteen accessions, provided by the international centre for agricultural research (icarda) of ankara (turkey), were used, in addition to “blanco sinaloa”, a commercial variety from mexico. based on previous experiments carried out in central italy (casini, 1989), the autumn-winter sowing period was not taken into consideration due to serious damage caused by anthracnose blight. as a result, the sowing dates were march 14, 2016 and february 22, 2017, respectively. plots were arranged according to a complete rcb design, with three replicates. the size of the plots were 2.0 x 4.0 m (four rows wide with 0.5 m row spacing). the sampling area was comprised of the two central rows of 3.0 m long. a seed quantity of 380 per plot was used. in order to obtain the correct planting density of 25 plants m-2, seedlings were thinned soon after complete emergence. plots were hand-weeded twice (45 and 65 days after emergence casini new kabuli chickpeas for canned seeds production 571 [dae]) during the growth cycle. the agricultural interventions performed during the two-year experimental period are reported in table 1. plant height, number of stems, height of the first pod and number of pods per plant were determined at maturation stage, using a total of 10 plants per sample plot. yield calculation and the number of seeds amounting to 100 g were performed using seed samples at standard humidity of 12%. grading of seeds was carried out according to the standard tables of conserve italia (2015) for the italian market as follows: grade a [<220 seeds per 100 g (caliber 6)]; grade b [220-250 seeds per 100 g (caliber 8)] and non standard or off-type (>250 seeds per 100 g). both grades a and b are considered suitable for processing, but b is the preferred grade. data collected in the experiments were processed utilizing a mixed-model analysis of variance (anova), where accession was considered as a fixed effect factor, and year as random effect factor. statistical differences were tested at p≤0.05, p≤0.01 or p≤0.001. the tukey’s hsd test was used to stress significant d i f f e r e n c e s b e t w e e n m e a n s a n d h o m o g e n o u s groups. 3. results, discussion and conclusions the climatic trends over the two-year experimental period were very different, especially with regard to rainfall. in the first year, the crop benefited from 562 mm of rainfall, of which 142 mm were evenly distributed during the period may-mid-june. in the second year, total rainfall over the cultivation cycle was 290 mm. in 2017 the maximum temperatures were particularly high with an average of 24.8°c (31.0°c in the summer). principle biometric characteristics of the lines (table 2) were significantly different at either p≤0.01 or p≤0.001, with the exception of the number of empty pods per plant. as regards the main biometric characteristics, the higher rainfall of 2016, contributed to a greater vegetative growth of the plants, compared to that in 2017. the height of the plants 2016 2017 previous crop wheat sunflower plowing september 6, 2015 rippering and rolling october 22, 2016 harrowing september 28, 2015 november 2, 2016 grubbering january 10 harrowing march 14 february 22 pre-sowing fertilization march 14 february 22 n 52 and p 2 o 5 114 kg ha-1 n 52 and p 2 o 5 114 kg ha-1 sowing march 15 february 23 emergence april 11 march 14 harvesting august 22 august 19 table 1 agronomic technique, date of sowing, date of emergence and harvesting of the two field trials source of variation plant height (cm) stems per plant (n) height of first pod (cm) filled pods (n) empty pods (n) seeds per pod (n) lines (l) flip05-69c 58.3 a 5.1 bcd 34.3 a 43.5 bc 3.5 a 1.2 ab flip05-156c 55.1 ab 5.3 a-d 28.2 bcd 45.6 bc 4.9 a 1.1 ab flip05-157c 53.9 ab 6.7 ab 29.2 bc 39.1 cd 3.4 a 1.1 ab flip07-230c 58.7 a 6.9 a 30.7 ab 45.1 bc 5.0 a 1.1 ab flip07-318c 52.7 ab 5.5 a-d 30.0 abc 42.0 cd 3.0 a 1.3 a flip08-69c 55.7 ab 6.6 ab 28.9 bcd 48.5 bc 3.9 a 1.2 ab flip08-160c 52.6 ab 6.2 abc 29.3 bc 47.2 bc 4.4 a 1.1 ab flip08-170c 56.8 a 5.1 bcd 27.5 bcd 58.5 a 4.5 a 1.2 ab flip08-200c 55.4 ab 4.7 cd 25.8 cde 43.7 bc 4.9 a 1.2 ab w6-12861 59.0 a 6.0 a-d 31.3 ab 63.3 a 4.9 a 1.1 ab w6-9484 54.9 ab 5.1 bcd 24.3 de 37.2 cd 4.3 a 1.0 b w6-30 54.0 ab 5.7 a-d 25.4 cde 46.2 bc 4.1 a 1.0 b w6-25 52.4 ab 4.5 d 28.0 bcd 57.2 ab 4.6 a 1.2 ab blanco sinaloa 49.1 b 5.1 bcd 21.4 e 29.4 d 2.6 a 1.2 ab f ** *** *** *** ns ** year (y) 2016 42.1 6.7 31.8 65.8 5.8 1.2 2017 42.9 4.6 24.5 28.0 2.4 1.2 f ** ** *** *** *** ns l x y *** ** *** *** *** ns table 2 principle biometric characteristics of the lines ns= not significant; ** significant at p≤0.01; ***significant at p≤0.001. means followed by the same letter(s) are not different for p≤0.05 according to tukey test. adv. hort. sci., 2018 32(4): 569-573 572 was almost 20 cm higher (average of 64.7 cm) with a corresponding increased height of first stage pods (31.8 cm from the ground), more favorable for mechanical harvesting. increased vegetative growth was also expressed in the greater ramification number of 6.7 stems per plant in 2016 as compared to 4.6 in 2017, respectively. the height of the plants was both positively and significantly correlated to the number of full pods per plant (r2 = 0.799**), which in the first year was more than double than that observed for the second year: 65.8 vs. 28.0 cm, respectively. the average seed yield was 2.8 and 3.4 t ha-1, respectively, in 2016 and 2017 (fig. 1). the yield increases in 2017 were observed for all varieties. varieties showing a yield increase that exceeded 25% included flip 69c, w6 12861, w6 9484 and w6 25, respectively. these increases were attained despite a lower production of full pods per plant in 2017. this yield increase was likely attributable to the more favorable climatic conditions of 2017, in comparison to those of 2016, which induced increased vegetative growth. table 3 shows an increase in the number of seeds per each 100 g weight for almost all varieties, with the exception of w6 25, in which the number seeds (195-196 seeds per 100 g) is maintained. table 3 also provides the commercial classification values for varieties and their suitability for processing into precooked products. in this regard, all the lines were found to be suitable for both years with a grade classification of either a or b, with the prevalence of the more desired latter classification in 2017. the results of this experiment highlighted a good production capacity of the majority of the lines tested. some varieties, such as flip08-69c, flip-160c and flip05-157c, are particularly suitable, as based on production stability over the two-year trial. moreover, among these lines, flip05-157c was also characterized by seed gradation stability, suitable for industrial transformation. conditions of balanced rainfall, evident in 2017, favored an 8 caliber (b) seed yield, which is preferred by industry. in conclusion, with some exceptions, all the lines tested are considered suitable for use in selection and/or for breeding programs, aimed at obtaining varieties dedicated to the production of pre-cooked seeds for the italian market. references casini p., 1987 confronto varietale di cece (cicer arietinum l.) in semina primaverile. agricoltura ricerca, 9 (80): 33-42. casini p., 1989 comportamento di alcune varietà di cece (cicer arietinum l.) in semina autunnale. agricoltura ricerca, 11(95): 27-42. conserve italia, 2015 allegato capitolati di acquisto prodotti di classe 1, pp. 16-18. dixon r.a., sumner l.w., 2003 legume natural products: understanding and manipulating complex pathways for human and animal health. plant physiol., fig. 1 seed production of tested lines in 2016 and 2017 lines 2016 2017 seeds per 100 g (n) grade seeds per 100 g (n) grade flip05-69c 241 ab b 232 ab b flip05-156c 236 ab b 243 ab b flip05-157c 233 ab b 238 ab b flip07-230c 229 bc b 224 bcd b flip07-318c 219 cd a 231 bc b flip08-69c 202 e a 240 ab b flip08-160c 200 ed a 245 a b flip08-170c 215 cd a 238 ab b flip08-200c 219 cd a 246 a b w6-12861 216 cd a 231 bc b w6-9484 198 ed a 229 bc b w6-30 195 d a 196 ed a w6-25 225 bcd b 231 bc b blanco sinaloa 198 ed a 229 bc b table 3 number of seeds per 100 g and the corresponding grade for canned products means followed by the same letter(s) are not different for p≤0.05 according to tukey test. casini new kabuli chickpeas for canned seeds production 573 131: 878-885. fratianni f., cardinale f., cozzolino a., granese t., a l b a n e s e d . , d i m a t t e o m . , z a c c a r d e l l i m . , coppola r., nazzaro f., 2014 polyphenol composition and antioxidant activity of different grass pea (lathyrus sativus), lentils (lens culinaris), and chickpea (cicer arietinum) ecotypes of the campania region (southern italy). j. functional foods, 7: 551-557. limonta m., 2016 la crescita dell’alimentare tra opportunità e rischi. iri. tuttofood, milano, april 19, italy. muehlbauer f.j., sarker a., 2017 economic importance of chickpea: production, value, and world trade, p p . 5 1 2 i n : v a r s h n e y r . k . , m . t h u d i , a n d f . muehlbauer (eds.) the chickpea genome. springer international publishing ag, berlin, germany, pp. 142. palumbo m.g., 2017 il cece da industria si coltiva così. ortive da industria. terra e vita, 25: 48-52. radicetti e., mancinelli r., campiglia e., 2012 combined effect of genotype and inter-row tillage on yield and weed control of chickpea (cicer arietinum l.) in a rainfed mediterranean environment. field crops research, 127: 161-169. watson a., reckling m., preissel s., bachinger j., bergkvist g., kuhlman t., lindström k., nemecek t., topp c.f.e., vanhatalo a., zander p., murphybokern d., stoddard f.l., 2017 grain legume production and use in european agricultural systems. advances in agronomy, 144: 235-303. impaginato 479 adv. hort. sci., 2018 32(4): 479-485 doi: 10.13128/ahs-21833 factors affecting in vitro propagation of some genotypes of himalayan cedar [cedrus deodara (roxb. ex lamb) g. don.] z. nazemi rafi, h. salehi (*) department of horticultural science, school of agriculture, shiraz university, shiraz, iran. key words: auxin, conifers, cytokinins, micropropagation, pinaceae. abstract: four genotypes of himalayan cedar were grown in vitro for assessing shoot proliferation. this experiment consist three parts. initially, the explants (leafy and defoliated shoot-tips) of mature plants were disinfected and cultured on different basal media (lp, ms and wpm) that supplemented with benzyladenine (ba) at different concentrations for 6 weeks. leafless explants produced the highest number of shoots and the longest shoots for four genotypes. there was no significant difference between the culture mediums and benzyladenine concentrations. in second phase, the influence of benzyladenine (2.5, 5, 10, 20 µm) and thidiazuron (tdz) (0.4, 0.8, 1.6 µm) with combination of different auxin (naa) concentrations (0, 1, 2, 3 µm) was determined on axillary shoot proliferation of the leafless explants of four genotypes grown on wpm. for all thidiazuron concentrations, significant differences between genotypes were detected. in general, with all genotypes, the use of 0.8 μm thidiazuron in the absence or presence of auxin (2 μm) led to the highest length and number of axillary shoots per explant, respectively. finally, in another experiment, the following cytokinin treatments were investigated for axillary shoot multiplication of the cd1 genotype: thidiazuron (0, 0.1, 0.2, 0.4, 0.8 μm) and n6-[2isopentenyl] adenine (2ip) (0, 0.1, 0.2, 0.3, 0.4, 0.5 μm) in combination with benzyladenine (2.5 µm). the best results were obtained in thidiazuron (0.8 µm) with combination of benzyladenine (2.5 µm). this protocol is considered as the first successful report on culture establishment of some genotypes of mature c. deodara trees. 1. introduction the himalayan cedar [cedrus deodara (roxb. ex lamb) g. don.], which belongs to the pinaceae family, is a beautiful, evergreen, and ornamental tree growing widely on the gradient of the western himalayas (champion et al., 1965). commercial seed bearing of c. deodara begins about 30 to 45 years of age, and good seed crops are borne every 3 years (tewari, 1994). regeneration through seeds in cedrus deodara is quite slow and undependable. generally, there is a preference for propagation of mature trees, which helps improve afforestation management, breeding projects and production of elite tree genotypes. however, maturation of most tree species is a major limiting factor for the use of micropropagation in (*) corresponding author: hsalehi@shirazu.ac.ir citation: nazemi rafi z., salehi h., 2018 factors affecting in vitro propagation of some genotypes of himalayan cedar [cedrus deodara (roxb. ex lamb) g. don.]. adv. hort. sci., 32(4): 479-485 copyright: © 2018 nazemi rafi z., salehi h. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 26 october 2017 accepted for publication 18 may 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(4): 479-485 480 afforestation projects (lin et al., 1991). there are a few reports on in vitro propagation of mature conifers in the last 20 years (gupta and durzan, 1985; dumas and monteuuis, 1995; parasharami et al., 2003; andersone and ievinsh, 2005; malabadi and van staden, 2005; cortizo et al., 2009; de diego et al., 2010). few reports on in vitro propagation of c e d a r s ( c e d r u s l i b a n i a . r i c h . a n d c . a t l a n t i c a manetti) are available (piola and rohr, 1996; piola et al., 1998, 1999; renau-morata et al., 2005). there are only two reports on the in vitro culture of deodar cedar with the sole use of the seeds (bhatnagar et al., 1983; tamta and palni, 2004). among regeneration methods, axillary bud induction is preferable for most maintenance of genetic stability and less mutation risk (vasil and vasil, 1980; pierik, 1987). the enhancement of reliability of tissue culture system is being achieved through improving of medium component or correction of the conditions of environment, or both. however, the genotype has a significant impact on the accuracy and repeatability of tissue culture and its effect must be evaluated (sul and korban, 1994). in this domain, cytokinins and auxins play important roles for maintenance and acceptable growth of cultures. during the recent decades, several synthetic compounds have been introduced for induction of regeneration potential of plants (guo et al., 2011). among the compounds, tdz (tang and newton, 2005) and ba (datta et al., 2006) are highly effective on pinaceae family such as eastern white pine and lodgepole pine, virginia pine, red spruce, and taxus wallichiana zucc., respectively. to our best knowledge, no report document the in vitro vegetative regeneration of this species from mature tissues. our goal was to build methods for the in vitro propagation of c. deodara from adult trees, and to show the effect of genotype on its micropropagation. 2. materials and methods experiment 1. plant materials actively growing shoots (4-6 cm long) were collected from mature (20-25 years old) c. deodara trees (genotype not specified) in a seed orchard near the school of agriculture, shiraz university, iran. this was done from september 2015 to november 2016. the shoots were wrapped with wet paper toweling, enclosed in plastic bags and then kept at 4°c until 1 day before use. surface sterilization of explants in this experiment, two kinds of explants with the similar length were used. the first type of explants retained their needles (leaves) to full length (fig. 1 a). in the second type of explants, the leaves were trimmed to a quarter of their initial length (fig. 1 a). both types of explants were soaked in tap-water for at least 2 h. then, they were submerged for 30 minutes in an aqueous solution of 2% benomyl to reduce fungal contamination. afterwards, they were treated with 70% ethanol for 2 minutes followed by 15% clorox (containing 5.25% sodium hypochlorite) with 0.2% ‘tween-20’ for 15 minutes for surface sterilization. finally, they were rinsed three times with sterile distilled water. both kinds of explants were cut into 1-2 cm pieces under sterile conditions and subsequently cultured on different nutrient media. culture establishment the explants were cultured on three different culture media: lp (quoirin and lepoivre, 1977), woody plant medium [wpm] (lloyd and mccown, 1980) and ms (murashige and skoog, 1962), that were supplemented with benzyladenine (ba) at different concentrations (0, 0.63, 1.25, 2.5, 5 µm). then, the cultures were placed in a growth chamber at 25±2°c under a 16 h photoperiod provided by cool white fluorescent lamps (30 μm m-2 s-1) for 6 weeks. at the end, the number and length of proliferated shoots were measured. experiment 2. plant materials and aseptic culture this experiment consisted of two phases. in the first phase, the effect of different growth regulators on axillary shoot proliferation of four genotypes was fig. 1 in vitro proliferation of cedrus deodara (roxb. ex lamb) g. don. (a) different types of explants: explants retained their needles (leaves) and, defoliated explants; (b) axillary shoot proliferation on wpm medium containing 0 . 8 μ m t d z a n d 2 μ m n a a ( a f t e r 4 w e e k s ) ; ( c ) elongation of axillary bud on em medium after 90 days. nazemi rafi and salehi in vitro propagation of himalayan cedar 481 studied. in the second phase, axillary shoot multiplication of genotype cd1 was studied. to examine the effect of genotype on proliferation rate, 110 to 130 actively growing shoots with 4-6 cm length were collected from four mature 20-25 year-old c. deodara trees (genotypes cd1 to cd4) in a seed orchard near the school of agriculture, shiraz university, iran. the shoots were defoliated, cut into 1-2 cm in length pieces and used as initial explants (n=114 per genotype). surface sterilization was carried out in the same manner as in experiment 1. for culture establishment, the explants were cultured on wpm medium supplemented with growth regulators [ba, naa and thidiazuron (tdz)] for shoot induction and proliferation. the medium was supplemented with ba (0, 2.5, 5, 10, 20 µm) and tdz (0, 0.4, 0.8, 1.6 µm) alone or in combination with naa (1, 2, 3 µm). the effect of tdz and ba on shoot proliferation of four genotypes (cd1 to cd4) was investigated. the conditions, under which the cultures were incubated, were same to those of experiment 1. elongation of induced shoots axillary shoots formed on explants (from genotype cd1) were isolated and transferred to elongation media (em). the em was growth regulator-free half strength wpm, and it was supplemented with 3 g l-1 activated charcoal (ac), 15 g l-1 sucrose and 8 g l-1 agar. the explants were maintained in the culture medium, and then subcultured into glass jars (150 ml) containing 40 ml of the fresh em. the environmental conditions were same as that in culture establishment. the subculturing procedure was repeated every 4 weeks for 3 months. the culture conditions (temperature and light) were same as those used for culture establishment experiment. shoot multiplication after 3 month, the shoots grown on em were cut to the same length and then transferred to shoot multiplication medium containing tdz (0, 0.1, 0.2, 0.4, 0.8 µm) and 2ip (0, 0.1, 0.2, 0.3, 0.4, 0.5 µm) in combination with 2.5 µm ba for 6 weeks. statistical analysis all experiments were conducted as factorial based on a completely randomized design with four replications, each replicate comprised of four jam glasses, four explants per glass. all experiments were repeated twice. shoot proliferation rate and shoot length were recorded at the end of the fourth week. spss statistical software was used for analyzing data, and the one-way anova with tukey’s test (p<0.05) was used for comparing means. three-way anova was applied to examine the interactions of tdz, ba and genotype. 3. results experiment 1 the effects of different culture media (lp, ms and wpm) on two types of explants were investigated. the defoliated explants showed the best results, and there was a significant difference for shoot proliferation between defoliated and leafy explants, in all three culture media. the lowest frequency in shoot proliferation belonged to the leafy explants in ms medium (0.06). according to the results, wpm medium showed the highest number and length of proliferated shoots, but without significant difference as compared with lp. moreover, leafless explants produced higher and longer proliferated shoots than leafy explants. there was no significant difference between different amounts of ba on length and number of proliferated shoots (data not shown). experiment 2 when all four genotypes (cd1, cd2, cd3, cd4) were grown at different levels of cytokinin treatments, shoot proliferation from leafless explants was observed. the highest mean number of shoots per explant was observed at 0.8 μm thidiazuron for genotypes cd1 (4.6), cd2 (4.4), cd3 (2.4), and at 0.4 μm thidiazuron for cd4 (2.4) (fig. 2). genotypes cd1 and cd2 were the most responsive genotypes over all cytokinin treatments. furthermore, the highest fig. 2 effect of tdz concentrations on shoot proliferation of four genotypes of cedrus deodara. bars with the same letters indicate no significant difference at tukey test (p=0.05). adv. hort. sci., 2018 32(4): 479-485 482 mean number of axillary shoots per explant of all genotypes (4.31) was obtained when 0.8 μm tdz was used in combination with 2 μm naa and the highest mean length of axillary shoots of all genotypes (11.2 mm) was obtained when 0.8 μm tdz was applied alone (tables 1, 2, fig. 1 b). naa had a positive effect on quality attributes (like color, vitality, and size). however, increasing the concentration of naa to 3 μm reduced the number and length of proliferated shoots (tables 1, 2). then, axillary shoot multiplication of genotype cd1 was studied (fig. 3). the highest mean number of axillary shoots per explant (4.13) and mean length of axillary shoots (10.38 mm) were obtained when 0.8 μm tdz was applied (table 3). the best result of shoot proliferation obtained for cd1 genotype on wpm medium supplemented with 0.8 μm tdz (fig. 2). the same result was obtained for cd1 genotype on wpm medium supplemented with 2.5 μm ba (fig. 4). 4. discussion and conclusions propagation of conifers from mature explants has always been difficult. in general, most studies on the induction of embryogenesis and/or organogenesis in conifers includes the culture of seed or zygotic tismeans with the same letters (small letters for interactions and capital letters for main effects) indicate no significant difference at tukey’s test (p=0.05). table 1 effects of different concentrations of tdz, ba and naa on the number of proliferated shoots in cedrus deodara treatments naa mean values 0 1 2 3 control 2.41± 0.02 lm† 2.16±0.02 pq 2.31±0.05 mno 1.97±0.01 r 2.21 f tdz (µm) 0.4 3.75±0.00 b 3.31±0.03 e 3.44±0.03 de 3.06±0.03 f 3.39 b 0.8 3.38±0.03 de 3.50±0.00 cd 4.31±0.03 a 3.63±0.03 bc 3.70 a 1.6 2.25±0.00 nop 2.50±0.00 jkl 2.63±0.03 ij 2.00±0.00 r 2.34 ef ba (µm) 2.5 2.69±0.03 hi 3.06±0.03 f 3.40±0.03 de 2.50±0.00 jkl 2.91 c 5 2.38±0.03 lmn 2.44±0.03 klm 2.44±0.03 klm 2.56±0.03 ijk 2.45 e 10 2.44±0.03 klm 2.88±0.03 g 2.81±0.03 gh 2.31±0.03 mno 2.61 d 20 2.25±0.00 nop 2.38±0.03 lmn 2.19±0.03 opq 2.06±0.03 qr 2.22 f mean values 2.66 b 2.71 ab 2.87 a 2.45 c 2.67 means with the same letters (small letters for interactions and capital letters for main effects) indicate no significant difference at tukey’s test (p=0.05). table 2 effects of different concentrations of tdz, ba and naa on the length of proliferated shoots in cedrus deodara treatments naa mean values 0 1 2 3 control 0.0 5.80±0.06 h 5.55±0.06 hij 4.88±0.04 klm 4.50±0.09 mn 5.18 d tdz (µm) 0.4 8.75±0.05 c 6.92±0.01 fg 8.12±0.09 d 7.12±0.12 fg 7.73 b 0.8 11.20±0.14 a 7.75±0.07 de 9.56±0.05 b 6.81±0.22 g 8.83 a 1.6 4.96±0.07 klm 5.24±0.08 ijk 5.20±0.08 i-l 4.67±0.02 lm 5.02 d ba (µm) 2.5 6.82±0.16 g 7.32±0.10 efg 5.57±0.10 hij 5.08±0.08 jkl 6.20 c 5 7.15±0.19 fg 7.43±0.10 ef 5.93±0.11 h 5.18±0.15 i-l 6.42 c 10 5.65±0.16 hi 5.93±0.08 h 4.07±0.09 no 3.12±0.08 q 4.69 d 20 3.44±0.12 pq 3.84±0.17 op 3.90±0.13 op 2.16±0.07 r 3.34 e mean values 6.62 a 6.17 ab 5.79 b 4.79 c 5.84 fig. 3 multiple shoot formation on subculture of cedrus deodara on wpm with 0.8 μm tdz in combination with 2.5 μm ba. nazemi rafi and salehi in vitro propagation of himalayan cedar 483 sues (tang et al., 2006; humánez et al., 2011). to our best knowledge, this is the first report of successful induction of axillary shoots on explants taken from adult c. deodara. there have been few reports on the effect of different kinds of basal media on the axillary shoot proliferation in cultures of explants from mature conifer trees (andersone and ievinsh, 2002; de diego et al., 2010; renau-morata et al., 2005). in the present investigation, shoot proliferation rates were generally greater on basal wpm than on basal lp and ms media. shoot buds cultured on ms medium presented the lowest organogenic response, and this was perhaps as a result of the comparatively high nitrate concentration as compared to the lp medium or wpm. tuskan et al. (1990) showed that the extra nitrate had a negative effect on the organogenic response during micropropagation. therefore, low nitrogen content of the medium is a major factor for promoting organogenesis in conifer species (tang et al., 2001; schestibratov et al., 2003). explant type was an important factor affecting axillary bud proliferation in in vitro culture. piola et al. (1998) demonstrated that the accumulation of aba in needles seems to be the major cause of bud dormancy in micropropagation of c. libani. in this experiment, defoliated explants of c. deodara also showed better response on proliferation medium. in in vitro bicentennial cedar micropropagation, it was found that the accomplishment of the protocol depends on genotype (renau-morata et al., 2005). among two cytokinin treatments examined, tdz induced the highest number of proliferated shoots for all genotypes. after investigating the effect of three tdz concentrations on shoot proliferation of four genotypes, the significant difference for their interaction was found. however, in our investigation, ba showed no significant genotypic differences for shoot proliferation. tdz has gotten more attention in recent years due to its ability to assist in vitro regeneration of conifers (mathur and nadgauda, 1999; sul and korban, 2004; renau-morata et al., 2005; tang and newton, 2005; cortizo et al., 2009; de diego et al., 2010; humánez et al., 2011). tdz can decrease the enzyme activity related to oxidative stress during formation of adventitious shoots (tang and newton 2005). recent reports on mature stone pine displayed the superiority of tdz over other cytokinins in advancing axillary shoot proliferation (cortizo et al., 2009). it was shown that high concentrations of cytokinin in the medium, particularly ba led to the low regeneration response, which may be attributed t o t h e t o x i c e f f e c t s o f h i g h c o n c e n t r a t i o n s o f cytokinins (sarmast et al., 2012). in this report, the presence of naa with either tdz or ba improved the incidence of shoot organogenesis in adult tissues of c. deodara. this has been also observed in other conifer species (sul and korban, 2004; zhu et al., 2010). as this study shows, the type of explants and culture media has a very important role in success of culture establishment of cedrus deodara. it was proven that defoliation of the explants has positive effect on shoot proliferation. the effects of genotype and growth regulators on proliferation of axillary in each column, means with the same letters indicate no significant difference at tukey’s test (p=0.05). table 3 effects of different concentrations of tdz and 2ip in combination with 2.5 µm ba on shoot multiplication of cedrus deodara treatments number of proliferated shoots length control 0.0 2.44± 0.07 e 6.69± 0.12 e tdz (µm) 0.1 3.38± 0.16 bc 7.26± 0.16 de 0.2 3.44± 0.21 bc 7.80± 0.27 cd 0.4 3.88± 0.16 ab 9.93± 0.24 a 0.8 4.13± 0.13 a 10.38± 0.11 a 2ip (µm) 0.1 2.50± 0.20 e 6.58± 0.11 e 0.2 2.69± 0.12 de 7.38± 0.18 de 0.3 3.00± 0.10 cde 7.93± 0.21 cd 0.4 3.25± 0.18 bcd 8.38± 0.15 bc 0.5 3.56± 0.12 abc 8.88± 0.14 b fig. 4 effect of ba concentrations on shoot proliferation of four genotypes of cedrus deodara. bars with the same letters indicate no significant difference at tukey test (p=0.05). 484 adv. hort. sci., 2018 32(4): 479-485 shoots have been studied and low concentration of tdz combined with naa has positive effects on the number of proliferated axillary shoots. furthermore, two of the four genotypes showed better response to cytokinins. acknowledgements we are grateful to shiraz university for supporting this study. also, we would like to thank prof. morteza khosh-khui for his guidance and advice. references andersone u., ievinsh g., 2002 changes of morphogenic competence in mature pinus sylvestris l. buds in vitro. ann. bot., 90: 293-298. andersone u., ievinsh g., 2005 in vitro regeneration of mature pinus sylvestris buds stored at freezing temperatures. biol. plant., 49: 281-284. bhatnagar s., singh m., kapur n., 1983 preliminary investigations on organ differentiation in tissue cultures of cedrus deodara and pinus roxburghii. indian j. exp. biol., 21: 524-526. champion s.h., seth s.k., khattak g., 1965 forest types of pakistan. pakistan forest institute, peshawar, pakistan, pp. 238. cortizo m., de diego n., moncaleán 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development of pinus ponderosa (dougl. ex law.) cotyledon explants. plant cell, tiss. org. cult., 20: 47-52. vasil i.k., vasil v., 1980 clonal propagation. int. rev. cytol. suppl., 11a: 146-173. z h u l . h . , w u x . q . , q u h . y . , j i j . , y e j . r . , 2 0 1 0 micropropagation of pinus massoniana and mycorrhiza formation in vitro. plant cell, tiss. org. cult., 102: 121-128. impaginato 97 adv. hort. sci., 2019 33(1): 97-104 doi: 10.13128/ahs-22618 morphological traits and yield of ajowan affected by different irrigation intervals and growth regulators s. ghassemi 1 (*), s. zehtab-salmasi 1, k. ghassemi-golezani 1, s. alizadehsalteh 2 1 department of plant eco-physiology, faculty of agriculture, university of tabriz, tabriz, iran. 2 department of horticulture, faculty of agriculture, university of tabriz, tabriz, iran. key words: carum copticum, field, grain yield, growth regulator, stem diameter, water stress. abstract: two field experiments were carried out to evaluate the effect of salicylic acid and abscisic acid applications on some morphological traits and yield of ajowan (carum copticum l.) under different watering conditions. irrigation intervals (irrigation after 70, 100, 130 and 160 mm evaporation from class a pan) were located in main plots and exogenous applications of water (control), salicylic acid (0 and 1 mm) and abscisic acid (0 and 50 micro-molar) were allocated to sub plots. water stress negatively affected the morphological traits of the ajowan. stem diameter, branches per plant, leaves number per plant, biological yield and grain yield per plant of ajowan were considerably improved by application of salicylic acid and abscisic acid. salicylic acid treatment was more effective than abscisic acid treatment, in this study, salicylic acid treatment increased the stem diameter and leaves number per plant more than abscisic acid and caused a yield enhancement in ajowan plants. in general, foliar spray of salicylic acid and abscisic acid could alter morphological traits and yield of ajowan. 1. introduction medicinal plants have been used for centuries as remedies for diseases and human health, because they contain secondary metabolites of medicinal value. ajowan (carum copticum l.) is an annual herbaceous plant belonging to the umbelliferae family, which grows in the east of india, iran, and egypt, with white flowers and small, brownish seeds. its fruit has been widely consumed as a food flavoring agent and spice. during the past centuries in the iranian traditional medicine, several therapeutic effects including anti-vomiting, antiasthma and anti-spasm, is postulated for ajowan fruits (boskabady et al., 2005). water deficit, defined here as an unbalance between soil water availability and evaporative demand which can naturally occur in the field and causes a decrease in carbon assimilation, tissue expansion and actually (*) corresponding author: saeid.ghassemi67@gmail.com citation: ghassemi s., zehtab-salmasi s., ghassemigolezani k., alizadeh-salteh s., 2019 morphological traits and yield of ajowan affected by different irrigation intervals and growth regulators. adv. hort. sci., 33(1): 97-104 copyright: © 2019 ghassemi s., zehtab-salmasi s., ghassemi-golezani k., alizadeh-salteh s. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 6 february 2018 accepted for publication 23 november 2018 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(1): 97-104 98 cell number. each of these macroscopic processes involves a large number of genes, enzymes, hormones and metabolites (skirycz and inze, 2010). abiotic stresses such as drought cause metabolic changes ranging from synthesis of limited quantities of specialized metabolites to large shifts in primary metabolite composition as well as many other physiological responses. during drought there is a need for osmoticum to accumulate inside the plant cell to retain water and maintain positive turgor pressure (verslues and juenger, 2011). it is generally accepted that, at whole plant level, adaptation to water depletion starts with a change in stomatal conductance, leading to reduced net co2 assimilation and impaired photosynthesis, which result in shoot growth termination. the result of nassiri et al. (2014) showed that seed yield, water use efficiency and harvest index of ajowan reduced in water stress treatment. also, fresh and dry weight of ocimum sp. significantly reduced in water stress conditions (khalid, 2006). adaptation to water stress can be highly controlled by plant growth regulators (popko et al., 2010). salicylic acid is a phenolic compound capable of enhancing plant growth and yield in some plants (arfan et al., 2007). salicylic acid can regulate various stress responses and development such as abiotic stress responses, flowering, senescence, thermogenesi s an d resi stan ce to p ath o gen s ( v i cen te an d plasencia, 2011). among abiotic stresses, this growth regulator has been reported to counter low temperature (tasgin et al., 2003), water stress (abbaspour and ehsanpour, 2016), salinity stress (el tayeb, 2005) and high temperature (he et al., 2005). salicylic acid plays diverse physiological roles in most of plants, which include nutrient uptake, stomatal movements, enzyme activities, thermogenesis, ethylene biosynthesis, photosynthesis, flower induction and plant growth (hayat and ahmad, 2007). applications of 1 or 2 mm sa significantly reduced chilling injury and fruit decay of apricot fruit as well as membrane electrolyte leakage and ascorbic acid content. fruits treated with sa resulted in high total polyphenolic content, antioxidant capacity and carotenoids content (ezzat et al., 2017). also, application of sa significantly enhanced activity of phenylalanine ammonialyase (pal) and content of hydrogen peroxide in apricot fruit. treated fruits showed significantly lower activity of catalase and ascorbate peroxidase but higher activity of superoxide dismutase and peroxidase than those in control fruits (wang et al., 2015). exogenous application of salicylic acid or its derivates affects diverse plant processes. since, this growth regulator is heavily involved in crosstalk with other plant hormones; its effect on some of these processes may be indirect (pieterse et al., 2009). abscisic acid plays critical roles in numerous biological processes, such as gene transcription, seed dormancy and stomatal closure (cutler et al., 2010). this growth regulator appears to be a major player in mediating the adaptation of the plant to water stress (pal et al., 2011). abscisic acid enhances drought tolerance in wheat and many of the other plant species (travaglia et al., 2010). that plays an important role in the regulation of abiotic stress resistance in plants and orchestrates complicated signalling pathways involved in the response to reduced water availability as well as in multiple developmental processes (kim et al., 2010). based on the aforementioned studies and other researchers, it is acceptable that exogenous application of salicylic acid and abscisic acid can improve abiotic stress tolerance in plants. thus, this study was designed to investigate the effect of salicylic acid and abscisic acid applications on some morphological traits of ajowan plants under water stress. 2. materials and methods a t t h e r e s e a r c h f a r m o f t h e f a c u l t y o f agriculture, university of tabriz, tabriz, iran, during the growing seasons of 2014 and 2015, two field experiments were carried out to evaluate the effect of salicylic acid and abscisic acid applications on some morphological traits and yield of carum copticum l. under different irrigation treatments. irrigation intervals [i1, i2, i3, i4: irrigation after 70, 100, 130 and 160 mm evaporation from class a pan (a cylinder with a diameter of 120.7 cm that has a depth of 25 cm) as normal irrigation and mild, moderate and severe water deficit, respectively] were assigned to main plots and foliar applications of water (control), salicylic acid (0 and 1 milli-molar) and abscisic acid (0 and 50 micro-molar) were allocated to subplots. average maximum and minimum temperatures and rainfall during the experiment in 2014 and 2015 are shown in table 1. each plot had 6 rows of 4 m length, spaced 25 cm apart. seeds of this plant were treated with benomyl (benlat wp 50%; shanghai bosman industrial co. ltd, china) at a rate of 2 g kg-1 before sowing. then sown by hand on april 2014 and 2015 at a depth of about 1 cm in a sandy-loam soil. all plots were regularly irrigated after sowing until seedling establishment, and ghassemi et al. morphological traits of ajowan affected by watering and growth regulators 99 thereafter irrigations were carried out according to the treatments. during plant growth and development, weeds were controlled by hand as required. salicylic acid and abscisic acid were sprayed on plants at vegetative (once) and reproductive (once) stages. leaf number per plant was measured by hand at grain filling stage. also, at maturity stage, other traits such as plant height (by meter), stem diameter (by digital caliper) and branches per plant (count by hand) were determined. at maturity, 10 plants of the middle part of each plot were harvested and grain yield per plant were determined. then above ground biomass was oven-dried at 80°c for 48 hours and weighed and subsequently plant biomass was calculated. measurements for all morphological traits were normally distributed and confirmed through the kolmogorov-smirnov test. randomness was confirmed using the run test, and the descriptive statistics were calculated. mstatc software used to the data analyzed and the means of traits were compared using duncan multiple range tests at p≤0.05. 3. results and discussions combined analyses of variance showed significant effects of irrigation and growth regulators on stem diameter, branches per plant and leaf number per plant, biomass and grain yield per plant. plant height was affected by irrigation treatments and plant biomass was significantly influenced by year. the interaction of year × irrigation, year × growth regulator and year × irrigation × growth regulator for plant biomass were significant. grain yield per plant significantly affected by interaction of irrigation × growth regulator (table 2). plant height of ajowan decreased with increasing water stress. there were no significant differences between hormonal treated and control plants (fig. 1). stem diameter decreased with increasing irrigatable 1 averages of maximum and minimum temperatures and rainfall during the work in tarbiz, iran, 2014-2015 ns,*,**= no significant and significant at p≤0.05 and p≤0.01, respectively. month temperature (°c) rainfall (mm) 2014 2015 2014 2015 april 23.7 12.6 50.2 43.2 may 29.5 30.45 10.7 1.5 june 37.1 38.9 18 0.9 july 38.9 40.2 1.3 0 august 34.75 31.1 0 26.4 september 20.65 23.45 84.2 24.1 source df mean square plant height stem diameter branches per plant leaf number per plant plant biomass grain yield per plant year (y) 1 0.056 ns 0.001 ns 0.68 ns 74.01 ns 65.532 ** 10.178 ns repeat 4 213.63 0.001 7.94 233.44 24.849 5.580 irrigation (i) 3 1465.38 ** 0.169 ** 47.90 ** 5662.23 ** 709.482 ** 294.030 ** y×i 3 0.05 ns 0.001 ns 1.71 ns 24.82 ns 15.325 * 2.631 ns error 12 48.69 0.004 1.35 190.24 3.320 2.420 growth regulator (g) 2 0.72 ns 0.05 ** 6.12 * 1762.16 ** 80.355 ** 18.847 ** y×g 2 0.05 ns 0.001 ns 0.10 ns 73.39 ns 5.137 ** 0.296 ns i×g 6 72.50 ns 0.001 ns 2.40 ns 90.44 ns 16.430 ** 3.944 ** y×i×g 6 0.05 ns 0.001 ns 1.07 ns 24.93 ns 2.724 * 0.316 ns error 32 54.43 0.002 1.25 67.27 0.834 0.498 cv (%) 14.32 9.86 10.44 16.81 8.53 12.03 table 2 combined analysis of variance of morphological traits of ajowan under different irrigation and growth regulators in tarbiz, iran, 2014-2015 fig. 1 means of plant height of ajowan for different irrigation levels. different letters in each column indicate significant difference at p≤0.05 (duncan test). i1, i2, i3, i4: irrigation after 70, 100, 130 and 160 mm evaporation, respectively. adv. hort. sci., 2019 33(1): 97-104 100 tion intervals. hormonal application, especially treatment with salicylic acid significantly increased this trait (fig. 2a and b). with increasing water deficit branches per plant of ajowan decreased. however, difference between i2 and i3 was not significant. salicylic acid treatment increased this trait but, there was no difference between salicylic acid and abscisic acid (fig. 2c and d). similarly, leaf number per plant decreased as water deficit increased. salicylic acid treatment significantly increased this trait. however, there was no significant differences between abscisic acid treated and control plants (fig. 2e and 2f). in both years plant biomass of ajowan decreased with decreasing water supply. salicylic acid application enhanced this trait under i1 and i2 in 2014 and under i1 in 2015, but abscisic acid treatment only increased plant biomass under i1 in both years. salicylic acid was advantage that of abscisic acid on this trait (fig. 3). with increasing water deficit, grain yield of salicylic acid treated plants significantly decreased, compared with control plants. exogenous application of salicylic acid under i1 and i2 and also abscisic acid application under i2 treatment improved grain yield of ajowan plants. however, under i3 and i4 irrigation treatments there was no significant difference between hormonal treated and non-treated plants (fig. 4). reduction in plant height due to water stress (fig. 1) is related with the competition of plants for nutrients and water availability (ghassemi-golezani et al., 2010). water stress during vegetative stages has the g r e a t e s t i m p a c t o n b i o m a s s a n d p l a n t h e i g h t (ghassemi-golezani et al., 2008). it has been config. 2 means of stem diameter (a and b), branches per plant (c and d) and leaf number per plant (e and f) of ajowan for different irrigation and growth regulators. different letters in each column indicate significant difference at p ≤ 0.05 (duncan test). i1, i2, i3, i4: irrigation after 70, 100, 130 and 160 mm evaporation, respectively. sa= salicylic acid; aba= abscisic acid. fig. 3 means of plant biomass of ajowan for year × irrigation × growth regulator. different letters in each column indicate significant difference at p ≤ 0.05 (duncan test). i1, i2, i3, i4: irrigation after 70, 100, 130 and 160 mm evaporation, respectively. sa= salicylic acid aba= abscisic acid. ghassemi et al. morphological traits of ajowan affected by watering and growth regulators 101 firmed by many researchers that water stress lead to growth reduction, which was reflected in leaf area, plant height, dry mass, and other growth functions (fischer et al., 1980; kriedemann et al., 1981). the impact of water stress on plant growth can be explained as a method of adaptation to the conditions of water shortage to limit the rate of transpiration (lu and neumann, 1998), in order to maintain the water supply in the soil around plant roots to i n c r e a s e s t h e c h a n c e o f s u r v i v a l o f t h e p l a n t (passioura, 2002). the mechanism, by which plant height is reduced under water stress, is through the reduction of cell elongation, which leads to the reduction of cell size and therefore the reduction of plant height (schuppler et al., 1998). a reduction in leaf turgor and photosynthesis under water stress condition suppresses cell expansion and growth, leading to the diminution of stem diameter (fig. 2a) (anjum et al., 2011). the decrease in growth parameters under drought stress could be considered as an avoidance mechanism which minimizes water losses (rodriguez et al., 2005). stem diameter fluctuates daily because of transpirationinduced tension changes in the stem sap (irvine and grace, 1997; perämäki et al., 2001). salicylic acid application promotes cell division and enlargement (hayat et al., 2005). it has been reported that salicylic acid significantly enhances the average growth speed of tomato stems (stevens and senaratna, 2006), which is in line with the results of the present study. salicylic acid was reported to increase cytokinins in corn and these hormones increased the stem diameter (shakirova et al., 2003). foliar spray of abscisic acid is favouring vegetative growth of plants as shown for ajowan (fig. 2b) and soybean (travaglia et al., 2009). exogenous application of abscisic acid was able to increase plant adaptive response to various environmental conditions (abraham et al., 2008). n u m b e r o f b r a n c h e s a n d l e a v e s p e r p l a n t decreased under drought condition (fig. 2c and 2e). this might be related with the suppression of cell expansion and cell growth due to the low turgor pressure. reduced number of leaves by moisture stress is in line with the finding of stolf-moreira et al. (2010) who found that water stress affected number of leaves for soybean. this is because water stress leads to decreased rate of leaf initiation and reduction in leaf area of already formed leaves. this can be related with lower photosynthetic activity in the affected leaves. the overall effect is a decrease in the rate of new leaf initiation and increase in leaf shedding thereby resulting to reduction in number of green leaves per plant (yunusa et al., 2014). salicylic acid stimulatory effect on growth estimated characteristics could be related with the effect of this growth regulator on the endogenous phytohormones such as growth promoters i.e. cytokinins, gibberellins and auxins (waffaa et al., 1996; shehata et al., 2000). application of these growth regulators increased number of branches per plant (fig. 2d) and leaves (fig. 2f), which could lead to increment of number of flowers and subsequently seed yield. in agreement with these results fathy et al. (2003) on eggplant and gharib (2007) on basil and marjoram they mentioned that salicylic acid increased plant height, number of branches and leaves per plant and dry mass as well, respectively. abscisic acid by induction of genes that encode enzymes and other proteins involved in cellular dehydration tolerance, plays a critical role in regulating plant water status through guard cells and growth as well as (luan, 2002; zhu, 2002) which might be the main reason for increased branches per plant. treatment with abscisic acid in sesamum indicum l. increased number of branches to a large extent (abraham et al., 2008). plant biomass was reduced under water stress (fig. 3) due to leaf senescence and decline in the cell enlargement resulting from reduced turgor pressure (shao et al., 2008). severe water stress may result in arrest of photosynthesis and disturbance of metabolism (liang et al., 2006). water stress inhibits cell enlargement and that reduces various biochemical and physiological processes (shao et al., 2007). fig. 4 mean grain yield of ajowan for interaction of irrigation × growth regulator. different letters indicate significant difference at p ≤ 0.05 (duncan test). i1, i2, i3, i4: irrigation after 70, 100, 130 and 160 mm evaporation, respectively. sa= salicylic acid, aba= abscisic acid. 102 adv. hort. sci., 2019 33(1): 97-104 drought stress leading to stomata closure and reduction in photosynthesis rate and leaf growth (ozturk, 1999), which ultimately decreases plant biomass. this reduction in plant biomass resulted in decreasing the plant height (fig. 1), stem diameter, branches per plant and leaf number per plant (fig. 2) and consequently grain yield per plant (fig. 4). salicylic acid improved the production of plant biomass (fig. 3) by increasing the stem diameter, branches per plant and leaf number per plant (fig. 2). salicylic acid influences a wide variety of plant processes, including stomatal regulation, chlorophyll content and photosynthesis (yildirim et al., 2008). also, by increasing ribulose 1,5-bisphosphate (rubp) content under drought condition, protecting the photosynthetic machinery from reactive oxygen species produced during drought stress (shehata et al., 2001) and maintaining lai and photosynthetic activity under stress (bayat and sepehri, 2012) increased plant biomass. abscisic acid plays a critical role in regulating plant water status through guard cells and growth as well as by induction of genes that encode enzymes and other proteins involved in cellular dehydration tolerance (zhu, 2002), which might be resulted in increasing dry mass under drought stress (fig. 3). reduction of grain yield under water stress (fig. 4) has been attributed to reduced plant height (fig. 1), stem diameter, branches per plant, leaf number per plant (fig. 2) and plant biomass (fig. 3). water stress severely limits growth and yield of plants by reducing chlorophyll content of leaves, photochemical efficiency of photosystem ii (ghassemi-golezani and lotfi, 2012), photosynthesis (munns et al., 2006) and g r o u n d g r e e n c o v e r ( g h a s s e m i g o l e z a n i a n d ghassemi, 2013). water stress during vegetative stages largely reduces plant height and biomass, while during reproductive stages it has the greatest negative impact on grain yield (ghassemi-golezani et al., 2008). grain yield was improved by growth regulators ( f i g . 4 ) . t h e i n c r e a s e i n y i e l d m i g h t b e d u e t o increased sink size mainly number and mass of grains. this could be related to increased photosynthetic efficiency by stabilization of chlorophyll and higher production and translocation of organic material from source to sink. salicylic acid stimulates physiological processes that were reflected on improving vegetative growth (fig. 2, 3) followed by active translocation of the photosynthesis products from source to sink. abscisic acid is also able to activate metabolism of carbohydrates temporally stored in the plant stem (yang et al., 2003; 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pistachio. in this research, the effect of salinity stress on morphological and physiological traits as well as the concentration of nutrition elements in some pistachio cultivars was investigated based on completely randomized design (crd), with two factors cultivars and irrigation water salinity. studied cultivars were ghazvini, shahpasand, akbari, khanjari, jandaghi, italiyayi, fndoghi 48, sabz pesteh tohg, ahmad aghaee, rezaie zood res, mousa abadi, ebrahimi, kaleh ghochi and badami zarand and levels of salinity were 0.5, 4.9, 9.8, 14.75 and 19.8 ds/m. each treatment had nine replicas. the results showed that increasing salinity reduced branch height, branch diameter, number of total leaves, and percentage of green leaves, relative humidity content, chlorophyll a, chlorophyll b and total chlorophylls in all cultivars. but percentage of necrotic leaves, percentage of downfall leaves, relative ionic percentage and cell membrane injury percentage were increased. the results showed that salinity stress affected the young trees through increasing the amount of minimum fluorescence (f0) and decreasing the maximum fluorescence (fm) and reducing variable fluorescence (fv) as well as the ratio of variable fluorescence to maximum fluorescence from 0.83±1 in the control plants to 0.59±0.015 in rezaie zood res cultivar and 0.61±0.009 in mousa abadi cultivar. the results also showed that in the total cultivars studied, the highest amount of na+ in leaves and roots (2.09±0.04% and 3.04±0.06%), and the lowest amount of k+ in leaves and roots (0.40±0.02% and 0.34±0.01%), were observed in treatment 19.75 ds/m. overall, ghazvini was found to be the most tolerant cultivar to salinity stress. this cultivar could well tolerate salinity 14.75 ds/m. 1. introduction pistachio (pistacia vera l.) is one of the important commercial crops in iran. majority of pistachio orchards are located in areas with saline soil and are irrigated with low quality and salty waters. although pistachio trees are classified as tolerant to salinity, researches have demonstrated (*) corresponding author: a.momenpour@areeo.ac.ir citation: momenpour a., imani a., 2018 evaluation of salinity tolerance in fourteen selected pistachio (pistacia vera l.) cultivars. adv. hort. sci., 32(2): 249-264 copyright: © 2018 momenpour a., imani a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 7 december 2017 accepted for publication 18 april 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(2): 249-264 250 that growth rates of pistachio trees decrease with increasing sodium chloride (nacl) concentration in soil and there is a positive correlation between sodium (na+) as well as chloride (cl−) concentration in plant tissue and soil (sepaskhah and maftoun, 1988; noitsakis et al., 1997; munns and tester, 2008; zrig et al., 2015). salinity stress also negatively affects photosynthesis rate, morphology of leaves, and nutrie n t b a l a n c e i n p i s t a c h i o t r e e s ( p i c c h i o n i a n d myamoto, 1990; saadatmand et al., 2007; karimi et al., 2011). walker et al. (1987) and karimi et al. (2009) reported that the highest chloride concentrations were observed in lamina and petiole of pistachio seedlings irrigated with salty water, whereas highest sodium concentration was observed in roots. ferguson et al. (2002) suggested that the decrease of water potential in plant in higher salinity levels is one of the main reason for decrease pistachio yield. it has been reported that salinity stress is one of the most important environmental factors limiting photosynthesis in the majority of worldwide cultivated crops, including pistachio crop (maxwell and j o h n s o n , 2 0 0 0 ; r a n j b a r f o r d o e i e t a l . , 2 0 0 6 ) . chlorophyll fluorescence (cf) has been used to study plant responses to different kinds of stress (baker and rosenqvist, 2004). chlorophyll (chl) fluorescence yield (chl fy) such as minimal chl fy (f0) and variable chl fy (fv) can be used for evidencing stress and damage of the photosynthetic apparatus, and characterizing the environment where plants grow (herda et al., 1999; deell and toivonen, 2003; kodad et al., 2010). fv/fm ratio has been used in many studies related to stress in plants. in most of plants, when ratio fv/fm is around 0.83 means that stress has not been introduced to the plant. values lower than this will be seen when the plant has been exposed to stress, indicating in particular the phenomenon of photo inhibition (herda et al., 1999; deell and toivonen, 2003; kodad et al., 2010). selecting nutrient sources that do not add harmful ions and salinity to irrigation water to avoid compounding salinity problems would be the best option. in areas affected by soil and water salinity, nevertheless, it is more convenient to use salt-tolerant rootstocks for the species characterized by a certain degree of salt tolerance, i.e. pistacia sp. an important characteristic of pistacia sp. is their ability to store large quantities of na+ in roots, which might make pistachio tolerant to na+ (picchioni and myamoto, 1990; karimi et al., 2011). sepaskhah and maftoun (1988) reported that 50% reduction in shoot growth was observed when the average root-zone salinity was between 7.9 and 10 ds/m (ece). saadatmand et al. (2007) postulated that salinity stress had more negative influence than drought stress on pistachio growth. they reported that sarakhs variety showed higher sensitivity to soil salinity than qazvini variety, but with increasing in irrigation intervals, sarakhs was more-tolerate to salinity than qazvini. although, other researchers have studied the influence of soil and water salinity on the growth indices and chemical composition of pistachio cultivars, but in before researches a low number of cultivars was investigated. therefore, in this research, the effects of five levels of irrigation water salinity on morphological and physiological traits as well as the concentration of nutrition elements in fourteen selected pistachio (pistacia vera l.) cultivars have been investigated in order to find most tolerant cultivars to salinity. 2. materials and methods plant material and natural salt treatments in this research, the effects of salinity stress on morphological and physiological traits and on the concentration of nutrition elements in 14 pistachio cultivars such as ghazvini, shahpasand, akbari, khanjari, jandaghi, italiyayi, fndoghi 48, sabz pesteh tohg, ahmad aghaee, rezaie zood res, mousa abadi, ebrahimi, kaleh ghochi and badami zarand were investigated. the experiment was carried out in the research greenhouse of temperate fruit research center, horticultural research institute in karaj-iran in years of 2013 and 2014 based on completely randomized design (crd), with two factors; cultivars with 14 levels and irrigation water salinity by 5 levels (control= 0.5 ds/m, a= 4.8 ds/m, b= 9.8 ds/m, c= 14.75 ds/m and d=19.8 ds/m) and with nine replications for each treatment, for a total of 630 pots. seeds were germinated according to the method described by karimi et al. (2009). seeds were pret r e a t e d w i t h b e n o m y l ( w e t t a b l e p o w d e r 5 0 % ; dupont, wilmington, de, usa) for 24 h, and then incubated at 30°c within layers of sterile moist crisped cloth. after radicle emergence, seeds were p l a n t e d i n j i f f y p o t s ( j i f f y g r o u p , m o e r d i j k , netherlands) and grown in a greenhouse for three months. seedlings with 10 to 15 cm height were transplanted to pots 2 kg filled with soil series of fine loamy mixed, which its characteristics are listed in table 1. salinity treatment was started and continued for momenpour and imani salinity tolerance in fourteen selected pistachio cultivars 251 two and half months. for salinity treatments, salts were collected of salt lake shore in qom-iran. then, salinity treatments were obtained by solving 0, 2.4, 4.8, 7.2 and 9.6 g of salt in 1 l of water (treatments composition is reported in table 2). also, to avoid sudden shock and plasmolysis, salt treatments were gradually added and reached to the final concentration within a week (2 stages of irrigations). field capacity (fc) of soil in pots was determined before transferring plants to units by a pressure plate (model f1, make usa). irrigation schedule was organized according to pots changes in weight and leaching requirement. electric conductivity and ph rate were regularly measured in drainage water to maintain the electric conductivity of both input and soil solutions in a stable range. at the end of the experiment, the soil of pots in each level salinity was mixed together. then three samples of each treatment (in total 15 samples) were analyzed (table 3). growth parameters at the end of the experiment, growth characteristics including main-shoot length, trunk diameter and number of leaves, were measured as well as percentage of necrotic leaves, downfall leaves and green leaves were calculated (papadakis et al., 2007). fresh weight of leaves, main-shoots and roots were measured immediately after removing, using a digital scale. dry weight of the samples was measured using an oven at 75°c for 48 h (papadakis et al., 2007). physiological parameters for determination of leaf chlorophyll, 0.2 g of leaf was extracted (in total 630 samples, means nine replicas for each cultivar and for each salinity treatment), with ethanol 80% and chlorophyll a, chlorophyll b and total chlorophyll content were calculated with the method described by arnon (1949). leaf greenness (chlorophyll index) was evaluated on the same leaves used for gas exchange and fluorescence using a spad (minolta, 502, made in japan) after 75 days since treatments introduction. leaf relative water content (rwc) was determined with nine replicas (made by four leaves each) for each treatment and for each cultivar, for a total of 630 samples. fresh weight (fw) was recorded and then samples were put into distilled water and kept at 4°c for 24 h in the dark. after the emission of extra humidity, samples were weighed again to obtain the total weight (tw). subsequently, samples were kept in the oven at 105°c for 24 hours and dry weight (dw) was recorded. finally, relative water content w a s c a l c u l a t e d v i a f o r m u l a e ( y a m a s a k i a n d title value saturation percentage (%) 39 field capacity (%) 27.33 permanent wiltering point (%) 14.8 ds/m (ec) 1.28 ph 7.5 n (%) 0.15 organic carbon (%) 1.49 p (ppm) 104.9 sand (%) 46 silt (%) 34 clay (%) 20 texture loam ca (ppm) 1230 mg (ppm) 316.2 total neutralizing value (%) 13.8 cu (ppm) 2.12 zn (ppm) 4.86 fe (ppm) 27.34 k (ppm) 690 mn (ppm) 16.26 na (ppm) 93.15 table 1 physical and chemical characteristics of soil mixture table 2 salt solution characteristics treatments electrical conductivity (ds/m) (ph) na (mg/l) cl (mg/l) ca (mg/l) mg (mg/l) hco3 -(mg/l) control 0.50 7.30 22.1 35.5 62 17.1 98 a 4.90 7.60 809 1386 79 23.01 137 b 9.80 7.78 1653 2836 99 25.7 159 c 14.75 7.87 2443 4199 123 28.5 186 d 19.80 7.95 3276 5610 151 31.9 214 treatments ec (ds/m) ph control 1.2 7.4 a 5.7 7.65 b 10.9 7.87 c 15.95 7.96 d 21.3 8.05 table 3 ec and ph soil treated with different levels of salinity adv. hort. sci., 2018 32(2): 249-264 252 dillenburg, 1999). rwc= [(fw-dw) / (tw-dw)] ´100 for the determination of relative ionic content was determined with nine replicas (made by four leaves each) for each treatment and for each cultivar, for a total of 630 samples. the amount of 0.5 g of each sample was put in tubes with 25 ml of distilled water at 25°c for 24 h on a shaker with speed 120 in/min. electrical conductivity (ec) of the medium was then read using a conductivity meter (conduct meter; radiometer, copenhagen). following the initial reading (lt), samples were autoclaved for 20 min to kill leaf tissues and then kept at 25°c for 2 h on shaker with speed 120 in/min and a final reading (lo) was obtained. finally, relative ionic percentage was calculated via formulae: relative ionic percentage = (lt/lo)/100 as described in lutts et al. (1995). after calculation relative ionic percentage, cell membrane injury in samples’ treatment with natural salt ratio samples control was performed as follows: % injury = 1[1(t1/t2)/ 1(c1/c2)] × 100 where t and c refer to the ec values of stresstreated and control tubes and 1 and 2 refer to the initial and final ec, respectively (lutts et al., 1995). chlorophyll fluorescence parameters chlorophyll fluorescence of leaves was measured using a portable fluorometer pam-2000 (h. walz, effeltrich, germany). before measuring chlorophyll fluorescence parameters, three leaves on mainbranch of each plant were put in dark-adapted state (das) for 30 min using light exclusion clips (maxwell and johnson, 2000). maximum quantum efficiency of photosystem ii (fv/fm) was determined as fv/fm= (fm-f0)/fm; where fm and f0 were maximum and minimum fluorescence of dark-adapted leaves, respectively. concentration of na+ and k+ concentration of na+ and k+ in leaves and roots was determined with nine replicas for each treatment and for each cultivar, for a total of 630 samples. leaves and roots of each plant, oven-dried at 75°c for 48 h, and then milled to a fine powder to pass through a 30-mesh screen. the amount of 0.5 g of each sample was dry-ached for 6h at 550°c, dissolved in 3 ml of 6 mol l−1 hcl and diluted to 50 ml with deionized water. subsequently, concentration of na+ and k+ were determined using atomic absorption spectroscopy (papadakis et al., 2007). statistical analysis this experiment was carried out based on completely randomized design (crd), with factors cultivars in 14 levels and irrigation water salinity in 5 levels and with nine replicas for each treatment in research greenhouse of temperate fruit research center, horticultural research institute in karaj-iran in years 2013 and 2014. finally, data were analyzed using analysis of variance (anova) using sas software. means were also compared by duncan’s multiple range test at 1% level. 3. results as reported in table 4, salinity treatments negatively affected plant height, trunk diameter and number of leaves. with increasing salinity concentration in irrigation water, final height, trunk diameter and n u m b e r o f l e a v e s i n a l l s t u d i e d c u l t i v a r s w e r e decreased. the lowest branch height, trunk diameter and leaf number were observed in salinity level d. the rate of decrease branch height, trunk diameter and number of leaves among the cultivars showed a significant difference with each other. the height of khanjari, jandaghi, italiyayi, fndoghi 48, sabz pesteh tohg, ahmad aghaee, rezaie zood res, mousa abadi, ebrahimi and kaleh ghochi cultivars were decreased in salinity level b compared to control plants. while the height of akbari, shahpasand and badami zarand cultivars in salinity level c and in qazvini cultivar only in salinity level d was decreased significantly compared to control plants. as reported in table 4, as the salt concentration increases, the trunk diameter and its growth were decreased during the application of salinity stress in all cultivars. the decrease in trunk diameter in the cultivars showed a significant difference with each other. the trunk diameter of khanjari, fndoghi 48, rezaie zood res, mousa abadi and kaleh ghochi cultivars was decreased in salinity level b compared to control plants. while the trunk diameter of italiyayi, j a n d a g h i , e b r a h i m i , s a b z p e s t e h t o h g , a h m a d aghaee, akbari, shahpasand and badami zarand cultivars in salinity level c and in qazvini cultivar only in salinity level d was decreased significantly compared to control plants. the results showed that number of leaves with increasing salinity concentrations were reduced, but the amount of reduction in the number of leaves in different cultivars had significant differences. the maximum number of leaves were observed in control momenpour and imani salinity tolerance in fourteen selected pistachio cultivars 253 table 4 effect of interaction between salinity and cultivar on some of the morphologic traits means in each column and for each factor, followed by similar letter(s) are not significantly different at the 1% probability level, using duncan’s multiple range test. *= a/ is less than z. given that variety of data was very wide, duncan’s test grouped data between a to z and less than z such as a/, b/, c/, d/ and e/. cultivars treatments no. of green leaves green leaves (%) trunk diameter (mm) branch height (cm) khanjari control 27.33±1.0 h-n 100.00±0.0 a 8.38±0.13 a 32.50±1.56 f-g a 25.00±1.93 n-r 100.00±0.0 a 8.38±0.09 a 31.47±0.37 g-j b 22.55±1.42 s-v 97.02±3.81 a-d 8.07±0.09 b 28.67±1.17 k-n c 20.00±1.22 t-w 76.21±5.82 m-o 7.57±0.09 cd 25.55±0.52 r-s d 16.33±1.32 x-z 51.41±6.87 q 6.94±0.05 f-h 21.31±0.54 w-x akbari control 24.67±1.80 o-s 100.00±0.0 a 7.53±0.10 c-d 25.05±2.78 r-s a 22.89±0.78 s-u 100.00±0.0 a 5.55±0.09 c-d 24.38±2.69 s-u b 22.11±0.70 s-v 100.00±0.0 a 7.31±0.05 d-e 23.55±1.26 s-v c 18.45±0.72 u-y 96.86±4.23 a-d 7.14±0.06 e-g 22.67±1.29 t-y d 16.45±0.88 x-z 89.43±4.92 g-i 6.65±0.08 h-j 20.18±1.79 w-y ghazvini control 27.23±0.77 h-n 100.00±0.0 a 5.82±0.07 p-u 22.57±1.08 t-w a 27.07±0.26 i-m 100.00±0.0 a 5.81±0.07 p-u 22.55±0.57 t-w b 26.52±0.67 j-o 99.55±0.0 a 5.70±0.05 s-w 21.67±0.50 u-x c 25.77±0.67 m-r 97.72±5.63 a-c 5.46±0.04 u-x 20.7±1.0 w-y d 23.81±2.24 q-u 90.88±7.07 e-h 5.02±0.06 x-y 18.11±1.21 y-z italiaie control 31.67±2.34 e 100.00±0.0 a 6.71±0.03 h-j 33.00±2.95 e-h a 31.06±2.0 e-f 100.00±0.0 a 6.74±0.16 h-j 32.30±1.21 f-h b 29.22±1.39 f-i 98.85±2.40 a-b 6.53±0.12 j-m 30.17±0.94 i-k c 25.22±1.71 n-r 90.72±8.49 e-h 6.15±0.29 n-q 26.43±1.19 o-r d 22.33±2.34 q-u 84.88±5.04 i-k 5.60±0.24 t-x 22.71±1.90 t-w kaleh ghochi control 27.00±1.93 i-n 100.00±0.0 a 6.63±0.05 f-h 32.09±0.87 f-h a 26.85±0.86 i-n 10000±0.0 a 6.59±0.04 j-k 31.87±0.86 f-i b 23.44±0.72 q-u 97.66±3.04 a-c 6.28±0.03 l-p 28.87±0.86 k-n c 19.33±1.0 u-w 89.60±5.26 f-i 5.78±0.04 p-u 25.43±0.71 r-s d 15.33±1.0 y-z 72.92±7.64 o 5.25±0.03 w-x 20.28±0.48 x-y jandaghi control 27.89±1.05 g-j 100.00±0.0 a 6.05±1 p-r 23.56±0.73 s-v a 27.11±2.26 i-m 100.00±0.0 a 6.04±1 p-r 23.45± 1.05 s-w b 25.33±0.70 n-r 95.80±3.46 a-e 5.97±1 q-s 21.03±0.53 w-x c 20.67±1.93 t-w 80.96±6.21 k-m 5.33±1 u-x 18.73±0.68 y-z d 15.44±1.42 y-z 64.71±7.56 p 4.81±1 y-z 16.45±0.51 a mousa abadi control 32.00±1.22 e 100.00±0.0 a 5.78±1 p-u 30.00±1.87 j-l a 30.44±1.74 e-f 100.00±0.0 a 5.61±1 s-w 29.48±0.87 k-m b 26.88±1.69 i-n 93.80±3.13 b-e 5.33±1 w-x 26.65±0.75 n-r c 22.11±2.02 s-v 78.71±5.28 l-n 5.12±1 x-y 21.75±0.72 u-x d 15.00±1.22 z 52.47±4.98 q 4.57±1 z 14.90±1.07 b ebrahimi control 29.33±1.93 f-h 100.00±0.0 a 6.07±0.09 p-r 33.67±1.58 e-f a 28.77±1.20 f-i 100.00±0.0 a 6.08±1.29 p-r 33.55±1.26 e-f b 27.44±1.33 g-k 91.85±2.60 d-h 5.75±0.66 p-u 31.26±1.29 h-j c 23.67±1.32 q-u 84.68±4.53 i-k 5.44±0.09 u-x 26.56±1.44 n-q d 19.33±1.32 u-w 62.98±8.62 p 5.17±0.09 w-y 22.15±1.34 s-v badami zarand control 25.00±1.22 n-r 100.00±0.0 a 6.73±0.05 h-j 29.32±1.09 k-m a 24.67±1.32 o-s 100.00±0.0 a 6.69±0.06 h-j 29.35±1.30 k-m b 23.33±1.0 q-u 97.39±0.0 a-c 6.56±0.08 j-l 27.40±0.92 m-q c 21.44±1.23 t-v 93.00±3.37 c-h 6.25±0.06 m-p 24.51±0.81 r-t d 17.78±1.48 v-y 83.71±4.45 j-l 5.80±0.12 p-u 21.19±0.90 w-x fandoghi 48 control 35.00±1.87 d 100.00±0.0 a 6.62±0.19 i-j 36.53±1.11 d a 34.33±1.58 d 100.00±0.0 a 6.53±0.11 j-m 36.23±0.92 d b 31.11±1.69 e-f 94.55±4.30 b-e 6.30±0.10 k-o 33.17±1.26 e-g c 26.77±1.85 j-o 87.96±2.29 h-j 5.90±0.15 q-s 29.34±1.65 k-m d 21.11±1.69 t-w 75.97±6.88 o 5.67±0.09 s-w 23.45±1.02 s-v sabs pesteh togh control 32.00±1.50 e 100.00±0.0 a 6.43±0.09 j-n 28.22±1.21 l-o a 31.67±0.86 e 100.00±0.0 a 6.44±0.05 j-n 27.73±1.10 m-p b 28.00±1.87 g-k 93.78±0.05 b-g 6.13±0.08 n-p 25.70±0.92 p-r c 24.33±1.32 p-t 83.08±3.09 h-j 5.73±0.08 s-w 22.91±0.29 s-v d 20.11±1.45 t-w 67.29±5.39 no 5.33±0.09 u-x 21.01±0.64 w-x ahmad aghaee control 30.00±1.87 e-g 100.00±0.0 a 5.71±0.11 s-w 23.11±1.16 s-w a 27.88±1.16 g-k 100.00±0.0 a 5.67±0.08 s-w 21.33±1.32 w-x b 25.00±1.22 n-s 95.55±3.16 a-e 5.35±0.11 u-x 18.33±0.96 y-z c 22.11±1.26 s-v 82.32±4.41 kl 5.11±0.08 x-y 17.21±0.54 z d 19.04±0.72 u-x 72.01±2.50 o 4.73±0.08 y-z 16.22±0.66 a/* rezaie zodres control 30.67±1.32 e-g 100.00±0.0 a 5.81±0.18 p-u 34.63±2.23 e a 29.22±1.30 f-h 100.00±0.0 a 5.50±0.26 u-x 32.22±1.27 e-h b 25.77±1.71 m-r 91.76±5.73 d-h 5.17±0.16 w 28.11±1.32 m-p c 20.55±1.01 t-w 75.51±6.87 no 5.05±0.33 x-y 23.46±1.48 s-v d 16.00±0.86 y-z 53.48±4.82 q 4.49±0.24 z-a 17.95±1.08 y-z shahpasand control 45.00±1.65 a 100.00±0.0 a 7.77±0.11 c 47.51±1.40 a a 43.67±1.0 a 100.00±0.0 a 7.65±0.10 c 47.07±1.29 a b 41.67±0.70 a-b 98.55±0.0 a-b 7.52±0.10 cd 45.01±0.98 a-b c 39.11±1.16 c 93.89±1.81 b-g 7.21±0.06 e-f 42.19±3.33 c d 35.33±1.58 d 83.01±4.73 j-l 6.90±0.09 g-i 37.01±1.06 d adv. hort. sci., 2018 32(2): 249-264 254 plants of shahpasand cultivar (45±1.65 leaves), and the lowest amount of them were observed in mousa abadi, kaleh ghochi, jandaghi and rezaie zood res cultivars in salinity level d (15±1.22, 15.33±1.00, 15.44±1.42 and 16±0.86 leaves), respectively. the results showed that with increasing salinity of irrigation water, the percentage of green leaves in all cultivars was decreased. in control plants and also plants treated with salinity level a, all leaves of plants were green and were not observed any necrotic leaves. necrosis and fallen leaves in all cultivars were observed in salinity levels b (except for akbari cultivar), c and d. the lowest percentage of green leaves was found in salinity level d and in mousa abadi (52.47±4.98%), and rezaie zood res (53.48±4.82%), cultivars, respectively. as reported in table 5, in all the cultivars as the salinity increases, the percentage of necrosis leaves were increased and the first symptoms of necrosis except for akbari cultivar were observed in salinity level a. in all cultivars, the highest incidence of necrosis leaves was observed in salinity level d. the percentage of fallen leaves also were increased with increasing salinity levels while in all cultivars except for akbari and ghazvini cultivars were observed fallen leaves in salinity levels c and d. the results showed that leaves and shoots fresh and dry weights in all studied cultivars significantly decreased by applying salinity stress and increasing its concentration. shoots and leaves fresh and dry weights in kaleh ghochi, italiyayi, jandaghi, fndoghi 48, ebrahimi, mousa abadi and rezaei zood res cultivars in salinity levels b, c and d, and in khanjari, sabz pesteh tohg, ahmad aghaee and badami zarand cultivars in salinity levels c and d, and in akbari, shahpasand and qazvini cultivars only in salinity level d, were decreased significantly compared to control plants. based on the results of this study, as the salinity increases, the amount of minimum chlorophyll fluorescence (f0) was increased significantly. the highest amount of f0 in all cultivars was observed in salinity level d. the highest amount of f0 was observed in the leaves of khanjari cultivar treated with salinity level d (table 7). also, maximum chlorophyll fluorescence (fm) in all cultivars was decreased significantly as the salinity increased. the highest amount of fm was observed in control plants while the lowest amount of fm was observed in rezaie zood res (365.22± 20.90), jandaghi (380.67±11.69) and mousa abadi (387.67±29.06) cultivars that was treated with salinity level d, respectively (table 7). the results showed that in all studied cultivars, (fv/fm) ratio was reduced significantly by applying salinity stress and increasing its concentration. furthermore, there was a significant difference fv/fm values in different levels of salinity among tested cultivars. in the leaves of the control plants fv/fm was 0.83±1 indicating the existence of ideal and nonstressed environmental conditions for the growth of all cultivars throughout the experimental period. regarding changes in (fv/fm) ratio the stress intensity in rezaie zood res and mousa abadi cultiv a r s w a s m o r e s e v e r e t h a n o t h e r c u l t i v a r s , (0.59±0.015 and 0.61±0.009 respectively). therefore, the susceptibility of these cultivars to salinity stress in levels c and d were higher than other cultivars. on the contrary, gazvini and akbari cultivars were less damaged, (0.76±0.003 and 0.75±0.007, respectively) (table 7). in other words, fv/fm in this cultivars, showed the lowest decrease. results on chlorophyll a, b and total chlorophyll content of the leaves treated in different salinity levels are reported in table 8. chlorophyll a content was reduced significantly in all of the studied cultivars in salinity level d compared to control plants while chlorophyll b content in salinity levels c and d was reduced significantly compared to the control plants. total chlorophyll content was decreased significantly in ghazvini cultivar only in salinity level d, and in akbari and badami zarani cultivars in salinity levels c and d while total chlorophyll content in other cultivars decreased significantly in salinity levels b, c and d (table 8). chlorophyll index was decreased significantly under salinity stress. the lowest chlorophyll index was observed in the leaves of the plants that were irrigated with salinity level d. the highest reduction in chlorophyll index was observed in mousa abadi (22.60±1.50), jandaghi (27.83±0.98) and kaleh ghochi (31.47±3.08) cultivars. the lowest reduction in chlorophyll index was observed in shahpasand (55.35±1.35), akbari (53.70±1.24) and ghazvini (49.78±1.10) cultivars (table 8). according to the results reported in table 9, the content of relative humidity of leaves decreased significantly as the salinity increased. the content of relative humidity in leaves of control plants were higher than 79.83% (of 79.83±0.24% in control plant leaves of shahpasand cultivar to 85.25±0.64% in control plant leaves of khanjari cultivar), while relative humidity content in leaves of mousa abadi, rezaie zood res and sabz pesteh togh plants in salinity level d, were 64.17±0.52%, 66.49±0.57% and 66.95± 0.77%, respectively. in ghazvini and akbari cultivars momenpour and imani salinity tolerance in fourteen selected pistachio cultivars 255 table 5 effect of interaction between salinity and cultivar on the morphologic traits measured means in each column and for each factor, followed by similar letter(s) are not significantly different at the 1% probability level, using duncan’s multiple range test. cultivar treatments leaf dry weight (g) leaf fresh weight (g) downfall leaves (%) necrosis leaves (%) khanjari control 2.77±0.02 d-f 6.28±0.06 e-f 0.00±0.0 l 0.00±0.0 q a 2.77±0.01 d-f 6.30±0.02 e-f 0.00±0.0 l 0.00±0.0 q b 2.67±0.02 f-g 6.05±0.06 f-g 0.00±0.0 l 2.98±3.81 n-q c 2.54±0.01 g-i 5.71±0.03 h 11.72±3.47 d 16.07±4.47 d-e d 2.47±0.03 h-k 5.04±0.06 k-m 19.24±3.22 b 29.35±4.24 a akbari control 1.73±0.12 s-u 3.67±0.26 q-t 0.00±0.0 l 0.00±0.0 q a 1.61±0.05 t-u 3.41±0.11 r-u 0.00±0.0 l 0.00±0.0 q b 1.48±0.04 u-x 3.16±0.10 s-v 0.00±0.0 l 0.00±0.0 q c 1.43±0.06 v-x 2.95±0.14 t-w 0.00±0.0 l 3.14±4.23 m-q d 1.34±0.06 w-y 2.69±0.13 u-x 0.00±0.0 l 10.57±4.34 f-h ghazvini control 2.45±0.06 h-l 5.44±0.15 h-j 0.00±0.0 l 0.00±0.0 q a 2.44±0.02 h-l 5.41±0.05 h-j 0.00±0.0 l 0.00±0.0 q b 2.40±0.06 h-l 5.30±0.13 i-k 0.00±0.0 l 0.45± 0.30 q c 2.31±0.06 k-o 5.02±0.13 j-l 0.00±0.0 l 3.28±5.63 m-q d 2.25±0.21 m-p 4.76±0.44 l-n 0.00±0.0 l 9.12±3.68 g-i italiaie control 2.52±0.18 h-j 5.70±0.42 h 0.00±0.0 l 0.00±0.0 q a 2.54±0.15 h-j 5.75±0.35 g-h 0.00±0.0 l 0.00±0.0 q b 2.30±0.10 k-o 5.14±0.24 j-l 0.00±0.0 l 1.15±2.40 p-q c 1.98±0.13 o-s 4.38± 0.29 o-p 3.78±3.50 g-l 5.50±5.62 i-n d 1.73±0.18 s-u 3.77±0.39 q-t 5.13± 3.05 f-j 8.99±3.09 g-j kaleh ghoch control 2.36±0.16 h-m 5.13±0.36 i-k 0.00±0.0 l 0.00±0.0 q a 2.37±0.07 h-m 5.13±0.16 i-k 0.00±0.0 l 0.00±0.0 q b 2.01±0.06 o-s 4.29±0.13 o-p 1.19±2.50 kl 1.15±3.04 p-q c 1.58±0.08 t-u 3.32±0.17 r-u 4.91±2.70 f-k 5.49±2.78 i-n d 1.18±0.11 y-z 2.43±0.15 v-y 17.10±7.08 b-c 9.98±6.03 f-h jandaghi control 2.92±0.23 c-d 6.41±0.24 e 0.00±0.0 l 0.00±0.0 q a 2.89±0.06 c-e 6.32±0.50 e-f 0.00±0.0 l 0.00±0.0 q b 2.55±0.18 g-h 5.53±0.14 hi 1.87± 1.90 i-l 2.33±2.95 n-q c 1.95±0.12 p-t 4.13±0.38 p-r 11.54± 4.80 d 7.50±4.54 g-l d 1.41±0.04 v-x 2.90±0.26 t-w 17.74± 6.60 b-c 17.55±6.71 cd mousa abadi control 1.83±0.02 r-t 4.07±0.09 p-r 0.00±0.0 l 00.00±0.0 q a 1.91±0.09 p-t 3.99±0.06 p-s 0.00±0.0 l 00.00±0.0 q b 1.51±0.10 u-x 3.22±0.20 s-u 2.65±1.30 i-l 3.55±1.50 m-q c 1.18±0.05 y-z 2.43±0.22 v-y 8.15± 2.50 d-f 13.04±2.74 e-f d 0.72±0.12 z 1.42±0.11 z 27.06± 4.90 a 20.47±4.70b-c ebrahimi control 1.94±0.07 p-s 4.52 ±0.29m-o 0.00±0.0 l 00.00±0.0 q a 1.87±0.10 r-t 4.32±0.18 o-p 0.00±0.0 l 00.00±0.0 q b 1.61±0.04 t-u 3.61±0.23 q-t 3.82±2.33 g-l 5.33±2.11 j-o c 1.22±0.12 x-y 2.70±0.09 u-x 5.02±2.89 f-k 9.29±3.24 f-h d 0.71±0.12 z 1.50±0.25 y-z 15.05±2.95 c 21.96±3.11 b badami zarand control 2.47±0.13 h-k 5.25±0.25 i-k 0.00±0.0 l 0.00±0.0 q a 2.43±0.09 h-k 5.15±0.27 j-k 0.00±0.0 l 0.00±0.0 q b 2.29±0.11 k-o 4.91±0.20 k-m 0.00±0.0 l 2.61±1.11 n-q c 2.08±0.14 o-r 4.28±0.24 o-p 3.50±2.70 h-l 3.50±2.51 m-q d 1.71±0.16 s-u 3.41±0.28 r-u 5.64±2.99 f-i 10.65±3.80 f-h fandoghi 48 control 3.11±0.14 c 7.03±0.37 d 0.00±0.0 l 0.00±0.0 q a 3.06±0.14 c 6.87±0.31 d 0.00±0.0 l 0.00±0.0 q b 2.73±13 e-f 6.07±0.32 f-g 0.00±0.0 l 5.45±2.37 l-p c 2.29±0.15 k-m 4.98±0.34 k-m 3.74±2.50 g-l 8.28±2.30 g-k d 1.73±0.13 s-u 3.67±0.29 q-t 18.15±3.70 b-c 15.88±3.53 de sabs pesteh togh control 2.34±0.04 j-n 4.99±0.09 k-m 0.00±0.0 l 0.00±0.0 a a 2.35±0.05 j-n 4.99±0.12 k-m 0.00±0.0 l 0.00±0.0 a b 2.29±0.06 k-o 4.76±0.13 m-n 1.11±1.17 j-l 5.01±1.39 k-p c 2.13±0.08 o-r 4.33±0.16 o-p 6.88±2.70 e-h 10.04±2.84 f-h d 1.98±0.08 o-s 3.96±0.17 p-s 14.46±3.67 cd 18.25±3.53 cd ahmad aghaee control 1.89±0.05 p-u 3.85±0.11 p-s 0.00±0.0 l 0.00±0.0 q a 1.87±0.03 p-t 3.68±0.06 q-t 0.00±0.0 l 0.00±0.0 q b 1.78±0.04 s-t 3.48±0.08 r-u 0.50±1.10 l 3.95±2.73 l-q c 1.66±0.04 t-v 3.18±0.08 t-v 6.56±2.12 e-h 11.12±2.64 f-g d 1.42±0.05 v-x 2.66±0.09 u-x 11.37±2.50 d 16.62±2.56 de rezaie zodres control 2.27±0.05 k-m 4.52±0.10 m-o 0.00±0.0 l 0.00±0.0 q a 2.25±0.03 k-m 4.45±0.05 m-o 0.00±0.0 l 0.00±0.0 q b 2.15±0.04 m-p 4.21±0.07 p-s 1.31±0.70 j-l 6.93±3.84 h-m c 1.85±0.067 r-t 3.56±0.13 q-t 9.31±3.12 de 15.18±3.82 de d 1.58±0.05 t-u 2.97±0.09 t-w 27.41±4.45 a 29.11±4.80 a shahpasand control 4.34±0.05 a 8.36±0.10 a 0.00±0.0 l 0.00±0.0 q a 4.34±0.02 a 8.32±0.05 a 0.00±0.0 l 0.00±0.0 q b 4.29± 0.03 a 8.17±0.06 a-b 0.00±0.0 l 1.45±0.5 o-q c 4.23±0.05 a 7.93±0.10 b 3.06±1.50 h-l 3.05±1.62 m-q d 4.02±0.08 b 7.41±0.16 c 7.54±3.01 e-g 9.45±3.08 f-h adv. hort. sci., 2018 32(2): 249-264 256 table 6 effect of interaction between salinity and cultivar on the morphologic traits measured means in each column and for each factor, followed by similar letter(s) are not significantly different at the 1% probability level, using duncan’s multiple range test. *= a/ is less than z. given that variety of data was very wide, duncan’s test grouped data between a to z and less than z such as a/, b/, c/, d/ and e/. cultivar treatments branch fresh weight (g) root fresh weight (g) root dry weight ratio to aerial organ dry weight root fresh weight ratio to aerial organ fresh weight khanjari control 4.42±0.02 g-i 8.12±0.13 i-l 0.65±0.01 m-v 0.76±0.01 o-y a 4.38±0.01 g-j 8.13±0.07 h-l 0.65±0.01 m-v 0.76±0.01 o-y b 4.16±0.05 h-m 7.75±0.28 l-o 0.67±0.02 m-u 0.78±0.02 o-x c 3.69±0.05 j-s 6.69±0.11 r-s 0.69±0.01 m-u 0.81±0.01 m-u d 3.01±0.06 r-z 6.14± 0.06 t-u 0.73±0.01 k-s 0.88±0.01 i-r akbari control 3.25±0.36 o-x 8.58±0.51 f-i 0.99±0.08 c-j 1.24±0.10 c-f a 3.17±0.34 o-x 8.51±0.25 f-j 1.03±0.05 c-h 1.28±0.06 c-e b 2.99±0.16 s-z 8.01±0.14 j-n 1.03±0.04 c-h 1.28±0.05 c-e c 2.76±0.15 u-a/ 7.95±0.05 j-n 1.04±0.04 c-g 1.31±0.06 c-e d 2.28±0.20 a/-c/ 7.79±0.04 l-o 1.11±0.06 c-d 1.42±0.08 a-c ghazvini control 3.44±0.16 n-v 9.46±0.15 a-b 0.91±0.02 d-l 1.06 ±0.02e-n a 3.38±0.08 o-w 9.42±0.35 a-b 0.91±0.03 d-l 1.07±0.04 e-n b 3.18±0.07 o-x 9.35±0.03 a-b 0.94±0.01 d-k 1.10±0.01 e-k c 2.66±0.14 v-a/ 9.29±0.03 a-c 1.03±0.01 c-h 1.21±0.03 c-g d 2.42±0.16 y-b/ 9.15±0.04 a-e 1.09±0.07 c-e 1.28±0.08 c-e italiaie control 4.35±0.39 g-k 9.60±0.07 a 0.82±0.06 g-n 0.96±0.06 g-p a 4.23±0.15 h-l 9.58±0.05 a 0.83±0.03 g-n 0.96±0.03 g-p b 3.89±0.12 h-o 9.41±2.98 a-b 0.85±0.28 f-n 0.99 f±0.33-o c 3.30±0.14 o-x 9.19±0.06 a-d 1.02±0.05 c-i 1.19±0.06 c-h d 2.72±0.22 v-a/ 8.96±0.07 b-f 1.20±0.09 b-c 1.37±0.10 b-d kaleh ghochi control 3.21±0.08 o-x 5.46±0.07 w-y 0.56±0.01 p-w 0.65±0.01 r-y a 3.12±0.08 p-y 5.46±0.03 w-y 0.57±0.01 o-w 0.67±0.01 q-y b 2.74±0.08 v-a/ 5.41±0.04 w-z 0.66±0.01 m-u 0.77±0.02 o-y c 2.23±0.06 a/-c/ 5.20±0.05 w-a/ 0.80±0.03 h-p 0.93±0.04 h-q d 1.62±0.03 c/ 4.90±0.03 z-b/ 1.03±0.04 c-h 1.21±0.05 c-g jandaghi control 3.49±0.10 m-u 5.24±0.06 w-a/ 0.46±0.01 u-w 0.53±0.01 v-y a 3.42±0.15 n-v 5.22±0.04 w-a/ 0.47±0.02 t-w 0.54±0.02 u-y b 2.90±0.07 t-z 5.01±0.07 y-b/ 0.52±0.01 r-w 0.59±0.01 t-y c 2.34± 0.08 z-b/ 4.78±0.05 a/-c/ 0.65±0.03 m-v 0.74±0.04 o-y d 1.81±0.05 b/ 4.28±0.03 d/ 0.79±0.03 i-p 0.91±0.04 i-r mousa abadi control 3.30±0.20 o-w 5.16±0.18 w-a/ 0.63±0.03 n-w 0.70±0.03 p-y a 3.18±0.09 o-w 5.08±0.10 y-a/ 0.63±0.01 n-w 0.71±0.01 p-y b 2.46±0.07 xa/ 4.97±0.07 y-b/ 0.75±0.02 k-r 0.84±0.03 k-t c 1.96±0.06 a/d/ 4.53±0.05 b/-d/ 0.95±0.03 d-k 1.08±0.04 e-l d 1.16±0.08 d/ 4.13±0.07 d/e/ 1.39±0.06 a-b 1.60±0.07 a-b ebrahimi control 5.05±0.23 e-g 7.51±0.09 n-p 0.76±0.03 j-q 0.79±0.03 n-w a 5.04±0.18 e-g 7.49±0.06 n-q 0.77±0.02 j-p 0.80±0.02 n-v b 4.41±0.18 f-j 7.32±0.04 o-q 0.87±0.02 e-m 0.91±0.02 i-r c 3.50±0.19 m-t 7.04±0.07 p-r 1.07±0.03 c-f 1.13±0.03 d-j d 2.68±0.16 w-a/ 6.69±0.06 r-s 1.47±0.13 a 1.61±0.16 a badami zarand control 5.57±0.20 d-e 8.80±0.09 c-g 0.78±0.02 j-p 0.81±0.02 m-u a 5.54±0.24 d-e 8.73±0.13 d-g 0.78±0.03 j-p 0.82±0.03 l-t b 5.10±0.17 e-f 8.65±0.13 e-h 0.84±0.02 g-n 0.87±0.03 j-s c 4.41±0.14 g-j 8.32±0.26 g-k 0.92±0.03 d-l 0.96±0.04 g-p d 3.64±0.15 k-s 8.06±0.17 i-m 1.09±0.05 c-e 1.14±0.06 d-i fandoghi 48 control 7.71±0.23 a-b 7.62±0.11 l-o 0.41±0.01 w 0.51±0.01 x-y a 7.60±0.19 a-b 7.58±0.05 m-o 0.42±0.01 v-w 0.52±0.01 w-y b 6.81±0.25 c 7.30±0.09 o-q 0.46±0.01 u-w 0.57±0.01 t-v c 5.78±0.32 d 6.99±0.08 q-s 0.52±0.02 r-w 0.65±0.033 r-y d 4.36±0.19 g-j 6.50±0.14 s-t 0.65±0.03 m-v 0.82±0.04 l-t sabs pesteh togh control 3.81±0.16 i-p 6.02±0.11 t-v 0.69±0.02 l-u 0.68±0.02 q-y a 3.74±0.14 i-r 5.99±0.07 u-v 0.70±0.01 l-t 0.69±0.01 p-y b 3.21±0.11 o-x 5.71±0.18 u-w 0.73±0.01 k-s 0.71±0.01 p-y c 2.61±0.03 x-a/ 5.38±0.14 w-z 0.78±0.01 j-p 0.78±0.01 o-x d 2.14±0.06 a/-c/ 4.95±0.11 y-a/ 0.82±0.02 g-n 0.81±0.02 m-u ahmad aghaee control 3.83±0.07 i-p 5.72±0.06 u-w 0.76±0.01 j-q 0.75±0.01 o-y a 3.76±0.03 i-q 5.73 ±0.07u-w 0.79±0.01 j-p 0.77±0.01 o-y b 3.55±0.07 l-t 5.61±0.07 v-x 0.82±0.01 g-n 0.80±0.01 m-v c 3.35±0.07 o-w 5.47±0.08 w-y 0.86±0.01 f-n 0.84±0.01 k-t d 2.85±0.09 t-z 5.02±0.07 y-a/ 0.91±0.01 d-l 0.91±0.01 i-r rezaie zodres control 4.56±0.09 f-h 4.56±0.12 b/-d/ 0.49±0.01 t-w 0.50±0.01 y a 4.46±0.12 f-i 4.41±0.11 b/-d/ 0.50±0.02 s-w 0.50±0.02 y b 4.13±0.09 i-n 4.28±0.16 d/ 0.51±0.01 s-w 0.51±0.01 x-y c 3.78±0.09 i-q 3.98±0.15 e/ 0.53±0.02 q-w 0.54±0.02 u-y d 3.05±0.10 q-z 3.53±0.09 f/ 0.58±0.01 o-w 0.59±0.01 t-y shahpasand control 8.01±0.07 a 9.44±0.11 a-b 0.47±0.01 t-w 0.57±0.01 t-y a 7.98±0.08 a 9.43±0.08 a-b 0.48±0.01 t-w 0.58±0.01 t-y b 7.83±0.12 a-b 9.21±0.05 a-d 0.48±0.01 t-w 0.58±0.01 t-y c 7.49±0.07 a-b 8.98±0.13 b-f 0.49±0.01 t-w 0.59±0.01 t-y d 7.18±0.10 b-c 8.53±0.16 f-i 0.50±0.01 s-w 0.60±0.01 s-y momenpour and imani salinity tolerance in fourteen selected pistachio cultivars 257 table 7 effect of interaction between salinity and cultivar on chlorophyll fluorescence parameters means in each column and for each factor, followed by similar letter(s) are not significantly different at the 1% probability level, using duncan’s multiple range test. cultivar treatments (fv/fm) maximum florescence (f m ) minimum florescence (f o ) khanjari control 0.82±0.003 b-c 603.67±3.46 b-d 107.22±1.71 r-u a 0.82±0.003 b-c 606.44±3.39 b-d 110.67±1.58 o-r b 0.80±0.005 d-e 586.00±8.74 e-f 116.55±1.66 k-m c 0.74±0.015 j-k 540.33±5.50 k-m 139.88±8.52 e d 0.65±0.025 q 467.77±5.65 r 163.11±11.20 a akbari control 0.83±0.002 a-b 625.44±6.72 a 106.11±2.31 s-u a 0.82±0.004 b-c 617.67±5.85 a-c 109.77±2.16 p-s b 0.82±0.007 b-c 614.11±5.01 a-c 110.55±3.77 o-s c 0.78±0.003 f-g 607.11±7.09 b-d 134.22±2.38 f-g d 0.75±0.007 i-j 555.44±11.58 h-k 141.33±1.58 e ghazvini control 0.82±0.003 b-c 602.44±11.54 c-d 106.11±3.51 s-u a 0.82±0.003 b-c 602.55±17.00 c-d 108.67±4.09 q-t b 0.81±0.004 c-d 591.00±2.87 d-f 109.77±2.27 o-s c 0.80±0.003 c-e 578.33±5.61 d-g 118.67±1.93 j-m d 0.76±0.003 h-i 538.11±4.59 l-m 129.33±2.39 h italiaie control 0.83±0.004 a-b 600.77±7.41 c-e 101.22±3.89 v-w a 0.83±0.003 a-b 604.22±9.31 b-d 103.77±3.70 t-v b 0.81±0.004 c-d 576.44±3.71 f-g 112.00±2.39 n-q c 0.78±0.008 f-g 532.11±13.50 m 117.11±2.14 k-m d 0.73±0.009 l 488.67±6.61 o-q 133.55±5.12 f-g kaleh ghochi control 0.83±0.002 a-b 538.44±9.46 l-m 93.22±1.98 y-z a 0.82±0.005 b-c 542.88±5.94 j-m 93.33±2.64 y-z b 0.80±0.005 d-e 529.88±7.18 m 106.44±2.29 s-u c 0.76±0.005 h-i 489.00±8.38 o-q 119.55±3.20 i-k d 0.69±0.009 m 444.11±12.31 s 137.00±3.46 e-f jandaghi control 0.83±0.005 a-b 580.33±18.67 f 100.67±2.64 v-w a 0.82±0.006 b-c 557.00±3.80 h-j 101.44±3.46 v-w b 0.77±0.006 g-h 479.88±6.73 p-r 110.33±2.54 p-s c 0.7±0.0043 l 417.33±9.04 t-u 113.89±3.25 m-p d 0.66±0.004 p 380.67±11.69 w 129.44±3.08 h mousa abadi control 0.84 a±0.005 551.78±7.15 h-l 91.00±2.87 z a 0.82±0.007 b-c 530.00±7.29 m 94.00±3.57 x-z b 0.79±0.002 e-f 495.22±14.77 op 104.55±3.46 t-v c 0.73±0.008 kl 447.22±21.89 s 122.44±4.97 i-j d 0.61±0.009 s 387.67±29.06 w 151.00±10.34 b-c ebrahimi control 0.83±0.003 a-b 608.11±8.08 b-c 104.55±2.50 t-v a 0.83±0.004 a-b 609.67±8.17 a-c 105.44±3.77 t-v b 0.80±0.006 d-e 586.44±10.27 e-f 114.44±3.35 m-p c 0.77±0.006 g-h 539.67±14.41 k-m 123.67±4.24 i d 0.73±0.011 kl 512.44±12.28 n 140.67±4.44 e badami zarand control 0.82±0.003 b-c 602.44±9.83 cd 105.55±2.12 s-u a 0.82±0.002 b-c 606.33±8.81 a-c 106.55±2.45 s-u b 0.81±0.003 c-d 597.11±10.32 d-f 108.55±1.58 q-t c 0.77± 0.007 g-h 542.55±13.92 j-m 124.44±4.92 i d 0.73±0.004 k-l 512.67±12.79 n 137.44±3.16 e-f fandoghi 48 control 0.84±0.006 a 585.44±10.90 f 95.11±2.97 x-z a 0.83±0.004 a-b 585.48±9.48 f 98.11±2.47 w-x b 0.79±0.004 e-f 579.00±8.95 f 118.89±3.33 j-m c 0.73±0.010 k-l 491.44±10.87 o-q 131.55±4.63 g-h d 0.64±0.018 r 404.55±14.39 u-v 147.33±4.74 d sabs pesteh togh control 0.83±0.005 a-b 562.00±14.96 g-h 95.67±1.73 x-y a 0.82±0.007 b-c 539.00±21.68 l-m 97.77±1.78 w-x b 0.76±0.009 h-i 470.89±14.88 r 110.88±2.52 o-r c 0.72±0.007 l 427.11±15.39 t 118.55±4.15 j-l d 0.67±0.009 o 409.11±14.58 u 134.00±3.93 f-g ahmad aghaee control 0.83±0.003 a-b 610.33±12.40 a-c 106.44±2.24 s-u a 0.82±0.004 b-c 603.22±19.07 cd 111.22±3.07 o-r b 0.78±0.011 f-g 543.89±20.01 i-m 117.11±2.61 k-m c 0.75±0.009 i-j 510.33±12.10 n 130.00±3.27 g-h d 0.67±0.022 o 469.89±30.25 r 153.33±4.66 b rezaie zodres control 0.84±0.008 a 562.89±13.27 g-h 92.67±3.04 y-z a 0.82±0.006 b-c 538.55±14.52 k-m 95.67±2.00 x-y b 0.76±0.012 h-i 467.77±16.20 r 105.33±3.60 t-v c 0.68±0.016 n 391.00±10.14 v-u 123.55±5.41 i d 0.59±0.015 t 365.22±20.90 x 147.77±7.52 cd shahpasand control 0.83±0.003 a-b 531.00±9.73 m 86.67±2.00 a/ a 0.83±0.004 a-b 530.88±8.26 m 88.00±1.58 a/ b 0.82±0.007 b-c 527.44±17.25 m-n 95.11±2.52 x-z c 0.79±0.012 e-f 499.67±18.36 o-p 105.77±2.81 s-u d 0.75±0.011 i-j 461.00±13.79 r 115.00±3.42 l-o adv. hort. sci., 2018 32(2): 249-264 258 table 8 effect of interaction between salinity and cultivar on the physiologic traits measured means in each column and for each factor, followed by similar letter(s) are not significantly different at the 1% probability level, using duncan’s multiple range test. cultivar treatments total chlorophyl (mg/g) chlorophyll b (mg/g) chlorophyll a (mg/g) chlorophyll index (spad) khanjari control 1.13±0.03 k-n 0.36±0.02 g-j 0.77±0.02 g 57.58±1.52 f-h a 1.11±0.02 m-n 0.36±0.04 g-j 0.75±0.02 g-i 56.62±1.62 g-j b 1.06±0.02 p-r 0.34±0.02 i-l 0.72±0.05 i-k 53.60±0.82 i-l c 0.88±0.01 w 0.31±0.01 l-o 0.57±0.02 s 48.55±1.50 o-q d 0.59±0.02 a/ 0.22±0.01 t 0.37±0.04 w-x 40.00±1.73 y-z akbari control 1.45±0.007 c 0.51±0.008 a 0.95±0.003 c 61.27±0.98 c-d a 1.45±0.11 c 0.52±0.008 a 0.94±0.009 c 61.40±1.57 c-d b 1.40±0.02 c-d 0.51±0.1 a 0.89±0.03 c-d 60.15±1.55 c-e c 1.23±0.01 f 0.46±0.03 cd 0.77±0.01 g 56.97±1.04 g-i d 1.05±0.009 q-s 0.38±0.02 e-h 0.67±0.02 m-o 53.70±1.24 k-m ghazvini control 1.20±0.01 f-g 0.46±0.007 c-d 0.74±0.01 h-j 55.17±1.27 g-l a 1.21±.005 f-g 0.47±0.01 c-d 0.74±0.008 h-j 54.67±1.25 i-l b 1.17±0.006 g 0.45±0.007 c-d 0.72±0.005 j-k 54.04±1.09 j-m c 1.14±0.02 g-m 0.43±0.008 d 0.71±0.003 j-l 51.98±0.85 l-n d 0.97±0.004 t 0.35± 0.01 h-k 0.62±0.008 q-r 49.78±0.82 n-o italiaie control 1.10±0.01 m-o 0.36±0.004 g-j 0.74±0.003 h-j 49.95±1.10 n-o a 1.09±0.01 n-p 0.36±0.004 g-j 0.73±0.01 i-j 50.02±1.38 n-o b 1.04±0.007 q-s 0.32±0.01 k-n 0.72±0.003 j-k 45.17±1.36 r-u c 0.91±0.008 v 0.26±0.009 q-r 0.6±0.0075 op 43.95±1.43 t-w d 0.70±0.01 z 0.20±0.004 t 0.50±0.005 u 40.41±1.46 x-z kaleh ghochi control 1.07±0.007 o-q 0.38±0.008 e-h 0.69±0.005 l-m 45.17±0.60 r-u a 1.06±0.008 p-r 0.37±0.004 f-i 0.69±0.003 l-m 43.95±0.16 t-w b 1.03±0.01 q-s 0.37±0.004 f-i 0.66±0.009 m-o 41.50±0.28 u-z c 0.80±0.009 x 0.31±0.008 l-o 0.49±0.02 u-v 36.24±0.40 a/ d 0.60±0.003 a/ 0.22±0.01 t 0.38±0.01 w-x 31.47±3.08 b/ jandaghi control 1.04±0.007 q-s 0.36±0.04 g-j 0.69±0.002 l-m 42.77±0.82 u-x a 1.04±0.008 q-s 0.36±0.04 g-j 0.68±0.004 l-n 42.77±0.76 u-x b 0.95±0.01 t-u 0.32±0.03 k-n 0.63±0.006 p-q 38.54±1.37 y-z c 0.74±0.004 y 0.26±0.03 q-r 0.48±0.010 u-v 35.45±0.85 a/ d 0.53±0.004 b/ 0.17±0.004 u 0.36±0.01 x 27.83±0.98 c/ mousa abadi control 1.05±0.007 q-s 0.36±0.008 g-j 0.69±0.002 l-m 42.40±0.95 v-y a 1.04±0.008 q-s 0.36±0.004 g-j 0.68±0.003 l-n 42.65±0.51 u-y b 0.95±0.01 t-u 0.30±0.005 m-p 0.63±0.005 p-q 38.11±1.00 z-a/ c 0.72 ±0.01y-z 0.23±0.01 s-t 0.49±0.009 u-v 32.33±1.10 b/ d 0.48±0.03 c/ 0.15±0.004 u 0.33±0.008 y 22.60±1.50 d/ ebrahimi control 1.20±0.04 f-h 0.45±0.03 d 0.74±0.01 h-j 55.21±1.22 g-l a 1.19±0.008 g-i 0.45±0.02 cd 0.74±0.01 h-j 54.97±1.65 h-l b 1.12±0.02 l-n 0.42±0.01 e-g 0.70±0.008 j-l 49.97±1.98 no c 0.95±0.01 t-u 0.35±0.01 h-k 0.60±0.01 r 45.51±1.88 r-t d 0.74±0.01 y 0.27±0.01 p-r 0.47±0.01 v 40.27±1.54 x-z badami zarand control 1.15±0.02 j-l 0.38±0.008 e-h 0.77±0.008 g 54.86±1.32 h-l a 1.15±0.02 j-l 0.39±0.01 e-g 0.76±0.01 g-h 54.80±1.47 i-l b 1.10±0.02 l-p 0.37±0.01 f-i 0.73±0.01 g-l 52.89±0.70 l-m c 0.93±0.01 u-v 0.30±0.01 m-p 0.63±0.01 p-q 49.39±1.53 n-p d 0.73±0.02 y-z 0.20±0.01 t 0.53±0.01 t 45.55±1.19 r-t fandoghi 48 control 1.23±0.02 f 0.41±0.03 e 0.83±0.01 e 56.88-i±1.16 g a 1.23±0.02 f 0.40±0.02 e-f 0.83±0.01 e 56.00±1.22 g-k b 1.16 ±0.009h-j 0.36±0.01 f-i 0.80±0.01 e-f 51.67±1.32 m-n c 1.02±0.03 s 0.32±0.01 k-n 0.70±0.01 j-l 47.22±1.48 p-r d 0.73±0.02 y-z 0.25±0.01 r-s 0.49±0.01 u-v 40.00±1.87 y-z sabs pesteh togh control 1.19±0.03 g-i 0.44±0.05 d 0.74±0.02 h-j 50.36±1.14 n-o a 1.16±0.02 i-k 0.44±0.02 d 0.73±0.007 i-j 46.93±7.47 o-r b 1.05±0.02 q-s 0.35±0.01 h-k 0.70±0.009 j-l 46.77±1.28 q-s c 0.81±0.008 x 0.28±0.02 o-q 0.53±0.01 t 42.47±0.90 v-y d 0.58±0.01 a/ 0.21±0.01 t 0.36±0.005 x 36.92±1.03 a/ ahmad aghaee control 1.04±0.01 q-s 0.37±0.01 f-i 0.67±0.007 m-o 46.48±3.78 q-t a 1.02±0.02 s 0.35±0.01 h-k 0.67±0.007 m-o 44.15±0.22 s-v b 0.97±0.01 t 0.32±0.009 k-n 0.64±0.006 o-q 41.37±0.44 w-z c 0.83±0.01 x 0.29±0.01 n-q 0.53±0.01 t 36.60±0.30 a/ d 0.62±0.01 a/ 0.23±0.01 s-t 0.39±0.01 w 31.23±0.75 b/ rezaie zodres control 1.12±0.02 l-n 0.36±0.008 g-j 0.76±0.02 g-h 57.72±1.04 e-g a 1.11±0.02 m-n 0.37±0.01 f-i 0.75±0.01 g-i 56.01±0.84 g-k b 1.02±0.02 s 0.32±0.01 k-m 0.70±0.01 j-l 53.25±1.63 lm c 0.86±0.02 w 0.29±0.01 n-q 0.57±0.01 s 48.56±1.41 o-q d 0.58±0.03 a/ 0.21±0.005 t 0.37±0.02 w-x 40.24±1.61 x-z shahpasand control 1.54±0.007 a 0.50±0.008 a-b 1.04±0.007 a 65.52±1.16 a a 1.54±0.01 a 0.50±0.01 a-b 1.03±0.009 a-b 64.52±1.19 a-b b 1.50±0.03 a-b 0.48±0.02 b-c 1.02±0.008 a-b 62.57±0.99 a-c c 1.33±0.03 e 0.40±0.02 e-f 0.93±0.01 c 59.98±0.79 d-f d 1.10±0.02 m-o 0.29±0.02 n-q 0.81±0.01 e-f 55.35±1.35 g-l momenpour and imani salinity tolerance in fourteen selected pistachio cultivars 259 table 9 effect of interaction between salinity and cultivar on the physiologic traits measured means in each column and for each factor, followed by similar letter(s) are not significantly different at the 1% probability level, using duncan’s multiple range test. cultivar treatments cell membrane injury (%) relative ionic leakage (%) relative water content (%) khanjari control 37.70±0.010 m-o 85.25±0.64 a a 2.98±1.28 w-y 39.14±0.008 k-o 84.16±0.46 a-b b 8.09±0.91 t-u 44.34±0.005 i-o 81.31±0.51 b c 23.09±3.10 k-m 51.75±0.019 d-k 77.60±0.62 c d 46.47±0.70 b 64.42±0.004 a-c 70.36±1.04 f akbari control 38.05±0.007 l-o 85.08±0.40 a a 0.83±1.21 x-y 38.41±0.016 l-o 84.52±0.60 a-b b 5.27±1.07 u-w 42.30±0.006 j-o 82.90±0.60 a-b c 16.37±1.78 o-q 45.96±0.011 g-o 82.08±0.68 a-b d 27.83±2.18 h-i 50.09±0.013 d-k 80.22±0.56 b ghazvini control 35.62±0.013 o 83.19±0.57 a-b a 0.42±0.19 y 36.59±0.006 m-o 82.98±0.64 a-b b 2.67±2.28 x-y 38.37±0.014 l-o 81.58±0.42 b c 5.89±2.81 u-w 39.73±0.018 k-o 80.88±0.36 b d 16.17±0.62 o-q 45.50±0.16 g-o 78.95±0.45 b-c italiaie control 40.85±0.012 j-o 84.43±0.35 a-b a 0.75±0.79 x-y 40.88±0.005 j-o 84.21±0.38 a-b b 12.16±3.06 r-s 47.75±0.018 e-n 81.51±0.36 b c 22.24±2.62 l-m 52.75±0.015 c-i 78.40±0.37 c d 33.65±3.51 j-l 60.54±0.020 a-d 74.06±0.50 d-e kaleh ghochi control 39.43±0.015 k-o 83.41±0.32 a-b a 3.89±2.81 v-y 40.81±0.017 k-o 82.41±0.29 a-b b 11.97±0.59 r-s 43.67±0.003 i-o 81.45±0.32 b c 21.88±1.09 l-n 48.89±0.006 d-l 78.35±0.33 c d 38.43±2.46 de 59.29±0.015 b-f 73.59±0.30 e jandaghi control 37.68±0.015 m-o 82.57±0.32 a-b a 3.06±1.36 w-y 38.85±0.008 l-o 81.45±0.34 b b 10.47±1.29 s-t 45.53±0.008 h-o 79.35±0.25 b-c c 18.68±2.28 n-p 55.70±0.014 b-i 74.67±0.29 d-e d 31.21±3.47 g-h 66.61±0.021 a-b 67.38±0.41 g mousa abadi control 37.59±0.007 l-o 80.42±0.27 b a 4.47±1.83 u-x 39.39±0.011 k-o 79.53±0.28 b-c b 18.68±2.77 n-p 49.41±0.017 d-k 75.55±0.35 d c 33.47±2.27 f-g 57.80±0.014 b-g 70.52±0.66 f d 54.83±4.50 a 71.34±0.028 a 64.17±0.52 i ebrahimi control 40.04±0.010 k-o 81.51±0.25 b a 4.88±4.12 u-w 42.77±0.027 j-o 80.40±0.45 b-c b 12.92±4.01 q-s 46.57±0.024 f-o 78.49±0.31 b-c c 18.34±2.62 n-p 49.90±0.016 d-l 76.34±0.34 c-d d 27.70±4.05 h-i 56.65±0.024 b-h 69.95±0.45 f-g badami zarand control 35.90±0.018 n-o 80.56±0.26 b-c a 2.36±3.55 w-y 37.60±0.028 l-o 79.41±0.24 b-c b 6.96±2.50 t-v 41.66±0.016 j-o 78.13±0.30 c c 14.18±2.66 q-r 45.00±0.017 h-o 76.38±0.32 c-d d 21.21±2.04 l-n 49.51±0.013 d-l 73.21±0.47 e fandoghi 48 control 41.44±0.012 j-o 82.51±0.33 a-b a 4.18±3.07 v-y 43.52±0.022 j-o 81.64±0.30 b b 10.24±2.12 s-t 47.33±0.012 f-o 79.22±0.47 b-c c 26.48±4.68 i-k 56.86±0.027 b-h 75.25±0.54 d d 41.04±3.79 c-d 65.40±0.022 a-b 69.12±0.47 f-g sabs pesteh togh control 41.06±0.014 j-o 81.34±0.31 b a 5.35±3.95 u-w 44.64±0.029 h-o 79.60±0.41 b-c b 14.57±6.87 q-r 50.94±0.039 d-k 77.18±0.32 c-d c 28.48±4.47 h-i 57.93±0.025 b-g 73.67±0.59 e d 42.89±6.21 c 66.20±0.035 a-b 66.49±0.77 g-i ahmad aghaee control 39.50±0.013 k-o 85.02±0.32 a a 3.27±1.44 v-y 41.35±0.008 j-o 84.39±0.36 a-b b 9.63±0.93 s-t 44.05±0.005 i-o 83.26±0.42 a-b c 19.71±1.64 m-o 50.30±0.010 d-j 80.1±0.38 b d 35.57±2.85 e-f 60.11±0.017 a-e 75.38±0.38 d rezaie zodres control 40.12±0.006 k-o 81.05±0.42 b a 2.18±1.61 w-y 40.97±0.010 j-o 80.50±0.25 b-c b 10.48±1.51 s-t 48.81±0.009 d-l 77.17±0.51 c-d c 26.78±3.43 i-j 55.85±0.020 b-i 74.34±0.45 d-e d 47.45±2.95 b 68.31±0.017 a-b 66.95±0.57 g-i shahpasand control 39.92±0.006 k-o 79.83±0.47 b-c a 3.61±3.10 v-y 40.43±0.021 k-o 79.49±0.22 b-c b 5.98±1.87 u-w 42.63±0.011 j-o 78.89±0.51 b-c c 15.32±3.43 p-r 46.68±0.020 f-o 77.15±0.53 c d 27.77±2.98 h-i 53.23±0.018 c-j 74.41±0.39 d-e 260 adv. hort. sci., 2018 32(2): 249-264 was observed the least decrease in the relative humidity content of the leaves. relative ion leakage percentage in all studied cultivars was increased by increasing salinity concentration. the increase in the relative ion leakage percentage was significant between the studied cultivars. the highest relative ion leakage percentage was observed in mousa abadi cultivar in salinity level d. after this cultivar, rezai zod res, jandaghi, sabz pesteh togh, fndoghi 48, kanjari and italiyayi cultivars had the highest relative ion leakage percentage. the increase in relative ion leakage percentage was not significant in ghazvini cultivar compared to the control plants (table 9). the results showed that the cultivars had a significant difference in cell membrane injury percentage. the highest cell membrane injury percentage was observed in the leaves of mousa abadi (54.83±4.50%), and the lowest cell membrane injury percentage was observed in the leaves of ghazvini (16.17±0.62%). results reported in table 10 assessed that with increasing salinity concentration in irrigation water, the sodium concentration in the leaves and roots of total cultivars increased. the increase in sodium concentration in the leaves of ghazvini cultivar was only significant when plants were treated with salinity l e v e l d , w h i l e i n a k b a r i , b a d a m i z a r a n d a n d shahpasand cultivars was observed a significant increased when treated with salinity levels c and d, compared to the control plants. while the increase of sodium concentration in the leaves of other cultivars was significant different salinity levels b, c and d, compared to the control plants (table 10). the highest sodium concentration in leaves was observed in the salinity level d and in mousa abadi (2.09± 0.045%), rezaie zood res (2.05±0.030%), khanjari (2.03±0.115%) and jandaghi (1.90±0.035%) cultivars treated. also the highest sodium concentration in roots was also observed in salinity level d, and in mousa abadi (3.04±0.06%) and rezaie zood res (2.99±0.05%) cultivars. with increasing salinity levels (to 14.75 ds/m), potassium concentration increased in leaves and roots of akbari, ghazvini, shahpasand, badami zarand and ebrahimi cultivars while potassium content in the leaves and roots of other cultivars except mousa abadi and rezaie zood res increased to salinity level c. potassium content in the leaves and roots of mousa abadi and rezaie zood res cultivars was increased only in salinity level b. overall, the highest potassium content in leaves and roots was observed in salinity level c and in ghazvini (1.81±0.02%) and akbari (1.38±0.02%) cultivars. 4. discussion and conclusions based on the results of this study, with increasing salinity concentration in irrigation water, final height, trunk diameter and number of leaves in all studied cultivars decreased. plant height is heavily dependent on growth environment. since the growth phenomenon gained vital activities in which condition the plant must be in possession of enough water, reduction in the height occurs in case of failure to provide the required water due to the reduction of cell turgor pressure and length of the cells would be negatively affected (munns, 2002; munns and tester, 2008). the osmotic effects of salinity stress can be observed immediately after salt application and are believed to continue for the duration of exposure, resulting in inhibited cell expansion and cell division (munns 2002; munns and tester, 2008). in this r e s e a r c h , t r u n k d i a m e t e r a n d i t s g r o w t h w e r e decreased during the application of salinity stress in all cultivars. these results are consistent with other results (sepaskhah and maftoun, 1988; munns and tester, 2008; zrig et al., 2015). it has been reported that growth rates of pistachio trees decrease with increasing sodium chloride (nacl) concentration in soil. it has been also reported that there is a positive correlation between sodium (na+) as well as chloride (cl-) concentration in plant tissue and soil (sepaskhah and maftoun, 1988; munns and tester, 2008; zrig et al., 2015). based on the results of this study, number of leaves with increasing salinity concentrations reduced. our results are consistent with studies reporting that increasing salinity levels negatively affect morphology and number of leaves in pistachio trees (picchioni and myamoto, 1990; saadatmand et al., 2007; karimi et al., 2011). the results of this research showed that with increasing salinity, percentage of green leaves, leaves and shoots fresh and dry weights in all cultivars decreased but the percentage of necrotic leaves and percentage of downfall leaves increades. the cultivars showed different responses to salinity levels. these results are consistent with the results of karimi et al. (2009 and 2011). in these studies, effect of salinity levels on pistachio cultivars was investigated and was reported that pistachio cultivars showed different responses to salinity levels. although pistachio trees are classified as tolerant to salinity, but amount of their tolerance to salinity is differently (sepaskhah and maftoun, 1988; momenpour and imani salinity tolerance in fourteen selected pistachio cultivars 261 table 10 effect of interaction between salinity and cultivar on root and leaf k+ and na+ contents means in each column and for each factor, followed by similar letter(s) are not significantly different at the 1% probability level, using duncan’s multiple range test. cultivar treatments root na+ (%) leaf na+ (%) root k+ (%) leaf k+ (%) khanjari control 0.55±0.03 s-y 0.43±0.027 q-v 0.60±0.03 z-a/ 1.35±0.03 k-m a 0.59±0.01 q-y 0.46±0.023 q-v 0.76±0.05 p-w 1.44±0.11 d-i b 0.70±0.06 o-x 0.64±0.023 m-r 0.73±0.05 q-y 1.56±0.03 c-f c 1.57±0.11 g-i 1.25±0.055 g-h 0.50±0.04 b/ 1.30±0.06 i-o d 2.59±0.06 c-d 2.03±0.116 a 0.37±0.04 d/ 1.01±0.04 u-x akbari control 0.42±0.01 y 0.37±0.013 t-v 0.80±0.03 m-u 1.08±0.03 r-x a 0.44±0.008 x-y 0.39±0.005 t-v 0.99±0.03 f-i 1.21±0.05 m-r b 0.48±0.03 v-y 0.42±0.007 r-v 1.37±0.03 a 1.58±0.04 c-e c 0.97±0.03 l-n 0.73±0.035 k-n 1.38±0.02 a 1.59±0.02 b-d d 1.86±0.04 f 1.40±0.066 e-g 0.77±0.02 o-v 1.11±0.03 q-v ghazvini control 0.46±0.003 w-y 0.41±0.014 s-v 0.79±0.02 n-v 1.31±0.02 i-n a 0.47±0.004 w-y 0.41±0.007 s-v 0.84±0.02 k-r 1.48±0.02 d-h b 0.49±0.006 v-y 0.43±0.004 q-v 0.95±0.02 g-k 1.55±0.03 c-g c 0.52±0.006 u-y 0.46±0.007 q-v 1.08±0.02 d-f 1.81±0.02 a d 1.05±0.02 k-m 0.82±0.023 k-m 0.85±0.02 j-q 1.35±0.02 h-m italiaie control 0.57±0.02 r-y 0.34±0.006 v 0.93±0.02 h-l 1.08±0.02 r-x a 0.61±0.01 q-y 0.36±0.007 t-v 0.98±0.03 f-i 1.19±0.02 n-t b 0.78±0.03 n-t 0.43±0.007 q-v 1.13±0.02 cd 1.23±0.02 m-p c 1.21±0.02 j-k 0.82±0.027 k-m 0.88±0.02 i-p 0.99±0.02 v-x d 1.81±0.04 f 1.55±0.035 c-e 0.60±0.02 z-a/ 0.67±0.02 a/ kaleh ghochi control 0.67±0.03 p-y 0.45±0.005 q-v 0.89±0.02 h-o 1.10±0.02 r-w a 0.70±0.02 o-x 0.47±0.003 p-v 0.95±0.02 g-k 1.18±0.04 n-t b 0.75±0.009 n-u 0.49± 0.003 p-v 1.14±0.02 b-d 1.57±0.02 c-e c 1.38±0.02 i-j 0.76±0.029 k-n 1.11±0.02 de 1.55±0.02 c-g d 2.55±0.04 d 1.50±0.044 d-f 0.82±0.02 l-t 1.17±0.02 n-t jandaghi control 0.49±0.006 u-y 0.41±0.005 s-v 0.68±0.02 u-z 0.82±0.02 y-z a 0.60±0.005 q-y 0.46±0.004 q-v 0.72±0.03 r-z 0.93±0.02 x-y b 0.82±0.01 m-q 0.57±0.017 n-u 0.75±0.05 q-x 1.08±0.02 r-w c 1.52±0.02 g-i 1.22±0.035 g-h 0.69±0.02 u-z 0.71±0.02 z-a/ d 2.73±0.03 cd 1.90±0.035 a-b 0.35±0.03 d/ 0.46±0.02 b/ mousa abadi control 0.53±0.007 t-y 0.44±0.002 q-v 0.60/±0.01 z-a 1.05±0.02 s-x a 0.65±0.01 q-y 0.47±0.005 p-v 0.85±0.02 j-q 1.13±0.02 p-v b 0.83±0.03 m-p 0.71±0.034 l-o 0.63±0.02 a/ 1.07±0.02 r-x c 1.76±0.02 f-g 1.33±0.027 f-h 0.45±0.02 c/ 0.95±0.02 w-y d 3.04±0.06 a 2.09±0.045 a 0.34±0.01 d/ 0.40±0.02 c/ ebrahimi control 0.46±0.005 w-y 0.39±0.005 t-v 0.68±0.02 u-z 1.26±0.03 k-q a 0.48±0.005 v-y 0.41±0.004 s-v 0.77±0.02 o-v 1.37±0.02 h-l b 0.53±0.007 t-y 0.45±0.006 q-v 0.82±0.02 l-t 1.48±0.02 d-h c 1.14±0.04 k-l 0.87±0.020 j-l 0.83±0.02 k-s 1.50±0.02 d-h d 2.11±0.03 e 1.62±0.027 c-d 0.60±0.02 z-a/ 1.15±0.01 o-u badami zarand control 0.51±0.007 u-y 0.48±0.005 p-v 0.71±0.02 s-z 1.04±0.01 t-x a 0.53±0.003 t-y 0.49±0.004 p-v 0.74±0.02 q-y 1.16±0.02 n-u b 0.56±0.008 r-y 0.51±0.019 o-v 0.97±0.02 f-j 1.43±0.01 e-j c 0.83±0.02 m-q 0.64±0.027 m-r 1.01±0.01 e-h 1.47±0.01 d-h d 1.73±0.04 f-g 1.23±0.027 g-h 0.70±0.02 t-z 1.05±0.01 s-x fandoghi 48 control 0.60±0.006 q-y 0.42±0.002 r-v 0.82±0.02 l-t 1.19±0.02 n-t a 0.64±0.008 q-y 0.44±0.005 q-v 0.91±0.02 h-n 1.28±0.03 j-p b 0.99±0.01 l-n 0.79±1.21 k-m 0.92±0.02 h-m 1.35±0.10 h-m c 1.43±0.02 h-j 0.93±0.01 j-k 0.90±0.02 h-n 1.30±0.02 i-o d 2.74±0.04 c-d 1.73±0.04 b-c 0.64±0.02 x-z 1.01±0.02 u-x sabs pesteh togh control 0.60±0.007 q-y 0.48±0.005 p-v 0.62±0.09 y-z 1.07±0.02 r-x a 0.64±0.01 q-y 0.51±0.005 o-v 0.88±0.01 i-p 1.28±0.02 j-p b 0.79±0.04 n-s 0.57±0.02 n-u 1.01±0.02 e-h 1.44±0.02 d-i c 1.55±0.03 g-i 1.02±0.05 i-j 0.67±0.009 v-z 1.19±0.01 n-t d 2.81±0.07 b-c 1.83±0.06 b 0.44±0.01 c/ 0.83±0.01 y-z ahmad aghaee control 0.62±0.006 q-y 0.55±0.004 n-v 0.95±0.02 g-k 1.37±0.02 h-l a 0.65±0.006 q-y 0.58±0.005 n-t 1.08±0.02 d-f 1.54±0.03 c-g b 0.81±0.02 n-p 0.63±0.02 m-s 1.39±0.02 a 1.65±0.03 b-c c 1.64±0.02 f-h 1.17±0.02 h-i 1.35±0.01 a 1.50±0.05 d-h d 2.60±0.03 c-d 1.83±0.04 b 0.64±0.01 w-z 1.17±0.02 n-t rezaie zodres control 0.55±1.41 s-y 0.42±0.005 r-v 0.82±0.02 l-t 1.22±0.02 l-r a 0.60±0.008 q-y 0.45±0.005 q-v 1.08±0.02 d-f 1.41±0.02 f-k b 0.93±0.02 l-o 0.68±0.03 l-p 0.85±0.01 j-q 1.21±0.02 m-r c 1.74±0.03 f-g 1.31±0.02 f-h 0.62±0.03 y-z 0.98±0.03 v-x d 2.99±0.05 a-b 2.05±0.03 a 0.39±0.02 d/ 0.64±0.02 a/ shahpasand control 0.43±0.004 y 0.35±0.005 u-v 0.97±0.02 f-j 1.40±0.03 g-k a 0.45±0.003 x-y 0.36±0.004 t-v 1.05±0.01 d-g 1.48±0.02 d-h b 0.48±0.006 v-y 0.38±0.006 t-v 1.23±0.01 b-c 1.68±0.03 a-c c 0.91±0.02 l-p 0.65±0.02 m-q 1.24±0.02 b 1.72±0.03 a-b d 1.76±0.02 f-g 1.28±0.07 g-h 0.90±0.03 h-n 1.37±0.03 h-l 262 adv. hort. sci., 2018 32(2): 249-264 munns and tester, 2008). based on the results of this study, fv/fm ratio was 0.83±1 in the leaves of the control plants indicating the existence of ideal and non-stressed environmental conditions for the growth of all cultivars throughout the experimental period. in many plant species, when fv/fm ratio is about 0.83, it means that stress hasn’t been introduced to the plant and, lower levels indicate stress condition in plants (maxwell and johnson, 2000). regarding changes in fv/fm values the stress intensity in rezaie zood res and mousa abadi cultivars were more severe than other cultivars (0.59±0.015 and 0.61±0.09, respectively). on the contrary, gazvini and akbari cultivars were less damaged (0.76±0.003 and 0.75±0.007, respectively). these results are consistent with the results of (herda et al., 1999; starck et al., 2000; deell and toivonen, 2003; kodad et al., 2010). it has been reported that salinity stress is one of the most important environmental factors limiting photosynthesis. symptoms of salinity stress are expressed at both stomatal and non-stomatal levels. at stomatal level, the plant closes its stomata to prevent injuries (maxwell and johnson, 2000, ranjbarfordoei et al., 2006). as a result, net photosynthesis is unavoidably reduced due to a decrease in co2 availability, which potentially damages the photosynthetic apparatus (lawlor and cornic, 2002). most of the decrease in photon flux energy used for photochemistry can be explained as an increase in non-photochemical dissipation of excitation energy (lawlor and cornic, 2002). the results of this research indicated that under salinity stress amount of chlorophyll b was reduced more than amount of chlorophyll a. these results are consistent with the results of dejampour et al. (2012). these researchers investigated the effect of nacl on the amount of chlorophyll a, b and total chlorophyll in some of the prunus genus, and they reported that amount of chlorophyll b and total chlorophyll significantly decreased under salinity stress. however, reduction in amount of chlorophyll a in these plants was not significant. also, total chlorophyll content was decreased significantly in all studied cultivars with increasing salinity that are consistent with the results of karimi et al. (2009 and 2011). researcher reported that salinity stress leads to reduction chlorophyll content and photosynthesis capacity in plants which are the major reasons of decreases growth and yield in plants (levitt, 1980; munns, 2002; munns and tester, 2008). the results showed that content of relative humidity were decreased significantly as the salinity increased. the highest reduction in relative humidity content was observed in leaves mousa abadi, rezaie zood res and sabz pesteh togh cultivars under salinity level of 19.8 ds/m. the results are consistent with the data reported by shibli et al. (2000) and massai et al. (2004). salinity, through the gradual accumulation of sodium ions, reduces the relative water content and osmotic potential of the leaf in full turgor state. relative ion leakage percentage and cell membrane injury percentage in all studied cultivars were increased by increasing salinity concentration. the highest relative ion leakage percentage and cell membrane injury percentage were observed in mousa abadi cultivar under treatment 19.8 ds/m. these results are consistent with the results of other studies. it has been reported that using a relative ionic leak test is one way to find out the extent to which cell membranes are damaged. recording the relative ion leakage rate allow for tissue damage estimation. this method was used for the first time by dexter et al. (1930 and 1932) to investigate the resistance to cold in plants and, over time, was used to measure cell membrane damage in relation to other environmental stresses, including salinity stress (chen et al., 1999). with increasing salinity concentration in irrigation water, the sodium concentration in the leaves and roots of total cultivars studied increased. the highest sodium concentration in leaves and roots were observed in salinity level 19.8 ds/m and in mousa abadi and rezaie zood res cultivars which had the highest percentage of leaves necrosis and loss, and at the end of the experiment, only 52.47±4.98% and 53.48±4.82% of leaves were greens. in researches on various plants under salt stress, it has been reported that the loss of water availability, toxicity of na+ and ion imbalance leads to growth limitation in plants (mahajan and tuteja, 2005; szczerba et al., 2009). it is repeatedly reported that k+ deficiency and na+ toxicity are major restrictors of crop production worldwide (mahajan and tuteja, 2005; szczerba et al., 2008, 2009). the results indicated that the type of cultivar is effective in potassium absorption and its transmission to the aerial part. in this research, ghazvini and akbari cultivars with increasing the amount of potassium in its leaves and roots could reduce the negative and destructive effects of sodium better than other cultivars. potassium plays an important role in vital metabolites in salinity stress conditions, so that the k+ can counteract na+ stresses, thus the potential of plants to tolerate salinity is strongly dependent on their potassium nutrition 263 (aleman et al., 2011; nieves et al., 2016). generally, the results of this study showed that by applying salinity stress and increasing its concentration, growth indices including branch height, branch diameter, number of total leaves, percentage of green leaves, fresh and dry weight of leaves, shoots and roots, relative humidity content, chlorophyll a, chlorophyll b and total chlorophyll content, have been reduced in the all cultivars studied. but the percentage of necrotic leaves, percentage of downfall leaves, relative ionic percentage and cell membrane injury percentage were increased. however, the reduction and increase of measured traits were significantly different among studied cultivars. the results also showed that salinity stress affected the young trees through increasing the amount of minimum fluorescence (f0) and decreasing the maximum fluorescence (fm) and reducing variable fluorescence (fv) as well as fv/fm ratio from 0.83±1 in the control p l a n t s t o 0 . 5 9 ± 0 . 0 1 5 i n r e z a i e z o o d r e s a n d 0.61±0.009 in mousa abadi cultivar. based on the results mentioned above, reducing fv/fm ratio was symptoms of the damaging stress in plants. the results of method chlorophyll fluorescence in this research are consistent with the results of morphological and physiological traits and therefore, it can be said that chlorophyll fluorescence technique (fv/fm indicator) is a rapid, sensitive and non-destructive method to check the intensity of stress that induced to plants. overall, the result showed that type of cultivar and level of salinity was affected on concentration of na+ and k+ in leaves and roots. ghazvini cultivar was recognized as the most tolerant cultivar to salinity. this cultivar could tolerate salinity 14.75 ds/m. after this cultivar, akbari, badami zarand and shahpasand cultivars had more tolerance to salinity, respectively. in contrast, rezaie zood res and mousa abadi cultivars were recognized as the most sensitive cultivars to salinity stress. after these cultivars, khanjari, jandaghi and fndoghi 48 cultivars had more sensitive to salinity. references alemán f., nieves-cordones m., martínez v., rubio f., 2011 root k+ acquisition in plants: the arabidopsis thaliana model. plant cell physiol., 52(9): 1603-1612. arnon d.i., 1949 copper enzymes in isolated chloroplast polyphenol oxidase in beta 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h., 2015 differential responses of antioxidant enzymes in salt-stressed almond tree grown under sun and shade conditions. j. plant sci. res., 2(1): 1-10. adv. hort. sci., 2018 32(2): 249-264 impaginato 495 adv. hort. sci., 2018 32(4): 495-501 doi: 10.13128/ahs-21934 onion crop response to different irrigation and n-fertilizer levels in dry mediterranean region i. mubarak (*), a. hamdan department of agriculture, atomic energy commission of syria, p.o. box 6091, damascus, syria. key words: bulb shape index, bulb yield, deficit irrigation, water productivity. abstract: due to the water scarcity in dry mediterranean condition, determination of water and nitrogen (n) fertilizer needs is a major challenge for crop production and environment protection. pot experiments under open field conditions were conducted for two consecutive years (2016 and 2017) to assess the effects of various levels of n-fertilizer and irrigation on onion crop, following a 4×3 factorial experiment arranged in a randomized block design with four nfertilizer rates (0, 40, 80, and 120 kg n ha-1), and three irrigation levels (100, 80, and 60% of the seasonal water use), with three replications. results indicated that the initial soil n-content (about 60 kg n ha-1) was sufficient to meet crop nitrogen requirements. however, results indicated that onion crop was sensitive to water stress, so that the highest total bulb yield (by, 19.1 t ha-1), dry matter in bulbs (dm, 2.97 t ha-1), and water productivity (wp, 1.9 kg m-3) were found under full irrigation compared to the deficit conditions. by, dm, and wp were predicted to be increased linearly with increasing levels of irrigation. the developed equations could be used for predicting onion crop yields under similar agro-pedo-climatic context, and as a tool for rational management of limited irrigation water. 1. introduction onion (allium cepa l.) is one of the most important horticultural crops worldwide. many studies have been carried out regarding the water and nitrogen fertilizer requirements of onion crop and the effects of deficit irrigation on yield and yield components (abdissa et al., 2011; igbadun et al., 2012; patel and rajput, 2013; tsegaye et al., 2016). a nitrogen fertilizer level of less than 100 kg ha-1 was found to be sufficient for onion crop production as in abdissa et al. (2011) and tsegaye et al. (2016). russo (2008) reported that nitrogen fertilizer had no significant effect on onion yield. moreover, the onion crop was found to be more moderately responsive to water deficit during the total growing season; and it is better to partition the water stress throughout the growing season (regulated deficit irrigation, rdi) rather than creating a stress during the (*) corresponding author: ascientific4@aec.org.sy citation: mubarak i., hamdan a., 2018 onion crop response to different irrigation and n-fertilizer levels in dry mediterranean region. adv. hort. sci., 32(4): 495-501 copyright: © 2018 mubarak i., hamdan a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 26 october 2017 accepted for publication 28 may 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(4): 495-501 496 critical stages of crop growth (kirda, 2000; kadayifci et al., 2005; patel and rajput, 2013). the deficit irrigation given at different levels (up to 40%) was found to be economically recommended. this wide range in allowable deficit levels could be due to the various agro-pedo-climatic context of their studied regions. in other word, onion grown in different soil and crop management factors responded differently to the application of both deficit irrigation and n-fertilizer. therefore, there is a continuous need to select both optimum n-fertilizer rate and irrigation level for onion crop in ever changing agro-pedo-climatic conditions. in dry areas of the mediterranean region, scarcity of water is the most limiting factor for onion crop production, due to the lack of rainfall over the production period between april and august (ragab and prudhomme, 2002; turner, 2004). the onion crop is grown in arid and semi-arid area in that region, with many cultivars. in the eastern mediterranean, as in syria, the ovalto elongated-shape onion, referred to as ‘selmouni red’, is the famous variety used for its long-day storage capacity. growers have targeted the larger bulb size and the higher marketable yield. they imagined the greater yields would require increased n fertilizer and water amount. high nitrogen fertilizer and irrigation water amounts may allow nitrate and other components to be most likely deeply percolated. so, research findings are an urgent need to determine water and n-fertilizer requirements for the onion crop in the production areas. in this context, the study reported herein has been designed to predict onion crop response to various levels of regulated deficit irrigation and n-fertilizer rates in dry mediterranean areas. results may contribute to introduce practical alternatives that would sustain onion productivity while using less water and fertilizer in the context of water scarcity and environmental protection. 2. materials and methods pot experiments under open field conditions were c a r r i e d o u t a t t h e d e i r a l h a j a r a g r i c u l t u r a l experiment station, damascus, syria (33°20′ n, 36°26′ e, altitude 600 m), for two consecutive growing seasons 2016 and 2017. table 1 shows some climatic data for the studied site during the course of these experiments. no rainfall was recorded during both growing seasons. the chemical and physical soil properties are: ph 8.0; ece 0.58 ds m-1; organic matter 1.19%; available p 6 ppm; total n 0.06%; no3 51.8 ppm; nh4 + 39.2 ppm. particle-size distribution analysis showed that the soil contains on average 27.8% sand, 42.7% silt and 29.5% clay, and therefore classified as a clay loam. average volumetric soil water contents at field capacity (fc) and permanent wilting point (pwp) are 0.36 and 0.18 m3 m-3, respectively. each pot was of diameter and depth of 25×30 cm, and contained 8 kg of natural soil from the field. three small bulb sets (also called as bulbils or bulblets) of onion (allium cepa l. ‘selmouni red’) were grown in each pot. after germination, plants were thinned to two bulb sets per pot, making a plant density of about 400000 plants ha-1. the pots were set outdoors under natural climatic conditions. the experiment was started on the planting day (march 31st and april 2nd for the 2016 and 2017 seasons, respectively) with the soil water content (swc) of all pots at field capacity (measured by pot’s weight). the experiment was laid out following a 4×3 factorial experiment arranged in a randomized complete block design (rcb design) with four n-fertilizer rates (n0, n40, n80, and n120), and three irrigation levels (fi, di80, and di60), with three replications, making a total of 36 pots. the n-fertilizer rates composed of n0, n40, n80, and n120 with 0, 40, 80, and 120 kg n ha-1 added to the soil, respectively. the three distinct irrigation treatments were: fi treatment (full irrigation) in which plants received 100% of accumulated crop evapotranspiration (100% of etc) and the root zone was replenished to field capacity; di80 and di60 treatments (regulated deficit irrigation) were irrigated at the same frequency as fi treatment but with season variable apr. may jun. jul. aug. 2016 tmin (°c) 11.6 14.9 18.7 19.9 21.0 tmax (°c) 29.2 30.5 36.6 38.1 37.8 taverage (°c) 21.2 23.6 30.6 28.9 29.5 rh (%) 67.0 58.0 69.0 64.0 65.0 precipitation (mm) 0.0 0.0 0.0 0.0 0.0 2017 tmin (°c) 9.7 14.4 17.3 20.6 20.0 tmax (°c) 26.2 31.6 35.8 40.6 38.5 taverage (°c) 19.2 24.9 28.4 31.1 28.9 rh (%) 63.1 57.9 56.3 55.6 59.3 precipitation (mm) 0.0 0.0 0.0 0.0 0.0 tmin= minimum temperature, tmax= maximum temperature, taverage= average temperature, rh= relative air humidity. table 1 some climatic data of the experimental site during both growing seasons mubarak and hamdan irrigation and n-fertilizer levels for onion crop 497 water amounts equal to 80 and 60% of the accumulated etc, respectively. in other words, the three watering treatments received at each irrigation event 1.0, 0.8 and 0.6 times the amount of soil water depleted under fi conditions, respectively. irrigation w a s a p p l i e d 3 t i m e s p e r w e e k . t h e p o t s w e r e weighed before and after each irrigation event. the water amounts were regulated by weight (eq. 1). the depleted water amount (crop evapotranspiration, etc) (mm) between two successive irrigations was calculated as: etc = w1 w2 [1] pw x s where w 1 was the weight of the pot (kg) after irrigation; w 2 was the weight of the pot (kg) just before the next irrigation; ρ w is the water density (g cm-3); and s is the soil surface area in the pot (m2). the daily crop evapotranspiration (mm day-1) was estimated by dividing the crop evapotranspiration estimated from eq. (1) by the number of days between two successive irrigation events. the seasonal crop evapotranspiration, i.e., the total crop water use during the growing season, was the summation of the daily etc. full doses of phosphorous and potassium were a p p l i e d a s b a s a l d o s e a t t h e t i m e o f p l a n t i n g . nitrogen was divided into two equal doses (according to the studied n-fertilizer rate) and applied with the irrigation water during early vegetative growth stage. irrigation was stopped at the end of july when over 50% dropping of leaf-tops was observed as signs of maturity. the onions were lifted to field cure about two weeks after (up to mid-august). after the leaves were completely dried, they were cut leaving about 2.0 cm above the bulb. the length, diameter, and weight of both matured onion bulbs from each pot were measured. the total bulb yield (by, t ha-1) was estimated. the dry matter in bulbs (dm, t ha-1) was also estimated by drying bulbs at 50°c to a constant weight. water productivity (wp, kg m-3) was computed by dividing the total bulb yield by the seasonal evapotranspiration. bulb shape index (sh i) was also calculated as the relationship between bulb length and diameter. no multi-centred bulbs were observed at harvest even under deficit irrigation. with two factors (n-fertilizer rate and irrigation level), the two-way analysis of variance (anova) was conducted using the dsaastat add-in version 2011 (onofri, 2007). a combined analysis of data over both years was performed to verify if n-fertilizer rate and irrigation level may have a significant and stable effect over year. mean comparison was made only for data after combined analysis using duncan’s multiple range test (dmrt) at the 1% level of significance. trend analysis (regression analysis) was also used to examine the relationship between measured variables and the quantitative factors showing a significant effect. the trend analysis was done based on the method of orthogonal polynominals as described by gomez and gomez (1984). 3. results and discussion the effects of years, n-fertilizer rates, and irrigation levels on the measured variables of onion crop (by, dm, wp, and sh i) were summarized in table 2. since no significant interaction year × treatments was observed, data are shown as the averages of the two years (table 3 and figs. 1-3). total bulb yield the combined analysis of data over years indicated that only the main effect of irrigation was significant, and highly influenced by (p<0.01) (table 2 and 3). no significant change in by was recorded with the addition of n fertilizer (table 3), indicating that the initial soil nitrogen content (about 60 kg n ha-1) was sufficient to meet the crop nitrogen requirements, and no extra n-application was needed. this is in agreement with the results of abdissa et al. (2011) who found that a rate of 69 kg n ha-1 was sufficient * = significant at 5% level, ** = significant at 1% level, ns = nonsignificant at 5% level. b = the degree of freedom of reps. within year is not adequate for valid test of significance (gomez and gomez, 1984). df = degree of freedom, by = total bulb yield, dm = dry matter in bulbs, wp = crop water productivity, and sh i= shape index. table 2 analysis of variance of the combined data of measured variables as affected by years, n-fertilizer rates, and irrigation levels (f-test values) source of variation df by dm wp sh i year (y) 1 b b b b reps. within year 4 irrigation level (i) 2 213.4 ** 75.44 ** 41.35 * 12.30 ns n-fertilizer rate (n) 3 <1 <1 <1 1.90 ns y x n 3 2.20 ns 1.24 ns 1.65 ns 1.30 ns y x i 2 <1 <1 1.03 ns <1 n x i 6 <1 1.82 ns <1 <1 y x n x i 6 2.89 ns 1.47 ns 2.58 ns 2.61 ns residual (pooled error) 44 total 71 cv (%) 18.6 22.8 18.9 14.9 adv. hort. sci., 2018 32(4): 495-501 498 for onion crop production. tsegaye et al. (2016) reported that a n level of 100 kg ha-1 was economically recommended for onion in southern ethiopia. according to russo (2008), nitrogen fertilizer did not affect onion yield. the effect of irrigation level resulted in the highest by (19.1 t ha-1) in fi treatment, while under di80 conditions, total bulb yield decreased by 36.4%, reaching 63.0% of decrease (7.1 t ha-1) under di60 c o n d i t i o n s ( t a b l e 3 ) . s i m i l a r r e s u l t s w e r e a l s o obtained by kumar et al. (2007) and bekele and tilahun (2007). trend analysis indicated that the relationship between total bulb yield and irrigation level was linear within the range of irrigation levels tested (r2=0.991 with p<0.01) (fig. 1). a significant linear decrease in the total bulb yield was predicted with increasing water deficit. in other word, by increased with increasing irrigation level (fig. 1). this is in agreement with other published findings (kadayifci et al., 2005; kumar et al., 2007; bekele and tilahun, 2007; nagaz et al., 2012). for instance, nagaz et al. (2012) observed that applying 60% of crop evapotranspiration (etc) caused significant decreases in fresh yield, dry matter, bulbs per hectare and bulb weight, compared to full irrigation (100% etc). this result confirmed the sensitivity of the onion crop to water deficit during the total growing season. on the contrary, other studies showed that onion crop is responsive to deficit conditions as compared with full irrigation. for example, tsegaye et al. (2016) and igbadun et al. (2012) found that deficit irrigation given at 75% of etc was economically recommended. also, nagaz et al. (2012) found no significant differences between regulated deficit irrigation at 80% etc and full irrigation (100% etc). these differences in onion crop response to deficit irrigation levels could be due to the various agro-pedo-climatic conditions. dry matter yield in onion bulbs (dm) also dm was found to be highly affected only by the main effects of irrigation levels (table 2 and 3). the mean values of dm were 2.97, 2.16, and 1.27 t ha-1 under fi, di80 and di60, respectively. that is to say, significant decreases of 27.2 and 57.1% in dm could be attained when onion crop was under di80, and di60, respectively, compared with fi (p<0.01) (table 3). trend analysis designated that the response of dm to various irrigation levels followed a linear relationship (r2=0.999 with p<0.01). dry matter yield increased with increasing levels of irrigation as can be seen in figure 2. thus, dry matter yield could be maximized whatever the n-fertilizer rate used in this study, when full irrigation was applied. several studied reported similar results, showing that dry matter yield was maximized under full irrigation rather than under deficit conditions (e.g., nagaz et al., 2012). concerning fertilizer rates, as mentioned above, anova did not detecte any significant change in dm yield with the addition of nitrogen fertilizer (table 3). this is in agreement with the findings of russo fig. 1 response of onion total bulb yield (by) to irrigation levels. a regression equation is fitted and coefficient of determination (r2) is given. ** = significant at 1% level. each experimental point represents the data of by averaged over both years and all levels of n-fertilizer, at the specific irrigation level. in each column and for each studied factor, means followed by different letters are significantly different according to the dmr test. table 3 effect of n-fertilization rate and irrigation level on total bulb yield (by), dry matter in bulbs (dm), crop water productivity (wp), and shape index (sh i) in onion ‘selmouni red’ (data are the averages of the two years of experimentation) studied factor by (t ha-1) dm (t ha-1) wp (kg m-3) sh i (cm cm-1) n-fertilization rate n0 (0 kg n ha-1) 13.47 a 2.26 a 1.59 a 1.78 a n40 (40 kg n ha-1) 12.26 a 2.14 a 1.47 a 1.86 a n80 (80 kg n ha-1) 12.73 a 2.06 a 1.52 a 1.86 a n120 (120 kg n ha-1) 12.61 a 2.08 a 1.47 a 1.65 a irrigation level full irrigation (100% etc) 19.08 a 2.97 a 1.90 a 1.72 a deficit irrigation at 80%of etc 12.14 b 2.16 b 1.50 b 1.67 a deficit irrigation at 60%of etc 7.08 c 1.27 c 1.14 c 1.98 a mubarak and hamdan irrigation and n-fertilizer levels for onion crop 499 (2008). this result newly indicated that the initial soil nitrogen content was sufficient to meet the n-fertilizer needs of onion crop. irrigation water applied and water productivity with no rainfall received during both growing seasons (table 1), large water amounts were applied to meet the high crop water demand. the irrigation water applied to fi, di80, and di60 were, 1014, 822, and 634 in the 1st season, and 1039, 849, and 566 mm in the 2nd one, respectively. seasonal crop evapotranspiration (etc), as calculated using eq. (1), during the 2016 season was 993, 803, and 615 mm, and during the 2017 season was 1013, 823, and 630 mm, for fi, di80, and di60, respectively. as it can be seen, the seasonal etc values were very close to the irrigation water amounts even in di80 and di60 treatments. therefore, wp can be considered also a good estimate of irrigation water use efficiency (iwue). the combined analysis over years detected that wp was highly significantly influenced by the irrigation levels. it was unchanged with the addition of n fertilizer (table 2 and 3). the mean value of wp under fi (1.90 kg m-3) was significantly higher than both deficit irrigation levels, i.e., di80 (1.50 kg m-3) and di60 (1.14 kg m-3) (table 3). trend analysis indicated that the relationship between wp and irrigation level was linear with values of r2 of 0.998 at the 1% level (fig. 3). results indicated that wp was not ameliorated under deficit irrigation. this could be due to high onion crop sensitiveness to the water deficit during the total growing season. thus, the water savings under deficit irrigation could not balance the huge decrease in the total bulb yield under the dry conditions. this result is in agreement with similar results obtained by kadayifci et al. (2005), who found that high water use efficiencies were o b s e r v e d w i t h i n c r e a s i n g l e v e l s o f i r r i g a t i o n . contrariwise, this result was in disagreement with the findings of other researchers (fereres and s o ri a n o , 2 0 0 7 ; ku m a r et a l . , 2 0 0 7 ; b ekel e a n d tilahun, 2007; patel and rajput, 2013; tsegaye et al., 2016). for example, patel and rajput (2013) reported that with 40% deficit irrigation throughout the growing season, water productivity can be significantly improved with 272-mm water saving which may be used to irrigate additional cropped area (half a hectare). kumar et al. (2007) found that irrigation water use efficiency and water productivity both were highest under 80% of etc and then declined with the increase in irrigation with microsprinkler irrigation system. igbadun et al. (2012) showed that higher water productivity in terms of water supplied could be obtained by irrigating onion crop at 50 and 75% of etc. these different results could be due to fig. 2 response of onion dry matter yield in bulbs (dm) to irrigation levels. a regression equation is fitted and coefficient of determination (r2) is given. ** = significant at 1% level. each experimental point represents the data of dm averaged over both years and all levels of n-fertilizer, at the specific irrigation level. fig. 3 response of onion crop water productivity (wp) to irrigation levels. a regression equation is fitted and coefficient of determination (r2) is given. ** = significant at 1% level. each experimental point represents the data of wp averaged over both years and all levels of n-fertilizer, at the specific irrigation level. 500 adv. hort. sci., 2018 32(4): 495-501 the differences in tested onion cultivars, soil type, and climatic conditions. they indicated that onion crop produced in different agricultural managements responded differently to water stress. bulb shape index no significant effects of both tested factors were observed on this variable (table 2 and 3). as above mentioned, the ovalto elongated-shape onion, referred to as ‘selmouni red’, was tested. its shape index generally varies from 1.5 to 3.0, according to several environmental conditions including planting dates, plant density, planting depth, and soil water availability. the lower the shape index, the better the bulb shape for marketing purposes (appearance and ease of packaging). the mean values of sh i, found in this study, were 1.72, 1.67, and 1.98 under fi, di80, and di60, respectively (table 3). the average sh i under severe water stress (di60) was 14.7% larger than that under non-water stress conditions (fi). in o t h e r w o r d , s h a r p d e f i c i t i r r i g a t i o n t e n d e d t o increase the bulb length, although this increase not significant, compared to its diameter. thus, the recommended agricultural management to produce better shape index of onion bulbs, is to irrigate onion plants using irrigation level of 100 or 80% of etc. 4. conclusions the following conclusions can be drawn from the results obtained in onion subjected to different nfertlizer rates and irrigation levels in the studied agro-pedo-climatic context: no additional n application over the initial soil n c o n t e n t ( a b o u t 6 0 k g n h a 1) w a s f o u n d t o b e required for the tested onion cultivar. the highest total bulb yield, dry matter, and water productivity were recorded under full irrigation compared to the deficit conditions, indicating that onion crop was sensitive to water stress. total bulb yield, dry matter, and water productivity were predicted to be increased linearly with the increment in irrigation water amount. the developed equations could be useful for predicting onion crop yields under similar agro-pedo-climatic context and for rational management of limited irrigation water. with huge crop water requirements, further studies should focus on how to improve the regulated deficit irrigation practices in order to address water shortage and sustainable crop production in dry areas of the mediterranean region. acknowledgements the authors would like to thank the aec of syria for financial support. references abdissa y., tekalign t., pant l.m., 2011 growth, bulb yield and quality of onion (allium cepa l.) as influenced by nitrogen and phosphorus fertilization on vertisol. i. growth attribute biomass production and bulb yield. afr. j. agric. res., 6(14): 3252-3258. bekele s., tilahun k., 2007 regulated deficit irrigation scheduling of onion in a semiarid region of ethiopia. agric. water manag., 89: 148-152. fereres e., soriano m.a., 2007 deficit irrigation for reducing agricultural water use. j. exp. bot., 58: 147159. gomez k.a., gomez a.a., 1984 statistical procedures for agricultural research. second edition. wiley, new york, ny, usa, pp. 680. igbadun h.e., ramalan a.a., oiganji e., 2012 effects of regulated irrigation deficit and mulch on yield, water use and crop water productivity of onion in samaru, nigeria. agric. water manag., 109: 162-169. kadayifci a., tuylu g.i., ucar y., cakmak b., 2005 crop water use of onion (allium cepa l.) in turkey. agric. water manag., 72(1): 59-68. kirda c., 2000 deficit irrigation scheduling based on plant growth stages showing water stress tolerance. deficit irrigation practices, fao water reports, rome, italy, 22: 3-10. kumar s., imtiyaz m., kumar a., singh r., 2007 response of onion (allium cepa l.) to different levels of irrigation water. agric. water manag., 89: 161-166. nagaz k., masmoudi m.m., ben mechlia n., 2012 yield response of drip-irrigated onion under full and deficit irrigation with saline water in arid regions of tunisia. isrn agronomy, 2012: 1-8. onofri a., 2007 routine statistical analyses of field experiments by using an excel extension. proceedings 6th national conference italian biometric society “la statistica nelle scienze dellavita e dell’ambiente”, pisa, italy, june 20-22, pp. 93-96. patel n., rajput t.b.s., 2013 effect of deficit irrigation on crop growth, yield and quality of onion in subsurface drip irrigation. inter. j. plant prod., 7(3): 417-436. ragab r., prudhomme c., 2002 climate change and water resources management in arid and semi arid regions: prospective and challenges for the 21st century. biosyst. eng., 81: 3-34. russo v.m., 2008 plant density and nitrogen fertilizer rate on yield and nutrient content of onion developed from greenhouse-grown transplants. hortscience, 43(6): 1759-1764. 501 mubarak and hamdan irrigation and n-fertilizer levels for onion crop tsegaye b., bizuayehu t., woldemichae a., mohammed a., 2016 yield and yield components of onion (allium cepa l.) as affected by irrigation scheduling and nitrogen fertilization at hawassa area districts in southern ethiopia. j. agric. sci. food technol., 2(2): 15-20. turner n.c., 2004 sustainable production of crops and pastures under drought in a mediterranean environment. ann. appl. biol., 144: 139-147. impaginato 511 adv. hort. sci., 2018 32(4): 511-516 doi: 10.13128/ahs-20537 effects of cold stratification and chemical treatments on seed germination in four hazelnut cultivars z. bakhtshahi-dizgahi 1, m. alizadeh 1 (*), e. seifi 1, d. javadi 2, a. hosseinpour 1 1 gorgan university of agricultural sciences and natural resources, faculty of plant production, department of horticulture, gorgan, iran. 2 guilan research center of agriculture and natural resources, rasht, iran. key words: chemical treatment, cold stratification, corylus avellana l., filbert, germination. abstract: propagation of european hazelnut by seed is influenced by some seed treatments. in this investigation, effect of stratification period and some chemicals on seeds of four hazelnut cultivars were studied. ga3 and four months of stratification, each individually resulted in the highest germination percentage at 82.73% and 83.75%, respectively. there were significant differences between cultivars and treatments in terms of germination percentage and rate. the highest germination percentage and rate were observed in the local cultivar gerd under ga3 treatment at 100 mg/l and also after four months of stratification. 1. introduction propagation by seeds is a conventional method to produce new plants. this is one of the most recognized efficient methods which is widely applied for different plant species. although sexual reproduction do not result in true to type plants, in breeding programs it is inevitable to apply it to grow hybrid seedlings. for example, in hazelnut, interspecific hybridization is necessary to transfer superior characters from wild species to the commercial european hazelnut (corylus avellana l.) (erdogan and mehlenbacher, 2000). seed germination is a main step in plant life cycle, and is influenced by various biotic and abiotic factors (yuan and wysocka-diller, 2006). it is essential to investigate different aspects in sexual propagation for all plant species. however, there are some common difficulties in using such approach to propagate many plants such as hazelnut, including seed dormancy and inconsistent seed germination which make some problems and retard improvement programs and sometimes end in hybrids loss. therefore, studying beneficial treatments to remove dormancy and subsequent uniform seed germination is considered of great importance (*) corresponding author: mahdializadeh@gau.ac.ir citation: bakhtshahi-dizgahi z., alizadeh m., seifi e., javadi d., hosseinpour a., 2018 effects of cold stratification and chemical treatments on seed germination in four hazelnut cultivars. adv. hort. sci., 32(4): 511-516 copyright: © 2018 bakhtshahi-dizgahi z., alizadeh m., seifi e., javadi d., hosseinpour a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 14 april 2017 accepted for publication 22 july 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(4): 511-516 512 (wang and berjak, 2000; copeland and mcdonald, 2001). generally, germination process is controlled through a balance between inducing and inhibiting factors. provided that concentration of inducers is higher than inhibitors, seed dormancy will predominate. some stimulants such as temperature and light are necessary to lower the effect of inhibitors in seed. in such case, an inducing factor such as gibberellic acid (ga3) could have cumulative influence so that germination process will commence (bradbeer, 1988). in many temperate zone species, dormancy prevents the seeds from germinating (derkx, 2000). a dormant seed would not germinate even in favorable environmental conditions. several approaches have been suggested in literature to overcome this phenomenon, including cold stratification (bewley and black, 1994), and seed treatment by some chemicals such as gi bberel l i c aci d (ga 3), pol yami nes and thiourea (frankland, 1961; çetinbaş and koyuncu, 2006; mello et al., 2009). cold stratification plays a major role as a stimulant to break seed dormancy. also its effect is accelerated in combination with chemicals or physical removing of seed coat (bewley and black, 1994). this technique is usually performed at temperatures between 0 and 10°c; which vary depending on different species. however, the best reported temperature for this kind of seed treatment is 5°c (bewley and black, 1994). aygun et al. (2009) suggested that hazelnut seeds need two to six months of pre-germination cold stratification. dormancy in hazelnut seeds is diminished through cytological, hormonal and biochemical changes during cold stratification period. for example, mobilization of phytic acid and phosphate was observed during this treatment (vasilios et al., 2005). ga3 treatments could remove various seed physiological dormancies and induce germination of dormant seeds (frankland, 1961). aygun et al. (2009) showed that in hazelnut seeds treated by ga3 at concentrations from 0 to 200 mg/l, the highest seed germination percentage was obtained by 100 mg/l ga3. the main polyamines existing in plant cells are putrescine, spermine and spermidine (davies, 2004). based on some evidences, polyamines have a role in seed dormancy process. sinska and lewandowska (1991) found that putrescine, spermine and spermidine decreased in apple seeds during cold stratification. in fact, putrescine and spermidine had inducing effect and spermine had inhibiting effect on apple seed germination. although thiourea is not applied commonly in seed germination experiments, it is able to enhance germination of some kinds of seeds (gul and weber, 1998; çetinbaş and koyuncu, 2006). stidham et al. (1980) showed that thiourea had an inducing effect on germination of 18 shrub species. according to cetinbaş and koyuncu (2006), this property of thiourea is attributed to its cytokinin-related effect in removing inhibitors. this investigation aimed to study the effects of some treatments including some chemicals and cold stratification on percentage and rate of seed germination in four hazelnut cultivars grown in iran. 2. materials and methods seeds of four hazelnut cultivars including a local c u l t i v a r g e r d a n d t h r e e i n t r o d u c e d c u l t i v a r s barcelona, ronde (= ronde du piemont) and segorbe were collected from astara hazelnut research station, in astara, guilan province, iran. defected seeds were discarded and proper seeds were separated to study. treatments treatments applied in this study included ga3 (100 and 200 mg/l), putrescine (0.01 and 0.1 mm), thiourea (1000 and 2000 mg/l) and stratification for two and four months. the control treatment was distilled water. germination test the seeds were soaked for 24 hours in ga3 (100 and 200 mg/l), putrescine (0.01 and 0.1 mm) and thiourea (1000 and 2000 mg/l), and also some seeds were soaked in distilled water. in order to prevent seeds from rotting, the treated seeds were surface sterilized with sodium hypochlorite (10% v/v for 5 min.) and then rinsed by sterilized water. afterwards, the seeds were cultured in plastic pots containing pre-autoclaved sand as the medium (diameter of 2 mm). germination test was conducted in a factorial completely randomized design with three treatments including “no stratification”, “two months of stratification” and “four months of stratification”. emerging radical was considered as the index of germination. in chemical treatments, the number of germinated seeds up to 40 days from the culture date, and in stratification treatments, the number of germinated seeds up to 40 days from outing from refrigerator were recorded. stratification treatments were just performed by keeping them in refrigerator (5°c) for two to four months. cultured seeds were inspected regularly and in case of moisture decrease of media, bakhtshahi-dizgahi z. seed germination in four halzenut cultivars 513 autoclaved water was sprayed on them. the measured characteristics were percentage and rate of seed germination. the following formula was used to calculate germination percentage. germination percentage = (number of germinated seeds / total number of seeds) × 100 germination rate is defined as the time to reach 50 percent of germination, which was calculated by the formula below: germination rate = (1/time of reaching 50 percent of germination) for each cultivar, 210 seeds were used. the data were analyzed by analysis of variance (anova) and the software germin-g was applied to measure the target parameters (soltani et al., 2004). 3. results and discussion according to the analysis of variance, there was a significant difference among cultivars and chemical treatments with regard to germination percentage (p < 0.01) (fig. 1). even so, no significant difference was observed among cultivars and chemical treatments regarding germination rate (fig. 2). in addition, effect of stratification on germination rate and percentage was significantly different between the cultivars (p<0.01) (fig. 3 and 4). f or germi n ati on p ercen tage, th e d i fferen ce between mutual effects of “cultivar × chemical treatment” (p<0.05) (fig. 1), “cultivar × stratification” (p<0.05) (fig. 4) and “chemical treatment × stratification” (p<0.01) (fig. 5) was significant. for germination rate, the mutual effect of “cultivar × chemical treatment” showed a significant difference (p<0.05) (fig. 2). of the four hazelnut cultivars, ‘gerd’ had the highest germination percentage (82.73%) at 100 mg/l ga3, which showed 50.76% increase compared to the control (31.97%). although applying both ga3 concentrations was resulted in higher germination percentages, but no difference was observed between these two levels (fig. 1). between stratification and cultivar, the highest germination percentage was exhibited after four fig. 1 effect of chemical treatments and cultivars on seed germination percentage of hazelnut cultivars. fig. 2 effect of chemical treatments and cultivars on seed germination rate of hazelnut cultivars. fig. 3 effect of stratification treatments on seed germination rate of hazelnut cultivars. fig. 4 effect of stratification treatments and cultivar on seed germination percentage of hazelnut cultivars. fig. 5 effect of stratification and chemical treatments on seed germination percentage of hazelnut cultivars. adv. hort. sci., 2018 32(4): 511-516 514 months of stratification in the local cultivar gerd. no difference existed in four months of stratification between two local cultivars and barcelona (fig. 6). figure 5 reveals that between mutual effect of chemicals and stratification treatments, the highest germination percentage occurred through four months of stratification and in the control treatment. nevertheless, in treatments of two months of stratification and no stratification, both levels of ga3 resulted in the highest germination percentage. in all cultivars, the highest germination rate was recorded in ‘gerd’ (fig. 6). four months of stratification had the greatest influence on germination rate (fig. 3). of chemical treatments, the fastest germination was obtained by using the first level of ga3 (100 mg/l) in the cultivar gerd (fig. 2). various studies have demonstrated a direct relationship between cold stratification length and increasing in germination percentage. as can be clearly seen in figures 4 and 5, when stratification period increased, as a result, germination percentage of hazelnut seeds increased in all treatments and all cultivars. similar results were observed by aygun et al. (2009) which suggested that 120 days of cold stratification ended in higher germination percentage i n h a z e l n u t s e e d s i n c o m p a r i s o n w i t h c o n t r o l . furthermore, bradbeer (1988) reported that three months of cold stratification led to a rise in hazelnut seed germination percentage. in other studies, three m o n t h s o f s t r a t i f i c a t i o n w i t h o u t a n y w a r m i n g i m p r o v e d g e r m i n a t i o n p e r c e n t a g e i n s e e d s o f jasminum fruticans (pipinis et al., 2009). another example for such impact has been reported by chin et al. (1992) in kiwifruit. in addition, there are many other studies that support the influence of applying some chemicals and plant growth regulators on rising germination percentage of seeds, which could be used singly or in combination with chilling. as a matter of fact, these chemicals are considered as substitutes for chilling requirement of seeds, and also can decrease length of chilling period. overall, gibberellins and cytokinins are able to promote seed germination in plants (davies, 2004; miransari and smith, 2014). in contrast, abscisic acid (aba) plays an inhibiting role in seed germination (miransari and smith, 2014). among types of gibberellins, ga3, ga4 and ga7 have the most impact on germination enhancement. however, cytokinins and auxins have so lower effect compared to gibberellins and cytokinins in terms of stimulating germination. in f a c t , t h e e f f e c t o f a l l t h e s e g r o w t h r e g u l a t o r s depends upon other factors such as light, temperature and oxygen; and also there are some mutual effects between them. gibberellins and cytokinins are capable of neutralizing the inhibiting effect of abscisic acid. since all these growth regulators natur a l l y e x i s t i n s e e d i n d i f f e r e n t r a t i o s , s o t h e i r observed effects on seed germination could be interpreted by the state of hormone balance (kucera et al., 2005). gibberellins are able to enhance seeds of different species to germinate through different ways. that is, external use of gibberellins could induce germination in seeds in which lack of germination is due to seed coat (e.g. legumes), or seed dormancy is because of seed embryo (e.g. apple, birch, hazelnut) (davies, 2004; miransari and smith, 2014). besides, in seeds that their germination depends on exposure to light (e.g. arabidopsis, lettuce), ga3 could promote seed germination even in the dark (cao et al., 2005). based on the results obtained in this study, the highest germination percentage and rate were gained through ga3 treatment at 100 mg/l (fig. 1, 2), which corresponded with results reported by aygun et al. (2009) that showed higher germination percentage after treatment by 100 mg/l gibberellin. in addition, there are some reports on gibberellin application on hazelnut seeds in order to enhance germination results (bradbeer and pinfield, 1967; jarvis and wilson, 1977; pinfield and stobart, 2006). in addition to gibberellins, other substances such as thiourea can also overcome seed dormancy. several investigations have demonstrated the effect of thiourea on dormancy breaking and increasing germination percentage of seeds in different plant s p e c i e s ( g u l a n d w e b e r , 1 9 9 8 ; ç e t i n b a ş a n d koyuncu, 2006). also ojha et al. (2010) showed that after using four treatments including gibberellin, potassium nitrate, ascorbic acid and thiourea on seeds of abrus precatorius, despite increasing germination percentage in all treatments, the highest and fig. 6 seed germination rates in four hazelnut cultivars. bakhtshahi-dizgahi z. seed germination in four halzenut cultivars 515 lowest germination percentage were obtained by gibberellin and thiourea treatments, respectively. polyamines are also a group of growth regulators and could improve seed germination in some species (szczotka and lewandowska, 1989; sinska and lewandowska, 1991). furthermore, in this research, putrescine had a significant difference compared to the control, in terms of germination percentage and germination rate of hazelnut seeds (fig. 2). 4. conclusions the results of this study revealed that the hormone ga3 and four months of cold stratification resulted in the highest germination percentage in hazelnut seeds at 82.73% and 83.75%, respectively. the cultivars and chemical treatments had significant effect on seed germination which were the highest in the local cultivar gerd under ga3 treatment at 100 mg/l and also after four months of cold stratification. acknowledgements the authors would like to express their gratitude to gorgan university of agricultural sciences and natural resources for financial and technical support. references aygun a., erdogan v., bozkurt e., 2009 effect of some pretreatments on seed germination of turkish hazel (corylus colurna l.). acta horticulturae, 845: 203-206. bewley j.d., black m., 1994 seeds: physiology of development and germination. 2nd ed. plenum press, new york/london. bradbeer j.w., 1988 seed dormancy and germination. chapman and hall, new york. bradbeer j.w., pinfield n.j., 1967 studies in seed dormancy iii. the effects of gibberellin on dormant seeds of corylus avellana l. new phytol., 66(4): 515-523. cao d.n., hussain a., cheng h., peng j.r., 2005 loss of function of four della genes leads to lightand gibberellin-independent seed germination in arabidopsis. planta, 223(1): 105-113. cetinbaş m., koyuncu f., 2006 improving germination of prunus avium l. seeds by gibberellic acid, potassium nitrate and thiourea. hortic. sci., 33: 119-123. chin k.l., blanche c.a., bachireddy v.r., 1992 gibberellic acid and cold stratification treatments affect kiwi seed germination and root elongation. hortic. sci., 27(6): 689. copeland l.o., mcdonald m.b., 2001 principles of seed science and technology. kluwer academic publishers, norwell, massachusetts, usa, pp. 488. davies p.j., 2004 plant hormones: biosynthesis, signal transduction, action! kluwer academic publishers, dordrecht, the netherlands, pp.750. derkx m.p.m., 2000 pre-treatment at controlled seed moisture content as an effective means to break dormancy in tree seeds, pp. 69-78. in: viemont j.d., and j. crabbe (eds.) dormancy in plants. cab international, wallingford, oxforshire, uk, pp. 400. erdogan v., mehlenbacher s.a., 2000 interspecific hybridization in hazelnut (corylus). j. amer. soc. hort. sci., 125(4): 489-497. frankland b., 1961 effect of gibberellic acid, kinetin and other substances on seed dormancy. nature, 192: 678-679. gul b., weber d.j., 1998 effect of dormancy relieving compounds on the seed germination of non-dormant allenrolfea occidentalis under salinity stress. ann. bot., 82: 555-560. jarvis b.c., wilson d., 1977 gibberellin effects within hazel (corylus avellana l.) seeds during the breaking of dormancy. i. a direct effect of gibberellin on the embryonic axis. new phytol., 78(2): 397-401. kucera b., cohn m.a., leubner-metzger g., 2005 plant hormone interactions during seed dormancy release and germination. seed sci. res., 15: 281-307. mello a.m., streck n.a., blankenship e.e., paparozzi e.t., 2009 gibberellic acid promotes seed germination in penstemon digitalis cv. husker red. hortic. sci., 44(3): 870-873. miransari m., smith d.l., 2014 plant hormones and seed germination. environ. exp. bot., 99: 110-121 ojha s., mondal p., konar j., 2010 effect of promoters and pretreatment with chemicals on seed germination of abrus precatorius l. plant archives, 10(2): 641-645. pinfield n.j., stobart a.k., 1969 gibberellin-stimulated nucleic acid metabolism in the cotyledons and embryonic axes of corylus avellana (l.) seeds. new phytol., 68(4): 993-999. p i p i n i s e . , m i l i o s e . , a s l a n i d o u m . , m a v r o k o r dopoulou o., smiris p., 2009 the effect of stratification on seed germination of jasminum fruticans l. (oleaceae): a contribution to a better insight on the species germination ecology. int. j. botany, 5(2): 181185. sinska i., lewandowska u., 1991 polyamines and ethylene in the removal of embryonal dormancy in apple seeds. physiol. plantarum, 81: 59-64. soltani a., ghorbani m.h., galeshi s., zeinali e., 2004 salinity effects on germinability and vigor of harvested seeds in wheat. seed sci. technol., 32: 583-592. stidham n.d., ahring r.m., powel j., claypool p.l., 1980 chemical scarification, moist prechilling, and 516 adv. hort. sci., 2018 32(4): 511-516 thiourea effects on germination of 18 shrub species. j. range manag., 33(2): 115-118. szczotka z., lewandowska u., 1989 polyamines in dormancy breaking of tree seeds. ann. sci. for., 46: 95-97. vasilios m.e., susan b., james d., 2005 phytic acid mobilization is an early response to chilling of the embryonic axes from dormant oilseed of hazel (corylus avellana). j. exp. bot., 56(412): 537-545. wang b.s.p., berjak p., 2000 beneficial effects of moist chilling on the seeds of black spruce (picea mariana [mill.] b.s.p.). ann. bot., 86: 29-36. yuan k., wysocka-diller j., 2006 phytohormone signalling pathways interact with sugars during seed germination and seedling development. j. exp. bot., 57(12): 3359-3367. impaginato 541 adv. hort. sci., 2018 32(4): 541-548 doi: 10.13128/ahs-22211 yield, quality, antioxidants and mineral nutrients of physalis angulata l. and physalis pubescens l. fruits as affected by genotype under organic management n.a. golubkina 1 (*), h.g. kekina 2, m.r. engalichev 1, m.s. antoshkina 1, s.m. nadezhkin 1, g. caruso 3 1 agrochemical research center, federal scientific center of vegetable production, 143080 moscow region, odintsovo district, vniissok, selectsionnaya 14, russia. 2 medical academy of postgraduate education, 123995 moscow, russia. 3 dipartimento di scienze agrarie, università degli studi di napoli federico ii, via università, 100, 80055 portici (na), italy. key words: antioxidants, mineral nutrients, physalis angulata l., physalis pubescens l., production, sugars, taste. abstract: introduction and selection of unconventional plants with high concentration of biologically active compounds is one of the worthy ways for producing functional food, which is beneficial to human health. research was carried out in northern europe (russia) with the purpose to assess yield, quality and biologically active compounds concentration in physalis angulata and physalis pubescens fruits. p. angulata cultivars konditer and konditer 2 gave the highest yield (11.3 and 11.0 t·ha-1 respectively), due to the highest mean fruit weight (80 and 70 g respectively); p. pubescens variety zolotaya rossip had the worst outcome due to the very small berries (3 g), in spite of their highest number per plant (165). ‘zolotaya rossip’ fruits overall attained higher values of quality indicators compared to all p. angulata cultivars. positive correlations were recorded between dry matter and polyphenols as well as between total sugars and polyphenols. physalis fruits showed to be a good source of antioxidants, k, mg and p for human beings. taste index turned out highly reliable in evaluating fruit quality and it was dependant on dry matter, total sugars, polyphenols and ca. 1. introduction the availability of new functional food has been becoming more and more important for human health regulation and protection against the major diseases of the millennium. physalis fruit is mostly an exotic product, imported from tropical and subtropical regions, particularly from central america, where this genus is characterized by the highest biodiversity (medina-medrano et al., 2015). the popularity of this genus plants has been increasing in several world (*) corresponding author: segolubkina45@gmail.com citation: golubkina n.a., kekina h.g., engalichev m.r., antoshkina m.s., nadezhkin s.m., caruso g., 2018 yield, quality, antioxidants and minearl nutrients of physalis angulata l. and physalis pubescens l. fruits as affected by genotype under organic management. adv. hort. sci., 32(4): 541-548 copyright: © 2018 golubkina n.a., kekina h.g., engalichev m.r., antoshkina m.s., nadezhkin s.m., caruso g. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 1 december 2017 accepted for publication 12 july 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(4): 541-548 542 countries, due not only to its high nutritional value and exclusive taste (puente et al., 2011), but also to its effectiveness as a medical plant. indeed, all plant parts are useful in modern medicine for their properties: antiasthma, antihepatitis, antimalaria and antidermatitis (chang et al., 2008), antiallergenic, anticarcinogenic, anti-inflammatory, antihyperglycemic, antimicrobial, antiseptic, antiviral, cardio-protective, diuretic, expectorant, febrifuge (bastos et al., 2006; sharma et al., 2015). in this respect, a negative correlation has been shown between the level of physalis consumption and the risk of cardiovascular, pulmonary and gastrointestinal diseases (leenders et al., 2014; hjartaker et al., 2015). moreover, the high antioxidant activity of water and alcoholic extracts of physalis is reportedly beneficial to patients suffering from alzheimer’s disease as well as from memory and concentration deficit (susanti et al., 2015). among the tens of physalis species, p. angulata and p. pubescens are the most suitable for production in the northern europe, due to their frost resistance and tolerance to fungal and bacterial diseases (mamedov et al., 2017). notably, the genetic selection carried out in russia allowed to create interesting varieties, suitable for cultivation both in central russia (kondratieva and engalichev, 2013; mamedov et al., 2017) and in siberia (makarov, 2002), mainly belonging to p. angulata. however, a few results concerning the variability of interspecies quality characteristics and even no cultivar comparisons of biologically active compounds accumulation in physalis fruits have been published so far. taking into account the scarcity of literature reports, the present research was aimed at evaluating the varietal differences in yield, quality and antioxidant content of p. angulata and p. pubescens fruits grown in northern europe. 2. materials and methods growth conditions six p. angulata l. cultivars (violet, lakomka, konditer, konditer 2, lezhky, korolek) and one p. pubescens l. cultivar (zolotaya rossip) were compared in a research carried out, under the organic farming management, in the experimental fields of the federal scientific center of vegetable production (moscow region, 55° 39’ 23’’ n, 37° 12’ 43’’ e) in 2015 and 2016. the trial was conducted on a clay-loam soil, with рн 6.8, 2.1% organic matter, 108 mg·kg-1 n, 450 mg·kg-1 p2o5, 357 mg·kg-1 k2o, exchangeable bases sum as much as 95.2%. the air temperature values, recorded at plant level, were: 14.2°c and 13.5°c in may, 16.9°c and 17.1°c in june, 21.2°c and 20.4°c in july, 19.3°c and 18.0°c in august, 12.1°c and 12.2°c in september, in 2015 and 2016 respectively. a randomized complete blocks design, with three replicates, was used for treatments distribution in the field; each treatment had a 19.25 m2 (5.5 × 3.5 m) surface area, including 50 plants. physalis seeds were sown in peat boxes on 14 april and the seedlings were transplanted in the field on 23 may, spaced 55 cm along the rows, the latter being 70 cm apart. as for p. angulata, the six cultivars chosen for the trial are the most spread in russia, whereas within p. pubescens only the cultivar zolotaya rossip tested in our research is cultivated in this country. the organic farming practice complied with ec regulation 834/2007. physalis crops were preceded by pea and each year the fertilization supplied the crops with 56 kg ha-1 of n, 16 of p2o5 and 98 of k2o. half of the fertilizers dose was given just before transplanting and the remaining 50% on dressing at two week intervals. drip irrigation was practiced for watering the crops when needed. plant protection from fungal diseases and insects was achieved by adopting trichoderma suspensions, copper, sulphur, azadirachtin. harvests were carried out from mid-august to the end of september. general analytical methods ripe, undamaged and regularly shaped fruits were classified as “marketable”. at each harvest, the weight and number of marketable fruits in each plot was recorded and the mean weight was assessed on random samples of 50 fruits per plot. cumulative plant biomass was calculated as the sum of the above-ground plant biomass at the end of the experiment plus the total fruit production from the beginning of the harvest period. dry weight was assessed after dehydration of the fresh samples in an oven at 70°c until they reached constant weight. in each plot, a sample of twenty-five fruits was collected and transferred to the laboratory for analysis. samples preparation prior to analyses, physalis fruits were extracted from papery husk and homogenized with a stainless steel blender for 1 min. the resulting homogenates golubkina et al. physalis spp. under organic management 543 were immediately subjected to the analysis. total soluble solids (tss) and sugars determination of total soluble solids was carried out by a refractometer (irf-22, russia). the results were reported as °brix at 20°c. monoand disaccharides were determined using cyanide method (kidin, 2008). titratable acidity (ta) t a w a s m e a s u r e d u s i n g 2 0 m l o f t h e w a t e r physalis extract (1:1), titrated to ph 8.1 using 0.1 n naoh (gost, 1996). the following formula was used for calculation: ta= v 1 x 0.1x0.064x100:20 where v-1 is the volume of naoh used; 0.1 is the naoh normality; 0.064 is the weight of a citric acid milliequivalent in g; 20 is the volume of the physalis extract used. mineral nutrients k, mg, mn, ca, na and p contents in dried homogenized fruit samples were assessed using icp-ms on q u a d r u p l e m a s s s p e c t r o m e t e r n e x o n 3 0 0 d (golubkina et al., 2017). nitrate content in fresh fruits of physalis species was determined using ionselective electrode on ionomer expert-001 (russia), as previously described (golubkina et al., 2017). antioxidants total polyphenols were assessed using folinciocalteu colorimetric method (sagdic et al., 2011). the ascorbic acid content was determined by visual titration of fruit extracts in 6% trichloracetic acid with tillmans reagent (aoac, 2012). taste the assessment of physalis taste was performed using two methods: chemical and organoleptic. taste index was calculated starting from the brix degree and acidity values, using the equation proposed by navez et al. (1999) and nielsen (2003) for tomato fruits: ti = (brix/20 ta) + ta where ti is the taste index and ta is the titratable acidity calculated referring to citric acid. though organoleptic perception of taste depends on the cultural background of judges and cannot be considered universally objective, physalis fruit taste was evaluated additionally by 10 experts, via sensory analysis using 5 balls scales (krueger and casey, 2000). statistical analysis data were processed by analysis of variance and mean separations were performed through the duncan multiple range test, with reference to 0.05 probability level, using spss software version 21. data expressed as percentage were subjected to angular transformation before processing. notably, the factor “year of research” had no significant effects on the variables examined, both in terms of main effects and of interactions with the other experimental factor. therefore, we have reported the results obtained from the data statistical processing as means of the two years of research. 3. results and discussion plant growth and yield as it can be seen in table 1, a correspondence between plant height and dry matter was recorded in p. pubescens variety zolotaya rossip, which had the smallest plants and the lowest dry matter, whereas within p. angulata the cultivar korolek showed the table 1 biometrical, growth and yield parameters of p. angulata and p. pubescens cultivars within each column, means followed by different letters are significantly different according to the duncan multiple range test at p≤0.05. cultivar plant height (cm) plant dry weight (g·m-2) planting to harvest beginning (days) fruits per plant (no.) yield (t·ha-1) mean fruit weight (g) violet (p. angulata) 90 d 296.6 c 105 bc 58 cd 9.0 b 60 c korolek (p. angulata) 130 a 320.3 c 108 b 64 b 9.3 b 56 cd lakomka (p. angulata) 105 c 200.7 d 90 d 47 f 7.3 c 60 c konditer (p. angulata) 125 ab 402.5 a 115 a 62 bc 11.3 a 70 b konditer 2 (p. angulata) 120 b 368.3 b 102 c 53 e 11.0 a 80 a lezhky (p. angulata) 110 c 223.6 d 118 a 55 de 7.7 c 54 d zolotaya rossip (p. pubescens) 80 e 133.3 e 85 e 165 a 1.3 d 3 e adv. hort. sci., 2018 32(4): 541-548 544 tallest plants but ‘konditer’ and ‘konditer 2’ produced the highest dry matter amounts. the latter cultivar was characterized by the longest crop cycle, similarly to lezhky, and p. pubescens zolotaya rossip resulted in the earliest fruit ripening; among p. angul a t u m v a r i e t i e s , o n l y ‘ l a k o m k a ’ s h o w e d e a r l y r i p e n e s s c o m p a r a b l e t o ‘ z o l o t a y a r o s s i p ’ o n e . correspondently to plant dry matter production, p. angulata cultivars konditer and konditer 2 also gave the highest yield (11.3 and 11.0 t·ha-1 respectively), due to the highest mean berry weight (80 and 70 g respectively), whereas p. pubescens variety zolotaya rossip had the worst outcome, in spite of the huge number of fruits per plant which were, however, very small (3 g). among p. angulatum varieties, lakomka and lezhky showed the lowest values (7.3 and 7.7 t·ha-1 respectively), due to the lowest prolificity and the smallest fruits respectively. quality indicators and mineral nutrient content as reported in table 2, the fruits of p. pubescens cultivar zolotaya rossip overall attained higher values of quality indicators than the six p. angulata varieties examined; compared to the average of the latter six cultivars, p. pubescens fruits showed 1.6 and 1.95 times higher content of total sugars and monosaccharides respectively as well as 1.27 times higher titratable acidity. among p. angulata cultivars, in ‘violet’ fruits the highest levels of dry residue, soluble solids and total sugars were recorded and in ‘lezhky’ the lowest. otherwise, ‘lakomka’ showed the highest monosaccharide content (32.2% out of total sugars) and cultivar violet the lowest (18.8%); the variation coefficient relevant to monosaccharide content in p. angulata species attained 18.3%. as for quality indicators, the physalis cultivars tested showed high variability in the content of dry matter (15.0%) and sugars (28.2%) as well as in juice acidity (16.7%), whereas the juice ph variability was low (2.1%). in our research, dry matter and sugar content in p. pubescens are similar to those detected in physalis berries grown in tropical and subtropical countries (yildiz et al., 2015). notably, soluble sugars highly affect flavour quality of tomato fruits (doras et al., 2001) and, according to olivares-tenorio et al. (2016) reports, the main carbohydrates of physalis fruits are sucrose and glucose, whereas fructose content is neglectable. the analysis of mineral nutrient content performed in our research showed the close element concentrations of p. pubescens and p. angulata fruits grown in the same environmental conditions (table 3). as for varietal differences in mineral nutrient table 2 quality indicators of physalis angulata and p. pubescens cultivars fruits table 3 mineral nutrient concentrations in physalis angulata and p. pubescens cultivars fruits (mg kg-1 d.w.) within each column, means followed by different letters are significantly different according to the duncan multiple range test at p≤0.05. cultivar dry matter (%) soluble solids (°brix) total sugars (%) reducing sugars (%) titratable acidity (%) рн violet 10.5 b 8.1 b 7.8 b 1.8 d 0.79 c 4.82 ab korolek 9.3 d 5.9 c 5.6 d 1.8 d 0.70 d 4.65 bc lakomka 8.7 e 6.1 c 6.0 c 2.9 b 0.45 f 4.94 a konditer 10.0 bc 6.0 c 5.9 cd 1.6 d 0.88 b 4.62 bc konditer 2 9.7 cd 6.1 d 5.9 c 2.2 c 0.79 c 4.51 c lezhky 8.5 e 4.9 d 4.7 e 1.7 d 0.63 e 4.70 ac zolotaya rossip 15.5 a 9.7 a 9.6 a 3.9 a 0.90 a 4.72 ac mean 9.5 6.2 6.0 2.0 0.71 4.71 within each column, means followed by different letters are significantly different according to the duncan multiple range test at p≤0.05. cultivar ca k mg na p no3 violet 415 b 2465 ab 1449 c 51 b 3483 c 2390 a korolek 722 a 2013 d 1740 ab 48 b 3825 b 2108 b lakomka 763 a 1284 f 1845 a 53 b 4345 a 2365 a konditer 720 a 2300 bc 1809 a 34 b 4277 a 1990 bc konditer 2 739 a 1723 e 1614 b 6 c 3503 c 1876 c lezhky 797 a 2152 cd 1286 d 61 b 3509 c 2287 a zolotaya rossip 414 b 2561 a 1708 ab 293 a 3572 c 1181 d golubkina et al. physalis spp. under organic management 545 accumulation, in p. angulata fruits the variability was high in na concentration (33%) and low in mg, p and nitrates (11.6, 8.5 and 6.9%). recently, the element composition of vegetable edible parts has been drawing attention as one of the most important factors affecting human being ingestion of mineral nutrients. unfortunately, the few literature reports available do not give the opportunity to perform correct interspecific or varietal comparisons, due to research carried out under different environmental conditions and species (el-sheikha et al., 2010; eken et al., 2014). referring to recommended dietary allowance values (institute of medicine, 2001), the consumption of 300 g fresh physalis fruits per day results in the ingestion of 24.8% potassium, 14.4% phosphorous and 12.3% magnesium needed by human organism. the latter benefits from these mineral nutrients in terms of optimization of carbohydrates, protein and lipid metabolism, bone integrity and brain activity, protection against cancer as well as cardiovascular diseases, obesity and diabetes. in this respect, p. angulata and p. pubescens fruits as well as the p. peruviana ones (zhang et al., 2013) may be considered as good sources of several elements. antioxidants polyphenols. in the present research, p. pubescens and p. angulata cultivars growing in the same geochemical environment (central russia) resulted in fruit polyphenols accumulation ranging between 18.7 and 25.1 mg gae/g d.w. (fig. 1). notably, the top concentration detected in the cultivar violet was 34.2% higher than the lowest level recorded in ‘korolek’, both belonging to p. angulata species; p. pubescens cultivar zolotaya rossip ranked third (20.5 mg gae/g d.w.). compared to our findings, in previous research (medina-medrano et al., 2015) higher phenolic values were detected in the fruits of five physalis wild species grown in mexico (32 to 86 mg age/g d.w.), with the lowest concentrations recorded in p. angulata berries. moreover, p. peruviana fruits produced in colombia accumulated 400 to 600 mg gae/g f.w. of polyphenols (narvaez-cuenca et al., 2014), but unfortunately this species is not suitable for cultivation in northern europe (kondratieva and engalichev, 2013). indeed, among natural secondary plant metabolites, polyphenols are considered to be the strongest antioxidants, which are able to inhibit carcinogenesis at initial and development stages, thus suggesting the great importance of their accumulation in agricultural plants (yang et al., 2001). the correlations between polyphenols and total sugars concentrations and between polyphenols and dry matter content were positive and highly significant (r= 0.99 and r= 0.91, respectively, at p≤0.001). the first one is supposed to reflect the existance of phenolic glycosides, identified as the main phenolics in wild physalis species (medina-medrano et al., 2015). the second correlation explains the higher dry matter and phenolics contents detected in physalis fruits produced in southern countries compared to those obtained in central russia. the high statistical significance of the correlations between the above parameters gives the opportunity to highlight the q u a l i t y p e r f o r m a n c e s o f c u l t i v a r v i o l e t , w h i c h showed the highest concentrations of polyphenols as well as the highest carbohydrates and dry matter content among p. angulata varieties and p. pubescens cultivar zolotaya rossip, with the highest content of dry matter and carbohydrates. these parameters may be used in physalis breeding for increasing fruit nutritional quality. ascorbic acid. in our research, vitamin c concentration in p. angulata cultivars ranged from 0.96 mg/g d.w. (cultivar violet) to 1.33 mg/g d.w. (cultivar korolek); however, the lowest value was recorded in the berries of p. pubescens cultivar zolotaya rossip (fig. 2). these results show that the synthesis of this antioxidant in russia is much lower than that reported for p. peruviana and p. pubescens in tropical and subtropical areas: i.e. 10 to 30 vs 20 to 50 mg/100 g f.w. (el sheikha et al., 2008, 2010; olivares-tenorio et al., 2016). indeed, the higher light intensity occurring at lower latitudes enhances ascorbic acid accumulation (bartoli et al., 2006), whereas in previous investifig. 1 polyphenol concentration as a function of physalis cultivar. means followed by different letters are significantly different according to the duncan multiple range test at p≤0.05. 546 adv. hort. sci., 2018 32(4): 541-548 gations carried out in colombia, kenia and southern africa (fischer et al., 2000), no relationship of this antioxidant with the altitude was found. moreover, the low vitamin c variability obtained in the present intestigation prove the predominance of environmental effects on the genetic one in affecting this antioxidant accumulation in physalis fruits (fig. 1). notably, the outer husk of physalis fruits is known to prevent ascorbic acid oxidation (valdenegro et al., 2012) and, despite the relatively low vitamin c content, 100 g of fresh physalis fruits produced in the northern hemisphere can supply human organism with 14 to 21% of the required vitamin c consumption (70 mg per day). taste among p. angulata and pubescens cultivars examined, the highest taste index (ti) and nutritional values were recorded in ‘zolotaya rossip’ (p. pubescens) and ‘violet’ (p. angulata). as far as taste assessment is concerned, it is a critical point in nonconventional plants produce and, indeed, this determination has never been performed on physalis fruits up to date. a general approach based on tomato berry testing suggests several significant factors affecting taste: dry matter, soluble solids, juice electrical conductivity, carbohydrates and organic acids contents (adams and ho, 1989; clement et al., 2008). in our research, we have assessed that the taste index (ti) used for tomato fruits can be successfully used for physalis berries. indeed, the organoleptic analysis and ti approach in evaluating physalis fruit quality show a good convergence of the results and suggest significant prospects of ti utilization in determining new varieties quality (fig. 3). in this respect, several correlations arose between the taste index and the following quality parameters of physalis fruits: total sugar content (r=+0.98; p<0.001), dry matter (r = +0.92; p<0.001), polyphenol content (r= +0.96; p<0.001); calcium (r= -0.91; p<0.001). notably, the two latter correlations had never been reported previously, neither in physalis nor in tomato. 4. conclusions the present research, carried out in central russia, allowed to assess interspecies and varietal differences in yield and quality characteristics of p. angulata and p. pubescens fruits grown under organic management. in this respect, this investigation provided with interesting clues, mainly concerning the nutritional and antioxidant properties of the cultivars tested and their growing prospects by organic farming procedures in northern europe. the variability of biologically active compounds, macroelement content and taste index, as well as their significant correlations may serve as the basis for enhancing the high potential of the physalis varieties examined for functional food production. acknowledgements we would like to thank dr. m. mamedov for his effective help with the research team arrangement. fig. 2 ascorbic acid concentration as a function of physalis cultivar. means followed by different letters are significantly different according to the duncan multiple range test at p≤0.05. fig. 3 correlation between taste index (ti) and organoleptic evaluation of physalis taste. golubkina et al. physalis spp. under organic management 547 references adams p., ho l.c., 1989 effects of constant and fluctuating salinity on the yield, quality and calcium status of tomatoes. j. hort. sci., 64(6): 725-732. aoac, 2012 the official methods of analysis of aoac 22 vitamin c. association official analytical chemists gaithersburg, md, usa. bartoli c.g., yu j., gomez f., fernandez l., mcintosh l., foyer c.h., 2006 inter-relationships between light and respiration in the control of ascorbic acid synthesis and accumulation in arabidopsis thaliana leaves. j. exp. bot., 57(8): 1621-1631. bastos g.n., santos a.r., ferreira v.m., costa a.m., bispo c.i., silveira a.j., do nascimento j.l., 2006 antinociceptive effect of the aqueous extract obtained f r o m r o o t s o f p h y s a l i s a n g u l a t a l . o n m i c e . j . ethnopharmacol., 103: 241-245. chang j.c., lin c.c., wu s.j., lin d.l., wang w.w., miaw c.l., ng lt., 2008 antioxidative and hepatoprotective effects of physalis peruviana extract against acetaminophen-induced liver injury in rats. pharm. biol., 46: 724-731. clement a., dorais m., vernon m., 2008 multivariate approach to the measurement of tomato maturity and gustatory attributes and their rapid assessment by visnir spectroscopy. j. agr. food chem., 56: 1538-1544. doras m., papadopoulos a.p., gosselin a., 2001 greenhouse tomato fruit quality. hort. rev., 26: 239319. eken a.e., unlu-enderlik b., baldemir a., ilgen s., soykut b., erdern o., kayseri c., 2014 antioxidant capacity of physalis peruviana l. fruit sold in markets. j. clin. anal. methods., 104328/gcam 2709. el sheikha a.f., piombo g., goli t., montet d., 2010 main composition of physalis (physalis pubescens l.) fruit juice from egypt. fruits, 65: 255-265. el sheikha a.f., zaki m., bakr a., el-habashi m., montet d., 2008 physico-chemical properties and biochemical composition of physalis (physalis pubescens) fruits. food global science books, 2(2): 124-130. fischer g., ebert g., ludders p., 2000 provitamin a, carotenoids, organic acids and ascorbic acid content of cape gooseberry (physalis peruviana l.) ecotypes grown at two tropical altitudes. acta horticulturae, 531: 263268. golubkina n.a., kosheleva o.v., krivenkov l.v., dobrutskaya h.g., nadezhkin s., caruso g., 2017 intersexual differences in plant growth, yield, mineral composition and antioxidants of spinach (spinacia oleracea l.) as affected by selenium form. sci. hortic., 225: 350-358. gost р., 1996 fruit and vegetable juices. method for determination of titratable acidity. european standard ен 12147. 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university of kentucky, 2015), owing to several breeding programs that have resulted in a rich diversity of colors (cardoso and teixeira da silva, 2013). although gerbera can be propagated in vivo using seeds or rhizome division (son et al., 2011), micropropagation using apical shoots or through organogenesis is the only way that can provide realistic large scale clonal propagation with genetic fidelity and disease free plantlets (kanwar and kumar, 2008; bhatia et al., 2009) to meet the current demand of highly technological floriculture market. multiplication stage in micropropagation is one of the most crucial steps to establish in vitro propagation. in this regard, most of the earlier studies on gerbera micropropagation were focused on establishment of different types and concentrations of plant growth regulators. several factors affected gerbera in vitro shoot multiplication, as cult u r e m e d i u m , p l a n t g r o w t h r e g u l a t o r s ( e s p e c i a l l y c i t o k i n i n s a s benziladenine), growth conditions and the propagation method. however, genotype is the most important factor influencing in vitro development, including the rate of multiplication of gerbera (son et al., 2011; cardoso and teixeira da silva, 2013). the basal culture medium ms (*) corresponding author: jeancardosoctv@gmail.com citation: cardoso j.c., 2018 in vitro responses of gerbera (gerbera jamesonii) genotypes multiplied under different photoperiods. adv. hort. sci., 32(4): 557-561 copyright: © 2018 cardoso j.c. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 13 november 2017 accepted for publication 21 may 2018 ahs advances in horticultural science short note adv. hort. sci., 2018 32(4): 557-561 558 (murashige and skoog, 1962) added 3% sucrose and benziladenine (0.5 to 3.0 mg l-1) is the most used for gerbera shoot multiplication (kanwar and kumar, 2008; cardoso and teixeira da silva, 2013). although the chemical factors have been well studied in relation to the micropropagation for several species, including gerbera, the physical factors such as temperature and light (wavelength, ppfd and photoperiod) conditions are among the less discussed ones, despite their great biological and economic influence on the different development stages of micropropagation. the electrical energy used in laboratories, especially in growth rooms, is the second most important cost factor of in vitro plantlet production, after labor cost (cardoso and teixeira da silva, 2012). therefore, an optimum light source is significant for this cost factor (cardoso et al., 2013). in this study, we tested different photoperiodic regimes at multiplication stage of micropropagation of gerbera cultivars to determine its effect on plant development and energy cost reduction in growth room conditions. 2. materials and methods five cultivars of gerbera were used in this study: ‘basic’ and ‘kiserian’ with both rose and pink inflorescences, and ‘orange dino’, ‘orca’ and ‘onedin’ with orange, white and yellow inflorescences, respectively. the plant material used as donor was cultivated u n d er green h o u se co n d i ti o n s an d p rep ared as described by cardoso and teixeira da silva (2012). briefly, the plants in reproductive phase were completely defoliated and the rhizomes were maintained in cold chamber at 4°c for seven days, followed by transplanting these rhizomes to plastic pots containing organic substrate consisting of coconut fiber. after 14 days of transplantation, the first shoots were observed, and 21-28 days old shoots (5-10 cm in length and 2-3 leaves) were excised from the mother plants and were taken to the laboratory for surface disinfestations. the shoots were reduced to 1.0-1.5 cm and washed for 5 min with tap water, followed by immersion in alcohol 75% (v/v), then in sodium hypochlorite with 1-1.25% of active chlorine for 20 min. finally, the explants were washed three times in autoclaved distilled water. shoot-tips of 2-3 mm length were used as explants to start gerbera micropropagation. the plants were multiplied to obtain the required quantity for the experiment. the experiment was carried out during the multiplication stage of micropropagation. the culture medium used was murashige and skoog (1962) supplemented with 30 g l-1 of sucrose, 0.1 g l-1 of myoinositol, 0.5 mg l-1 of benzyladenine (ba) and 6.0 g l-1 of agar-agar (algagel, type 900, barueri, brazil). the flasks containing the culture medium and the individual shoots were maintained in growth room at 25±1°c and cultivated under cool white fluorescent lamps (philips®, barueri, brazil) as light source with photon flux density of 20-25 µmol m–2 s–1. the plants were subjected to three different photoperiods as follows: 10-h light/14-h dark, 12-h light/12-h dark and 16-h light/8-h dark. the last photoperiod condition was taken as control, as this photoperiod condition is regularly used for many species, including gerbera, for in vitro micropropagation (cardoso and teixeira da silva, 2013). in total, five borosilicate flasks (10 cm height x 5.5 cm diameter) containing four shoots, and covered with transparent polypropylene caps (repetitions), for each treatment were used. the experimental design was 5 × 3 factorial with five cultivars and three photoperiods. t h e e x p e r i m e n t s w e r e c o n d u c t e d f o r t h r e e months with time of transplanting of shoots each time for 30 days, resulting in three cultivation periods. in each pricking time, the multiplication factor was determined by counting the number of shoots obtained for the next subcultivation divided by the number of plantlets used at the start of the experiment. five homogenized flasks with four plantlets in each turn were separated for the next evaluation, maintaining the plantlets in the same photoperiodic regime used in the previous experiment. the data were subjected to the analysis of variance (anova) and the shapiro-wilk test; if necessary, the data were normalized before comparison of means. the means were compared by the scott-knot test at 5% of probability. 3. results, discussion and conclusions around 20% of the explants were successfully established in vitro, regenerated into plantlets, and transferred to the multiplication medium. shoot apices with first leaf developed and without signals cardoso photoperiods in gerbera micropropagation 559 of bacterial or fungal contamination in culture medium were transferred to the light condition and used to obtain the first shoots for the experiment. around 60% of the explants were contaminated and around 2 0 % f o r m e d c a l l u s w i t h n o r e g e n e r a t i o n i n t o plantlets. the shoots so obtained were successfully in vitro multiplicated and used for the experiments of multiplication. these results were similar to those observed in other works at the establishment stage (cardoso and teixeira da silva, 2012, 2013). genotype is one of the most important factors that affect the micropropagation, including the rate of multiplication and the responses to the factors affecting in vitro development of explants being micropropagated. this was also observed in our work with a positive interaction between the genotypes and photoperiods (fig. 1). under 16-h photoperiod, the cultivars ‘onedin’, ‘orange dino’ and ‘orca’ showed a higher multiplication rate (5.5, 5.0 and 4.8 shoots/explant, respectively) than the rose inflorescences cultivars basic and kiserian, which showed a lower multiplication rate of 3.6 and 2.4 shoots/ e x p l a n t , r e s p e c t i v e l y . i n t e r e s t i n g l y , w h e n t h e plantlets were subjected to the photoperiod of 12-h (4-h less than control photoperiod), only the cultivar kiserian showed the multiplication rate less than 7.0 shoots/explant. thus, it can be concluded that the response to the photoperiods has a direct correlation with the genotype used for micropropagation (fig. 1). in fact, some cultivars of gerbera with rose and red inflorescences showed recalcitrance characteristics during in vitro multiplication, limiting their propagation and requiring protocol modification to accelerate the production of shoots and the efficiency of multiplication stage, compared with other cultivars. as example, a historical mean of multiplication along three years of in vitro cultivation showed that gerbera cv. lamborghini (red inflorescences) was 3.8 s h o o t s / e x p l a n t c o m p a r e d w i t h 6 . 8 a n d 4 . 6 shoots/explant for ‘suzzane’ (orange) and ‘dino’ (yellow) (cardoso, personal observations in commercial lab). the influence of genotype toward shoot multiplication was also observed by hartl et al. (1993). they classified gerbera cultivars according to the rate of multiplication into three multiple-shoot induction cultivars: high (8 shoots/inoculum), moderate (6-7 shoots/inoculum) and low (4-5 shoots/inoculum). we also observed all these classes in our cultivars. however, the different photoperiodic conditions had different effects on the position of the cultivars in different classes of multiplication as proposed by hartl et al. (1993). in our work the classification of cultivars using the scott-knot test with the best photoperiod for multiplication (12-h photoperiod) showed at least two classes of multiplication: (a) easily multiplied with number of shoots/explant >7.0; and (b) recalcitrant for multiple shoot induction, with the number of shoots/explant <4.0. an intermediary classes can also be considered as (ab), where the multiple shoot induction rates was 4 to 7 shoots/explant, but this was observed only in 10-h and 16-h photoperiod conditions. the photoperiod had significant effects on gerbera in vitro multiplication (fig. 1 and 2). normally, the multiplication at 25±2°c associated with 16-h photoperiod is most used for gerbera (cardoso and teixeira da silva, 2013), but in our actual work, we observed that for all five cultivars tested, the use of 12-h photoperiodic regime resulted in significant increase in multiple shoot induction when compared with 16-h standard photoperiod conditions (fig. 1). the maximum positive effects (97.2%) were obtained in one of the most recalcitrant gerbera cultivar basic, followed by ‘orca’ (68.8%), ‘kiserian’ (62.5%), ‘orange dino’ (54.0%) and ‘onedin’ (41.8%). similar results were observed in micropropagation of ornamental pineapple annanas comosus var. erecfig. 1 in vitro multiplication of gerbera (gerbera jamesonii) cultivars under different photoperiodic regimes. the means were obtained from three multiplication times (experiments) considering five replicates (flasks) with four plants. different small and capital letters show the differences among genotypes and photoperiod regimes, respectively, by the scott-knot test at 5% probability. the coeficient of variation is 13.52. the data were considered normal by shapiro-wilk normality test and the f was significative for all factors, including the interaction genotype x photoperiod. adv. hort. sci., 2018 32(4): 557-561 560 tifoilius, in which the reduction of photoperiod for 12-h resulted in better multiplication rate (6.6 s h o o t s / e x p l a n t s ) t h a n 1 6 h p h o t o p e r i o d ( 4 . 2 shoots/explants) (santos et al., 2015). the influence of photoperiod on in vitro development was also noted for other species and found affecting other in vitro plantlet characteristics of development, such as micro-tuberization in potato cultivars (seabrook et al., 1993). in amaryllis (hippeastrum johnsonii), the authors observed that 16-h photoperiodic regime resulted in better in vitro root length and number, bulblet diameter, and leaf length, compared with 14h and 12-h of photoperiod (zakizadeh et al., 2013). several amendments to reduce the cost have been proposed for gerbera micropropagation, such as chemical sterilization in place of autoclaving during all stages of micropropagation (cardoso and teixeira da silva, 2012) and the use of greenhouse rooting and elongation, called pag culture, in place of laboratory growth conditions (cardoso et al., 2013). the reduction in photoperiodic regime resulted in low energy consumption and so the low cost for the multiplication stage of gerbera. this might be useful for the production of low cost plantlets from micropropagation of this important cut flower. the major conclusion can be drawn from this study is that a reduction in photoperiod by 4-h of light per day along with increase in multiplication rate may increase the efficiency of the propagation system. chen (2016) concluded that the electrical energy for control environmental conditions on growth room, associated with the low efficiency of multiplication, is the most important factor that leads to increase cost of micropropagated plantlets of phalaenopsis. this author showed that increase in multiplication rate from 1.5 to 2.5 could lead to a 50% cost reduction in micropropagated phalaenopsis. photoperiod has a direct effect on the development of greenhouse cultivated gerbera genotypes, affecting plant width, height, shoot dry weight and number of flowers in a condition of 16-20-h of photoperiod (gangnon and dansereau, 1990). however, the interaction of photoperiod and the cultivation and development of gerbera could be more complex involving possible role of other environmental factors (pettersen and gislerød, 2003), and only few of them have been understood. our results showed for the first time that the in vitro development of gerbera plantlets can be affected by photoperiod regimes (fig. 1 and 2). increases in axillary shoot production was observed in reduced photoperiods (12 and 10-h) compared with 16-h. in addition, the use of white fluorescent lamps in the in vitro cultivation of gerbera resulted in a higher multiplication rate compared to different colors of light-emitting diodes (leds) lamps (gök et al., 2016). our actual experiments showed that reducing the photoperiod to 12-h produced slightly increased shoot height with no visual effect on the size of leaves (fig. 2). on the other hand, a further reduction of photoperiod to 10-h reduced the size of leaves (diameter and length) and resulted in etiolated development, showing negative effects on quality of gerbera shoots produced for rooting stage (fig. 2). as example, the fresh weight of gerbera shoots in 10-h photoperiod is 20.0% (‘onedin’), 29.7% (‘orca’) and 11.6% (‘orange dino’) less than compared with shoots produced in 12 or 16-h photoperiodic regimes (data not showed). only gerbera cv. kiserian showed practically the same fresh weight in 10-h and 12-h photoperiod. plantlets obtained from all treatments were successfully in vitro rooted (95-100% of rooting) in ½ms with 30 g l-1 sucrose and 0.5 mg l-1 of indol-butyric acid and were acclimatized in greenhouse conditions using polypropylene trays with coconut powder as organic substrate. no morphological alterations were observed in acclimatized plantlets obtained, independent to the cultivar or photoperiod used, and the plantlets maintained the main characteristics of the donor plants until the flowering stage. fig. 2 morphological characteristics of in vitro cultivation gerbera (gerbera jamesonii) cv. onedin under different photoperiodic regimes: 10-h and 12-h. shoots from 10-h photoperiod presented smaller leaves (sl) and smaller and etiolated shoots (ses), compared to 12 and 16-h photoperiod. bar = 1.0 cm cardoso photoperiods in gerbera micropropagation 561 acknowledgements special thanks to uniplant co. for the infrastruct u r a l s u p p o r t t o t h i s r e s e a r c h . j c c t h a n k s t h e conselho nacional de desenvolvimento científico e tecnológico (cnpq) for the research grants (process number 304174/2015-7). references bhatia r., singh k.p., jhang t., sharma t.r., 2009 assessment of clonal fidelity of micropropagated gerbera plants by issr markers. sci. hortic., 119: 208211. bhatia r., singh k.p., sharma t.r., jhang t., 2011 evaluation of the genetic fidelity of in vitro propagated gerbera (gerbera jamesonii bolus) using dna-based markers. plant cell tiss. organ cult., 104: 131-135. cardoso j.c., rossi m.l., rosalem i.b., teixeira da silva j.a., 2013 pre-acclimatization in the greenhouse: an alternative to optimizing the micropropagation of gerbera. sci. hort., 164: 616-624. c a r d o s o j . c . , t e i x e i r a d a s i l v a j . a . , 2 0 1 2 micropropagation of gerbera using chlorine dioxide (clo2) to sterilize the culture medium. in vitro cell. dev. biol. plant, 48: 362-368. cardoso j.c., teixeira da silva j.a., 2013 gerbera micropropagation. biotech. adv., 31: 1344-1357. chen c., 2016 cost analysis of micropropagation of phalaenopsis. plant cell tiss. organ cult., 126: 167175. gangnon s., dansereau b., 1990 influence of light and photoperiod on growth and development of gerbera. acta horticulturae, 272: 145-151. gök k.m., san b., bayhan a.k., 2016 micropropagation of gerbera (gerbera jamesonii bollus) under different color of light-emitting diodes. j. nat. appl. sci., 20(3): 468-474. hartl d., kuzmicic i., jug-dujakovic m., jelaska s., 1993 the effect of genotype on gerbera shoot multiplication in vitro. acta bot. croat., 52: 25-32. kanwar j.k., kumar s., 2008 in vitro propagation of gerbera-a review. hort. sci., 35: 35-44. murashige t., skoog t., 1962 a revised medium for rapid growth and bioassays with tobacco tissue cultures. physiol. plant., 15: 473-497. pettersen r.i., gislerød h.r., 2003 effects of lighting period and temperature on growth, yield and keeping quality of gerbera jamesonii bolus. europ. j. hort. sci., 68: 32-37. santos p.b., barbosa f.s., vieira c.f., carvalho a.c.p.p., 2015 number of explants, culture medium and photoperiod in ornamental pineapple micropropagation. rev. ciênc. agron., 46(4): 749-754. seabrook j.e.a., coleman s., levy d., 1993 effects of photoperiod on in vitro tuberization of potato (solanum tuberosum l.). plant cell tiss. organ cult., 34: 43-51. son n.v., mokashi a.n., hegde r.v., patil v.s., lingaraju s., 2011 response of gerbera (gerbera jamesonii bolus) varieties to micropropagation. karnataka j. agric. sci., 24: 354-357. university of kentucky, 2015 field-grown cut flowers. cooperative extension service of university of kentucky, university of kentucky, usa. zakizadeh s., kaviani b., onsinejad r., 2013 role of photoperiod on some growth characters of amaryllis (hippeastrum johnsonii), a bulbous plant. eur. j. exp. biol., 3(1):289-291. impaginato 77 adv. hort. sci., 2019 33(1): 77-85 doi: 10.13128/ahs-23298 apple seed stocks affected scion tree vigor and performance based on maternal self(in)compatibility h. hajnajari temperate and cold fruits research institute (karaj), horticulture sciences research institute, ag. research education and extension organization (areeo), karaj, iran. key words: breeding, dwarfness, genetic purity, inductive effect, malus × domestica borkh., seed rootstock. abstract: in a breeding program to increase uniformity of apple saplings size, shape and vigor the genetic improvement of seed rootstocks was considered as a key point. the half-sib seeds of two selected native crab parents, self-compatible “morabbaei” and self-incompatible “azayesh”, both dwarf, were considered as the main sources of variances into the six grafted scions. the inductive effects of the two seed masses were assessed on vegetative traits of 6 and 7-year-old scion trees. according to the results, shoot length, shoot thickness and internode length as main components of tree vigor showed significant differences at 1% level in all the six scions. the shoot length mean of the grafted scions on “azayesh” seed progeny was higher than “morabbaei” seed stock. the grafted scions on self-compatible “morabbaei” seed stock gave place to the shoots with higher thickness than the same grafted on “azayesh” seed source. combinations of “red delicious”-“morabbaei” and “braeburn”-“azayesh” demonstrated max and min values of internodes length during two consecutive years. both rootstock and scion trunk diameters on self-incompatible “azayesh” seeds resulted higher than seed progeny of self-compatible “morabbaei”. shoot number was not affected by rootstock type, whereas the effect of rootstock × scion, scion and year resulted significant. 1. introduction malus genus is characterized by great phenotypic and genetic diversity. high heterozygosity is reinforced by dominant self-incompatibility besides inbreeding depression (korban and skirvin, 1984). among 6000 documented apple cultivars, a large part of commercial production still relies on few cultivars including “golden delicious”, “granny smith”, “fuji”, “gala” and delicious group (o’rourke, 2003). rootstocks can influence vigor, habit and cropping of the scion cultivar as well as tolerance to unfavorable climatic or edaphic conditions (webster, 2005). rootstock performance is highly correlated with the genetic potential of rootstock to provide anchorage, adaptaion to pedo-climatic conditions and resist to abiotic stress efficiently (fazio, 2014). trees with limited height are requested due to facilitate orchard management. smaller trees are also (*) corresponding author: hassanhajnajari@yahoo.com citation: hajnajari h., 2019 apple seed stocks affected scion tree vigor and performance based on maternal self(in)compatibility. adv. hort. sci., 33(1): 77-85 copyright: © 2019 hajnajari h. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 28 may 2018 accepted for publication 12 december 2018 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(1): 77-85 78 easier to target with sprays, reducing undesirable spray drift and increasing efficiency of usage (jobir, 2016). good performance, dwarfness, precociousness, scion compatibility, free from suckers and burrknots are some of the attributes of an ideal rootstock (wertheim and webster, 2005). clonal rootstocks are being developed rapidly while seed rootstocks have gradually decreased. still today, many nurseries in canada, belarus and finland use apple seed rootstocks. “antonovka” was selected as a seed source due to the freeze resistance and its cytoplasmic heritability (żurawicz and lewandowski, 2014), the system for which some traits are transmitted to the next generation by maternal parent (lorenzetti and ceccarelli, 1980). seed rootstocks improvement is applied for dwarfing effect, and tolerance to dry soil conditions (brown and maloney, 2005). in iran seed rootstocks are used because of the alkaline and dry soils and also due to bad topography and exaggerated fragmentations. in absence of a specialized sector for pure seed production, the mixture of recycled seeds of processing industry characterized by high genetic diversity are used in saplings production (hajnajari and tarrahi, 2009; hajnajari, 2010). increasing genetic purity of seed rootstocks will enhance scion tree size and uniformity by taking advantage of their neglected aspects (hajnajari and mizani, 2015). similarly, almond trees are raised on wild peach and bitter almond rootstock (sharma et a l . , 2 0 0 4 ) . “ m a z z a r d ” s e e d l i n g s f o l l o w e d b y “mahaleb” are the most widely used rootstocks for cherry tree production (demirsoy et al., 2017). each open pollinated seed has its specific hormonal status due to the different genetic provenience different from the rest which will cause a gradient of tree growth habits and vigor within the same scion cultivar. while tree vigor is provoked by hybrid heterosis (lorenzetti et al., 2011), the mandatory inbreeding ruling the self-compatible cultivars as maternal seed source may cause vigor decline of scion trees. it was suggested that the inbred f1 seed stocks could weaken the vigor of scion. vigorous trees with higher canopy size need increased planting distance (kosina, 2010). selection of proper parents as seed source was the unique pass to way out from the emblematic situation (hajnajari, 2018). cultivar evaluation program led to selection of few native dwarf crabs “morabbaei”, “zinati” and “azayesh” as seed source parents (hajnajari, 2010). contemporaneous field screening program for determination of self-compatibility level of the 92 cultivars followed by fluorescent microscope studies showed complete self-compatibility of “morabbaei”, “zinati” and “azayesh” as selfincompatible (forughikia et al., 2014; hajnajari and moradi, 2014). using such maternal parents that profit high level of self-compatibility accompanied with dwarfness can minimize seed genetic variability leading to production of the f1 seed progeny marked by high genetic purity. both traits of dwarfism and low vigor within the seedlings can be used for production of uniform saplings. the same parents were used for production of clonal rootstocks resistant to the crown rot disease (hajnajari et al., 2012). the e f f e c t s o f t w o s e e d m a s s e s , a s f 1 p r o g e n y o f “azayesh” (self-incompatible) and “morabbaei” (selfcompatible) on growth characteristics of six apple scion cultivars were investigated. practically, the two open pollinated seed masses derived from two different maternal parents are assumed as two family’s genetically, diverse within the lots and among them, however two various sister masses belonging to different families. nevertheless, both parents as crab apples carry jointly dwarfness trait and are native to iranian habitat. here is shown that the seed rootstocks bred by selfcompatible parent incur more morphological uniformity and control of tree vigor in scion trees compared with the rival sister seeds produced by heterozygote parent “azayesh” which afford more vigorous and less homogenous tree architecture due to the need of self-incompatible parent for foreign pollen. 2. materials and methods t h e m a t u r e a p p l e t r e e s , 6 a n d 7 y e a r o l d ( “ g o l d e n d e l i c i o u s ” , “ r e d d e l i c i o u s ” , “ g r a n n y smith”, “braeburn”, “gala” and native “golab-e kohanz”) pruned in spindle form, grafted on two selected seed masses were studied to understand the effects of seed sources on vegetative characteristics of the scion cultivars. genetically, the two seed lots were identified as f1 half-sib progenies derived from two different maternal parents including the self-compatible cultivar “morabbaei” and the other self-incompatible “azayesh”, revealed as homozygote and heterozygote, correspondingly. the two seed lots were obtained from the maternal parents exposed equally to open pollination, so forming two genetically diverse seed masses as two families considered as the main treatments. the investigated traits included annual diametric growth of scion hajnajari seed rootstocks improvement in apple 79 trunk, graft point and rootstock. for each tree, 4 annual shoots were selected and labelled, and internodes number, internodes length, shoot length, shoot thickness and lenticels number were assessed. also, shoot numbers per tree and crotch angle were studied. the entire comparative inter and intra varietal measurements were achieved to distinguish the influences of the two seed rootstocks as the main treatments. interaction of scion per rootstock was recorded to study also the role of scion tree vigor against dwarfing effect of the seed source. six trees, 3 × 3 m, were assigned for each combination of “scion cv-seed progeny” within six replications, large enough to explain seed source inductive effects. growth traits of grafted scions were measured in two successive vegetative seasons (2014 and 2015). t h e e x p e r i m e n t a l o r c h a r d w a s e s t a b l i s h e d i n meshkin-abad station under temperate and cold f r u i t r e s e a r c h c e n t e r l o c a t e d i n k a r a j , a l b o r z province (iran). the experiment was set as according to a completely randomized blocks design (rcbd) with six replications for statistical analysis. anova was performed by combining the analysis of the collected data regarding all growth traits in exam, both seed rootstocks and cultivars. all statistical analyses were carried out using the general linear model (glm) procedure of the sas version 9.0. the duncan’ multiple range test (p≤0.01) was used to evaluate differences between treatments. 3. results shoot length and thickness shoot length and shoot thickness were affected significantly by rootstock, scion and year interactions (table 1). all variables are measured at annual shoots. shoot length in 2014, “red delicious”-”azayesh” combinations showed the longest shot lenght, while “golden delicious” on “morabbaei” had the lowest value, in 2015. comparing the treatments, no significant differences were observed for effects of the two seed progenies “azayesh” and “morabbaei” on the single cultivars, at the end of the first growing season. in t h e s e c o n d y e a r , s i g n i f i c a n t d i f f e r e n c e s w e r e observed by different sources of seed rootstocks for grafted cultivars (table 2). in 2014, it was found that shoot length value for all scions of “golab-e kohanz”, “gala”, “granny smith” and “braeburn” combined with “morabbaei” seed rootstocks showed lower values than “azayesh” f1 progeny; albeit “braeburn” d e m o n s t r a t e d s i g n i f i c a n t d i f f e r e n c e , “ g o l d e n delicious” and “red delicious” showed no significant differences on the 2 seed sources for shoot length. these results indicate that the maternal self-incompatible “azayesh” gave place to more vigorous seed progeny. whereas in 2015, the entire scions had lowered values of shoot length showing significant diff e r e n c e a f f e c t e d b y s e e d s o u r c e ( t a b l e 2 ) . considering the prevailing traits of dwarfness associated with homozygosis carried by “morabbaei” it becomes clear how these are transmitted to the relative f1 seed progeny whom could control the vigor of the combined scion trees. though both parents may produce seeds which reinforced vigor control of scion trees but the “morabbaei” seed progeny showed the capability both to induce dwarfism and uniformity in size and shape into the scion cultivars (table 2). parallel researches of artificial self-pollination followed by florescent microscopy studies put in evidence how the pollen tubes of “morabbaei” could penetrate to its own ovary confirming the total selfc o m p a t i b i l i t y o f s e l e c t e d m a t e r n a l p a r e n t (forughikia, et al., 2014). future investigations are source df mean square shoot length shoot thickness internode number internode length rootstock diameter graft diameter trunk diameter shoot number branch angle lenticels number year (y) 1 44376.9 ** 3.92 ** 7.65 ** 1.14 ** 1912.11 ** 4460.61 ** 0.22 ** 0.25 * 67.79 ns 0.007 ns block (year) 10 4982.37 ** 0.04 ** 0.17 ** 0.07 ** 223.66 ns 108.48 ns 0.01 ns 0.03 ns 420.71 ** 0.07 * rootstock (r) 1 39402.37 ** 0.67 ** 0.04 ns 0.56 ** 1005.01 * 911.83 * 0.03 ns 0.03 ns 3679.76 ** 1.29 ** scion (s) 5 10396.58 ** 0.063 ** 0.24 ** 0.12 ** 28.27 ns 111.47 ns 0.02 ns 0.14 * 833.27 ** 0.24 ** r × s 5 6614.24 ** 0.067 ** 0.06 ns 0.23 ** 409.78 * 143.15 ns 0.02 ns 0.17 ** 1749.75 ** 0.76 ** y × r 1 17580.61 ** 0.003 ns 0.05 ns 0.56 ** 56.99 ns 31.91 ns 0.03 ns 0.006 ns 667.52 * 0.10 ns y × s 5 7960.64 ** 0.06 ** 0.12 ** 0.08 ** 82.02 ns 79.06 ns 0.02 ns 0.10 ns 440.02 * 0.20 ** y × r× s 5 11061.31 ** 0.07 ** 0.12 ** 0.06 ** 52.05 ns 46.51 ns 0.01 ns 0.01 ns 668.91 ** 0.37 ** cv (%) 25.62 12.16 16.52 10.74 24.007 23.11 7.63 22.32 16.80 14.83 table 1 analysis of variance regarding the effects of year, rootstock and scion on vegetative traits **= significant differences at 1% level, *= significant differences at 5% level, ns= not significant. adv. hort. sci., 2019 33(1): 77-85 80 required to elucidate the mechanisms and biochemical pathways by which a self-compatible cultivar can accept insider pollen leading to fertilization, within a self-incompatible species. shoot thickness combination of “red delicious”-”morabbaei”, in the first year, showed the highest shoot thickness, while both combinations of “golden delicious” and “red delicious” on “azayesh”, in the second year, showed the lowest rates. it could be inferred that low vigor of “morabbaei” f1 progeny inhibited shoot length growth rising inversely shoot thickness. in 2014, converse growth rhythm was registered in “golden delicious” and “braeburn” on heterozygote seeds of “azayesh” inducing significant differences, while “golden delicious”, “red delicious” and “gala” were affected significantly by “azayesh” f1 progeny for the measured variables in 2015. moreover, in 2014 “morabbaei” seeds induced significant differences between “golden delicious” and “granny smith”. similar differences were observed among “golden delicious”, “red delicious” and “gala”, in 2015 (table 2). such differences in successive years within the combinations are attributed to the normal annual growth. internode number year and scion main effects and year × scion and year × rootstock × scion interaction significantly affected internode number (table 1). the highest number of internode was found in 2014 in combination of “golden delicious”-“morabbaei” confirming the role of homozygosity on lowering vegetative growth, changes in metabolic pathways and relative hormonal modifications at intra-cellular level. however, the lowest expression of the genes for internode number was found in “gala”-“azayesh” combination, in 2015. as a reason, the latter low vigor of “gala” occurred in “on” year could influence such result. in 2014, there were no significant differences among the cultivars for internode number on “azayesh” seeds, while in the second year significant differences were observed caused by annual growth. contrarily, “morabbaei” seed stocks induced significant differences in “gala”, “golden delicious” and “granny smith”, in 2014, which confirms relatively lower vigor of “gala”, whilst in 2015 significant differences was observed among the grafted cultivars (table 2). in 2014, there were no significant differences between rootstocks for each scion combination, but in 2015, “golden delicious” and “red table 2 the interaction effects of year × scion × rootstock on some vegetative traits of the apple scion cultivars year seed stock scion shoot length mm shoot thickness mm internodes number internodes length mm rootstock diameter mm graft diameter mm trunk diameter mm shoot number crotch angle lenticels number 2014 azayesh 'golden delicious' 179.24 b-g 5.70 ef 21.06 ab 14.66 b-e 45 a 43 a 30.2 a 7.75 a 61.67 cd 16.25 ij 'red delicious' 208.22 a 6.43 de 19.26 a-d 17.20 bc 43.6 a 47.2 a 38.5 a 10.2 a 68.33 bcd 24.23 cde 'granny smith' 177.39 b-h 6.87 bcd 20.17 abc 17.19 bc 57.5 a 58.25 a 52.25 a 17.66 a 73.75 a-d 33.12 a 'braeburn' 175.25 b-h 5.10 ghi 18.08 b-e 15.54 b-e 49.4 a 56 a 40.5 a 11.16 a 83.33 a-d 14.53 j 'golab-e kohanz' 168.75 b-h 6.80 cd 19.40 a-d 14.66 cde 47.33 a 51.66 a 50.83 a 8.5 a 87.50 ab 10.22 k 'gala' 178.45 b-g 6.86 bcd 18.16 b-e 17.83 b 62.83 a 60.83 a 47.66 a 10.5 a 85.00 abc 18.16 e-i morabbaei 'golden delicious' 193.63 abc 6.18 de 22.86 a 16.83 bcd 47 a 43.66 a 35.83 a 14.5 a 82.17 a-d 21.21 c-f 'red delicious' 195.06 ab 8.00 a 19.46 a-d 20.33 a 56 a 41 a 51 a 17.16 a 95.00 a 21.94 c-g 'granny smith' 174.61 b-h 6.36 de 20.20 abc 13.73 b-e 40.25 a 49.6 a 41.8 a 13 a 90.00 ab 21.40 c-h 'braeburn' 159.70 e-i 7.63 ab 19.58 a-d 15.75 b-e 43.4 a 49.6 a 43.4 a 9.33 a 60.00 d 23.80 bcd 'golab-e kohanz' 165.74 c-h 7.41 abc 19.56 a-d 15.30 b-e 44.6 a 54.5 a 43.16 a 6.33 a 82.50 a-d 29.27 ab 'gala' 171.44 b-h 7.86 ab 15.57 d-g 15.03 b-e 41.2 a 44.5 a 36 a 8.00 a 73.33 a-d 15.50 hij 2015 azayesh 'golden delicious' 176.64 b-h 3.94 k 13.39 fgh 13.24 ef 69.33 a 70.33 a 58.66 a 6.33 a 68.33 bcd 14.18 ij 'red delicious' 149.59 hi 4.03 k 9.52 hi 11.36 fg 51 a 62 a 52.33 a 10.16 a 70.00 bcd 22.65 c-g 'granny smith' 164.23 d-h 4.47 h-k 14.17 efg 14.89 b-e 63.2 a 70.2 a 58.8 a 10.66 a 69.00 bcd 19.34 c-h 'braeburn' 181.71 a-f 4.31 jk 11.32 ghi 9.97 g 62.16 a 69.16 a 61 a 9.4 a 59.00 d 16.30 hij 'golab-e kohanz' 187.42 a-e 4.30 ijk 14.11 efg 10.23 g 60.33 a 68.66 a 58 a 7.5 a 76.67 a-d 18.22 d-i 'gala' 185.61 a-e 4.63 g-j 8.25i 10.36 g 68.33 a 68.5 a 57.5 a 9.16 a 83.33 a-d 17.28 ghi morabbaei 'golden delicious' 143.23i 6.10 de 9.11i 14.67 cde 53 a 58.33 a 45.83 a 10.00 a 90.00 ab 21.08 c-g 'red delicious' 191.06 a-d 4.33 h-k 16.10 c-f 21.24 a 60 a 62 a 53 a 17.16 a 95.00 a 22.56 bc 'granny smith' 116.40 j 4.63 g-j 14.70 efg 13.79 ef 44.6 a 57.5 a 44.66 a 7.33 a 85.00 abc 17.26 hij 'braeburn' 151.71 ghi 4.68 g-j 11.31 ghi 16.79 bc 51.66 a 64 a 50 a 6.66 a 76.67 a-d 23.19 bc 'golab-e kohanz' 156.16 f-i 4.97 gh 14.43 efg 15.61 b-e 58.25 a 65.75 a 53 a 7.83 a 73.33 a-d 19.94 c-h 'gala' 150.88 ghi 5.24 fg 11.82 f-i 11.50 fg 44 a 57.66 a 44 a 9.33 a 83.33 a-d 17.50 f-i in each column, means with similar letters are not significantly different at the 1% probably level using duncan multiple range test. hajnajari seed rootstocks improvement in apple 81 d e l i c i o u s ” s h o w e d s i g n i f i c a n t d i f f e r e n c e s o n “morabbaei” and “azayesh” seed sources (table 2). internode length internode length was affected by year, scion cultivar and rootstock main effects and their interactions (table 1). the longest internode was found in “red delicious” on “morabbaei” rootstocks in both years and the lowest value was registered in combination of “braeburn”-“azayesh”, in 2015 (table 2). in the first year, among all the scions combined with “azayesh” rootstocks, there was significant difference between “golab-e kohanz” and “gala” and among the grafted cultivars on “morabbaei” sole “red delicious” showed significant difference. “golab-e kohanz” is the most vigorous iranian cultivar early and “gala” with moderate tree vigor. tree vigor, denoted as potential of vegetative growth, is measured based on height and spread of adult trees self-rooted, or relative to reference cultivars on the same rootstock in apple descriptors (watkins and smith, 1997). “golden delicious” and “red delicious” were used as reference cultivars to assess rootstocks efficiency for tree vigor, the trait that encompasses other components as shoot length, shoot thickness, internode number, internode length, shoot number, and crotch angel by which the tree overall size, shape and architecture will be reflected. fazio (2014), assumed that rootstock performance is highly correlated with the genetic potential to impart positive architectural properties to the scion like vigor control. considering the ruling genetic purity carried by “morabbaei” seed rootstocks, it imparted uniformity in the size of internode length in all the scion cultivars. in the second year, “golden delicious” and “granny smith” showed significant differences against other scions grafted on “azayash”, while “red delicious” and “gala” had significant difference relative to the other scions on “morabbaei” (table 2). such differences are attributed to the different genetic vigor of scion materials. rootstock, graft point and scion diameter according to the table 1, the effect of year was significant for rootstock, graft union and scion trunk diameters. overall, rootstock diameter, graft and trunk were higher in the second year. rootstock and graft point were affected equally by rootstock factor. interaction of rootstock × scion was significant for rootstock (table 1). interaction effect of scion × rootstock on rootstock demonstrated that combination of “granny smith” on “azayesh” and “morabbaei” rootstocks endured modifications on the trait, for example 60.66 mm to 42.66 mm, respectively. there w a s o n l y s i g n i f i c a n t d i f f e r e n c e b e t w e e n “ r e d delicious” (47.30 mm) and “gala” (65.58 mm) for rootstock limiting factor to “azayesh” rootstock. similar comparison within seed lots of “morabbaei” revealed significant difference in combination with “granny smith” (42.66 mm) and “gala” (60.22 mm). surprisingly, both seed rootstocks influenced “gala” increasing rootstock to the highest values of 65.58 mm and 60.22 mm (table 3). moreover, significant difference was observed between the combinations of “granny smith”-“azayesh” and “granny smith”“morabbaei” for the effect of rootstock type on each scion for rootstock, while no difference was observed between seed rootstocks for other combinations (table 3). this result is assumed positive for uniform apple tree saplings production on the improved seed rootstock. shoot number shoot number was not affected by rootstock type, while the effects of year, scion and rootstock × scion were significant (table 1). in 2014, more lateral branches were grown than 2015. among scionss t o c k s c o m b i n a t i o n s , “ m o r a b b a e i ” s e e d s t o c k showed both max. and min. shoot number, on “red delicious” (17.16) and on “golab-e kohanz” (7.08), respectively (table 3). no significant difference was found among the six scion cultivars for shoot number on “azayesh”, whilst “morabbaei” rootstock induced significant differences of shoot number on “golden delicious” and “red delicious”. crotch angle in this study crotch angle was affected by year, rootstock scion rootstock diameter (mm) shoot number 'azayesh' 'golden delicious' 55.43 abc 7.14 c 'red delicious' 47.30 bc 10.18 bc 'granny smith' 60.66 ab 13.00 bc 'braeburn' 56.36 abc 10.36 bc 'golab-e kohanz' 53.83 abc 8.00 bc 'gala' 65.58 a 9.83 bc 'morabbaei' 'golden delicious' 50.27 abc 12.25 ab 'red delicious' 58.00 abc 17.16 a 'granny smith' 42.66 c 10.16 bc 'braeburn' 46.50 bc 8.00 bc 'golab-e kohanz' 50.67 abc 7.08 c 'gala' 60.22 ab 9.25 bc table 3 the interaction effect of scion × rootstock on rootstock diameter and shoot number in each column, means with similar letters are not significantly different at the 1% probably level using duncan multiple range test. 82 adv. hort. sci., 2019 33(1): 77-85 rootstock and scion interaction (table 1). the combination of “red delicious”-“morabbaei” showed the widest value of crotch angle probably as a result of heavy cropping, while light bearing trees in the combination of “braeburn”-“azayesh” had the lowest crotch angle rates, in both years. in general, the scion cultivars grafted on “morabbaei” had wider crotch angle than on “azayash” progeny (table 2). except partial genetic role of the scions regarding growth habit, this effect might be result of higher tree vigor control by self-compatible parent “morabbaei” rootstocks and subsequent decrement of woody parts in favor of flower bud initiation and fruit set (wertheim and webster, 2005). clearly, bearing shoots under higher fruit weight were bent downward increasing crotch angle. lenticels number lenticels number was affected by year × rootstock × scion interaction (table 1). the max and min values of lenticels number were found in “granny smith” and “golab-e kohanz” both on half-sib seed stocks of “azayesh”, in the first year. it can be deduced that the increased leaf lenticels number of the “golab-e kohanz” as the most vigorous scion is a normal physiological trend appeared trough interaction of the grafting with as much vigorous seed stock “azayesh” in the year. more vegetative growth induced by vigorous seeds demands higher respiration rate for which a higher lenticels number equilibrate the defined biological status. in both years, significant difference was observed among the fruit scions on “azayesh” and “morabbaei”. this confirms significant effect of rootstock seed masses for lenticels number. in 2014, comparisons of rootstocks for each cultivar showed that “golden delicious”, “granny smith”, “braeburn” and “golab-e kohanz” were affected by seed stocks for lenticels number. also in 2015, “golden delicious” and “braeburn” were affected for this trait (table 2). the comparison between two seed sources put in evidence of high dominant uniformity of lenticels number trait for scions grafted on the seed stocks and originated from self-compatible “morabbaei”. 4. discussion and conclusions according to the results, shoot length, shoot thickness and internode length as 3 main components of tree vigor showed significant difference in all t h e s i x c u l t i v a r s g r a f t e d o n t w o s e e d m a s s e s . dominant homozygosity carried out by self-compatible “morabbaei” and relative progeny overcame heterosis effects which induced by “azayesh” seeds and tree vigor interaction. our results regarding control of tree vigor “morabbaei” seed stocks demonstrated that it could reduce shoot length; infact this trait decreased in all the cultivars except for “golden delicious”, in 2014. this cultivar showed the same opposite rhythm for all growth traits like crotch angle, internode thickness, internode number, roostock, and scion diameter on “morabbaei” seed stock. also, other four scion cvs. decreased shoot length value and were affected by dwarfing seeds in the range of 13 to 3 mm for “red delicious” and “golab-e kohanz”, 7 and 3 mm for “gala” and “granny smith”, respectively. these results confirm the reciprocal influences of two genomes, scion and seed rootstock within the same genus, each one specialized differently for which were selected in the selection procedure. there is no clear reason for reaction of “golden delicious” for measured trait when compared with five scion cultivars grafted on the same seed pure line progeny. this may not be related to the distant geographical origin of two genomes, iran-usa. with studying scion tree vigor was suggested that j-te-e as an acceptable rootstock for “rubin” among 14 roots t o c k s ( k o s i n a , 2 0 1 0 ) . t h e s c i o n c u l t i v a r s o n “morabbaei” seeds showed higher shoot thickness than the same scions on “azayesh” seed progeny. increased shoot thickness as the diametric growth of annual shoots on “morabbaei” seeds indicated accumulation of carbohydrates for productive functions. however the final canopy size is determined genetically by scion-rootstock interaction. higher dwarfing efficiency raised in “morabbaei” than “azayesh” progeny is in agreement with tworkoski and miller (2007 a), who indicated that malling rootstocks have different size-controlling effects on different apple scions. the lowest interned number (8.25) was found in “gala”-“azayesh” combination, in 2014, while the highest (22.86) was found in combination of “golden delicious”-“azayesh”, in 2015. the biennial comparisons evidenced that the higher mean of internode number was induced by “morabbaei” in both years, 22.86 and 9.11, related to “azayesh” equal to 21.06 and 8.25. these finding confirmed stronger dwarfing effects of the self-compatible parent. dwarfing mechanism acted through an increase of internode length and contrarily shortening internode length. highest v a l u e o f i n t e r n e d l e n g t h w a s f o u n d i n “ r e d hajnajari seed rootstocks improvement in apple 83 delicious”-“morabbaei” combination for 2014 and 2015 and the lowest in “braeburn” on “azayesh” in 2015 (table 2). others reported that wild peach rootstocks influenced shoot growth and leaf area for the scions only during the first year (sharma et al., 2004). in general, in the first year, the internode number and internode length were higher than the second year. it can be attributed also to the difference between orchard management during two years. interesting results were obtained in 2015 comparing effects of two seed stocks on major part of the scions. dwarfing property of “morabbaei” seed stocks overcame in the next season which caused shoot length shortening in appropriate growth rhythm and shoot thickness increasing and internode number and decreasing rootstock and scion diamet e r . a p a r t o f l e s s e x p e c t e d b e h a v i o r o f “ r e d delicious” on “morabbeai” stock which shoot length and internode length was both increased, but other traits like shoot thickness, internode numbe , and scion diameter followed normal dwarfing trend. all the other growth traits assessed in the rest of four scion cultivars were highly affected by dwarfing effect of “morabbeai” seed progeny, without exception (table 2). weibel et al. (2003) indicated that differences in peach shoot length were related primarily to the internode length rather than the node number, whereas seleznyova et al. (2003) attributed the differences in apple branch size to the reduction in neoformed internode length and node number. mean of internode length depended on node number. it means that shorter internodes are caused by lower node number (costes and garcia-villanueva, 2007). measured diametric values of graft union and scions on self-incompatible “azayesh” seed stocks s h o w e d h i g h e r t h a n t h o s e o n s e l f c o m p a t i b l e “morabbaei” f1 seed progeny. scion-rootstock discontinuity may lead to burrknot formation and high diametric difference between rootstock and trunk that may be a site where xylem may be affected by modified cell diameters or cell disorganization between different scions and rootstocks (atkinson et al., 2003; tworkoski and fazio, 2011). such morphological differences may in turn affect hydraulic conductivity, root hormones production, or nutrient transport and explain the differences in growth. tworkoski and fazio (2015) reported that the scion cultivars “gala”, “sm.9”, and “sm.27” reduced rootstock of “g.41” and “g.11” compared with “fuji” scion. in the second year, the most of fruit scions on “ m o r a b b a e i ” s e e d h a d s m a l l e r d i a m e t e r t h a n “azayesh” without significant difference, except for “red delicious” (table 2). this implies that rootstocks may affect scion. so, shoot number was not affected b y r o o t s t o c k t y p e , w h e r e a s t h e e f f e c t o f rootstock×scion, scion and year resulted significant. “red delicious” and vigorous “golab-e kohanz” on “morabbaei” seed rootstock had the highest and lowest brn, respectively (table 3). “morabbaei” f1 seeds induced more new shoots than “azayesh” seed stocks. generally, the mandatory cut back of leader in spindle pruning method activates the latent buds, forming new lateral shoots due to removal of apical dominance. this practice however was performed equally on the leader of all combinations. thus, it may be concluded that while, on one side, homozygosis affects canopy size and decreases shoot length, on the other side, the reserved carbohydrates might be used in activation of latent buds in absence of apical dominance. obviously, winter pruning obliges crossing out of adventitious shoots for flower initiation purposes. across all scions, the dwarfing m.9, and seedling rootstock induced the lowest and the highest tree height and scion diameter, respectively. significant interactions indicated that effects of sizecontrolling rootstock on components of shoot growth vary with apple tree growth habit (tworkoski and miller, 2007 a). in this study all the six scions carried the spread habit, for which crotch angle was investigated. meantime, it might be attributed to the hormonal factor due to the genetic factor derived from seed stocks, scions and relative interaction. seedling rootstock might affect growth habits by increasing the auxin/cytokinin ratio. changing in hormonal ratio might be a factor regulating the development of growth habit in apple scions, and rootstock might modify the hormone concentrations in shoot tips (tworkoski and miller, 2007 b). as prerequisite, crotch angle is mentioned as a stable trait and used in cultivar differentiation and cultivar/progeny evaluation (watkins and smith, 1997), and less affected by external factors which is agreed with our results of crotch angle among five scions on two diffrent stocks, except for “red delicious”. in general, the grafted scions on “morabbaei” seeds showed wider crotch angle than “azayash” progeny, probably for higher fruit weight as a result of higher set. the significant differences of crotch angle between “golden delicious” and “granny smith” on “azayesh” seed rootstocks, in the first year, can be attributed to the stability of this trait, whereas lack of differences in the second year would be justified by pruning effect. it is however very important that the single cultivars on “azayesh” didn’t show significant differences in adv. hort. sci., 2019 33(1): 77-85 84 terms of crotch angle with the same cultivar on “morabbaei”, except for “red delicious” (table 2). however, endogenous growth regulators mainly auxins and cytokinins play a major role in the control of lateral bud development (sachs and thimann, 1967). there are numerous reports of growth-regulating chemicals affecting branch crotch angle (verner, 1938; williams and billingsley, 1970; elfving and forshey, 1977). warner (1991) reported that rootstock affects primary scaffold branch crotch angle of apple trees, which is consistent with our results. also lenticels number of annual shoots unit, defined as stable trait like crotch angle, was affected by rootstocks and year, and interaction of rootstock × scion × year. comparisons of rootstocks for each cultivar, in 2014, showed that “golden delicious”, “granny smith”, “braeburn” and “golab-e kohanz” were affected by seed stocks for lenticels number, and in 2015, “golden delicious” and “braeburn” were affected for this trait (table 2). however, the use of lenticels number may be successfully applied for cultivar/hybrid evaluations when more cultivars are grafted on similar rootstocks. the self-compatible parents are able to supply seeds characterization with high genetic purity. furthermore, the improved seed rootstocks seem to be the preferred solution to combat genetic variability, suitable for heavy and alkaline soils and inappropriate slopes. conventional breeding remains the main method to release new clonal or seed rootstock (wertheim and webster, 2005). seed rootstock improvement program was started to impede use of a mixture of seeds with unknown origin and with high genetic variability and evolved into adequate genetic purity, which is the basic element for standard apple tree production. references atkinson c.j., else m.a., taylor l., dover c.j., 2003 root and stem hydraulic conductivity as determinants of growth potential in grafted trees of apple (malus pumila mill.). j. exp. bot., 54: 1221-1229. brown s.k., maloney k.e., 2005 malus × domestica apple, pp. 475-511. in: litz r.e. 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branches of apple trees with cytokinins and gibberellic acid. j. amer. soc. hort. sci., 95: 649-651 żurawicz e., lewandowski m., 2014 controlled freezing as a low-temperature tolerance test for apple rootstocks. acta horticulturae, 1058: 451-456. impaginato 379 adv. hort. sci., 2018 32(3): 379-387 doi: 10.13128/ahs-23129 yield, quality and antioxidants of peeled tomato as affected by genotype and industrial processing in southern italy f. de sio 1, m. rapacciuolo 1, a. de giorgi 1, a. trifirò 1, b. giuliano 2, l. vitobello 1, a. cuciniello 3, g. caruso 3 (*) 1 stazione sperimentale per l’industria delle conserve alimentari (ssica), via faustino tanara, 31, 43100 parma, italy. 2 associazione nazionale industriali delle conserve alimentari vegetali (anicav), viale della costituzione isola f/3, 80143 napoli, italy. 3 d i p a r t i m e n t o d i a g r a r i a , u n i v e r s i t à d i n a p o l i f e d e r i c o i i , v i a università, 100, 80055 portici (na), italy. key words: long-type fruit hybrids, lycopene, polyphenols, pre-processing and post-processing production, sensorial features, solanum lycopersicum l. abstract: research was carried out on processing tomato in san severo (tavoliere delle puglie, foggia, italy) in order to compare four long-type fruit hybrids oriented to peeled produce (abbundo, umex, superpeel, taylor), using a randomized complete block design with three replicates. the hybrid superpeel reached the highest marketable yield due to the highest fruit number and mean weight; along the peeling chain, umex and taylor showed the highest processing efficiency. titratable acidity and sodium were highest in taylor fruits, whereas the highest fiber content was detected in abbundo fruits. compared to pre-processing fruits, peeled tomatoes showed increased values of total and soluble solids as well as reducing sugars, but decreased sugar ratio and colour. the highest concentrations of antioxidants in processed fruits were recorded in umex for lycopene and in superpeel for β-carotene. compared to pre-processing fruits, in peeled tomatoes lycopene and β-carotene concentrations remained stable and polyphenols increased referring to fresh weight. the hybrids examined did not show univocal trends in terms of sensorial features. 1. introduction tomato is the most cultivated vegetable species worldwide with 5,023,810 ha (fao, 2014); italy is a major european producer of processing tomato with a surface area as much as 79,761 ha devoted to this crop (istat, 2017 dati.istat.it), of which 32% are located in emilia-romagna and 25% in apulia. the italian industry of tomato and its derivatives (peeled, diced and sauce) attained an export gross marketable yield as (*) corresponding author: gcaruso@unina.it citation: de sio f., rapacciuolo m., de giorgi a., trifirò a., giuliano b., vitobello l., cuciniello a., caruso g., 2018 yield quality and antioxidants of peeled tomato as affected by genotype and industrial processing in southern italy. adv. hort. sci., 32(3): 379-387 copyright: © 2018 de sio f., rapacciulo m., de giorgi a., trifirò a., giuliano b., vitobello l., cuciniello a., caruso g. a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 13 april 2018 accepted for publication 7 september 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(3): 379-387 380 much as about 1.5 billion of euros in 2017 (istat, 2017). these products are traditionally addressed to united kingdom, usa, france, australia, japan, germany, belgium, but also to growing demanding markets, i.e. eastern europe, south america (brazil and argentina), south africa, south corea, united arab emirates. the interest of processing factories in southern italy to valorize peeled-oriented tomato type matches the seed company goal to provide farmers with hybrids showing high yield, improved taste and flavour features, and better industrial requirements compared to the current genotypes. with these prospects, the new hybrids are evaluated with regard to the main traits, such as: plant architecture and parasite resistance; morphology, number, weight, ripeness uniformity, technological and quality performances of fruits. in order to carry out tomato genotype selection, some authors suggested to perform a comprehensive evaluation using synthetic agronomic and quality indexes (carli et al., 2011), upon assessing an appreciable number of related variables such as dry matter, soluble solids, sugars, acidity and antioxidants. notably, high dry matter and soluble solids are desirable characteristics for the canned tomatoes industry since they improve the quality of the processed product (de pascale et al., 2001). indeed, soluble solid content and titratable acidity are the main components responsible for tomato flavor (kader, 1986; flores et al., 2008) and they are most likely to match the consumer perception of the internal quality (baldwin et al., 1998; arazuri et al., 2007). in this respect, the balanced ratio between sugars and organic acids is important to sweetness, sourness and overall flavor intensity in tomatoes (de bruyn et al., 1971; stevens et al., 1977). in fact, high acids and low sugars will produce a tart tomato while high sugars and low acids will result in a tasteless tomato (kader, 1986). due to their antioxidant attributes in addition to sensorial appealing (raiola et al., 2016), tomatobased products are associated with a low risk of both cancer and incidence of coronary heart disease (pernice et al., 2010; ilahy et al., 2011). the present research was carried out since new hybrids for peeled tomato with improved features are needed for the cultivation in southern italy and, in this respect, they were compared with a widespread cultivar in southern italy, in terms of yield, technological, quality and sensorial characteristics. 2. materials and methods field conditions research was carried out on processing tomato at de maio farm, located in san severo (tavoliere delle puglie, foggia, southern italy) in 2017 on silty-sandy soil. the experimental protocol was based on the comparison between 4 long-type hybrids oriented to peeled produce: abbundo (hm clause italia spa., italy); umex (syngenta italia spa., italy); superpeel (united genetics italia spa., italy); taylor (nunhems italy srl, italy) as a control. a randomized complete block design was used for the treatment distribution in the field, with three replicates, and the elementary plot had a 67 m2 surface area. the transplant was performed on 28 april, arranging a double-row layout, with 35 cm between the plants spacing along the rows, 40 cm between the two rows in each couple and 150 cm between the outer rows of two adjacent couples, thus achieving a density of 3 plants per m2. the ordinary farming technique related to processing tomato in tavoliere delle puglie was followed a n d h a r v e s t s w e r e p r a c t i c e d b e t w e e n 3 a n d 7 august. yield, quality and technological determinations when the 90% fruits were ripe, the following agronomic determinations were made in each plot: weight of marketable fruits (red + colour turning point) and waste berries (green + rotten); mean fruit weight on a random 100 fruit sample; middle length and width on a random 20 fruit sample; percentage of fruit coverage exerted by plant canopy, referred to the ranges of 0-25%, 25-50%, 50-75%, 75-100%. quality analyses of pre-processing fruits sampled in each field plot, as well as technological and quality determinations of processed fruit samples of each h y b r i d w e r e p e r f o r m e d a t t h e l a b o r a t o r i e s o f stazione sperimentale per l’industria delle conserve alimentari in angri (salerno). as for technological determinations, the processi n g y i e l d w a s a s s e s s e d , r e p r e s e n t i n g t h e r a t i o between the canned tomato fruit amount, after selection and technological process, and the marketable yield obtained in the field. the fruit processing was performed using the pilot plant available at ssica. in this respect, tomato peeled production was carried out on a semi-industrial scale, with juice addition, packaged in painted tinplates of 1 kg; notably, the juice obtained by the same cultivar was added de sio et al. peeled tomato for processing industry 381 after partial concentration at 7.5°brix. in order to assess the processing yield, weights were recorded at each different step, the product to be peeled underwent selections and weighing of each fruit fraction such as yellow and necrotized, rotten, broken, undersized; moreover, skins were weighed after peeling. as concerns the product submitted to the juice chain, yellow, necrotized and rotten fruits, as well as skins and refining seeds were weighed. next, the drained fruit percentage was assessed, calculated as a mean of five cans; all the determinations were performed in triplicate and averaged. the fruit quality features assessed on both fresh and peeled fruits, referred to fresh weight, were: total solids (ts), soluble solids (ss), reducing sugars (glucose and fructose) and sucrose, colour (a/b ratio), antioxidants (lycopene, b-carotene, total polyphenols). in addition, the components included in the current european nutritional label, according to eu regulation 1169/2011, were determined: proteins, titratable acidity, lipids, fatty acids, fiber, salt and sodium. the analytical procedures were performed according to caruso et al. (2012) for total and soluble solids, sugars, proteins, fiber, ash and sodium; mipaf (1973) for titratable acidity and fats; golubkina et al. (2015) for fatty acids; conti et al. (2015) for colour; de sio et al. (2001) for carotenoids; golubkina et al. (2017) for polyphenols. briefly, total solids were assessed in oven at 70°c under vacuum until steady weight, whereas soluble solids by means of a digital refractometer. sugars were determined by hplc, using the 600e waters chromatographic system and a column sugar-pak waters at 85°c, edta-ca in water solution as eluent (50 mg l-1). proteins were assessed with the kjeldahl method, by a foss tecator digestor with a kjeltec 2300 distiller. fibre was determ i n e d a f t e r t h e s a m p l e s w e r e w e i g h e d , d r i e d (105°c), gelatinized in the presence of heat-resistant α-amylase and enzymatically digested by proteases and amydoglucosydase, to remove proteins and starch, whereas soluble fibre was precipitated by ethanol; the residue was filtered, washed with ethanol and acetone, dried, weighed and split into two fractions to determine proteins and ash, and fibre content as the difference to the residue weight. sodium was assessed by atomic adsorption spectrophotometry, after sulpho-nitric mineralization, with a model 1100 perkin-elmer spectrophotometer. fatty acids were detected by gas chromatography via appropriate methyl ethers chromatography on capill a r y g l a s s c o l u m n , u s i n g a n a g i l e n t 6 8 9 0 g a s cromatograph, equipped with a flame ionization detector; the peaks of fatty acids methyl ethers were identified by comparison to the retention times of reference standards. colour was assessed by a h u n t e r a s s o c i a t e l a b o r a t o r i e s d 2 5 a m o d e l colourmeter, using a suitable measurement cell with the standard bcr n. 1266 reference whose values are l= 25.7, a= 23.7, b= 14.8; chromatic parameter values are expressed in the hunter scale as a/b. carotenoids were assessed through hplc, using a waters alliance chromatograph equipped with photodiode array detector mod. 996, performing the determinations at 450 nm on a reversed phase column ymc-pack c30 (250 x 4.6 mm i.d.) filled with 5 µm average particle size. polyphenols were determined in water extract through a spectrophotometer (unico 2804 uv, usa); the concentration was calculated according to the absorbance at 730 nm, using 0.02% gallic acid as an external standard. sensorial determinations sensorial or organoleptic determinations were performed on processed tomato samples of each hybrid, which were coded and anonymously analyzed by a panel test team composed of ten specialists in tomato derivatives and five fellows at the first panel experience. each expert evaluated the samples under neutral light (4000 k) and his opinion was reported in a form including 11 sensorial variables. among the latter, five were considered of primary importance and the remaining as their detailing. with regard to p ri mary vari ab l es, th e sco re ran ged fro m zero (extremely unpleasant) to ten (extremely pleasant) and, in particular: colour score zero was matched to brick red and ten to bright red; firmness score zero was associated to chewiness resistance absence and ten to extremely tough product. as for secondary variables, the form delivered to the panel test team was elaborated in order to minimize the fluctuations caused by the first-experience fellows. moreover, the following perceptions and the related scores in brackets were taking into account: absence (2), mild presence (4), medium presence (6), strong presence (8); the scores were used to calculate the average value per each sample. statistical processing the data relevant to agronomic, technological, quality and sensorial determinations were statistically processed by analysis of variance, with the ensuing duncan’s multiple range test for mean separation at 0.05 probability level. the percentage values were subjected to angular transformation before processing. adv. hort. sci., 2018 32(3): 379-387 382 3. results and discussion the hybrids did not significantly differ in terms of crop duration, presumably due to the high temperatures and lack of rainfall during the crop cycles (fig. 1), which led to fruit ripeness and harvest anticipation. from yield and biometrical data reported in table 1, it arises that hybrid superpeel attained the highest marketable yield (175.2 t·ha-1), as much as 95.2% of the total yield, due to the very low waste production. the productive result derived from the combination of the fruit number per plant (90.7) and mean weight (77.4 g), with the berries showing higher values of diameter and length compared to abbundo and umex respectively, and higher thickness than abbundo and taylor. the hybrids abbundo and umex gave 32% lower marketable yield than superpeel due to lower values of the fruit number, size and weight as well as a higher waste production. the hybrid taylor ranked in the middle between the highest yielding superpeel and the least productive abbundo and umex. indeed, it provided with a 19.6% lower yield than superpeel but 15.4% higher production than the other two genotypes; this result was the consequence of the lowest fruit number (71) per plant but the highest berry dimensions and mean weight (79.3 g) as well as also the lowest fraction of waste produce. no stati sti cal l y si gn i fi can t d i fferen ces were recorded between the hybrids in terms of fruit covering by vegetation, which exceeded 75%. consistently with our findings, in previous investigation (portugal et al., 2015) hybrid productive performances ranged between 110 and 160 t·ha -1, whereas in other research (caruso et al., 2016; peixoto et al., 2017) tomato genotypes showed a wide range of yields under the 70 t·ha-1 threshold. with regard to processing efficiency (table 2), umex showed the highest value (87.0%) though not statistically different from the control. similarly, along the peeling chain umex and taylor showed the best performances (81.5% as an average), whereas fig. 1 ten-day means of temperatures and rainfall in san severo (foggia, italy) in 2017. ns= not significant; within each column, the values followed by different letters are statistically different according to duncan’s multiple range test at p≤0.05. table 1 yield and biometrical parameters of long-type processing tomato hybrids hybrid total fruits marketable fruits waste fruits weight (%/total) weight (t·ha-1) number per plant weight (t·ha-1) weight (%/total) number per plant mean weight (g) diameter (cm) length (cm) length/ width flesh thickness (mm) abbundo 128.7 c 93.0 b 119.3 c 92.7 82.7 b 57.8 b 4.1 c 7.8 ab 1.89 7.0 b 7.3 a umex 126.0 c 91.0 b 118.9 c 94.3 78.5 b 60.4 b 4.3 bc 7.7 b 1.79 7.5 ab 5.7 b superpeel 184.1 a 97.7 a 175.2 a 95.2 90.7 a 77.4 a 4.6 ab 8.1 ab 1.76 7.8 a 4.8 c control 146.8 b 79.0 c 140.8 b 95.9 71.0 c 79.3 a 4.7 a 8.4 a 1.77 7.0 b 4.1 d ns ns ns= not significant; within each column, the values followed by different letters are statistically different according to duncan’s multiple range test at p≤0.05. table 2 processing yield of four hybrids for peeled tomato hybrid processing yield (%) waste fruits along peeled chain (%) waste fruits along juice chain (%) total peeled juice yellow and necrotized rotten broken undersize skins yellow and necrotized rotten skins and seeds abbundo 81.5 b 72.4 b 90.1 13.3 a 0.6 a 6.3 a 1.3 6.3 c 5.0 a 3.0 b 2.0 c umex 87.0 a 82.0 a 92.2 4.0 d 0.0 b 5.8 b 0.8 7.4 b 2.8 c 2.2 c 2.9 a superpeel 82.7 ab 75.1 b 90.5 9.3 b 0.7 a 6.1 ab 0.6 8.2 a 4.3 b 2.9 b 2.3 b control 86.3 ab 80.9 a 91.8 7.9 c 0.2 b 3.8 c 0.7 6.5 c 2.9 c 3.4 a 1.9 c ns ns de sio et al. peeled tomato for processing industry 383 no stati sti cal l y si gn i fi can t d i fferen ces were recorded between the hybrids referring to: total solids (7% on average), soluble solids (6.1%), reducing sugars (3.8%), sugars ratio (54.3%), proteins (1.6%), lipids (0.3 %), energetic value (24.5 kcal·100 g1 or 104,5 kj·100 g-1), colour (1.82), glucose (1.5%), fructose (1.6%), sucrose (0.04%), fat acids expressed as saturated (0.07%), monounsaturated (0.06%) and polyunsaturated (0.14%). compared to pre-processing fruits, peeled tomatoes showed increased values of total and soluble solids as well as reducing sugars, but decreased levels of sugar ratio and colour; moreover, ph of fresh fruits was not significantly affected by the hybrid, ranging between 4.4 to 4.5. in previous research (raiola et al., 2018) the comparison between the values recorded before and after processing was genotype-dependent, except for titratable acidity and sugar ratio which always decreased and increased respectively, upon processing. high total solids content in fruits is an industrial target, as it reduces the processing costs; this parameter in tomato varies around the 5-6% average and it is affected by some factors such as cultivar, soil type and climate conditions during the growing and no significant differences arose as concerns the juice yield (91.2% as an average). i n t e r m s o f w a s t e a l o n g t h e p e e l i n g c h a i n , abbundo attained the highest occurrence of yellow and necrotized fruits, umex the lowest and taylor the lowest broken berry percentage. the undersized and rotten fruits were very few and superpeel had the highest skin fraction. as for waste along the juice chain, abbundo showed the highest percentage of yellow and necrotized fruits, which was 79% higher than the lowest one corresponding to umex; the latter hybrid also had the lowest value of rotten berries and the highest of skin and seeds. the results relevant to the quality indicators of processed tomato (tables 3 and 4) show that significant effects of the hybrid were recorded on the following parameters: titratable acidity and sodium attained the highest value in taylor fruits (0.37% and 7.4 mg·100 g-1 respectively) and lowest in abbundo; the fiber content recorded in abbundo fruits was 17% higher than taylor one; ash and salt concentrations showed the highest levels in umex (0.54% and 20.4 mg·100 g-1 respectively) though the latter variable was not significantly different from taylor. table 3 quality features (referred to fresh weight) of peeled tomato fruits obtained from four hybrids ns= not significant; within each column, the values followed by different letters are statistically different according to duncan’s multiple range test at p≤0.05. hybrid total solids (g·100 g-1 ) soluble solids (°brix ) reducing sugars (g·100 g-1) titratable acidity (g anhydrous citric acid 100 g-1) sugar ratio (%) proteins (g·100 g-1) fats (g·100 g-1) fiber (g·100 g-1) energetic value (kcal·100 g-1) colour (a/b) abbundo 7.0 6.1 4.0 0.27 c 56.7 1.56 0.27 1.32 a 24 1.82 umex 7.0 6.1 3.8 0.29 c 53.5 1.52 0.31 1.23 b 25 1.81 superpeel 7.0 6.1 3.7 0.33 b 53.5 1.59 0.26 1.19 bc 24 1.80 control 7.1 6.2 3.8 0.37 a 53.6 1.64 0.32 1.13 c 25 1.85 ns ns ns ns ns ns ns ns average 7.0 6.1 3.8 54.3 1.82 peeled/fresh (%) + 24.7 + 21.8 + 18.2 5.2 30.6 table 4 sugars, fatty acids and mineral components (refereed to fresh weight) in peeled tomato fruits produced by four hybrids ns= not significant; within each column, the values followed by different letters are statistically different according to duncan’s multiple range test at p≤0.05. hybrid sugars (g·100 g-1) fatty acids (g·100 g-1) ash (g·100 g-1) sodium (g·100 g-1) salt (g·100 g-1) glucose fructose sucrose saturated monounsaturated polyunsaturated abbundo 1.48 1.57 0.04 0.06 0.06 0.13 0.47 c 5.6 c 10.3 b umex 1.54 1.58 0.04 0.07 0.07 0.15 0.54 a 6.4 b 20.4 a superpeel 1.46 1.58 0.04 0.06 0.05 0.13 0.51 ab 5.9 c 9.8 b control 1.49 1.60 0.04 0.08 0.07 0.15 0.50 bc 7.4 a 19.5 a ns ns ns ns ns ns 384 adv. hort. sci., 2018 32(3): 379-387 harvesting season (siddiqui et al., 2015). other authors (majkowska-godomska et al., 2008) recorded a total solids content of tomato fruits ranging from 3.83 to 7.00%. campos et al. (2006) and kader et al. (1987) reported that values of soluble solids below 4.5% are considered low for industrial tomatoes; in this respect, turhan and seniz (2009) found this quality indicator ranging between 5.0 to 5.5 % in processing tomato fruits and in other studies (cramer et al., 2001; de pascale et al., 2001) soluble solids values of tomato fruits ranged from 4 to 6%. sugar content is positively and highly correlated with total soluble solids in tomato fruit and, notably, fructose has a large impact on the sweetness perception (tieman et al., 2012). in our research the sum of glucose and fructose accounted for the 80% of the total sugars, whereas it attained about 65% in previous investigations (stevens et al., 1977; jones and scott, 1984; malundo et al., 1995). other authors found the total sugar content of ripe tomato ranging from 1.7 to 4.7% (petro-turza, 1987; turhan and seniz, 2009) or from 0.54 to 3.44% (melkamu et al., 2008) of fresh weight. in previous research, titratable acidity (ta) in tomato fruits ranged from 0.22 to 0.40% (turhan and seniz, 2009) or even from 0.25 to 0.70 (george et al., 2004). according to beckles (2012), values of total soluble solids and titratable acidity as much as 5.0 and 0.4% respectively are considered desirable to produce a good-tasting tomato. titratable acidity is mainly affected by citric and malic acids which reportedly attain about 15% of the dry content in ripe fresh tomatoes (petro-turza, 1987). kamis et al. (2004) states that taste and flavour of tomato fruits are positively correlated to sugars and organic acids content. moreover, in addition to flavour the organic acids influence ph, the latter being an important factor in canned tomato products to control the growth of thermophilic microorganisms (yousef and juvik, 2001). the influence of ph on the thermal conditions applied along the tomato processing chain is mainly associated to producing safe products and, in this respect, values below 4.5 prevent microorganism proliferation in the final product (campos et al., 2006; garcia and barrett, 2006). notably, some authors did not detect varietal dependent ph differences in tomato berries (kerkhofs et al., 2005), whereas in other research ph showed differences among cultivars (benal et al., 2005; frusciante et al., 2007) even in the wide range of 3.78 to 5.25 (turhan and seniz, 2009). mineral element concentration in tomato fruits may reach 8% of dry matter and influences tomato fruit taste through the effect on ph and titratable acidity (siddiqui et al., 2015). with regard to antioxidants (table 5), lycopene attained a 16% higher concentration in umex compared to abbundo; superpeel showed a 43% higher β-carotene content than abbundo; polyphenols concentration was the lowest in the control fruits, but did not significantly differs between the three hybrids examined. compared to fresh fruits, in peeled tomatoes lycopene and β-carotene concentrations remained stable and polyphenols increased referring to fresh weight, whereas in relation to total solids lycopene had a 19.1% decrease whereas polyphenols just a slight reduction (5.2%). in previous research (binoy et al., 2004) significant differences were found in lycopene and phenolic contents between the different genotypes, with lycopene showing 1 to 4 fold and 1 to 2 fold variation on fresh and dry weight basis respectively. moreover, unlike our findings where lycopene remained stable and polyphenols increased upon industrial processing table 5 antioxidants concentration in peeled tomato fruits obtained from four hybrids within each column, the values followed by different letters are statistically different according to duncan’s multiple range test at p≤0.05. hybrid lycopene β-carotene mg·kg-1 total polyphenols mg·kg-1 mg·100 g-1 ts mg eq. gallic acid 100 g-1 mg eq gallic acid g-1 total solids abbundo 127.0 c 181.9 c 2.1 c 36.7 a 5.3 a umex 146.6 a 208.3 a 2.3 bc 36.0 a 5.1 a superpeel 136.2 b 195.3 b 3.0 a 35.7 a 5.1 a control 131.0 bc 184.7 bc 2.4 b 31.7 b 4.5 b average 135.2 192.6 2.5 35.0 5.0 peeled/fresh (%) 0.9 -19.1 1.0 17.6 -5.2 385 de sio et al. peeled tomato for processing industry and referred to fresh weight, dewanto et al. (2002) reported the increase of lycopene concentration with no changes in polyphenols content in processed tomato fruits compared to raw berries. however, in other research (pavlović et al., 2017) the antioxidants content in tomato fruits decreased upon processing thermal treatment, though the significance and amplitude of the differences are genotype dependent, and it is also affected by temperature settings (jabbari et al., 2018). the scores resulted from the panel test performed by an expert team in tomato organoleptic evaluation and their subsequent statistical processing allowed to obtain the sensorial profiles which convey the immediate and clear quantitative and descriptive perception of the analyzed products. the graphic representation of qda (quantitative descriptive analysis) obtained by processing the evaluation forms filled in by the experts is shown in figure 2 a. taking into account the high number of data and in order to make it easier to interpret the profiles, the sensorial variable data considered negative for the relevant hybrids were extrapolated. in particular, the data related to strange taste and flavour and to acidity were clustered (fig. 2 b): the profiles and the statistical processing performed show that there are no significant differences between the hybrids. the sensorial profiles of the positive variables are shown in figure 2 c. moreover, from data statistical processing reported in table 6, it can be observed that the peeled product obtained from the hybrids abbundo and umex is statistically different in terms of colour, aspect and flavor; in addition, abbundo is statistically different from taylor for the colour and from superpeel for the sensation of fresh flavor. 4. conclusions from research carried out on the comparison between long-type hybrids oriented to peeled produce in tavoliere delle puglie (foggia, southern italy), superpeel showed the best yield performances (175.2 t·ha-1) with about 21% higher production than the average value attained by abbundo, umex and taylor, as a consequence of the combined effects of fruit number and mean weight. however, the highest processing efficiency was recorded for the hybrid umex and taylor, whereas no hybrid displayed an overall superiority in terms of quality features and sensorial profile. acknowledgements the authors wish to thank: the seed companies h.m. clause italia, syngenta italia spa and united genetics italia for the financial contribution intended for carrying out the research; the plant nursery “aniello cerrato” in sarno (salerno) for producing the fig. 2 organoleptic evaluations of peeled tomato fruits: comparison of the sensorial profiles of the four hybrids (a), sensorial profiles of the undesired features, named negative (b); sensorial profiles of the desired features, named positive (c). variable cultivar δ s aspect abbundo umex -1.43 0.007 colour abbundo umex -1.30 0.013 colour abbundo control -1.10 0.035 flavour abbundo umex 1.00 0.050 flavour 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constituents of fruits of selected tomato genotypes grown in turkey. afr. j. agric. res., 4(10): 1086-1092. yousef g.g., juvik j.a., 2001 evaluation of breeding utility of a chromosomal segment from lycopersicon chmielewskii that enhances cultivated tomato soluble solids. theor. appl. genet., 103(6-7): 1022-1027. impaginato 295 adv. hort. sci., 2019 33(2): 295-298 doi: 10.13128/ahs-23986 tobacco dust waste as an alternative medium to grow geranium (pelargonium x hortorum) plants s. tzavara, a.i. darras (*), a. assimakopoulou department of agriculture, university of peloponnese, 24100 kalamata, greece. key words: agricultural waste, ec, ornamentals, peat, ph. abstract: tobacco dust (td) waste is the typical lignocellosic agricultural residue of cigarette processing. in the region of peloponnese, cigarette production is carried out by the leading company of karelias s.a. the production of td waste of approx. 2-3 tons/month is a major problem for the company. plant growth media containing peat (p) + 0, 5, 10, 25 or 50% td were prepared and tested on geranium plant growth and development. the use of td increased ec and ph of the final medium. plants of the cvs “ml diego” and “ml sailing ‘12” grown in p+5% or in p+10% td had similar height, number of leaves, number of flowers, photosynthetic activity and transpiration rates to the p alone (control) indicating that solid agro-industrial waste of tobacco could be used to partially substitute peat in growing medium for floricultural crop production. 1. introduction pelargonium x hortorum or ‘‘zonal geranium’’ is an ornamental species that originate from south africa, perfectly adapted to the mediterranean region. it is propagated by cuttings and it is a hybrid between p. inquisaus (l.) l’herit and p. zonale (l.) l’herit (dole and wilkins, 2005). zonal geraniums attract an increased commercial interest as they are extensively used in landscape designs and terrace gardens (berninger, 1993). flowering is strongly dependent on growth stage (i.e. juvenility), temperature (i.e. cold requirement) and sunlight (i.e. intensity and duration), but zonal geraniums are not described as longor short-day plants (fonteno, 1992; dole and wilkins, 2005). agricultural waste such as cotton gin trash, olive mill and green waste have been used in combination with peat for cultivation of ornamental plants (papafotiou et al., 2004; 2005; grigatti et al., 2007). the residue from tobacco processing (i.e. the tobacco dust; td) is buried to landfields, but in high w/w concentrations can be toxic due to its high tannin and alkaloid content (briski et al., 2003). compared to other waste material, td contains higher n and k and has ph values ranging between 5.0 and 6.0 (aderidan et al., 2003). the application of td waste in soil cultivated with lettuce increased yield compared to control plants (okur et al., (*) corresponding author: tassosdarras@yahoo.co.uk citation: tzavara s., darras a.i., assimakopoulou a., 2019 tobacco dust waste as an alternative medium to grow geranium (pelargonium x hortorum) plants. adv. hort. sci., 33(2): 295-298 copyright: © 2019 tzavara s., darras a.i., assimakopoulou a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 22 september 2018 accepted for publication 20 march 2019 ahs advances in horticultural science short note http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(2): 295-298 296 2008). it was suggested that incorporation of td waste as an alternative organic amendment might improve soil chemical and biological parameters, as well as crop yield in soils containing low organic matter content. apparently, no previous research has been conducted on utilization of tobacco byproducts as alternatives to grow ornamental plants. in the present study, we tested growing mediums of peat + increasing concentrations of td on growth and flowering response of zonal geraniums. 2. materials and methods plant material, media preparation and experimental lay-outs zonal geranium (pelargonium x hortorum) rooted cuttings (3-5 leaves; up to 12 cm height) of cvs ‘ml diego’ (red inflorescences) and ‘ml sailing’12’ (white inflorescences) were provided by selecta-one ltd (kavala, greece). single rooted cuttings were transplanted in plastic 2.5 l pots filled with growing mediums of peat + td. td was provided by karelias s.a. (kalamata, greece) and samples were analyzed in a continuous flow analyzer (cfa αα3; seal-analytical ltd., germany) (table 1). peat (hawita, germany) was mixed with 0, 5, 10, 25 and 50% (w/w) td (table 2). six-replicate pots per treatment with geranium plants were placed on the ground of a non-heated greenhouse at the premises of the university of peloponnese (lat. 37° 2’ 20’’ n, long. 22° 6’ 51’’ e) in a completely randomized design. two individual experiments were carried out (one for each cultivar) from november 2017 to february 2018. medium properties, plant assessments and statistical analysis medium ph and ec (ms/cm) were measured using a ph/mv meter (delta ohm hd 2105.2, padova, italy) and a conductivity handheld meter (eutech instruments, ecoscan con 5, singapore), respectively. plant height (cm), number of leaves and number of inflorescences were recorded weekly over the entire cultivation period of nine weeks. chlorophyll fluorescence, net co2 assimilation (as; μmol m-2 sec) and transpiration (e; mmol m-2 sec) were recorded using a handheld fluorimeter (os-30p, opti-sciences, inc. u.s.a.) and a lcpro+ portable photosynthesis system (adc bioscientific ltd. great amwell, herts, uk), respectively, on the 5th, 6th, 7th and the 8th week from transplanting (i.e. week-1). data means were separated using duncan’s multiple range test at p = 0.05. statistical analysis was performed in spss v. 21. 3. results tobacco dust contained nicotine, tss and small amounts of nitrates and ammonia (table 1). ec and p h v a l u e s r a n g e d b e t w e e n 8 . 9 2 a n d 9 . 4 6 , a n d between 5.3 and 5.6, respectively. after mixing peat with 0 50% td, ph and ec of the final growing medium increased linearly to reach the values of 5.42 and 6.18 ms/cm, respectively (table 2). table 1 means and range values of tobacco dust content tobacco dust samples were analyzed in a continuous flow analyzer tobacco dust content means range nicotine (mg/l) 158.18 157.84 -160.14 tss (mg/l) 583.12 581.36 -584.04 nitrates (mg/l) 32.06 30.15-33.02 ammonia (mg/l) 6.38 6.25 6.52 ec (ms/cm) 9.17 8.92-9.46 ph 5.5 5.3-5.6 table 2 ph and ec (ms/cm) values of growing media after mixing peat with tobacco dust at 0 (control), 5, 10, 25 and 50% (w/w) growing media ph ec (ms/cm) mixing peat 4.00 0.06 mixing peat + 5% tobacco dust 4.51 1.30 mixing peat + 10% tobacco dust 4.85 3.22 mixing peat + 25% tobacco dust 5.09 4.84 mixing peat + 50% tobacco dust 5.42 6.18 peat replacement with 5 or 10% td in the growing medium positively affected growth and flowering of cvs. “ml diego” and “ml sailing’12” geraniums (table 3). plants of cv. “ml diego” grown in p+5% or p+10% td maintained similar or higher height, number of leaves, number of inflorescences, net co2 assimilation and transpiration rates to the control plants (i.e. p+0% td) (table 3). however, plants grown in p+25 or p+50% td suffered reductions in growth and flowering compared to the controls. plants grown in p+25 or p+50% td showed reduced number of inflorescences and transpiration rates, compared to the plants grown in p+5 or p+10% td (table 3). stunting growth and reduced flowering of plants cultivated in p+25 or p+50% td were justified tzavara et al. tobacco dust an alternative medium to grow geranium 297 by decreases in chlorophyll fluorescence (fv/fm) ratios (table 3). the fv/fm ratios ranged between 0.571 and 0.893 for plants grown in p+25% td and between 0.538 and 0.835 for plants grown in p+50% td indicating damage in plants’ photosystem (ps ii) that induced stress responses. plants of cv. “ml sailing’12” grown in p+5, p+10 or p+25% td, showed similar or higher number of leaves, number of inflorescences, net co2 assimilation and transpiration rates to the control plants (table 3). however, plants grown in p+50% td showed reduced height, number of leaves, number of inflorescences, indicating damage in plants’ photosystem (ps ii) recorded as low fv/fm ratio ranging between 0.538 and 0.773 (table 3). plants in p+50% td failed to reach minimum growth requirements and standard commercial size. 4. discussion and conclusions td successfully replaced part of peat in growth medium for zonal geranium plant production. plants of cvs. “ml diego” and “ml sailing 12” responded well when grown in p+5, p+10 and in few cases in p+25% td and, therefore, could potentially replace part of peat in the growth medium. the concept of peat replacement with agricultural waste material for the cultivation of ornamental plants has been examined the past 20 years. papafotiou et al. (2004) showed that olive-mill waste composts (owc) could partially replace peat for the production of euphorbia pulcherrima (poinsettia), although, at concentration of >12.5% delayed growth compared to plants cultivated in peat/perlite medium. tropical potted plants such as syngonium podophyllum, ficus benjamina and codiaeum variegatum could be successfully grown in 75% owc without showing symptoms of toxicity or other negative effects on growth and development (papafotiou et al., 2005). cotton gin trash compost (cgtc) and rice hulls (rh) were tested as peat replacements for the production of nerium oleander, pelargonium zonale, dedranthema grandiflora and lantana camara (papafotiou et al., 2001). replacing peat with 60% of gctc resulted in plant height decrease of all species, except those of p. zonale and increase in number of flowers to all species, except those of d. grandiflora. the use of green waste and sewage sludge compost (wssc) at 80% 20% (v:v) as a 25%-replacement of white peat, had positive effects on growth and flowering of begonia semperflorens, mimulus hybridus, tagetes patula x erecta and salvia splendens (grigatti et al., 2007). all species grown in 25% wssc showed greater height, number of flowers and plant dry weight compared to plants grown in 100% white peat. td is a potent agricultural byproduct that could be used in concentrations of <25% without affecting growth and quality of ornamentals. replacing peat with byproducts of the agricultural sector, merits an eco-biological prospect of environmental-friendly ornamental production. further research is needed to test td as peat replacement for cultivation of other ornamental species. data are means ± se of 9-week recordings and letters indicate the statistical differences according to duncan’s multiple range test at p = 0.05. not measured. plants failed to reach minimum growth requirements. table 3 effect of growing medium of peat amended with 0, 5, 10, 25 and 50% td on number of leaves, plant height, number of inflorescences, chlorophyll fluorescence, net co2 assimilation and transpiration of p. x hortorum plants of cvs "ml diego" and "ml sailing'12" treatments plant height (cm) range (cm) number of leaves range number of inflorescences range net co 2 assimilation (μmol m-2.sec) range (μmol m-2.sec) transpiration (mmol m-2.sec) range (mmol m-2.sec) chlorophyll fluorescence (f v /f m ) range (f v /f m ) ml diego' 0 8.83±0.25 ab 4-16 11.82±0.41 c 4-24 0.85±0.07 a 0-3 2.18±0.26 a 0.34-6.11 1.00±0.087 a 0.61-1.92 0.805±0.001 a 0.778-0.834 5 8.89±0.27 ab 2-18 13.50±0.66 b 4-33 0.86±0.09 a 0-6 3.01±0.34 a 0.24-7.14 1.36±0.170 a 0.71-2.57 0.801±0.002 a 0.685-0.828 10 9.12±0.24 a 5-17 15.12±0.68 a 2-36 0.93±0.10 a 0-5 2.40±0.29 a 0.22-5.32 1.28±0.184 a 0.42-2.34 0.802±0.002 a 0.753-0.878 25 8.39±0.23 b 4-15 12.11±0.55 bc 4-28 0.60±0.08 b 0-3 2.08±0.20 a 0.28-4.00 0.65±0.060 b 0.33-1.06 0.786±0.004 b 0.571-0.893 50 7.01±0.19 c 4-18 5.79±0.32 d 2-20 0.31±0.05 c 0-2 2.23±0.68 a 0.14-4.90 0.37±0.082 c 0.13-0.61 0.731±0.006 c 0.538-0.835 ml sailing'12' 0 9.70±0.33 a 5-16 14.11±0.62 bc 5-24 0.83±0.10 ab 0-3 2.07±0.32 b 0.34-4.07 0.89±0.096 a 0.29-1.40 0.801±0.001 a 0.778-0.819 5 9.53±0.38 a 2-18 15.79±1.00 ab 4-33 0.76±0.11 ab 0-3 3.40±0.52 a 0.24-7.14 1.04±0.161 a 0.30-2.72 0.793±0.003 a 0.685-0.818 10 9.07±0.34 a 5-17 16.68±1.01 a 4-36 0.98±0.16 a 0-5 2.40±0.62 ab 0.22-5.32 1.17±0.232 a 0.32-2.27 0.799±0.001 a 0.777-0.824 25 7.64±0.30 b 4-14 12.94±0.83 c 4-28 0.53±0.11 b 0-3 1.77±0.40 b 0.28-3.69 0.48±0.057 b 0.26-0.71 0.779±0.005 a 0.668-0.893 50 6.25±0.14 c 4-9 5.01±0.16 d 3-7 0.20±0.06 c 0-1 0.678±0.008 b 0.538-0.773 adv. hort. sci., 2019 33(2): 295-298 298 acknowledgements we sincerely thank karelias s.a. for providing tobacco dust used in the present study references adediran j.a., baets n.d., mnkeni p.n., kiekens l., muyima n.y.o., thys a., 2003 organic waste materials for soil fertility improvement in the border region of the eastern cape, south africa. j. biol. agric. hortic., 20: 283-300. berninger l.m., 1993 status of the industry, pp. 1-2. in: white j.w. 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component for the production of poinsettia. scientia hortic., 102(2): 167175. impaginato 79 adv. hort. sci., 2018 32(1): 79-92 doi: 10.13128/ahs-20646 cotton flowering behavior, fiber traits and gene expression under watershortage stress d. jawdat 1 (*), a.w. allaf 2, n. taher 1, a. al-zier 2, n. morsel 1, z. ajii 2, b. alsafadi 1 1 department of molecular biology and biotechnology, atomic energy commission, po box 6091, damascus, syria. 2 department of chemistry, atomic energy commission, po box 6091, damascus, syria. key words: cotton, fiber quality, flowering, ftir, gene expression, tga, xrd. abstract: two accredited cotton (gossypium hirsutum l.) cultivars (aleppo 118 and deir al-zour 22) have been investigated to assess physiological, morphological, and molecular responses under water shortage conditions. both cultivars have shown early flowering. however, higher percentage of treated ‘aleppo 118’ plants kept flowering towards the end of the growing season compared to treated ‘deir al-zour 22’ plants. both cultivars kept consistent micronaire and cohesion under normal and water shortage conditions. the cultivar aleppo 118 displayed more consistent fiber quality between control and treated plants, while ‘deir al-zour 22’ showed variation in fiber length and strength between control and treated plants. results have demonstrated an increase in fold expression of dreb1a, tps and hspcb genes in the flowering stage of treated plants compared to the controls. results also showed a continuous activation of dreb1a gene in the two critical growing stages of a cotton life cycle, flowering and boll development in treated plants of ‘deir al-zour 22’. this work illustrates the different responses of two cotton cultivars under water shortage stress and its impact on flowering and fiber traits. 1. introduction the supply of groundwater in agriculture is reduced due to intersecting environmental, industrial and domestic sectors. groundwater-dependent agro-economies are mostly affected by drought stress, water deficits and aquifer depletion, specifically in the arid and semi-arid regions. drought is considered a major constraint to crop productivity and yield stability around the world. it is therefore, a major challenge to farmers in drought subjected regions. water saving and stable crop production are the main challenges that urge researchers to develop new cultivars and irrigation strategies for a sustainable use of water in agriculture. developing new cultivars that tolerate water stress has been assisted (*) corresponding author: ascientific2@aec.org.sy citation: jawdat d., allaf a.w., taher n., al-zier a., morsel n., ajii z., al-safadi b., 2018 cotton flowering behavior, fiber traits and gene expression under water-shortage stress. adv. hort. sci., 32(1): 79-92 copyright: © 2018 jawdat d., allaf a.w., taher n., al-zier a., morsel n., ajii z., al-safadi b. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 10 may 2017 accepted for publication 12 january 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(1): 79-92 80 and supported by wide range of strategies and procedures that enable the selection of candidate parents and an efficient screening of targeted progenies. genomics, marker-assisted selection, the manipulation of qtls using genetic engineering and transcriptomics can contribute in the release of improved, drought-tolerant cultivars (tuberosa and salvi, 2006). the metabolomics-assisted breeding is another candidate approach for the development of crops with increased tolerance to abiotic stresses (fernie and schauer, 2009). improving drought tolerance in crops requires understanding the biochemical responses under water deficit (reddy et al., 2004) and the characterization of drought patterns in the growing regions along with the physio-morphological traits of these crops under such environments (fukai and cooper, 1995; khan et al., 2010). plants have evolved their drought-adaptive strategies (meyre et al., 2001), between drought escape (early flowering) and drought tolerance. comprehending the flowering pattern of each crop under stressed and non-stressed environments is a key to which drought-adaptive strategy the crop plant follows. early flowering and maturity can help in saving groundwater for irrigation and can help in avoiding early rainfall that usually affects the harvest of certain crops such as cotton (jawdat et al., 2012). therefore, a vigilant assessment of crop responses to alteration in irrigation regimes is needed. at any rate, amendments and management of irrigation and water saving methods has a great potential in saving water sources in the arid and semi-arid regions (oweis et al., 2011; ünlü et al., 2011). cotton is a major cash oilseed and fiber crop with a world production estimated to be 25.01 million tonnes in 2013-2014 as announced at the official w e b s i t e o f t h e i n t e r n a t i o n a l c o t t o n a d v i s o r y committee (icac) (https://www.icac.org). the widely famous, allotetraploid gossypium hirsutum l. has a distinct growth manner and can be useful in monitori n g g r o w t h a n d d e v e l o p m e n t a l c h a n g e s u n d e r diverse conditions (jawdat et al., 2012). cotton is grown in around 70 countries where the irrigated system dominates in arid regions; and is used to supplement rainfall in the humid regions to ensure yield stability. seed cotton production in syria (mostly semi-arid where cotton is irrigated) was around one million tonnes in 2000. a decrease in production from around 670 k tonnes in 2009 to around 472 k tonnes in 2010 and then up to about 671 k tonnes in 2011 in the same cultivation area (~200 k hectares), was attributed to the drought wave that hit the country in 2010. a rapid decline in production was recorded between 2011 and 2014, from around 670 to around 162 k tonnes respectively. this was concurrent with the unfortunate events taking place in syria. the principal objective of this paper is to assess physiological, morphological, molecular, and biochemical responses of two accredited cotton cultivars in syria under deficit water regime, in benefits of future breeding programs. 2. materials and methods plant materials plant material consisted of cotton seeds of two g. hirsutum l. accredited local cultivars: aleppo 118 [aleppo 40 (aleppo 1 x acala sj1) x american cultivar bw 76-31] and deir al-zour 22 (a selected line from deltapine 41). drought stress treatment seeds were sown in rows, where the distance between rows and plants were kept at 75 and 25 cm respectively. experimental plots were designed according to a randomized complete block design (rcbd) with each treatment is replicated once in each block. the experiment site was located in doubaya in damascus country side (980 m above sea level, 33° 29´ 36´ n and 36° 04´ 57´ e). the experiment consists of two irrigation treatments (control with full irrigation and treated with 50% of control irrigation), two cultivars (aleppo 118 and deir al zour 22) and three replicates per treatment. the soil (total n: 0.08%, available p: 12 ppm, k: 1.30 meq/100 g, and organic matter: 1.19%) was prepared by tractor ploughing deep for 45 cm, followed by two surface ploughings for 15 cm deep. fertilization was conducted following the cotton research administration (cra) guidelines and recommendations. nitrogen fertilizer as urea 46% [co(nh2)2] was applied at a rate of 415 kg/ha in four unequally split applications, 20% before sowing, 40% 30 days after sowing, 20% at floral bud emergence, and 20% at bolls initiation phase. phosphorus fertilizer [ca(h2po4)2] was applied at a rate of 130 kg/ha before sowing. potassium fertilizer was applied at the rate of 170 kg/ha in the form of potassium sulphate (k2so4). drip irrigation was applied at 250 m3/ha, at first, for both control and treated blocks. consequent irrigations were applied during the growing season of cultivated plants once a week at 250 m3/ha (full irrigation) in control blocks, and once in every 10 days at 125 m3/ha (deficit irrigajawdat et al. cotton behaviour under water stress 81 tion; receiving water at 50% of control treatment) in the treated blocks. except for one week where temperatures reached around 45 degrees in the afternoon, we had to irrigate both control and treatment blocks with emergency full irrigation. the distance between emitters was 25 cm and the emitter discharge was 8 l/h. thinning of plants was conducted around 4 weeks of germination. physiological data chlorophyll a and b content. leaves samples (100 mg/sample) were each immersed in 4 ml of 95% acetone and incubated at 6-8°c for 24 hrs. chlorophyll a and b concentration was determined spectrophotometrically by measuring the absorbance (optical density-od) at 662 and 644 nm respectively. the concentrations of chlorophyll a and chlorophyll b (μg. g-1 fw) in leaf tissues were calculated using the following equations (cha-um et al., 2006): chlorophyll a= 9.784*d 662 0.99*d 644 chlorophyll b= 21.426*d 644 4.65*d 662 where di is an optical density at the wavelength i. osmotic potential op) and osmotic adjustment (oa). osmometer readings in mosm/kg, were taken using the freezing point osmometer, the microosmoettetm (precision systems inc., usa). frozen 100 mg of leaf samples in liquid nitrogen were ground (a pool of three leaves per treatment). two milliliters of autoclaved d.d. water were added to each sample and samples were vortexed briefly and centrifuged for one minute at 13000 rpm. fifty microliters of the supernatant were used for each reading. the osmotic potential ψs was calculated using the following formula: op (mpa)= {r * t * osmometer reading}/1000 where r is the gas constant (0.008314), and t is the laboratory temperature in kelvin (298). the osmotic adjustment (oa) was calculated as the difference between the osmotic potential of non-stressed plants and the water stressed plants. morphological data twenty plants of each replicate were randomly tagged and flowering behavior data for days after planting (dap) were recorded such as: first floral bud emergence, plant height at first flower, and nodes above white flower (nawf). the percentage of plants bearing flowers and plants bearing floral buds were recorded towards end of season. end of season data also included: plant height, root depth (randomly selected plants from each replicate were carefully pulled out of ground), and number of secondary roots. physical and chemical fiber properties fiber quality testing. thirty bolls of each replicate were picked and sent to the fiber quality lab at the cotton research administration (cra) for testing: micronaire, fiber length, fiber strength, fiber cohesion, and ginning percentage. cra fiber quality lab conducts its testing according to the international testing standards with a temperature of 22±2°c and relative humidity of 64%±4%. the lab uses the following instruments: micronaire 775, digital fibrograph, pressley tester and stelometer for fiber testing. fourier transform infrared (ftir) acquisition spectra of cotton fiber ftir was conducted to study the chemical variance, if any, between fibers from control and treated plants of the two studied cultivars. the infrared spectra were recorded on nicolet 6700 in the range 4000 to 400 cm-1 with a resolution of 4 cm-1 at 32 scans as direct measurements of the cotton fibers (three replicates per treatment) samples using universal attenuated total reflectance technique ftir-atr with krs-5 crystals. the instrument is equipped with dtgs detector and kbr beam splitter. the operating system used was the omnic software (version 7.3, thermo nicolet, usa). the infrared spectra of fiber samples for control and treated plants of aleppo 118 and deir al zour 22 cultivars were recorded. the recorded spectra were repeated three times for each investigated sample. measurement of crystallinity by conventional x-ray powder diffractometry (xrd) xrd was applied to determine the crystallinity of fiber samples from control and treated plants of ‘aleppo 118’ and ‘deir al-zour 22’ samples. x-ray powder diffraction patterns were obtained on a stoe t r a n s m i s s i o n s t a d i p d i f f r a c t o m e t e r w i t h monochromatic cu k α1 radiation (λ = 1.5406 å) selected using an incident-beam curved-crystal germanium ge (111) monochromator, with the stoe transmission geometry (horizontal set-up) and a linear position-sensitive detector (psd). the determination of crystallinity of the samples was performed with the program winxpow (stoe and cie, 1999) using single-sample method. this method requires defining the portions of the diagram due to air scatter and inelastic scattering, amorphous scattering and bragg or crystal scattering. if ic(2q) and ia(2q) are known for the whole diagram, the crystallinity index is calculated from the relation: xc=(∑ic(2θ)⁄(lp∙f∙t)⁄(∑[(ic (2θ)⁄t+ia (2θ)]⁄(lp∙f) eq. 1 adv. hort. sci., 2018 32(1): 79-92 82 t h e s u m m i n g i s o v e r b r a g g a n g l e ( 2 q ) . t h e lorentz-polarization factor lp, the average form factor f and the temperature factor t are all 2q-dependent functions: lp(2θ)= 1 + cos2 (2θ) ⁄ (sin2 (2θ) cosθ eq. 2 f(2θ)= ∑f (n, 2θ) eq. 3 t(2θ)= exp(-2b sin22θ) ⁄ λ2 ) eq. 4 the summing in eq. 3 is over all atoms in the formula unit. the sample’s overall formula and average temperature factor (b) have to be known in order to be able to apply this method. it’s noteworthy to mention that sample absorption is always neglected; therefore, this method works well for transmission data of organic samples within ± 3% error of the true crystallinity. the formula of cellulose (c6h12o5) and b = 4.0 å2 was used for our cotton fiber samples. thermogravimetry analysis (tga) and differential scanning calorimetry (dsc) the dynamic weight loss of fiber samples as a function of increasing temperature tests were conducted using a mettler instrument (tg50). the tests were carried out in a nitrogen atmosphere, purged (30 ml/min) using sample weights of 10-15 mg at a heating rate of 10oc/min. the resolution of the balance is given, as 1 microgram for weights less than 100 milligrams, and the temperature precision of the instrument is ± 2oc. dsc was used to determine the glass transition temperatures of the prepared samples. a mettler instrument (dsc20) was utilized to record the dsc spectra. all samples were tested in aluminum pans at a heating rate of 10oc/min over a temperature range from room temperature to 400oc. the precision of the used instrument is ± 0.2oc. scanning electron microscopy (sem) scanning electron microscopy (vegaii xmu, tescan, czech republic) with an accelerating voltage of 30 kv, was operated to capture sem images of cotton fibers coming from control and treated plants of both cultivars. gene expression analysis rna isolation and cdna synthesis. total rna was isolated from leaves of cotton plants using the modified hot borate method (jawdat and karajoli, 2012). quality of total rna was tested by agarose-formaldehyde gel electrophoresis using standard protocols. the iscripttm select cdna synthesis kit (biorad, usa) was used to obtain first strand cdna starting from 1 µg total rna following the manufacturer’s protocol. the cdna was diluted 4x and stored at -20°c. relative quantification of gene expression the cdna samples (control and treated) were u s e d a s a t e m p l a t e t o q u a n t i f y t a r g e t g e n e s (dreb1a, tps and hspcb) expression level. realtime pcr was performed in two replicates in a 25 µl of a reaction mixture composed of 12.5 µl iq sybr super mix (biorad, usa), 1 µl of cdna, 1 µl of each of the forward and reverse primer (10 µm), and 9.5 µl of d.d. water. the quantification of mrna levels was normalized with the level of mrna for ghef1α5 (artico et al., 2010). the relative expression (fold expression) was calculated using the -∆∆ct method as follows: 2-∆∆ct = 2[(ct treated target gene – ct treated reference gene ) (ct untreated target gene ct untreated reference gene )] specific target and reference gene primers are listed in table 1. 3. results physiological, morphological, molecular and chemical data were recorded to identify main changes in two cash cultivars of cotton. the physiological and molecular analysis were carried out on leaf material from control and treated plants of ‘aleppo 118’ and ‘deir al-zour 22’, harvested 45 dap table 1 target and reference genes accession numbers and primers sequences gene genbank accession no. forward and reverse primers (5’-3’) dehydration responsive element binding gene (dreb1a) ay321150.3 f-agctatagcactgagagggaag r-gcttcttcgtccaagtaaaacc trehalose-6-phosphate-synthase gene (tps) ay628139.1 f-ttcactacatgctgcccatgtg r-ggctgtggaggaaaaaaccaag heat-shock protein calmodulin binding gene (hspcb) ay819767.1 f-ctccttgaatgtatttactgcc r-gtgcgtcctctagtgtcttt elongation factor 1-alphagene (ef1α5) dq174254 f-tccccatctctggttttgag r-cttgggctcattgatctgggt jawdat et al. cotton behaviour under water stress 83 in the course of floral bud initiation stage. chlorophyll a and b content and osmotic potential the chlorophyll a content showed a non-significant decreasing trend in treated plants compared to control plants in both cultivars. however, the chlorophyll b content showed a small increment in the treated plants. the chlorophyll a/b ratio showed a reduction in treated plants of both cultivars. a larger reduction (18%) was observed in treated plants of ‘deir al-zour 22’ compared to the small reduction (4%) in treated plants of ‘aleppo 118’ (fig. 1 inset graph). a non-significant drop in the osmotic potential of leaves in treated plants was observed in both cultivars and the osmotic adjustment was higher (0.016 mpa) in ‘aleppo 118’ compared to ‘deir alzour 22’ (0.006 mpa) (fig. 1). plant morphology early floral bud emergence was observed in both treated cultivars compared to control plants (fig. 2 inset graph i). the control plants of aleppo 118 cultivars flowered earlier than control plants of ‘deir alzour 22’. nawf counts, another indicator of flowering earliness, was also recorded in the third week of f i r s t f l o w e r e m e r g e n c e ( f i g . 2 i n s e t g r a p h i i ) . r e c o r d e d n a w f c o u n t s s h o w e d a s i g n i f i c a n t decrease in treated plants of both cultivars. a final record of plants with flowers, and plants with floral bud percentages were also taken five months after planting towards the end of season (fig. 2). end of season flowering counts have shown ‘aleppo 118’ tendency to keep initiating floral buds (~ 21%) in treated plants compared to control plants (5.6%), and produce fully opened flowers (~ 31%) in treated compared to control plants (~ 2%). however, a similar but less potent flowering counts pattern was observed in deir al-zour 22 cultivar. end of season measurements have also included plant stem height, taproot depth and number of secondary roots (table 2). treated plants in both cultivars showed a slight increase in plant stem height and minor decrease in root depth. however, non-significant increase in number of secondary roots was observed in treated plants of both cultivars. physical and chemical fiber properties fiber quality testing. the two tested cultivars have presented slightly different behavior in terms of fiber properties. seed cotton yield showed no significant difference between treated and control plants in each cultivar. the cultivar deir al-zour 22 showed a significant increase in both fiber length and strength in the treated plants compared to control ones. fiber fig. 1 the inset graph is chlorophyll a/b ratio in leaves of control (c) and treated (t) ‘aleppo 118’ and ‘deir-al zour 22’ plants. main graph represents osmotic potential and osmotic adjustment in leaves of control and treated ‘aleppo 118’ and ‘deir al-zour 22’ plants. plants were under drip irrigation system, where the control plants were given full irrigation each week, and treated plants have been given deficit irrigation each 10 days. data represent means and standard error of three replications. fig. 2 the inset graph-i shows floral buds emergence in days after planting (dap) in control and treated ‘aleppo 118’ and ‘deir al-zour 22’ plants. the inset graph-ii represents nawf counts in control and treated plants of both cultivars. data were subjected to duncan’s test with a confidence level of 95% using statistica program. columns sharing a letter are not significantly different. data represent means and standard error of three replications. the original graph is the percentages of plants with flowers and plants with floral buds of both control and treated aleppo 118 and deir al-zour 22 cultivars, five months after planting. data were subjected to duncan’s test with a confidence level of 95% using statistica program. line markers sharing a letter are not significantly different (capital letters for plants with floral buds and small letters for plants with flowers. adv. hort. sci., 2018 32(1): 79-92 84 elongation showed no significant difference between c o n t r o l a n d t r e a t e d p l a n t s i n e a c h c u l t i v a r . furthermore, no significant difference was recorded in the cultivar aleppo 118. two fiber properties, fiber cohesion and micronaire, exhibited no significant difference between cultivars and between treatments. a significant increase in fiber maturity was observed in the treated plants of aleppo 118 cultivar, whereas, a non-significant increase was recorded in treated plants of ‘deir al-zour 22’ (fig. 3). ftir-atr spectra acquisition of the cotton fiber. the ftir-atr spectra of fiber samples from control and treated plants of aleppo 118 and deir al-zour 22 cultivars were recorded. the spectra showed a typical characteristic reflectance bands for common cotton fiber substances. the spectra of fiber samples from control and treated ‘aleppo 118’ plants were aligned for comparison and showed minimum variation (fig. 4 a). the spectra of fiber from ‘aleppo 118’ control plants showed 15 distinctive bands centered at 3310, 2890, 1740, 1605, 1429, 1372, 1315, 1203, 1160, 1103, 1055, 1020, 675, 554 and 431 cm-1. the vibration at 1429 cm-1 is designated and assigned as a “crystalline” absorption band of the fiber (abidi et a l . , 2 0 1 4 ) . t h e b a n d a t 1 3 7 2 c m 1 v i b r a t i o n i s assigned to c-h bending, and it may be most suitable for indicating cellulose crystallinity associated with the band at 2890 cm-1. the spectra of control and treated samples were highly similar except for the band at 1740 cm-1 which was absent in the treated sample. similar spectra range was observed in deir al-zour 22 cultivar. however, few noticeable new bands with distinctive structures were observed in the treated sample at 2890, 1740, and 1605 cm-1. the band centered at 2890 cm-1 contains two prominent peaks vibrations at 2910 and 2847 cm-1, which are assigned to ch2 asymmetrical and symmetrical stretching, cultivar plant height (cm) root depth (cm) no. secondary roots control treatment control treatment control treatment aleppo 118 99.3±3.5 ab 105.9±5.5 a 19.8±2.2 17.7±2.1 10.4±0.8 ab 13.1±0.8 a deir al-zour 22 80.3±3.8 c 87.6±5.1 bc 18.3±2.8 17.0±0.7 9.9±0.9 b 11.4±1.1 ab table 2 end of season measurements of plant stem height, taproot depth and number of secondary roots in control and treated plants of aleppo 118 and deir al-zour 22 cultivars data were subjected to duncan’s test with a confidence level of 95% using statistica program. numbers in rows and columns sharing a letter in each block (plant height and no. secondary roots) are not significantly different. data represent means and standard error of three replications. fig. 3 fiber properties of control and treated aleppo 118 and deir alzour 22 cultivars. fiber length (a), fiber strength (b), fiber cohesion (c), fiber elongation (d), m i c r o n a i r e ( e ) , a n d m a t u r i t y index (f). data were subjected to duncan’s test with a confidence level of 95% using statistica program. columns sharing a letter are not significantly different. data represent means and standard error of three replications. jawdat et al. cotton behaviour under water stress 85 respectively. as for the second band at 1740 cm-1, which is assigned to a carboxylic ester group (c=o stretching), a clearer structure is noticed in the treated sample in comparison with the control. another distinctive banding pattern is observed at 1605 cm-1 (adsorbed water), which is neither seen in the treated aleppo 118 cultivar and nor observed in deir alzour 22 cultivar control sample. this band contains two prominent peaks vibrations at 1577 and 1540 cm-1 (fig. 4 b). measurement of crystallinity by conventional x-ray powder diffractometry. fiber analysis using the xrd procedure showed a typical cellulose pattern in both control and treated plants for each cultivar (fig. 5). the amount of crystalline cellulose in studied samples was between 66-75% (table 3). as seen in table 3, there was a minor difference between the ci values of the treated and control ‘deir al-zour 22’ samples. however, there is no variation between control and treated samples of aleppo 118 cultivar. thermogravimetry analysis (tga) and differential scanning calorimetry (dsc). typical dynamic tga thermograms of the studied samples have been recorded and are shown in figure 6 a. the tga thermograms show a slight decrease in the weight and one significant step at high temperature. differential scanning calorimetry (dsc) was used to locate a possible glass transition temperature (tg) of the samples. the glass transition temperature could not be observed in all dsc thermograms. the endothermic peak at around 100oc could be due to evaporation of adsorbed water (fig. 6 b). scanning electron microscopy (sem). sem images reveal the longitudinal morphology of fibers coming from control and treated plants of aleppo 118 and deir al-zour 22 cultivars under three magnifications table 3 fiber crystalline index in control and treated aleppo 118 and deir al-zour 22 cultivars fig. 4 ftir-atr spectra of the cotton fibers obtained from (a) ‘aleppo 118’ control and treated plants and (b) ‘deir alzour 22’ control and treated at room temperature in the range 400-4000 cm-1. the recorded spectra were repeated three times and showed a typical and characteristic absorption bands for common cotton fibers substances. fig. 5 x-ray powder diffraction patterns of fibers in control and treated aleppo 118 and deir al-zour 22 cultivars. cultivar crystallinity index (%) control treatment aleppo 118 66.5±3.0 66.8±3.0 deir al-zour 22 73.7±3.0 68.5±3.0 adv. hort. sci., 2018 32(1): 79-92 86 (1.00 kx, 3.00 kx and 20.00 kx) (fig. 7). images of the two cultivars show the ribbon fiber shape rolled in a helicoid manner, a typical cotton morphological feature. the spiral and twisting fiber nature is present in both cultivars under both conditions. expression analysis of dreb1a, tps and hspcb drought-related genes fold expression of three drought-related genes in cotton (dreb1a, tps and hspcb) was analyzed, assisted by ghef1α5 gene expression for normalization (fig. 8). leaf samples of control and treated plants of both cultivars were harvested at two points (stages), s1 and s2. where s1 represents leaf material harvested at 40 dap (during floral bud to flower transition stage) and s2 represents leaf material harvested at 60 dap (bolls opening and maturation). gene expression analysis showed an activation of the studied genes in treated plants compared to the controls of both cultivars during s1. the s2 stage, experienced a drop in genes activity in both treated cultivars. analysis showed a major significant drop in gene activity (hspcb and tps) in treated ‘deir al-zour 22’ plants (s2) compared to samples of the earlier stage (s1). exceptionally, dreb 1a gene showed a slight decrease in gene expression keeping its high activity in treated plants ~4 fold higher than the config. 6 (a) tga thermograms of cotton fibers in presence of nitrogen. (b) dsc thermograms of cotton fibers in presence of nitrogen. fig. 7 sem images of cotton fibers. a, b and c. fibers of control ‘aleppo 118’ under 1.00, 3.00 and 20.00 kx magnifications. d, e and f. fibers of treated ‘aleppo 118’ under 1.00, 3.00 and 20.00 kx magnifications. g, h and i. fibers of control ‘deir al zour 22’ under 1.00, 3.00 and 20.00 kx magnifications. j, k and l. fibers of treated ‘deir al zour 22’ under 1.00, 3.00 and 20.00 kx magnifications. jawdat et al. cotton behaviour under water stress 87 trols. on the other hand, aleppo 118 cultivar showed an almost steady expression pattern of the three genes in the two stages (high expression in s1 and low in s2). this continuous high expression activity of dreb 1a needs to be further investigated in ‘deir alzour 22’ to study its relation to water deficit tolerance. 4. discussion and conclusions this study overlooks the behavior of two accredited cotton cultivars subjected to two irrigation regimes along their life cycle. it is anticipated that water scarcity in the mediterranean region will increase under climatic change and this can be addressed by evaluating the impacts of climate change on water resources and their management, the adaptive capacity and the policy responses (iglesias et al., 2011). improving water resources management is challenged by the physiology and biology of the crop, irrigation practices, environment, farmers’ perspectives, and government funding. increasing crop water productivity, i.e. producing more food, income, better livelihoods and ecosystem services with less water (molden et al., 2010), has been a major interest to agronomists, farmers, environmentalists and economists. our research is mainly interested in understanding the responses of two credited syrian cotton cultivars grown under water-shortage stress conditions, motivated by the concept of reaching a better quality and quantity of cotton fiber coupled with saving ground water resources in a cotton growing country. recently, drip irrigation systems have been widely employed, due to their improvement of water use efficiency (patanè et al., 2011). hence, our field experiments were irrigated using the drip irrigation system to reduce water runoff losses. two credited cotton local cultivars, aleppo 118 and deir al-zour 22, have been investigated in our study. the two cultivars are assigned by the ministry of agriculture to be grown in their pertinent environments in the syrian land. ‘deir al-zour 22’ is the featured cultivar for cultivation in deir al-zour province, along the euphrates, where high temperature is the dominant feature of the area especially during the growing season. ‘aleppo 118’, an early maturity cultivar, is cultivated in the arid and semi-arid northernwest regions of syria, mostly in aleppo province. control (full irrigation) and treated (deficit irrigation) plants of both cultivars were tested using some physiological, morphological, molecular, and biochemical parameters. the restricted water regime applied on our treated plants showed mild, non-significant effects on chlorophyll a and b content, which indicates a weak impact of the water-shortage stress regime on the photosynthesis process. however, the cultivar aleppo 118 showed a slight reduction in chlorophyll a/b ratio in treated plants compared to a larger reduction in treated plants of ‘deir al-zour 22’, which indicate the presence of higher chlorophyll b content in treated ‘deir al-zour 22’ plants. this in turn may suggest a slower degradation process of chl b coming from a decrease in the enzymatic activities that are known to render the conversion of chl b to chl a (folly and engel, 1999). it is suggested that drought stress has to be prolonged and severe before the chloroplast start to break down (jiang et al., 2 0 1 0 ) . a n o t h e r n o n s i g n i f i c a n t d r o p w a s a l s o observed in the osmotic potential values of treated plants in both cultivars. however, the cultivar aleppo 118 showed a greater osmotic adjustment value which may indicate its stronger turgor maintenance mechanism. osmotic adjustment has been also associated with maintenance of membranes and protein structure, protection against oxidative damage (dacosta and huang, 2006). it has been reported that the greater the stress duration (increasing the number of stress cycles), the larger the osmotic adjustfig. 8 fold expression of dreb1a, tps and hspcb genes in leaves of treated ‘aleppo 118’ and ‘deir al-zour 22’ plants. leaf samples were collected in two stages, s1 (during floral bud to flower transition stage) and s2 (bolls opening and maturation). data were subjected to duncan’s test with a confidence level of 95% using statistica program. significant fold expression change of tps and hspcb genes was observed between s1 and s2 treated ‘deir al-zour 22’. data represent means and standard deviation of three replications. adv. hort. sci., 2018 32(1): 79-92 88 ment in cotton leaves (oosterhuis and wullschleger, 1987). the morphology and phenology of plants can be significant indicators of drought tolerance. changes in plant growth rate and/or in flowering time are two common strategies that plants use to cope with drought (schmalenbach et al., 2014). the allotetraploid g. hirsutum l. species has a distinct growth manner in which the plant keeps a systematic morphological architecture (khan, 2003) that can be useful in monitoring growth and developmental changes under diverse environmental conditions (jawdat et al., 2012). in general, physiological processes are induced in plants under stress conditions to reduce the cellular damage and to alter developmental timing to complete their life cycle fittingly (yaish et al., 2011). stress-mediated flowering has been reported in several plant species; stresses like ultraviolet c, poor nutrition, low temperature and drought (wada and takeno, 2010). our results noted earliness in treated plants of both varieties and a tendency of ‘aleppo 118’ to bear higher percentage of flowers and floral buds toward the end of season. ‘aleppo 118’ has been reported to show excessive vegetative growth under excess water and nitrogen compared to other local cultivars (cra, personal communication). this may suggest that water status (excess and shortage) may hold back or trigger floral cues, and this needs to be investigated fully taking into consideration our result of the higher osmotic adjustment in ‘aleppo 118’ compared to ‘deir al-zour 22’. the small increase in stem height in treated plants of both cultivars can be explained that plants accumulate reserves in shoots and stems to cope with water stress (chavez et al., 2002). the water deficit regime, applied in our study through drip irrigation, encouraged the increase in number of secondary roots in treated plants of both cultivars on the expenses of a decrease in tap root length. fiber quality, which is a result of interactions between genetics and environment, controls fiber prices of cotton textile products (wang et al., 2014). the quest for cotton growers is the balance between fiber quality and quantity. achieving such a balance in cotton cultivation regions with growing water shortages is a main concern to the agricultural and industrial sectors. an escalating number of publications aim at understanding the mechanism of cotton fiber development and adaptation to abiotic stresses, such as drought, for the improvement of cotton fiber yields and quality (zhu et al., 2011; deeba et al., 2012; padmalatha et al., 2012; bowman et al., 2013; riaz et al., 2013; wang et al., 2013; zhang et al., 2013; sekmen et al., 2014; xie et al., 2015). cotton growth and yield are severely affected by excessive water-shortage stress, especially at critical growth stages such as the flowering phase (dağdelen et al., 2009). soil water was found to be correlated with fiber strength, elongation (johnson et al., 2002) and fiber maturity (davidonis et al., 2004). our results showed that two fiber properties, cohesion and micronaire, had no significant differences between control and treated plants of the two cultivars. it is with fiber cohesion, a property that causes materials to cling together, yarn spinning from staple fiber is possible (wakelyn, 2007). a previous study showed fiber cohesion stability of ‘deir al-zour 22’ between s e a s o n s a n d l o c a t i o n s ; w h e r e a s , ‘ a l e p p o 1 1 8 ’ showed cohesion stability between seasons in one location (jawdat et al., 2012). as for fiber micronaire, it reflects fiber fineness and is a measure of internal fiber thickness and deposits of cellulose. it has a desired value range in the international market between 3.5 and 4.9 (jawdat et al., 2012). fiber micronaire, showed stability in both ‘aleppo 118’ and ‘deir al-zour 22’ between seasons and locations (jawdat et al., 2012). this indicates that environmental factors including water deficit have weak impact on the two fiber properties, cohesion and micronaire. deficit irrigation showed no significant effect on fiber length and strength in aleppo 118 cultivar. however, it had significant impact on the two fiber properties, causing increment in both strength and length of fiber in treated plants of deir al-zour 22 cultivar. fiber strength is an indicator of fiber resistance to stretching (wang et al., 2014) and is determined by environmental conditions and cultivar traits (zhou et al., 2011). it has been found that fiber strength is correlated with daily mean temperature during fiber development (hanson and ewing, 1956; ma et al., 2006; wang et al., 2009). this has been also observed on a previous study where a difference of 4-5°c showed a significant effect on fiber strength in both ‘aleppo 118’ and ‘deir al-zour 22’ (jawdat et al., 2012). however, this temperature difference did not affect fiber length in both cultivars (jawdat et al., 2012). this shows the impact of each of water deficit and temperature on fiber strength of ‘deir al-zour 22’ compared to the impact of only temperature on fiber strength of ‘aleppo 118’. fiber maturity can be defined as the relative wall thickness and wall development (wakelyn, 2007). in our work, we observed an increase in fiber maturity factor values in treated jawdat et al. cotton behaviour under water stress 89 plants compared to the controls, which suggests that water deficit did not affect the maturity of cotton fiber in a negative way and hence their quality as related to dye-ability and ease of processing. t h e f t i r a t r s p e c t r a o f t h e c o t t o n f i b e r s obtained from control and treated aleppo 118 and deir al-zour 22 cultivars showed similar spectra range except for few noticeable bands. this can be due to a reduction in surface water adsorption which leads to stronger fibers. the minimized accessibility of water molecules to the internal hydroxyl groups can be mostly due to the formation of cellulose macromolecules that induce a re-organization of cellulose and also increase the ordered cellulose (or crystalline) portion to produce a stronger fiber (liu, 2013) in deir al-zour 22 treated cultivar. results of xrd showed minor ci variation between fibers from control and treated deir al-zour 22. both tga and dsc showed no variation between fibers of treated and control plants in both cultivars. our work has also investigated gene fold change i n e x p r e s s i o n o f t h e t r a n s c r i p t i o n f a c t o r g e n e (dreb1a), the molecular chaperone gene (hspcb) (sotirios et al., 2006), and the trehalose biosynthesis gene (tps) (kosmas et al., 2006). the two genes hspcb and tps have been found in few studies to be expressed differently between drought-tolerant and drought-sensitive cotton genotypes. while the hspcb showed differential expression during the drought period in leaves of drought tolerant genotypes and not in the sensitive ones (nepomuceno et al., 2002; voloudakis et al., 2002), the tps gene was induced during the water stress period in both tolerant and s en s i ti ve cu l ti va rs ( nep o mu cen o et a l . , 2002) . interestingly, our study showed a significant reduction in hspcb and tps genes activity in leaves during bolls opening and maturation in both cultivars. whereas, dreb 1a gene kept a high active mode in leaves of treated ‘deir al-zour 22’ plants compared to ‘aleppo 118’ treated plants during that stage. dehydration-responsive element binding proteins (drebs) are transcription factors known to activate the expression of abiotic stress-responsive genes in divergent species via specific binding to the dehydration-responsive element/c-repeat (dre/crt) cis-acting element in promoters of target genes (mizoi et al., 2012). in arabidopsis, the overexpression of dreb1a revealed both freezing and dehydration tolerance in transgenic plants (liu et al., 1998). a better drought and salt tolerance was observed in transgenic wheat plants with dreb1 from glycine max (shiqing et al., 2005). the overexpression of oryza sativa dreb1 in rice transgenic plants has also showed improved tolerance to drought, salt and low temperature stresses (dubouzet et al., 2003; ito et al., 2006). a group of researchers found that dreb 1a was induced at a high level in pistils, three days after drought treatment which suggests its possible role as a n e a r l y d r o u g h t r e s p o n s e r e g u l a t o r i n t h e arabidopsis flower (su et al., 2013). our study adds to such findings and points to dreb 1a constant upregulation in leaves of ‘deir al-zour 22’ under drought stress, during flowering and boll maturation. our study has presented the responses of two accredited cotton cultivars, aleppo 118 and deir alzour 22 which have shown different behavior under water shortage. both cultivars have shown early flowering. however, a larger percentage of treated ‘aleppo 118’ plants kept flowering towards end of season compared to treated ‘deir al-zour 22’ plants. this may indicate a potential survival mechanism and fruiting cycle extension in aleppo 118 cultivar under water deficit regime, compared to the cultivar deir al-zour 22. aleppo 118 cultivar displayed more consistent fiber quality between control and treated plants, while ‘deir al-zour 22’ showed variation in fiber length and strength between control and treated plants. both cultivars kept consistent micronaire and cohesion under normal and water deficit conditions. two critical stages in cotton life cycle, flowering and boll development, were screened for hspcb, tps and dreb 1a gene activity using leaf material. results have demonstrated an increase in fold expression of these genes in the flowering stage of treated plants compared to the controls. a large regression in genes activity was noticed during boll development except for dreb1a gene in treated ‘deir al-zour 22’ plants. dreb1a gene showed continuous activation in the two stages and can be considered a candidate gene to support water deficit stress tolerance mechanism in this cultivar. the question of which cultivar is more droughttolerant than the other is to be deeply investigated in both cultivars, since each cultivar has shown different approach towards water-shortage stress tolerance. acknowledgements the authors would like to thank the director general of aecs and the head of molecular biology and biotechnology department for their support. the adv. hort. sci., 2018 32(1): 79-92 90 authors would like to extend their thanks to mrs. intissar kara joli for lab assistance, dr. moufak roukaya for xrd analysis, the physics department for sem imaging and the agrarian office at the aecs for land and drip irrigation preparation. references abidi n., cabrales l., haigler c.h., 2014 changes in the cell wall and cellulose content of developing cotton fibers investigated by ftir spectroscopy. carbohydr. polym., 100(0): 9-16. artico s., nardeli s.m., brilhante o., grossi-de-sa m.f., alves-ferreira m., 2010 identification and evaluation 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y., chen b., zhao x., oosterhuis d.m., shu h., 2011 effect of planting date and boll position on fiber strength of cotton (gossypium hirsutum l.). am. j. exp. agri., 1(4): 331-342. zhu l.-f., he x., yuan d.-j., xu l., xu l., tu l.-l., shen g.x., zhang h., zhang x.-l., 2011 genome-wide identification of genes responsive to aba and cold/salt stresses in gossypium hirsutum by data-mining and expression pattern analysis. agri. sci. china, 10(4): 499-508. impaginato 311 adv. hort. sci., 2018 32(3): 311-318 doi: 10.13128/ahs-22302 postharvest performance of cut rose cv. lovely red as affected by osmoprotectant and antitraspirant compounds e. di stasio, y. rouphael (*), g. raimondi, c. el-nakhel, s. de pascale dipartimento di agraria, università degli studi di napoli federico ii, via università, 100, 80055 portici (na), italy. key words: β-aminobutyric acid, l-proline, rosa spp., stomatal conductance, transpirational flux, water balance. abstract: in cut flowers, the post-harvest turgor is a critical aspect in a system in which, in the absence of the root system, transpiration water losses must be compensated. two experiments were conducted in order to elucidate the effect of osmoprotectants (l-proline) as well as of molecules with antitranspirant behavior (ß-aminobutyric acid baba or pinolene) on the water relations and vase life of rose cut stems. applications of l-proline enhanced water fluxes, water conductivity, relative water content and stomatal conductance of rose cut stems in comparison to untreated plants, thus increasing the vase-life of rose cut flowers. baba treatment reduced the stomatal conductance in rose as well as the daily water consumption, on the other hand senescence phenomena occurred earlier. the water used by pinolene treated stems was lower compared to the control and this was associated with a medium increase of the vase life. overall, enhanced osmoregulation prolonged the vase life of cut flowers since the improved water status allowed the cut stem to partially continue its metabolic functions. on the other hand, the control of transpirational flux was functional in maintaining cellular turgor in pinolene treated cut stems, whereas with baba, senescence phenomena occurred probably due to the activation of biochemical pathway of senescence involving abscisic acid. taking all together, osmoregulation or direct control of transpirational fluxes may provide a promising avenue for improving the post-harvest longevity of cut roses. 1. introduction post-harvest efficiency is a crucial point of the cut flowers commercial value since it is related to growth and storage conditions interacting with the plant genetic background; those aspects will overall contribute to maximize the stems qualitative performance after cutting (fanourakis et al., 2013). cut flowers are subjected to a wide range of post-harvest losses as developmental senescence, leaf and petal abscission, leaf discolo rati o n , p rematu re wi l ti n g an d d i sease fro m mo u l d s an d fu n gal pathogens (scariot et al., 2014). however, among all, water balance is yet a major factor influencing the longevity of cut flowers, in a system in (*) corresponding author: youssef.rouphael@unina.it citation: di stasio e., rouphael y., raimondi g., elnakhel c., de pascale s., 2018 postharvest performance of cut rose cv. lovely red as affected by osmoprotectant and antitraspirant compounds. adv. hort. sci., 32(3): 311-318 copyright: © 2018 di stasio e., rouphael y., raimondi c., elnakhel c., de pascale s. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 14 december 2017 accepted for publication 18 april 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(3): 311-318 312 which water losses must be compensated by water uptake and transport in the absence of the root system (singh and moore, 1992; lu et al., 2010). in particular, the post-harvest life of cut flowers is strongly dependent on their ability to maintain tissues hydration overtime, and water deficit or wilting mainly occur if the amount of transpiration exceeds the volume of water uptake (halevy and mayak, 1981; van doorn, 2012). one of the first plant responses to abiotic stress (as for cutting) is the stomatal closure, and this mechanism provides protection against tissues dehydration by reducing transpiration from the leaf surface (hare et al., 1998). however, in cut stems stomata are not completely closed after cutting, leading to a residual stomatal transpiration that together with cuticular transpiration, determine additional water losses from the stem (van doorn, 2012). on the other hand, a phenomenon that can severely undermine the cut stems post-harvest performance, is the lowering of water uptake that is mainly due to occlusions located in the basal stem end (he et al., 2006). in rose, one of the main causes of the cut flower wilting is the vascular occlusion determined by bacteria, air e m b o l i a n d p h y s i o l o g i c a l r e s p o n s e s t o c u t t i n g (fanourakis et al., 2013). in rose cut flowers, the regulation of water balance has been in the past decades a key aspect in the improvement of the stems vase life (alaey et al., 2011; reid and jiang, 2012). among several mechanisms suggested that may improve water uptake in response to a stress, hydraulic conductivity variations and accumulation of compatible solutes are the most documented (chen and murata, 2002; ehlert et al., 2009). compatible solutes or osmolytes are organic compounds which help in raising the osmotic pressure and thereby maintaining both the turgor pressure and the driving gradient for water uptake (serraj and sinclair, 2002). common osmolytes found in plants mainly include proline, trehalose, fructan, mannitol and glycinebetaine and these compounds also help in maintaining the structural integrity of enzymes, membranes and other cellular components during the stress regime (zhao et al., 2007; chen and jiang, 2010). compatible solutes may be constitutively overproduced in transgenic plants (zhang et al., 2004) or directly applied on plants to improve stress tolerance under both open-field and protected cultivation (okuma et al., 2004; barbieri et al., 2011; cirillo et al., 2016). in addition to physiological mechanisms that can be exploited to improve water uptake, the reduction of transpirational flux has traditionally been one of the main objectives for controlling the cellular turgor after harvesting of fresh-cut vegetables and ornamentals (prakash and ramachandran, 2000). the reduction of transpiration can improve the water balance of cut flowers and extend their vase life, whereby artificial closure of the stomata might be an efficient strategy to reduce water losses (lu et al., 2010; van doorn, 2012). one method of limiting water loss involves the use of antitranspirants, which reduce plant transpiration forming a vapour-impermeable film on the leaf surface and, among these, the natural terpene polymer di-1-p-menthene (pinolene) is widely applied on different crops (francini et al., 2011; abdel-fattah, 2013). these polymers, also called “film forming antitranspirants”, sprayed on crops in a form of water emulsion, are generally employed to reduce weathering and extend pesticide efficacy, improving distribution and adherence of agrochemicals and decrease water loss and wilting of young transplants (gale and hagan, 1996; percival and boyle, 2009). research conducted on cut roses revealed that treatments with a film-forming antitranspirant are able to reduce the degree of fresh weight loss and water loss during transpiration, delay the process of flower opening and slow down the rate of stomatal conductance reduction (song et al., 2011). alternatively to traditional antitranspirants, the use of compounds which may induce a series of stress adaptation mechanisms, such as stomatal closure, could also be considered. beta-aminobutyric acid (baba) is a non-proteinogenic amino acid known for its ability to increase plant resistance to biotic (jakab et al., 2001; baider and cohen, 2003) and abiotic stresses (jakab et al., 2005). applications of baba may improve the plant tolerance to stress by activation of defense mechanisms mediated by abscissic acid (aba) and salicilic acid (sa) (zimmerli et al., 2000; jakab et al., 2005; baccelli and mauchmani, 2016). taking this background into consideration, it is clear that an efficient control of water balance is crucial to improve cut flowers vase-life and this can be achieved by using molecules that activate water transport in stem or inhibit transpiration. therefore, the aim of this study was to assess the influence of exogenous applications of osmolytes such as proline or anti-transpiring solutions like βaminobutyric acid and pinolene on the water balance, vase-life and also to shed light on the potential di stasio et al. postharvest performance of cut rose cv. lovely red 313 physiological mechanism(s) involved in cut stems of rose. 2. materials and methods plant material and growth conditions two experiments were carried out in order to assess the effects of exogenous applications of lproline (experiment 1) or antitranspirants [specifically: an active compound film forming (pinolene) and a stomatal closure inducing active compound βaminobutyric acid ; experiment 2] on water control in cut stems of rose plants (rosa spp. l.) cv. lovely red. cut flowers of rose were harvested from two years plants grown in closed soilless system in a heated greenhouse located in naples, south italy (40°51’55.5”n 14°20’30.1”e). rose plants were grown in plastic channels containing pumice and lapillus. the basic nutrient solution was supplied through a drip-irrigation system at a flow rate of 2 l h-1. irrigation frequency was regularly adjusted during the growing cycle based on the crop water requirements. at marketable harvest, cut stems were immediately transferred to the laboratory under room conditions, re-cut at the base (2-3 cm) and placed in graduated glass cylinders with 300 ml of deionized water and sodium hypochlorite (50 mg l-1). application of compatible solutes and antitranspirants compounds in the first experiment, two days before harvest, rose plants were treated with 10 mm l-1 of l-proline (sigma-aldrich, saint louis, missouri, usa) in 200 ml of deionized water per plant, applied in the growth substrate. control plants were treated with deionized water only. the treatment was performed at the end of the last daily irrigation and repeated after 24 h. in the second experiment, two days before harvest and at the end of the last daily irrigation, a substrate treatment was performed on rose plants, with of 0.5 mm l-1 of ß-aminobutyric acid (baba sigmaaldrich, saint louis, missouri, usa) in 200 ml of deionized water per plant, whereas control consisted of plants treated with deionized water. on a second plot of plants, the pinolene treatment was performed in post-harvest once transferred to the laboratory. stems were sprayed with a solution of 50 g l-1 of pinolene (96% poly-1-p-menthene, nu-film, intrachem bio, italy) in deionized water. control stems were sprayed with deionized water only. storage and physiological measurements part of the stems, weighted and sized based on length and diameters, were placed on ten precision balances (ek-410i, a&d instruments ltd, abingdon, uk) connected via usb to a computer for automatic monitoring of weight through a specific software (rscom®, corby, u.k.). these cylinders were sealed with parafilm to avoid water losses through evaporation. rs-com® software was set to record three daily weights in order to determine stems water consumptions over storage. cut stems were stored for 12 days under room conditions measuring daily mean temperature and relative humidity using a thermohygrometer (do 9847k, delta ohm srl, padova, italy). at storage days 2, 4 and 6, water flux measurements were recorded by using a scholander pressure chamber (3005f01 plant water status console, soil mosture equipment corp., santa barbara, california, usa). twenty centimeter stem segments (5 cm below the calix after measuring stems diameter) were immersed into a cylinder containing distilled water, placed in the pressure chamber, while the other extremity, out of the chamber, was connected to falcon tubes to collect and weight the water efflux. the system was then subjected to increasing pressure (p= 0.05, 0.1, 0.2, 0.3 mpa) and maintained at each pressure value for 5 minutes up to a constant outflow from the stem. water flux (jv) was expressed as jv= kg h2o m -2 s-1 m-1. water conductivity (lp) of stems was then expressed by the slope of the regression function of jv vs. p. volumes of collected efflux per unit of time (jv) were normalized to the cutting section surface. stomatal conductance (gs) was determined at storage days 2, 4 and 6 using a diffusion porometer (delta p-4, delta-t devices, cambridge, u.k.) in three daily measurements (h 9:00 am; h 1:00 pm; h 7:00 pm). osmotic potential (yπ) was measured using a dewp o i n t p s y c h r o m e t e r ( w p 4 , d e c a g o n d e v i c e s , washington) on frozen/thawed leaf samples. relative water content (rwc) value was calculated as: rwc= (leaf fresh weight leaf dry weight)/(leaf saturated weight leaf dry weight) (morgan, 1984). leaf area was estimated by scanning cut stems leaves and using the image j® software (abramoff et al., 2004) for image processing. the cut stems vase life was assessed visually using a “quality score” from 0 to 4. statistical analysis all data were statistically analyzed by anova u s i n g t h e s p s s s o f t w a r e p a c k a g e ( s p s s 1 0 f o r windows, 2001). the rwc data were transformed in adv. hort. sci., 2018 32(3): 311-318 314 arc-sin before anova analysis. 3. results experiment 1. effect of l-proline application on postharvest performance of cut rose exogenous applications of l-proline enhanced significantly water fluxes of rose cut stems in comparison to the untreated control, for all the 3 days of measurements (fig. 1a). during storage, water flux decreased from day 2 to day 6 in treated stems (fig. 1a). moreover, stems water conductivity (lp) was 3.05 [(kg h2o m -2 s-1 m-1) mpa-1] in control and 3.55 [(kg h2o m -2 s-1 m-1) mpa-1] in l-proline treatment (table 1). significant increase in stomatal conductance (gs) as well as in rwc were observed in l-proline treatment compared to the control (table 1). similarly to the physiological measurements, water consumptions normalized per leaf area were higher in treated stems (fig. 1b). as a result of cellular osmotic adjustment due to l-proline application, leaf osmotic potential was lower for treated stems in comparison to untreated control (table 1). the improved water status of l-proline treated stems influenced positively cut stems longevity extending their vase life by 2 days compared to the untreated control. experiment 2. effect of antitraspirants application on postharvest performance of cut rose in our current study, stomatal conductance was reduced by baba treatment respect to the control (table 2). consequently, daily water consumption, normalized per leaf area, was lower for baba treated stems (fig. 2b). the application of 0.5 mm of baba significantly reduced water fluxes during storage (fig. 2a) as well as the water conductivity (lp) of rose cut stems (2.6 [(kg h2o m -2 s-1 m-1) mpa-1] in control vs. 1.54 [(kg h2o m -2 s-1 m-1) mpa-1] in baba treatment). rwc was significantly higher in the control and it decreased during storage (table 2). furthermore, the water potential decreased over time and it was lower for rose stems treated with baba (table 2). the vase life, however, was not influenced by the treatment since in baba treated stems the improvement of the water balance was accompanied by premature yellowing of the leaves. as a consequence of the mechanical stomatal closure, the water use of pinolene treated stems was always lower compared to control (fig. 3). this was associated with a significant increase of the vase life by 1.5 days (fig. 4). 4. discussion and conclusions it is well established that osmotic adjustment contributes to maintain water uptake and cellular turgor (maggio et al., 2002; heuer, 2003). among all the osmolytes involved in this process, it has been sugfig. 1 effect of exogenous l-proline on water flux (a) and water consumptions per leaf area (b) of rose cut stems during storage. vertical bars indicate ± se of means. table 1 effect of exogenous l-proline on stomatal conductance (gs), relative water content (rwc), osmotic potential (ψπ) and water conductivity (lp) of rose cut stems treatment gs (cm s-1) rwc (%) ψπ (mpa) lp (kg h 2 o m-2s-1 m-1) control 0.38 b 84 b -0.17 b 3.05 b proline 0.42 a 89 a -0.34 a 3.55 a significance * * * * ns,*, not significant or significant at p≤0.05 respectively. within each column, different letters indicate significant differences. table 2 effect of exogenous β-aminobutyric acid (baba) on stomatal conductance (gs), relative water content (rwc), osmotic potential (ψπ) and water conductivity (lp) of rose cut stems ns,*, not significant or significant at p≤0.05 respectively. within each column, different letters indicate significant differences. treatment gs (cm s-1) rwc (%) ψπ (mpa) lp (kg h 2 o m-2 s-1 m-1) control 0.41 a 79 a -0,17 2.60 baba 0.25 b 74 b -0,27 1.54 significance * * * * di stasio et al. postharvest performance of cut rose cv. lovely red 315 gested that proline, exogenously applied via foliar spraying or through the irrigation water, could localize into the cytoplasm to reduce the cellular osmotic potential and to restore cellular hydration (gadallah, 1999; barbieri et al., 2011). in our experiment, lproline treatment on rose plants has shown to substantially improve the hydration state of the cut stems, with an observed decrease of the leaf osmotic potential and increased stomatal conductance and leaf rwc. consequently, the improved hydration state of tissues and stomatal conductance enhanced the water consumption in plants treated with lproline. the decline in stem water conductivity, is one of the main reasons for impaired water balance, as well as water stress is the most common reason for reduced cut roses vase life (halevy, 1976; joyce and jones, 1992). the increase in water fluxes and water conductivity for the l-proline treated flowers was an evidence of improved water status of the plant tissues that probably was the main factor involved in the extended vase life of l-proline treated stems. it h a s b e e n d e m o n s t r a t e d t h a t , i n c u t f l o w e r s , osmolytes are fundamental compounds in maintaining water balance, a key factor to extend their longevity (ichimura et al., 1997) as well as accumulation of these solutes, such as proline, is one of the main mechanisms to alleviate the detrimental effects of dehydration (morgan, 1984; anjum et al., 2011). in fact, the role of osmolytes includes mainly protection against the deleterious effects of the low water activity, preserving appropriate cellular volume (csonka and hanson, 1991). however, even though it is not yet clear if an extension of the cut flowers vase life may be more related to the ability of stem to maintain sustained water uptake rates or to control water losses, the control of the stomatal conductance is a fundamental determinant of the tissue water balance (fuchs and livingston, 1996; woodward et al., 2002). in nature, it is well known that plants control water losses by regulating transpiration in response to environmental factors (chaerle et al., 2005). in some respect, cut flowers respond to the same stimuli and the difference between the rate of water uptake and the tranfig. 2 effect of exogenous β-aminobutyric acid (baba) on water flux (a) and water consumptions per leaf area (b) of rose cut stems during storage. vertical bars indicate ± se of means. fig. 3 effect of pinolene on water consumptions per leaf area of rose cut stems during storage. vertical bars indicate ± se of means. fig. 4 effect of pinolene on the vase life of rose cut stems expressed as decay of 50% of the stems quality. different letters indicate significant differences at p≤0.05, vertical bars indicate ± se of means. 316 adv. hort. sci., 2018 32(3): 311-318 spiration rate is one of the parameters that will define their hydration state. as documented in different cases, baba acts through potentiation of aba-dependent signaling pathways (ton and mauch-mani, 2004) and for this reason, we supposed that applications of ß-aminobutyric acid (baba) may increase the tolerance to water shortage through the induction of functions associated to the synthesis of aba, such as stomatal closure (jakab et al., 2001; desikan et al., 2004). applied on rose plants, baba treatment has induced a decrease in stomatal conductance, with the consequent reduction of the stems water consumptions. along with this decrease of the transpirational flux, water fluxes and rwc decreased over time and they were generally lower in baba treated stems. in addition, lp was lower in baba-treated stems. the decreased rwc and water potential, together with a reduction of the cut stems hydration state, may be associated to senescence phenomena that occurred with the premature yellowing of the leaves, which is also mediated by aba (hunter et al., 2004; ferrante et al., 2006). accordingly, mayak and halevy (1972) reported that exogenous application of aba to rose cut flowers accelerate senescence phenomena. another strategy to control the plants transpiration and sustain a favorable plant water status is the utilization of antitranspirants compounds (del amor and rubio, 2009). our results confirmed that filmforming antitranpirants are effective in reducing water losses providing a thin coating on the leaves surfaces leading to an improved tissues water status in cut roses (moftah and al-humaid, 2005; song et al., 2011; mikiciuk et al., 2015). consistently, this mechanical effect on the transpirational flux regulation was observed on water consumptions normalized per leaf area rose cut stems, that were significantly reduced by pinolene application during the vase life. the reduced water use was correlated to an extended vase life compared to the water-treated control. in conclusion our results demonstrated that both osmoregulation and direct transpirational control were effective strategies in maintaining an enhanced hydration state of rose cut stems, leading to a prolongation of the stems vase life. treatment with 10 mm l-proline has allowed the maintenance of higher stomatal aperture and improved cut stems rwc and lp during storage. the positive effects on cut stems were measured as decrease of osmotic potential and increased stomatal aperture consequent to osmoregulation. these physiological conditions are crucial for prolonging the vase life of cut flowers because, despite the absence of the root system, allow the stem to partially continue its metabolic functions. on the other hand, the reduction of transpiration that is considered a functional target for controlling cellular turgor after harvest thus prolonging cut flowers. since aba is involved in the induction of physiological mechanisms that facilitate adaptation to abiotic stress, it has been hypothesized that the administration of baba, a mediator of aba functions, may c o n f e r a s t r e s s p r o t e c t i o n t h a t c o u l d r e s u l t i n enhanced turgor and vase life of cut stems. in fact, our results also demonstrated that applications of 0.5 mm baba on rose has reduced water consumption by inducing stomatal closure. however, this was associated with a more rapid decay of the cut stems quality probably for earlier oncoming of senescence phenomena. furthermore, pinolene treatment prolonged the vase life of cut stems, by reducing water losses through transpiration. this was likely due to the formation of a ‘film’ at the leaf surface that acts as a physical barrier to gas exchanges. t a k i n g a l l t o g e t h e r , w e c a n c o n c l u d e t h a t osmoregulation or direct control of traspirational fluxes may provide a promising avenue for improving the post-harvest longevity of cut roses. however, further investigations are required whenever other physio-chemical processes are involved such as the induction senescence phenomena. references abdel-fattah g.h., 2013 response of water-stressed rose of china 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attributes of garden cress (lepidium sativum l.) under greenhouse conditions a. jorkesh 1 (*) , m.h. aminifard 2 1 department of horticulture, faculty of agriculture, university of guilan, rasht, iran. 2 department of horticulture science, special plants regional research centre, faculty of agriculture, university of birjand, iran. key words: foliar spray, nitrogen content, phosphorus content, vegetative stage. abstract: in this study, the effect of foliar application of asparagine (asn) and casein (csn) during vegetative stage at four rates (0, 50, 100 and 150 mg l-1) was investigated on garden cress (lepidium sativum l.). the results showed that asparagine application, especially at a high level, could significantly increase the morpho-physiological traits such as plant height, leaf and stem fresh weights and leaf and stem dry weights, leaf pigments (chlorophyll a and chlorophyll b) and leaf nutrients content (nitrogen and phosphorus). also, the results indicated that casein application at 50 mg l-1 rate had the best performance through in stem and root fresh weights, stem dry weight and diameter of main stem traits. casein application at rate 100 mg l-1 had the highest leaf nitrogen and phosphorus content. generally, our findings suggest that the use of asparagine and casein can be considered as an appropriate growth regulator in garden cress cultivation. 1. introduction garden cress (lepidium sativum l.) is an edible herb and a member of the cruciferae (brassicaceae) family. it is commonly cultivated throughout the temperate regions of india and pakistan (nadkarni, 1954). the plant is cultivated as culinary vegetable all over asia (nadkarni, 1976). garden cress is an annual standing plant, growing up to 30 cm. it is a well known cookable herb and the leaves are widely used as a garnish in salads. in addition to its leaves that have medicinal properties, the seeds are aperients, diuretic, tonic, demulcent, aphrodisiac, carminative (chopra et al., 1986). moreover, the seeds, which are used in folk therapies, have many activities like thermogenic, depurative, rubefacient, tonic, aphrodisiac, abortive, ophthalmic, diuretic (gokavi et al., 2004; dugasani et al., 2009). intensive farming practices, which produce high yields and quality, require the extensive use of chemical fertilizers that are both costly and (*) corresponding author: a.jorkesh@gmail.com citation: jorkesh a., aminifard m.h., 2019 foliar application of asparagine and casein on biochemical and morphological attributes of garden cress (lepidium sativum l.) under greenhouse conditions. adv. hort. sci., 33(2): 227-233 copyright: © 2019 jorkesh a., aminifard m.h. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 10 august 2018 accepted for publication 18 february 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(2): 227-233 228 create environmental problems. therefore, there has been a recent resurgence of interest in environmentally friendly, sustainable and organic agricultural practice (orhan et al., 2006). thus, it is necessary to supply the plant requirement to nutrient through proper procedure. there are different ways to supply plant nutrient’s requirement such as soil feeding and foliar application. of these, foliar feeding is an effective method for improving soil deficiencies and overcoming the soils inability to transfer nutrients to the plant. it has reported the foliar feeding can be 8 to 10 times more effective than soil feeding and up to 90% of a foliar fed nutrient solution can be found in the smallest root of a plant within 60 minutes of application (garcia and hanway, 1976). amino acids are the major building element for proteins (andrews, 1986). amino acids is a wellknown biostimulant which has positive influence on plant growth, yield and significantly decrease the damages caused by abiotic stresses (kowalczyk and zielony, 2008). asn is widely used as a source of organic nitrogen in the media upon which certain bacteria are grown (long and seibert, 1926). asn is thought to play a clearly important role in the transportation and storage of nitrogen (lehmann and ratajczak, 2008), because of their relatively stable nature and high n⁄c ratio (ireland and lea, 1999; masclaux-daubresse et al., 2006). in plants, asn, one of the most prevalent amides, has been reported to be the primal source of nitrogen for protein synthes i s , p a r t i c u l a r l y i n a c t i v e l y g r o w i n g t i s s u e s (brouquisse et al., 1992). asn aggregation in plants in response to environmental stress could be an ammonium detoxification mechanism and a means to stock up nitrogen when protein synthesis is impaired in plants due to stressful environments (herrerarodríguez et al., 2007). csn is a very rich source of essential amino acids (sarode et al., 2016). there are four different csn proteins (αs1, αs2, β and k), which are different in their amino acid composition (dalgleish, 1989). among proteins, csn has been vastly used in artificial rearing diets because it contains all the essential amino acids, is soluble in water, and does not coagulate or precipitate after heating (parra, 1979). csn also contains important substances such as fatty acids, cholesterol, sugars, vitamins, and minerals (vanderzant, 1974). csn contains 0.7-0.9% phosphorus. so, csn is known as a phosphoprotein (sarode et al., 2016). several pre-harvest factors like climatic conditions and available nutrients can influence on yield and quality of vegetables (lee and kader, 2000). in spite of wide rang of properties, there are a few study about the effect of amino acids on medicinal plants. so this study was conducted to evaluate the effects of asn and csn foliar application on vegetative and reproductive growth, physiological and biochemical traits of garden cress. 2. materials and methods in order to investigate the response of lepidium sativum to foliar application of asparagine (asn) and casein (csn) two separate pot experiments were conducted at research greenhouse of university of guilan, iran. the experiments were carried out based on two randomized complete design with three replications. the treatments in this study were different levels of asn and csn (0, 50, 100 and 150 mg l-1). foliar application of experimental solutions was started from the four-leaf stage and done once every two weeks in the morning, up to the early may. in each spraying date, 20-25 ml of corresponding solution was applied per pot. garden cress seeds which were prepared from isfahan-pakanbazr company were planted in 6th january. the sowing was done in pots with the 33 cm diameter and 22 cm height. the seed bed was a mixture of soil, cow manure and sand with the ratio of 2:1:1. the main properties of soil used in the pots are shown in table 1. except for cow manure, no fertilizer was used during the plants growth cycle. plants were irrigated every 10 days. plant thinning was performed 15 days after emergence, so that, 6 plants were kept in each pot. during plant growth the greenhouse temperature was 25˚c on the day and 15°c during the nights. in addition, co2 concentration was 350 ppm, relative humidity was 40% and photoperiod adjusted as 16 hours’ light and 8 hours of darkness. organic carbon (%) nitrogen (%) calcium (%) phosphorous (%) potassium (%) ph texture 1.2 1 0.48 0.19 0.55 7.1 loam sandy table 1 some chemical indices of soil used in pots for garden cress cultivation jorkesh and aminifard foliar application on lepidium sativum under greenhouse conditions 229 in 4th may, four plants were lifted form the soil in each pot for determination of some aboveand below-ground vegetative indices. the measured indices were plant height, leaf weight, root weight, stem weight and main stem diameter (by a ruler). the remained two plants were used to measure some reproductive traits including number of florets. the pigments content (carotenoid, chlorophyll a and chlorophylls b) was measured by the method has been explained by minguez-mosquera and prezgalvez (1998). to determine the leaf nitrogen, phosphorous and calcium percentages, the method of jones (2001) was used. the method of bakhshi and arakava (2006) also was used for the extraction of leaf extract to determine phenolic compounds and antioxidant capacity. then, phenols were analyzed by using the method of folin-ciocalteau reported by tavarini et al. (2008). for this purpose, the absorbance at 760 nm was measured using a spectrophotometer (t80+pg instrument uv/vis spectrometer) and the values were expressed as mg gallic acid/100 g fresh weight. antioxidant capacity was determined using dpph free radical scavenging method which has been described by sanchez-moreno et al. (1999). finally, data analysis was done using sas 9.2 software and means were separated via tukey’s (honest significant difference) test at the 5% level of probability. 3. results and discussion vegetative and reproductive growth the analysis of variance of the investigated morphological traits was presented in table 2. the results revealed that both asn and csn significantly affected the plant length, leaf and stem fresh weight, leaf and stem dry weight. so that, the greatest plant height w a s o b s e r v e d a t a r a t e o f 1 5 0 m g l 1 o f a s n . moreover, applying asn at rate of 100 mg l-1 had the highest leaf and stem fresh weights and leaf and stem dry weights. casein application significantly affected the stem and root fresh weights, stem dry weight and diameter of main stem (table 2). so that, the plants grown under csn spray at rate of 50 mg l-1 had the best performance due to stem dry and fresh weight, root fresh weight and main stem’s diameter. asparagine and csn foliar spraying could improve the morphological characteristics in garden cress. these results are in agreement with the previously studies by kaya et al. (2013) on maize, rasmia et al. (2014) on palm, saeed et al. (2005) on soybean, akladious and abbas (2013) and el-desouky et al. (2011) on tomato, shafeek and helmy (2012) on onion. also, el-zohiri and asfour (2009) on potato found that spraying of amino acids at 0.25 ml/l significantly increased vegetative growth expressed as plant height and dry weight of plant. their finding indicated that amino acids could improve the vegetative and reproductive traits in plants. these ability my be due to their important role in plant metabolism and protein assimilation which is necessary for cell formation and consequently increase the fresh and dry matter (fawzy et al., 2012). amino acids contribute to the synthesis of growth hormones; therefore, it can be concluded that an increase in cell division and cell enlargement is the r e a s o n b e h i n d e n h a n c e d g r o w t h p a r a m e t e r s (shafeek and helmy, 2012). the positive effect of amino acids on growth was stated by goss (1973) who indicated that amino acids can serve as a source table 2 mean squares for the effect of different levels of aspargine (asn) and casein (csn) on vegetative and reproductive indices of garden cress traits source of variation treatment error cv asn csn asn csn asn csn plant height 24.22 * 5.86 ns 3.75 10.41 5.75 10.38 leaf fresh weight 0.66 * 1.008 ns 0.09 0.25 5.09 7.97 leaf dry weight 0.27 ** 0.04 ns 0.03 0.02 11.69 12.28 root fresh weight 0.17 ns 0.2 ** 0.06 2 8.52 5.22 root dry weight 0.002 ns 0.004 ns 0.01 0.005 8.54 5.94 stem fresh weight 2.65 ** 2.001 ** 0.04 0.16 3.62 6.83 stem dry weight 0.5 ** 0.22 * 0.02 0.04 9.66 14.13 diameter of main stem 0.04 ns 0.11 * 0.05 0.01 10.98 5.87 number of florets 0.22 ns 0.75 ns 0.66 0.5 11.66 10.47 ns, *, ** non-significant, significant at 5%, and 1% probability level, respectively. adv. hort. sci., 2019 33(2): 227-233 230 of carbon and energy when carbohydrates become deficient in the plant’s releasing the ammonia and organic acid form which the amino acid was originally formed (table 3). the organic acids then enter kerb’s cycle, to be broken down to release energy through respiration (goss, 1973). serna et al. (2012) found that the spray of pepper plants with a mixture of amino acids led to raise the efficiency of photosynthesis, and thus, give the best vegetative growth. biochemical traits and nutrients content the mean squares of physiological and mineral content are presented in table 4. total phenolic compounds and antioxidants activity were not affected by foliar spraying of asn and csn (table 5). chlorophyll a and b content significantly increased in response to foliar asn treatments (table 4). the highest contents of chlorophyll a and b observed in plants grown under spraying of asn at rate of 100 table 3 means comparison for the effect of different levels of asparagine and casein on some vegetative and reproductive growth parameters of garden cress means with the same letter(s) within a column are not significantly different (p≤0.05) based on tukey's test. table 4 mean squares for the effect of different levels of aspargine (asn) and casein (csn) on leaf nutrient and pigments content and some biochemical parameters in garden cress ns, *, ** non-significant, significant at 5%, and 1% probability level, respectively. traits source of variation treatment error cv asn csn asn csn asn csn chlorophyll a 0.13 ** 31.66 ns 0.006 0.008 4.9 6.75 chlorophyll b 0.05 ** 0.01 * 0.001 0.002 5.83 7.89 carotenoid content 0.0004 ns 0.003 ns 0.001 0.002 8.32 12.96 leaf nitrogen content 0.09 ** 0.18 ** 0.006 0.01 4.79 6.72 leaf phosphorous content 0.01 * 0.03 ** 0.003 0.002 8.86 6.66 leaf calcium content 0.007 ns 0.0007 ns 0.007 0.003 9.45 6.95 total antioxidants 21.41 ns 6.97 ns 12.75 6.16 6.64 4.7 total phenol 31.66 ns 6.97 ns 7.33 17.33 4.99 7.81 treatments (mg l-1) plant height (cm) leaf fresh weight (g plant-1) leaf dry weight (g plant-1) root fresh weight (g plant-1) root dry weight (g plant-1) stem fresh weight (g plant-1) stem dry weight (g plant-1) diameter of main stem (mm) number of florets aspargine 0 29.66 b 5.47 b 1.18 b 2.87 a 1.20 a 4.77 c 1.15 c 1.99 a 7.00 a 50 34.66 ab 6.05 ab 1.81 a 2.97 a 1.25 a 5.73 b 1.44 bc 2.08 a 6.66 a 100 34.00 ab 6.57 a 1.83 a 3.42 a 1.26 a 6.96 a 2.06 a 2.25 a 7.00 a 150 36.33 a 6.31 a 1.62 ab 3.12 a 1.26 a 6.38 ab 1.86 ab 2.02 a 7.33 a casein 0 29.66 a 5.47 a 1.18 a 2.87 b 1.2 a 4.77 b 1.15 b 1.99 b 7.00 a 50 31.00 a 6.75 a 1.45 a 3.39 a 1.22 a 6.64 a 1.80 a 2.37 a 6.33 a 100 30.66 a 6.23 a 1.38 a 2.82 b 1.15 a 6.22 ab 1.58 ab 2.01 b 6.33 a 150 33.00 a 6.65 a 1.43 a 3.14 ab 1.25 a 6.16 ab 1.55 ab 2.3 ab 7.33 a table 5 means comparison for the effect of different levels of asparagine and casein on biochemical parameters of garden cress means with the same letter(s) within a column are not significantly different (p≤0.05) based on tukey's test. treatments (mg l-1) chlorophyll a chlorophyll b carotenoid content leaf nitrogen content leaf phosphorous content leaf calcium content total antioxidants total phenol aspargine 0 1.38 b 0.56 c 0.39 a 1.45 b 0.57 b 0.89 a 51.6 a 50.3 a 50 1.53 b 0.73 b 0.4 a 1.6 ab 0.66 ab 0.83 a 53 a 52.6 a 100 1.87 a 0.89 a 0.37 a 1.81 a 0.71 ab 0.95 a 57.6 a 57.3 a 150 1.64 ab 0.71 b 0.35 a 1.81 a 0.73 a 0.87 a 52.6 a 56.3 a casein 0 1.36 a 0.56 b 0.39 a 1.45 b 0.57 c 0.89 a 51.66 a 50.3 a 50 1.41 a 0.73 a 0.37 a 1.62 ab 0.60 bc 0.86 a 52 a 54.3 a 100 1.45 a 0.70 a 0.44 a 2.01 a 0.78 a 0.82 a 55 a 54.6 a 150 1.38 0.65 ab 0.43 a 1.88 a 0.74 ab 0.87 a 52.3 a 53.6 a jorkesh and aminifard foliar application on lepidium sativum under greenhouse conditions 231 needs of the cells are provided with the lowest losses. results of the current study revealed that foliar application of asn and csn could increase the growth and yield of garden cress. it could be recommended that spraying garden cress plants by asn (100 and 150 mg l-1) increased the vegetative growth traits (plant height, leaf fresh weight, number of florets), nutrients uptake (leaf nitrogen and phosphorus content) and pigments concentration (chlorophyll a and chlorophyll a) and also application of csn at 50 mg l-1 level could improved some morphological and biochemical traits of garden cress such as: root fresh weight, stem fresh weight, chlorophyll b, leaf nitrogen and phosphorus content in comparison control treatment. therefore, it can be concluded that application of asn and csn is a good strategy in garden cress cultivation, but their effectiveness must be also evaluated under environmental stresses, in the future studies. also spraying different nutrient solution in combination with each other may show interaction and contrary behavior in plants in comparison applying them solely, so further research with higher concentrations and also combined spraying of these amino acids, applying on different vegetables in greenhouse and farm conditions is suggested. references abo sedera f., abd el-latif a., bader l., rezk s., 2010 effect of npk mineral fertilizer levels and foliar application with humic and amino acids on yield and quality of strawberry. j. appl. sci., 25: 154-169. akladious s.a., abbas 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amino acid under sandy soil conditions. j. appl. sci. res., 8: 5521. tavarini s., degli innocenti e., remorini d., massai r., guidi l., 2008 antioxidant capacity, ascorbic acid, total phenol and carotenoids change during harvest and after storage of hayward kiwifruit. food chem., 107: 282-288. vanderzant e.s., 1974 development, significance, and application of artificial diets for insects. annual rev. entom., 19: 139-160. y o u s s e f i f . , b r o w n p . , w e i n b a u m s . , 2 0 0 0 relationship between tree nitrogen status, xylem and phloem sap amino acid concentrations, and apparent soil nitrogen uptake by almond trees (prunus dulics). j. hort. sci. biotec., 75: 62-68. impaginato 153 adv. hort. sci., 2019 33(2): 153-160 doi: 10.13128/ahs-23517 diversity of morpho-physicochemical traits in iranian sour cherry genotypes using multivariate analysis s. aliyoun nazari 1 (*), j. hajilou 1, m. zeinalabedini 2, a. imami 3 1 department of horticultural sciences, faculty of agriculture, university of tabriz, iran. 2 agriculture biotechnology research institute of iran (abrii), karaj, iran. 3 horticultural departments of seed and plant improvement institute (spii), karaj, iran. key words: fruit quality traits, genetic diversity, prunus cerasus l. abstract: in this study, morpho-physicochemical characterization of sour cherry genotypes from iran was investigated. thirty-four morphological and eight physicochemical traits were recorded. sour cherry genotypes had a high variability in traits related to fruit characters such as fruit weight, stone volume, total anthocyanin content and total soluble solid. as a result, sour cherry genotypes exhibit total phenolic content and antioxidant activity higher than “ciganymeggy” and “erdi botermo” cultivars. principal component analysis (pca) suggested that leaf dimensions, fruit weight, stone weight, and stone volume could be sufficient for identification of genotypes. hierarchical cluster analysis classified sour cherry genotypes and “ciganymeggy” and “erdi botermo” cultivars into two main clusters. the first cluster was characterized by a upright tree vigour, depressed fruit pistil end, reniform shape of fruit, high sweetness, dark red juice, flower high length and diameter, fruit and stone weight and length and diameter, total soluble solid, low total phenolic content, high total flavonoid content and high total anthocyanin content. 1. introduction sour cherry, prunus cerasus l., is known as tetraploid (2n =4x = 32), originated through natural hybridization of the large statured, cold sensitive sweet cherry (p. avium l., 2n = 2x = 16), and the low growing, cold tolerant ground cherry (p. fruticosa pall., 2n = 4x = 32) (olden and nybom, 1968). this species originated around the black and caspian seas and were cultivated in temperate and cold regions. sour cherry spread slowly from its origin to other regions due to human and animal migrations (pérez-sánchez et al., 2008). sour cherry fruit is mostly used for industrial preserves (jams, purees, juices and concentrates), while only a small portion is assigned to fresh consumption. sour cherry is also used as a sweet cherry rootstock. this rootstock is more resistant to soil wetness and cold (*) corresponding author: saliyoun66@gmail.com citation: aliyoun nazari s., hajilou j., zeinalabedini m., imami a., 2019 diversity of morpho-physicochemical traits in iranian sour cherry genotypes using multivariate analysis. adv. hort. sci., 33(2): 153-160 copyright: © 2019 aliyoun nazari s., hajilou j., zeinalabedini m., imami a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 28 june 2018 accepted for publication 12 march 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(2): 153-160 154 climate than wild sweet cherry and mahaleb forms. in 2014, the total world production of sour cherry reached 1.1 million tons, being turkey, the russian federation, poland, ukraine, iran and serbia the most important producing countries (faostat, 2014). the main sour cherry producing areas in iran are the ardebil, azerbaijan, khorasan and alborz provinces. from the viewpoint of fruit quality, several studies for characterization of fruit traits have been accomplished recently. sour cherry is a valuable source of vitamins (a, b1, b2, c, e, k, and niacin), carotenoids like beta-carotene, minerals, fiber, various sugar like fructose, glucose, maltose, antioxidant agents such as caffeic acids, cyaniding-3-o-glucosylrutinoside and flavoids (mulabagal et al., 2009; ferretti et al., 2010). this products has positive effects on human health (ataie-jafari et al., 2008; saric et al., 2009; kuehl et al., 2010). analysis of flavonoids from p. cerasus identified kaempferol, quercetin, quercetin 3-o-glucoside, and isorhamnetin 3-o-rutinoside (piccolella et al., 2008). the main anthocyanins found in cherry are cyanidin-3-o-glucoside, cyanidin-3-o-rutinoside, cyanidin-3-o-glucosylrutinoside, cyanidin-3-osophoroside, pelargonidin-3-o-glucoside, peonidin-3o-rutinoside and cyanidin-3-o-arabinosylrutinoside (chaovanalikit and wrolstad, 2004). much of the genetic diversity is available in the wild types and natives from the center of origin. wild species are probable gene resources for the breeding objectives such as resistance to pests and diseases, more appropriate cultivars for table and industry, extending cherry season and developing new resistant and dwarfing rootstocks. therefore, it is necessary to characterize and preserve these species (demirsoy and demirsoy, 2004; aliyoun nazari et al., 2012). description of the morphological characteristics is the usual methodology accepted from a legal point of view for patenting and registration of varieties. several quantitative and qualitative evaluations showed a clear difference between sour cherry, with a more marked variability within the sour cherries group, probably due to the more intense domestication processes that have taken place. morphological characterization continues to be the first step for germplasm description and classification, and the statistical method of factor analysis is a useful tool for screening the accessions of a collection (badenes et al., 2000; hajilou and fakhimrezaei, 2011). several morphological characterization studies have been c a r r i e d o n t h e s o u r c h e r r y ( k r a h l e t a l . , 1 9 9 1 ; r o d r i g u e s e t a l . , 2 0 0 8 ; r a k o n j a c e t a l . , 2 0 1 0 ; najafzadeh et al., 2014). as an origin of the subgenus cerasus, iran has a rich cherry germplasm. the area of this study is part of a large growing area in north west of iran. sour cherry has been cultivated in this area for many years. however, the conservation and characterization of local cultivars is important to avoid the loss of genetic variability and as a potential source of genetic variation for future sweet and sour cherry breeding programs. these genotypes show distinctive agronomic characters such as low susceptibility to fruit cracking, high levels of soluble solids and early fruit maturity. the objective of this study was to survey, identify and characterize sour cherry genotypes existing in the province of east azerbaijan shabestar (iran) for their later introduction into a germplasm bank. 2. materials and methods plant materials the plant material was located on the shabestar town in west side of the east azerbaijan province, in north-west of iran. a total of 15 sour cherry genotypes and two cultivars, “ciganymeggy” and “erdi botermo”, were used in this study. evaluation of morphological and physicochemical traits characterization of vegetal material and fruits was based on sour cherry descriptors developed by the international union for the protection of new varieties of plants upov (upov, 2006). thirty four morphological (16 qualitative and 18 quantitative) and eight physicochemical traits were recorded as described in table 1 and 2. in this study, a total of 17 sour cherry genotypes, including 15 local sour cherry genotypes and two cultivars, “ciganymeggy” and “erdi botermo”, with three replicates for each genotype were evaluated. the evaluation for morphological characters was based on 30 measurements of each trait. for the analysis of physicochemical traits, fruits were picked at the commercial maturity stage. all fruits were collected from a single plant, randomly from all cardinally oriented branches with different directions around the canopy. all samples were stored in a freezer at -20°c. the frozen fruit material (5 g) was homogenized with a polytron (2 min on ice) with 10 ml of extraction solution, consisting of 0.5 n hcl in methanol/milli-q water (80% v/v). the mixture nazari et al. iranian sour cherry characterization 155 was incubated overnight at 4°c and then centrifuged for 20 min at 4°c and 20000 g. supernatant was recovered and the volume measured. this hydroalcoholic extract was used for total phenolics, anthocyanins, flavonoids, and antioxidant capacity assays (cantin et al., 2009). the content of phenolic comno. trait 1 2 3 4 5 6 7 9 11 1 tree vigour very weak weak medium strong very strong 2 tree habit upright semi-upright spreading drooping 3 tree branching weak medium strong 4 tree bud distribution along entire branch only on middle distal part of branch only on distal part of branch 5 flower arrengment of petal free intermediate overlapping 6 flower shape of petal circular medium obovate broad obovate 7 flower arrangement solitary double in clusters irregular 8 starting bloom from april 9-11 day 11-13 day 13-15 day 15-17 day 17-19 day ›19 day 9 fruit ripening time from june 5-10 day 10-15 day 15-20 day 20-25 day 25-30 day › 30 day 10 fruit pistil end pointed flat depressed 11 stone shape narrow elliptic broad elliptic circular 12 fruit shape reniform oblate circular elliptic 13 fruit color of skin orange red light red medium red dark red brown red blackish 14 fruit color of flesh yellowish pink medium red dark red 15 fruit sweetness low medium high 16 color of juice colorless light yellow pink medium red dark red table 1 sixteen qualitative traits and their states and codes studied of sour cherry genotypes no. trait unit min max mean cv (%) 1 flower diametr mm 23.5 39.5 28.1 12.4 2 petal length mm 10.8 13.7 11.9 8.1 3 petal width mm 9.6 14.0 11.6 11.9 4 pestil length mm 11.4 13.6 12.4 5.1 5 number of stamens 31.9 37.0 34.5 4.1 6 fruit length mm 13.5 19.1 15.2 8.8 7 fruit diameter mm 13.1 22.6 17.6 10.9 8 fruit length/ diameter -mm 0.8 1.0 0.9 6.1 9 length of stalk mm 41.0 54.0 46.8 7.3 10 fruit weight gr 12.4 54.3 21.4 55.7 11 stone length mm 5.6 9.5 7.1 11.7 12 stone diameter mm 5.5 9.0 7.4 13.2 13 stone volume cm3 0.1 0.4 0.2 36.0 14 stone weight gr 2.2 3.5 3.0 13.5 15 leaf blade length mm 67.0 96.8 78.9 10.2 16 leaf blade width mm 36.8 53.4 43.4 9.8 17 leaf blade length/ blade width 1.7 2.0 1.8 4.0 18 petiole length mm 13.6 18.4 16.2 8.2 19 ph 2.0 3.6 3.3 13.9 20 total soluble solid % 12.1 23.3 16.8 20.9 21 vitamin c mg/100g fw 10.5 13.2 11.7 7.7 22 titratable acidity % 1.9 2.7 2.3 9.0 23 total phenolic content mg gae/100 g fw 228.0 289.0 241.8 6.0 24 total anthocyanin content mg cyanidin 3-glucoside /100 g fw 60.1 130.3 98.0 21.5 25 antioxidant activity μg te/ 100g fw 52.3 67.7 60.5 7.5 26 total flavonoids content mg qe/ 100 g fw 141.8 155.8 145.8 2.5 table 2 the range of 26 quantity variability in sour cherry genotypes traits, mean and coefficient of variations (cv %) pounds in methanol extracts was determined according to the folin-ciocalteu method (waterhouse, 2001). absorbance was measured at 725 nm using a spectrophotometer (uv-2100 spectrophotometer). total anthocyanin content (tac) was determined using the ph differential method (giusti and adv. hort. sci., 2019 33(2): 153-160 156 wrolstad, 2001). the absorbances of the extracts at 510 and 700 nm were measured against a blank. tac was calculated and expressed as mg cyanidin 3-glucoside equivalent/100 g of fw. total flavonoid content of each extract was determined following colorimetric method (chang et al., 2002). the antioxidant capacity was measured using the dpph method adapted from brand-williams et al. (1995). titratable acidity was established by titration with 0.1 n naoh and sugar content was measured as total soluble solids (tss) using digital refractometer (atago pr 100, japan). vitamin c content was estimated according to the titration with 2, 6-dichlorophenolindophenol method (aoac, 2000). statistical analysis statistical analysis were performed using spss 17.0 (spss inc., chicago, il). to obtain basic statistics for the entire plant material studied, maximum and minimum values, mean, and coefficients of variation (cv %) were calculated for each trait. relationships among the species were investigated by principal component analysis (pca). pca was performed using spss statistics software. scatter plot of the first two pcs and the cluster analysis were created by past statistics software (hammer et al., 2001). 3. results and discussion characteristics of cultivars several researchers have reported the morphological variation between some prunus subgenus cerasus genotypes such as for sweet cherry (p. a v i u m ) , s o u r c h e r r y ( p . c e r a s u s ) , m a h a l e b ( p . mahaleb), marmareh (p. incana) and tomentosa cherry (p. tomentosa) (ganji-moghadam and khalighi 2007; khadivi-khub et al., 2008; perez-sanchez et al., 2008; zhang et al., 2008; rakonjac et al., 2010; aliyoun nazari et al., 2012). morphological characteristics of the studied genotypes are resumed in the table 1 and 2. results showed that high variation among studied genotypes was found for fruit weight (cv=55.7%) and stone volume traits (cv=36%). this result is compatible with zhang et al. (2008) report. they observed high morphological variation among populations, where the highest variations were in fruit weight, fruit width, and leaf width. tree habits of the studied genotypes are different. most of the genotypes have drooping, three have spreading and one has upright tree habit (“erdi botermo”). tpc values ranged between 228 and 289 mg gae/100 g fw of sour cherry genotypes, which is in good agreement with previously published results (dragovic-uzelac et al., 2007; khoo et al., 2011; alrgei et al., 2015). “erdi botermo” had the lowest tpc among the studied genotypes and n02 had the highest, that is consistent with the results of papp et al. (2010) that reported the “erdi botermo” have lowest tpc among all tested genotypes. behrangi et al. (2015) reported that tpc is versatile on the basis of fruit type, stage of growth, farm of landing, extraction method, component of tpc experiment and other factors. therefore tpc decreased by transforming fruit from first stages of growth to fully ripe form that is compatible with our results. as shown in table 2, total antioxidant capacity (ac) of sour cherries was between 52.3 and 67.7 μg te/100 g fw. the total ac of different sour cherry cultivars showed significant difference (blando et al., 2004; bonerz et al., 2007; khoo et al., 2011). the lowest tac was found in n02 genotype (60.1 mg cyanidin 3-glucoside/100 g fw), while “erdi botermo” had the highest tac (130.3 mg cyanidin 3-glucoside/ 100 g fw). these differences in tac showed that the plant growth region and the harvest period might have an impact on plant growth and metabolite concentration (premier, 2002). sour cherry is one of the richest source of flavonoid (marinova et al., 2005), that is consistent with our results. principals component analysis eighty percent of the variability observed was explained by seven components (table 3). for each trait, a factor loading of more than 0.51 was considered as being significant. pc1 represents mainly fruit pistil end, fruit color of skin, fruit length, fruit diameter, fruit weight, stone length, stone diameter, stone volume, leaf blade length, leaf blade width and total flavonoids content with significant positive effects, also tree habit, flower shape of petal, fruit ripening time with negative effects and account for 29.65% of the variance. the second principals component with 13.4% of total variance included traits of the tree branching, vitamin c, titratable acidity with negative impacts and the trait of flower diameter, petal length, petal width and stone weight with positive impacts. high absolute values of the correlations between variables related to the growth, fruit and leaf size, and pc1 or pc2 were also established by krahl et al. (1991) and rakonjac et al. (2010) in sour cherry, by lacis et al. (2010) and rakonjac et al. (2014) in sweet cherry, aliyoun nazari et al. (2012) in nazari et al. iranian sour cherry characterization 157 marmareh (p. incana) and by khadivi-khub et al. (2012) in prunus subgen. cerasus. pc3 was correlated with starting bloom, stone shape, pistil length, fruit length/diameter, ph and length of stalk. the remaining components explain less variability. grouping of cultivars hierarchical cluster analysis classified native sour cherry genotypes and “ciganymeggy” and “erdi botermo” cultivars in two main clusters (fig. 1). the first major cluster is divided into two subgroups; subgroups i consisted of erdi botermo cultivar and and subgroup ii contained ciganymeggy and some of the genotypes, indicating that these sour cherry genotype had high similarity to ciganymeggy cultivar. the second cluster included the native genotypes. the table 3 eigen values and cumulative variance for seven major factors obtained from principal component analysis (pca) and traits within each factor for sour cherry genotypes trait factors pc1 pc2 pc3 pc4 pc5 pc6 pc7 tree vigour -0.10 0.32 0.12 -0.62** 0.02 0.30 -0.52 tree habit -0.87** 0.05 0.19 0.14 0.14 -0.15 0.00 tree branching 0.14 -0.74** 0.21 0.21 0.02 -0.24 -0.08 tree bud distribution 0.32 0.02 0.15 -0.60** -0.04 -0.29 -0.39 flower arrangement of petal 0.32 -0.34 0.35 -0.02 -0.14 0.39 0.32 flower shape of petal -0.90** 0.10 0.19 -0.03 -0.19 -0.20 -0.04 flower arranement -0.36 -0.39 0.18 -0.22 0.13 0.64** -0.22 starting bloom from april 0.02 -0.15 0.61** -0.24 -0.48 0.14 0.27 fruit ripening time from june -0.87** 0.08 -0.10 0.15 0.20 0.02 -0.02 fruit pistil end 0.81** -0.13 -0.02 -0.29 -0.18 -0.13 -0.04 stone shape 0.18 0.34 0.62** 0.50 0.02 0.26 0.07 fruit shape -0.83** 0.22 0.26 0.25 0.07 -0.05 -0.06 fruit color of skin 0.80** -0.12 -0.27 0.10 0.34 0.11 0.09 fruit color of flesh 0.16 -0.50 0.14 0.26 -0.55** 0.26 -0.06 fruit sweetness 0.63** 0.02 -0.51 -0.08 0.28 0.15 0.16 color of juice 0.90** -0.10 -0.19 0.03 0.19 0.20 0.04 flower diameter -0.28 0.71** -0.15 0.17 0.05 0.15 0.39 petal length -0.10 0.77** -0.14 0.32 -0.32 -0.03 0.15 petal width -0.10 0.70** -0.12 0.14 -0.48 0.29 0.06 pestil length -0.18 0.08 0.70** 0.06 -0.36 0.02 -0.38 number of stamens -0.21 0.11 -0.46 0.46 0.40 -0.02 -0.19 fruit length 0.90** 0.33 0.09 0.03 0.04 -0.11 -0.07 fruit diameter 0.81** 0.23 -0.19 0.22 -0.13 -0.18 -0.15 fruit length/diameter -0.15 0.01 0.52** -0.38 0.33 0.24 0.28 length of stalk 0.01 -0.02 0.59** -0.13 0.40 0.07 0.11 fruit weight 0.86** 0.16 0.21 -0.14 0.28 -0.19 0.07 stone length 0.89** 0.11 0.25 0.14 -0.10 -0.03 0.09 stone diameter 0.60** -0.20 0.47 -0.02 -0.23 -0.34 0.05 stone volume 0.88** -0.05 0.31 0.01 -0.17 -0.16 0.09 stone weight 0.35 0.77** 0.26 -0.03 0.13 0.08 -0.05 leaf blade length 0.64** 0.50 0.11 -0.17 0.01 0.17 -0.28 leaf blade width 0.64** 0.41 0.03 -0.09 0.07 0.34 -0.49 leaf blade length/leaf blade widht 0.05 0.49 0.21 -0.23 -0.13 -0.39 0.43 petiol length 0.38 0.14 -0.39 -0.13 -0.12 0.18 0.54** total soluble solid 0.47 -0.17 0.31 0.45 0.45 -0.16 -0.13 ph 0.12 0.31 0.67** 0.59 0.09 0.25 0.01 vitam c -0.05 -0.56** -0.13 -0.09 -0.15 0.07 0.19 titratable acidity 0.21 -0.51** 0.47 0.04 0.39 0.21 0.31 total phenol content -0.10 0.14 0.40 0.12 0.27 -0.67** -0.07 total anthocyanin content 0.51 -0.20 -0.24 0.20 -0.59** -0.12 0.00 antioxidant activity 0.12 0.36 0.01 -0.42 0.34 -0.08 0.28 total flavonoids content 0.61** -0.32 -0.24 0.50 0.08 0.09 -0.25 eigen value 12.45 5.63 4.69 3.32 2.42 1.43 1.18 cumulative variance (%) 29.65 43.06 54.24 62.16 69.30 75.11 80.54 158 adv. hort. sci., 2019 33(2): 153-160 first cluster were characterized by a upright tree vigour, depressed fruit pistil end, reniform shape of fruit, high sweetness, dark red juice, flower high length and diameter, fruit and stone weight and length and diameter, total soluble solid, low total phenolic content, high total flavonoid content and high total anthocyanin content. perez-sanchez et al. (2008) suggested that dendrogram gained from morphological characteristics clearly showed the relationships among the cultivars of sweet, sour and duke cherries. in addition, khadivi-khub et al. (2012) reported that dendrogram obtained from morphological characteristics clearly separated some cerasus genotypes. the second cluster were characterized by small fruit and stone, drooping or spreading tree habit. scatter plot was prepared according to the pc1 and pc2 by past software (fig. 2). starting from the positive to the negative values of pc1, these genotypes indicated a gradual decrease in fruit pistil end, fruit color of skin, fruit length, fruit diameter, fruit weight, stone length, stone diameter, stone volume, l e a f b l a d e l e n g t h , l e a f b l a d e w i d t h a n d t o t a l flavonoids and an increase in tree habit, flower shape of petal, fruit ripening time. starting from the negative towards the positive values of pc2, the genotypes indicated a gradual increase tree branching, vitamin c, titratable acidity and a decrease flower diameter, petal length, petal width and stone weight. 4. conclusions morphological characterization continues to be the first step for the description and classification of germplasm and statistical methods like principal components analysis (pca) are useful tools for screening the accessions of a collection (cantini et al., 1999; badenes et al., 2000). pca is used for data reduction that transforms the original variables into a limited number of uncorrelated new variables. this technique, producing a smaller set of composite variables, account for much of the variance among the set of original variables and allows visualization of the differences among the individuals, identification of possible groups and finding relationships among individuals and variables (martinez-calvo et al., 2008). high correlations were found between some traits and principal components, which could reduce the number of traits to be studied in sour cherry germplasm. for instance, measuring the traits of pc1 (such as fw, sl, sd. sv, fpe, and fcs) is suggested for future studied in sour cherry genotypes. dependent on the trait, a certain number of genotypes were observed that showed lower or higher values than the commercially grown cultivars involved in this study. especially in reference to the fruit weight, a high portion of genotypes were characterized by smaller fruits than that of “ciganymeggy” and “erdi botermo”. in addition, native genotypes showed higher values of total phenolic content and antioxidant activity traits than the commercial cultivars. we conclude that this is the first study of sour cherry native genotypes, which deals with the morphological and physicochemical variation basis of genetic diversity. although these accessions does not fig. 1 dendrogram of 17 sour cherry genotypes based on morphological traits by past software. fig. 2 factor scores for the first two principle components (pcs) for sour cherry genotypes. nazari et al. iranian sour cherry characterization 159 represent the whole sour cherry germplasm in iran, considerable genetic diversity observed in both morphological and 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(ed.) current protocols in food analytical chemistry. john wiley & sons, new york, usa. zhang q., yan g., dai h., zhang x., li c., zhang z., 2008 characterization of tomentosa cherry (prunus tomentosa thunb.) genotypes using ssr markers and morphological traits. sci. hortic., 118: 39-47. impaginato 123 adv. hort. sci., 2019 33(1): 123-131 doi: 10.13128/ahs-22952 spiralling whitefly and its management practices in the south pacific. a review r.r. chand 1, 2 (*), a.d. jokhan 2, r. kelera 1 1 school of biological and chemical sciences, faculty of science, technology and environment, the university of the south pacific, private mail bag, suva, fiji. 2 school of science and technology, the university of fiji, private mail bag, lautoka, fiji. key words: abundance, aleurodicus dispersus russell, management, south pacific, spiralling whitefly. abstract: a few species of whiteflies are considered a serious insect pest of vegetation and ornamental plants across many countries. the spiralling whiteflies, aleurodicus dispersus russell has been recorded on many different plant species across countries. these whiteflies feed exclusively on leaves and have the ability to spread plant diseases. a great deal of research has been done on whiteflies in relation to which control methods would be most effective in pest management. the management of spiralling whitefly in the south pacific is heavily reliant on biological control (using the parasitic wasps, predators and entomopathogenic fungi). other control methods include physical, botanical, and chemical to keep the whitefly population at lower levels. in this paper, an overview of spiralling whiteflies and its management practices in the south pacific will be reviewed. 1. introduction spiralling whiteflies, aleurodicus dispersus russell, 1965 (hemiptera: aleyrodidae), are polyphagous pest of agricultural and horticultural crops in glasshouses and fields worldwide (oliveira et al., 2001; mani and krishnamoorthy, 2002; stansly and natwick, 2010). it is a native to the caribbean region and central america (waterhouse and norris, 1989). it was first noticed as a pest in hawaii in 1978 and since then has spread to the pacific islands and other continents (kumashiro et al., 1983; waterhouse and norris, 1989). whitefly adults and larvae feed on leaves, stems and fruits by inserting stylets into the plant. some specific plants that are usually attacked include cassava, pepper, papaya, mango, eggplant, citrus, guava, banana, coconut, breadfruit, tropical almond, sea grape, paper bark and rose (russell, 1965; jayma et al., 1993; neuenschwander, 1994; reddy, 2015). when the stylets are in the phloem, the whiteflies ingest large quantities of sap that contains a lot of sugar. they excrete the excess liquids and sugar which is called honeydew. the honeydew is deposited on leaves (*) corresponding author: s11074077p@gmail.com citation: chand r.r., jokhan a.d., kelera r., 2019 spiralling whitefly and its management practices in the south pacific. a review. adv. hort. sci., 33(1): 123-131 copyright: © 2019 chand r.r., jokhan a.d., kelera r. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 31 march 2018 accepted for publication 7 september 2018 ahs advances in horticultural science mini review http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(1): 123-131 124 and fruits, fostering the growth of black sooty mould fungi and falling of premature leaves. in addition, these moulds influence the rate of photosynthesis and transpiration as it hinders the light penetration, vapour movement and exchange, leading plants to exhibit yellowish specks on leaves, to wilt or die off (mcauslane et al., 2004; al-shareef, 2011; reddy, 2015). spiralling whiteflies have caused detrimental effects in the production of crops and ornamental plants. it is one of the most common pest that has the ability to spread diseases and influence the global food production (ecpa, 2015). over 300 plant species from approximately 77 families have been recorded as hosts of a. dispersus worldwide (lambkin, 1999). the spread of the spiralling whitefly continues to increase rapidly due to the general ineffectiveness of chemical control and other pest control measures (mani and krishnamoorthy, 2002). alternative measures for controlling spiralling whiteflies include physical, cultural and biological control. these approaches have been used in the south pacific but there is no data available (except biological control) that supports their practice and effectiveness in terms of whitefly management (waterhouse and norris, 1987). hence, the present review builds information on studies carried in the south pacific on a. dispersus russell alternative control methods, which would be valuable in terms of effective management of the pest. 2. origin, biology and whitefly-plant interactions aleurodicus dispersus is a tropical pest to a variety of horticultural crops, ornamental plants and shade trees (department of agriculture and fisheries, 2015). it was first reported in florida in late 1957 ( n a t i v e a r e a b e i n g t h e c a r i b b e a n a n d c e n t r a l america) and since then it has expanded its range to most regions around the world including north america, south america, asia, africa, australia and several pacific islands (russell, 1965; waterhouse and norris, 1987; reddy, 2015). in the south pacific, t h e p e s t h a s b e e n r e p o r t e d i n m a j u r o ( 1 9 8 6 ) (marshall is), cook islands (1984), fiji (1985), nauru (1987), papua new guinea (1987), kiribati (june 1988), tokelau (late 1988) and tonga (november 1988) (waterhouse and norris, 1987). the lifecycle stages of a. dispersus are eggs, four larval instars and adults. the eggs (0.3 mm long) are usually smooth surfaced, yellow and tan elliptical in shape (reddy, 2015). these are laid at an angle of 90° with spiralling deposits of white flocculence on underside of the leaves. the first instars are mobile and called crawlers. they can travel to a short distance to select their feeding sites (martin, 1987). they are usually 0.32 mm long and settle near the spiral pattern of the eggs from which they hatched. as the crawler grows, they develop mid-dorsal waxy tufts and the secretion of wax is usually from the narrow band of sub-margin. the second and third instars are 0.5-0.65 mm long and remains feeding at same place. the distinguishing feature about the third instar larvae is the presence of glass-like rods of wax (usually short and evenly-spaced) lined along the body. these cottony secretion is much less abundant than on the fourth instar [russell (1965) cited in the centre for agriculture and bioscience international (cabi, 2015)]. the fourth instar or puparium is 1.06 mm long and covered with numerous amounts of white materials and long glass-like rods (~8 mm in length); due to fragmentation some are shorter (fig. 1). the second to fourth instars are protected by waxy secretions making them sessile and scale-like (martin, 1987; banjo, 2010). the adults are mobile and most active during the morning. the bodies of males are fig. 1 mature puparium of spiralling whitefly glassy wax rods emanating from each compound spores. these glassy rods are whitish in colour, translucent and longer (3-4 times) than the width of the body from the dorusm (extending upwards and outwards) of mature puparium, a copious amount of white cottony substance is secreted chand et al. whitefly management in the south pacific 125 usually 2.28 mm and females are usually 1.74 mm (3-4 times longer than the body width). the adults develop white translucent powder covering on their bodies. these whiteflies also have a pair of antenna. the males have several pores on the abdomen scattered dorsally, laterally and ventrally on the first 2 segments posterior to wax plates while the females are without pores [russell (1965) cited in the centre for agriculture and bioscience international (2015)]. the eye is reddish-brown in colour. the spiralling whiteflies also have two characteristic dark spots on their forewings (fig. 2). after mating adult females lay eggs in irregularly spiralling patterns and it is where whiteflies derived their common name, spiralling whitefly (reddy, 2015). heavy occasional rains and cool temperatures result in a temporary reduction in a. dispersus population (mani, 2010). the mortality rate of immature stages increase between 40-45°c, and for adults the mortality rate increases at 35-40°c. temperature below 10°c also lead to increased mortality (cherry, 1979; waterhouse and norris, 1987). however, population of spiralling whiteflies will increase during droughts and presumably when the natural enemies decline. the spread of crawler and winged adult whiteflies usually occurs with short-distance movements whereby the crawlers are walking on the plant on which it hatched and the plants that touch it and adults fly to other parts of the same plant or to other nearby host plants. instead, the movement to long distance involves the international horticultural trade (pacific pests and pathogens, 2016). whitefly-plant interactions stomatal conductance whiteflies cause damage to plant productivity in terms of photosynthesis, respiration and transpiration performance (shannag and freihat, 2009). the whitefly a. dispersus is considered to be a major pest causing damages to crops (boopathi et al., 2015). these insect pests are reported to alter the chemical processes, growth and photosynthesis (schröder et al., 2005; fatouros et al., 2012). moreover the plants primary metabolism is also altered by this particular infestation. for example, egg deposition by a particular species of moth (podoptera frugiperda) demonstrated inhibitory effect towards the production of herbivore induced plant volatiles in maize plants (fatouros et al., 2012). the egg deposition has also shown to influence the rate of photosynthesis by reducing the amount of carbon dioxide diffusion in the mesophyll cells (fig. 3) (schröder et al., 2005; fatouros et al., 2012). for instance, the study by shannag and freihat (2009) concluded that gas exchange in cucumber plants infested by whiteflies (bemisia tabaci) caused photosynthesis to decline by up to 30%. the feeding behaviour of whiteflies also fig. 2 adult of spiralling whitefly. fig. 3 (a) the stomatal underneath the normal leaf surface and (b) the egg attached on the stomata of infected leaf. presence of dark spot on the forewings white waxy flocculants materials dark reddish-brown eye, where part the compound eye joined by 3 or 4 facets presence of a pair of antenna adv. hort. sci., 2019 33(1): 123-131 126 increased the respiration rate by 24-78% and the rate of water loss from infested leaves was 3-32% greater than that of control leaves. likewise, the increasing number of whiteflies (tobacco whitefly) led to increase in transpiration rate (shannag and freihat, 2009). the artificial infestation of spiralling whiteflies described by pitan (2003) on pepper led to increase in the damage of leaves. this simply means that the chlorophyll, sugar, protein and crude fibre contents of the leaves decreased as with the level of infestation. 3. status and damage in the south pacific the reproduction and dispersal rate of spiralling whiteflies are relatively high, posing great threat to vegetable, tropical fruit tree and ornamental industries around the globe (pacific pests and pathogens, 2016). the host range of these spiralling whiteflies covers at least a range of 27 plant families, 38 genera and more than 100 species (waterhouse and norris, 1989). despite there is no evidence in the measurement of economic loss by these spiralling whiteflies in the south pacific. heavy infestations on plants is more likely to result in economic loss (pacific pests a n d p a t h o g e n s , 2 0 1 6 ) . b e i n g a p o l y p h a g o u s , spiralling whitefly has been recorded on many plant species in different countries. according to surveys conducted from 1996 to 1997 (waterhouse and norris, 1989; barro et al., 1997), spiralling whitefly is an exotic pest in american samoa, cook islands, fiji, kosrae (fsm), pohnpei (fsm), truk (fsm), yap (fsm), g u a m , k i r i b a t i , m a r s h a l l i s l a n d s , n a u r u , n e w caledonia, northern mariana islands, palau, papua new guinea, solomon islands, french polynesia, tonga and western samoa (table 1). the survey also revealed the absence of a. dispersus from niue, tuvalu and vanuatu. 4. management of aleurodicus dispersus russell the management of whitefly has been difficult as a result of its many host plants. perennial plants such as ornamentals, fruit trees and shade trees were probably used successfully throughout the year by this coloniser (whiteflies) which sucks the sap of leaves (kajita et al., 1991). according to chandel et al. (2010), whiteflies must be dealt with a combination of environmental manipulations, natural enemy enhancement and area-wide control programme. t h i s t e c h n i q u e i s k n o w n a s i n t e g r a t e d p e s t management (ipm) which uses a combination of different strategies to control pests. the ipm program uses current, comprehensive information on the life cycles of pests and their interaction, combination with pest control methods to manage pest by the most economical means with least hazardous to the environment and the people (boopathi, 2013; epa, 2017). however, based on the literature available in the south pacific, there are no reported studies on chemical and physical or combined (ipm) control practices on a. dispersus. the only data available is on biological control (waterhouse and norris, 1987). there are various methods of biological control of whitefly techniques utilised in the south pacific, such as the use of parasitic wasps, predators (lacewings, big-eyed bugs and minute pirate bugs) and the use of entomopathogenic fungi of the genus aschersonia. biological control natural predators and parasitoid. spiralling whitefly is not regarded as a pest in its native area of caribbean and central america where it is assumed that populations are kept low by natural enemies (prathapan, 1996). biological control is perhaps the safest and most sound approach to pest control. it is an effective tool in programmes of integrated pest table 1 distribution of aleurodicus dispersus whiteflies in the pacific found during the whitefly survey (1996-1997) aleurodicus dispersus ams coi fij kos poh tru yap gua kir mai nau niu nmi pal png si frp ton tuv van wsa distribution in 1996/7 x x x x x x x x x x x x x x x x x x exotic x x x x x x x x x x x x x x x x x x serious pest potential x x x x x x x x x x x x x x x x x x ams= american samoa; coi= cook islands; fij= fiji; kos= kosrae (fsm); poh= pohnpei (fsm); tru= truk (fsm); yap= yap (fsm); gua= guam; kir= kiribati; mai= marshall islands; nau= nauru; nca= new caledonia; niu= niue; nmi= northern mariana islands; pal= palau; png= papua new guinea; si= solomon islands; frp= french polynesia; ton= tonga; tuv= tuvalu, van= vanuatu and wsa= western samoa. the letter ‘x’ indicates a.dispersus indication for presence and concern. information retrieved from barro et al. (1997). chand et al. whitefly management in the south pacific 127 management. utilising natural enemies reduces the risk of pesticide usage that results into environmental pollution increase. the importation of parasitoids of genera encarsia or eretmocerus and of various predators have been successfully used in greenhouses for whitefly control (gerling et al., 2001). the t h r e e p a r a s i t i c s p e c i e s , e r e t m o c e r u s m u n d u s ( m e r c e t ) , e r e t m o c e r u s e r e m i c u s r o s e a n d z o l n e r o w i c h a n d e n c a r s i a f o r m o s a g a h a n (hymenoptera: aphelinidae) have been used against whiteflies in japan (sugiyama et al., 2011). in fiji, the common predators of a. dispersus are coccinellids megalocarla (= archaioneda tricolor) fijiensis, serangiella and the neuropteran cbrysop [kumar et al. (1987) cited in waterhouse and norris (1989)]. some common predators of whiteflies are lacewings, big-eyed bugs, minute pirate bugs and several lady beetles (for example; scymnus or chilocorus species). a major outbreak of spiralling w h i t e f l i e s o n p a p a y a w a s r e p o r t e d i n s a m o a (pestnet, 2005). the outbreak of whiteflies usually happens when their natural enemies are disturbed or destroyed by pesticides, dust build-up and other factors. these outbreaks commonly affected guavas, palms, ground orchids, and poinsettias (ornamental). a recent study showed that a. swirskii (mite) is increasingly used for the biological control of thrips and whiteflies in many crops (messelink et al., 2008). three major predators that have been found to be most effective in attacking spiralling whiteflies are megalocaria fijiensis, serangiella and the neuropteran chrysopa species (waterhouse and norris, 1989). according to waterhouse and norris (1989), the establishment of pacific bridgehead in hawaii (1978) led to spread of spiralling whiteflies to many of the pacific nations. parasitic wasps encarsia (?) haitiensis, encarsia species and three coccinellid predators from trinidad were used as biological agents to reduce the damaging populations of whitefly in guam. it was noted in lanai island (hawaii) that e. (?) haitiensis and encarsia species could effectively control the spiralling whitefly. adult whiteflies were observed on buses, cars and near parking areas. by late 1979, a. dispersus was considered to be an economic pest and initiated a search for natural enemies in the caribbean. different species of coccinellid predators and aphelinid parasite were introduced in 1979 and 1980 to reduce the population of a. dispersus. in fiji, a. dispersus spread rapidly and became a serious pest since 1986. the introduction of encarsia (?) haitiensis, n. oculatus and n. bieolor from guam and hawaii became well established up to 2 km from their release sites. the three predators found to attack a.dispersus prior to 1986 were the coccinellids megalocarla fijiensis, serangiella sp. and the neuropteran chrysopa sp. similarly, in american samoa, a. dispersus were found in 1981 on a wide range of plants including ornamentals, citrus and other fruit trees. the introduction of coccinellid predators and the parasite encarsia (?) haitiensis in 1984 from hawaii rapidly reduced the a. dispersus population. table 2 provides detail as where the spiralling whiteflies were discovered, their host range and biological control in hawaii, american samoa, cook islands, fiji, pohnpei, guam, kiribati, palau, papua new guinea, tonga and western samoa. physical control of a. dispersus removal and traps. removal of leaves may be an environmental friendly approach, but it does not completely remove the pest, it rather lessens the level of whitefly population from the plant. a slight infestation can quickly spread to other plants. the removal of leaves is a good approach to get rid of non-mobile nymphal and pupal stages of whiteflies from highly dense leaves. in addition, yellow sticky traps are used to trap adults since whiteflies are attracted to yellow (barbedo, 2014). it is where a trap consisting strips of paper and sticky substances such as petroleum are placed in the greenhouse. the insects are caught as they fly. the drawback of this type of approach is that it only captures specimens that can fly. however it is generally ineffective for the insects that are in their early stages since they are not able to fly (barbedo, 2014). this method is not a full-proof control method for farmers since it does not eliminate damaging population, but aims to reduce the whitefly population (nakamura et al., 2007). botanicals. natural pesticides such as plant essential oils would represent an alternative in crop protection (coats, 1994; isman, 2000; koul et al., 2008). different plants have been used for the control of pests and the research has worked out well (gonzalezcoloma et al., 2010). medicinal plants can be an alternative to a lot of synthetic chemicals for human health and agriculture. however, people in fiji and the rest of the south pacific countries are not very cognizant of the presence of the great plant diversity surrounding them. the only study published in relation to botanical effects against a. dispersus were carried by chand et al. (2016). plant extracts such as essential oils from these medicinal plants could possibly be used in agriculture in the form of pest controls. the study focused 128 adv. hort. sci., 2019 33(1): 123-131 on five common medicinal plants randomly selected and screened for the insecticidal properties (fumigant and repellent test). these medicinal plants were m a k o s o i ( c a n a n g a o d o r a t a ) , l e m o n g r a s s (cymbopogon. citratus), curry leaves [murraya. koenigii (l.) spreng] tulsi [ocimum tenuiflorum (l.)] and uci (euodia. hortensis forma hortensis). the results revealed that tulsi essential oils showed strong fumigant toxicity (100% mortality in 3 hours) while lemon grass and curry leaves showed the best repellent effect with lc50 value of 0.004 and 0.113, respectively (chand et al., 2016). table 2 introductions for the biological control of a.dispersus country aleurodicus dispersus discovered place and year plants on which they were found first introduction of biological control hawaii honululu in september 1978 tropical almond coleoptera. delphastus pusillus (1980) from trinidad. nephaspis oculatus (1979) from trinidad. nephaspis oculatus (1979) from honduras. n. bicolor(1979) from trinidad. hymenoptera. e. ? haitiensis (1979) from trinidad. encarsia sp. (1980) from trinidad. american samoa tutuila in 1981 guava, ornamentals, citrus and other fruit trees. infestations were noted on banana leaves and later in vegetable gardens coleoptera. delphastus pusillus (1984) from hawaii. nephaspis oculatus (1984) from hawaii. nephaspis bicolor (1984) from hawaii. hymenoptera. e.? haitiensis (1984) from hawaii. cook islands rarotonga in 1984 frangipani, guava, hibiscus and mango coleoptera. ? nephaspis bicolor (1985) from hawaii. hymenoptera. ?encarsia ? haitiensis (1985) from hawaii. encarsia ? haitiensis (1988) from fiji. fiji suva in april 1986 -coleoptera. nephaspis oculatus (1987) from guam. nephaspis oculatus (1987) from hawaii. n. bicolor (1987) from hawaii. hymenoptera. e. ? haitiensis (1987) from guam. e. ? haitiensis (1987) from hawaii. pohnpei --hymenoptera. e.? haitiensis (1986) from guam. guam guam in 1981 coconut, frangipani, guava and mango coleoptera. nephaspis oculatus (1981) from hawaii. hymenoptera. encarsia ? haitiensis (1981) from hawaii kiribati bikenibeu, tarawa, in june 1988 chillies, bell peppers, tomatoes, pawpaw, guava, breadfruit, banana, ornarnentals -including, frangipani and coleus a biological control project is to be commenced in the near future palau --hymenoptera. e.? haitiensis (1986) from guam. papua new guinea october 1987 in the port moresby guava, mango leaves and coconut palms *coccinellids and spiders were seen preying on them tonga in november, 1988 -*attacked by unidentified wasps. western samoa first recognised in 1985 --chand et al. whitefly management in the south pacific 129 chemical control. the use insecticides are widespread among farmers. for instance the paper by kajita et al. (1991) provides a description of several insecticides against a. dispersus on soya beans in indonesia. however, because the whitefly has wide host-plant ranges in addition to the fact that insecticides also have impact on natural enemies, chemical control is usually considered impractical and uneconomic in the long-term (kajita et al., 1991; lambkin, 1998). carmichael et al. (2008) also discourage the use of chemical control for managing spiralling whiteflies, suggesting that soaps and detergents can provide effective control in small scale planting. in the south pacific, there are no recorded publications dealing with chemical control measures of a. dispersus yet (waterhouse and norris, 1987). according to reddy (2015), the common chemicals used for controlling whiteflies are dimethoate 30 ec at 0.05% and insecticidal soap at 2.5%, which deterred the adults. likewise, the following chemicals imidacloprid, buprofezin and pyridaben are also u s e d t o m a n a g e w h i t e f l i e s ( b i e t a l . , 2 0 0 2 ) . spiromesifen, a novel insecticide inhibited egg hatching in green house by 80% to 100% at the concentrations of 3.1, 3.0, and 10.0 µg ml-1. the insecticide also showed mortality of 100% for the first, second, and third instar nymphs of whiteflies (toscano and bi, 2007). chemical approach mostly kills those that come in contact with the insecticides (chemicals). the use o f t h e c h e m i c a l a p p r o a c h s h o w e d e f f i c i e n c y towards controlling pests in small and in large scale farms. for instance, farmers in colombia intensify the use of insecticides, as the whiteflies reduced the crop yield by 79% (carabalí et al., 2010). although, plant productions may have increased due to pesticidal applications at the same time these chemicals may have raised detrimental concerns for so many (aktar et al., 2009). chemical pollution is a major concern to the environment and to human health due to the bioaccumulation of chemicals through food chains, resulting in severe physiological disorders and diseases (oliva et al., 2001; baldi et al., 2003; briggs, 2003; saiyed et al., 2003; lemaire et al., 2004). the extensive use of synthetic chemicals has led to pests in developing resistance to chemicals and at the same time resulting into the accumulation of harmful chemical pollutants in the environment. these pollutants gradually affect the quality of air and water, on which many organisms rely on. 5. conclusions and future perspective whiteflies are considered serious pests to vegetation and ornamental plants in many countries and as such, spiralling whiteflies are fast becoming a concern for many farmers in the pacific and other parts of the world. in the south pacific the pest has been reported in majuro (1986) (marshall is), cook islands (1984), fiji (1985), nauru (1987), papua new guinea (1987), kiribati (june 1988), tokelau (late 1988) and tonga (november 1988) (waterhouse and norris, 1987). these whiteflies feed exclusively on leaves, which eventually damage the plant leading to diseases or plant death. whiteflies are very difficult to manage as a result of multi host plants that support their lifespan. the most common management practices used for the control of whiteflies are biological control using the parasitic wasps, predators and entomopathogenic fungi, physical method using removal and traps, botanicals such as plant extracts and essential oils, and chemical control. at present there is no available literature in the south pacific that provides data on chemical and physical practices. the only data available were on biological control methods and the use of botanicals (essential oils) for the management of a. dispersus (waterhouse and norris, 1987; chand et al., 2016). the salient findings gathered from this review indicate a general lack of information and research on the current status and the degree of damages that the spiralling whitefly has inflicted on agricultural and ornamental crops in the south pacific. acknowledgements the authors are grateful to mrs reema prakash for the support provided throughout the research journey especially in collecting data on whiteflies. the authors are also thankful to the university of the south pacific for providing research funding in form of graduate assistantship to the corresponding author. references aktar m.w., sengupta d., 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waterhouse d.f., norris k.r., 1989 biological control: pacific prospects. supplement 1. in: waterhouse d.f. and k.r.norris (eds.) biological control. acfia, inkata press, melbourne, australia, pp. 123. impaginato 353 adv. hort. sci., 2018 32(3): 353-362 doi: 10.13128/ahs-21927 influence of soil and soilless agricultural growing system on postharvest quality of three ready-to-use multi-leaf lettuce cultivars b. pace 1, 2, i. capotorto 1, 2, m. gonnella1, f. baruzzi 1, m. cefola 1, 2 (*) 1 istituto di scienze delle produzioni alimentari, consiglio nazionale delle ricerche (cnr), via g. amendola, 122/o, 70126 bari, italy. 2 istituto di scienze delle produzioni alimentari, consiglio nazionale delle ricerche (cnr), urt c/o cs-dat, via m. protano, 71121 foggia, italy. key words: ammonium, lactuca sativa l., nitrate, microbial quality, postharvest storage, soilless cultivation. abstract: in this study the influence of soil and soilless growing systems (substrate 3:1 v/v perlite:peat) on quality and microbial traits of three multi-leaf lettuce cultivars (two green, ‘eztoril’ and ‘ezabel’, and one red, ‘ezra’) was evaluated at harvest and after 7 and 13 days of storage at 8°c. at harvest, ‘ezra’ showed a respiration activity and a total phenol content respectively 2-fold and 25% significantly higher than the green cultivars. soil lettuces resulted more stressed than those grown in soilless, as indicated by their initial content in antioxidants. as for nitrate content, soilless grown lettuces at harvest showed an average concentration higher than soil-grown ones, although values are generally lower than limits imposed by the eu regulation (no. 1258/2011). during storage, soilless lettuces showed no ammonium accumulation, differently from those cultivated in soil. in addition, lettuce cultivars grown in soilless condition showed unchanged content in the antioxidant activity and total phenols, and lower microbial counts than soil lettuces. results of the present study showed that soilless growing system can positively affect qualitative and microbiological parameter of lettuces studied, and it can be considered a good soilless growing technique in order to obtain high quality multi-leaf lettuces for readyto-use industry. 1. introduction the consumer’s demand of ready-to-use fruits and vegetables, and in particular that for minimally processed leafy vegetables, is continuously growing. although iceberg lettuce is still the main lettuce used in the ready-to-use industry, consumers are requesting other types of lettuce with attractive colours and shapes combining the best quality characteristics from all varieties (rijk zwaan, 2009). the new baby-sized leaves, (*) corresponding author: maria.cefola@ispa.cnr.it citation: pace b., capotorto i., gonnella m., baruzzi f., cefola m., 2018 influence of soil and soilless agricultural growing system on postharvest quality of three ready-to-use multi-leaf lettuce cultivars. adv. hort. sci., 32(3): 353-362 copyright: © 2018 pace b., capotorto i., gonnella m., baruzzi f., cefola m. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 10 october 2017 accepted for publication 12 january 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(3): 353-362 354 babyand multi-leaf have been developed recently as high quality lettuce varieties for the ready-to-use market. some benefits of baby-sized lettuce, when compared with whole-head lettuce, include: i) greater efficiency with higher percentage of usable product; ii) easier and faster processing; iii) more attractive colour and shapes, and iv) minimal oxidation due to smaller stem diameter (martínez-sánchez et al., 2012). moreover, for both multiand baby-leaf lettuces, no physical wounding was undertaken, except that of the harvesting, avoiding the physical damage that occurs during preparation of fresh-cut lettuce that causes an increase in respiration activity, biochemical changes and microbial spoilage, which may result in degradation of colour, texture and flavour of the ready-to-use produce (cantwell, 2004). likewise, cultivar selection is of great importance in the ready-to-use industry since quality characteristics (such as leaf colour, shape, freshness, texture and browning potential) can change largely depending on the genotype (nicola et al., 2009). the quality and shelf-life of ready-to-use leaves depend on genotypic traits of raw material and on several aspects from preharvest to postharvest processing (clarkson et al., 2003; cantwell, 2004). some physical and chemical indicators can be used for objective assessment of visual quality (barrett et al., 2010; salinas-hernández et al., 2015). among these, ammonium (nh4+), produced during storage as a consequence of senescence in various vegetables (cefola et al., 2010; pace et al., 2014), might be used as predictors of shelf-life (cefola et al., 2017). in general, preharvest factors should be aimed to optimize their impact on postharvest quality (crisosto and mitchel, 2002). from this point of view, soilless system is becoming of high interest since it can improve both, preharvest and postharvest quality of vegetables (rodríguez-hidalgo et al., 2010). in particular, soilless agricultural growing system allows to set optimal conditions and nutrient concentration for plant growth (silberbush and ben-asher, 2001; selma et al., 2012) with the following advantages: higher yields (lopez-medina et al., 2004; recamales et al., 2007), better quality vegetables (recamales et al., 2007; cefola et al., 2011) and higher earliness (recamales et al., 2007; valenzano et al., 2008), compared to soil cultivation. the success of lettuce production depends to a great extent on the maintenance of a continuous growth rate by the optimal management of nutrients (luna et al., 2013). in addition, especially for leafy vegetables, the use of soilless system can avoid soil contaminants and improve the sanitary quality respect to traditional soil cultivation, leading benefits on raw materials for postharvest industry (selma et al., 2012). starting from these findings, the aim of this work was to evaluate the influence of two growing system (soil and soilless) on postharvest quality of three multi-leaf lettuce genotypes, including two green and one red, stored under refrigeration for 13 days. 2. materials and methods reagents extraction solvents (meoh, etoh), 2,2-diphenyl-1picrylhydrazyl (dpph), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox) and all standards used in the experiments were obtained from sigma-aldrich (st. louis, mo., usa). folin-ciocalteu’s p h e n o l r e a g e n t w a s p u r c h a s e d f r o m m e r c k (germany). plant material and growing system three types of dutch multi-leaf lettuces (lactuca sativa l.), two green (cv. eztoril and ezabel) and one red (cv. ezra) (enza zaden, enkhuizen, netherland) were cultivated in an unheated plastic tunnel under soil (s) or soilless (sl) agricultural growing system in the same greenhouse at the experimental farm “la noria” of the institute of sciences of food production (cnr-ispa) located in the south of italy (mola di bari). a split-plot design with three replications was applied, randomizing the growing systems (gs) in the main plots and cultivars in the subplots. main plots were of 3.6 m2 (0.9 m wide and 4 m long). the sl system consisted of three single benches (4 m long x 0.3 m wide x 0.1 m high, with a slope of 2%) each plot containing a 3:1 (v:v) perlite:peat mixture as substrate. the nutrient solution was supplied to the sl system without recirculation and had the elemental composition given in table 1, where the soil characteristics are reported too. the irrigation water had the following composition (expressed in mmol l-1): 0.3 n-no3, 0.23 k, 0 p, 1.73 mg, 1.82 ca, 7.39 cl, 4.05 na. nutrient solution and water were supplied to sl and s units based on a timer controlled schedule, using minimum substrate water content values, monitored by tensiometers. furthermore, as additional reference control taking into account two different threshold levels of -5 and -25 kpa to start the irrigation supply in sl and s, respectively. for sl system a nutrient supply level criterion was additionally pace et al. postharvest quality of three ready-to-use multi-leaf lettuce cultivars 355 applied. at transplant soil plots were fertilized with ammonium nitrate and monopotassium phosphate giving the equivalent of 50, 80, 50 kg ha-1 of n, p2o5 and k2o, respectively, and after a month a 30 kg ha-1 i n t e g r a t i o n o f n f r o m a m m o n i u m n i t r a t e w a s applied. seedlings were produced in greenhouse in polystyrene trays on peat and were transplanted 25 days after sowing on february 22. harvest was performed 55 days after transplanting (on april 18) for the sl and after 73 days (on may 6) for the s system. greenhouse ventilation temperature was 20°c. in figure 1 the climatic parameters measured in the greenhouse are reported. daily air temperature was on average 21°c, and minimum and maximum air temperature ranged from 0.2 to 17.5 and from 17.5 to 46.0°c, respectively (fig. 1a). air relative humidity was on average 50.5%; daily minimum and maximum relative humidity ranged from 6 to 56% and from 45 to 85% (fig. 1b). the average photosynthetically active radiation was 282 µmol m-2 s-1; its mean and maximum values changed from 80 to 420 and from 400 to 2,080 µmol m-2 s-1, respectively (fig. 1c). after harvest, lettuces were immediately transported under refrigerated condition in polystyrene boxes to the cnr ispapostharvest laboratory. processing and storage after harvest, for each multi-leaf lettuce cultivar (‘ezra’, ‘eztoril’ and ‘ezabel’), and for each agricultural growing system (s or sl), leafs were selected in order to avoid damaged samples, and no washing or pre-treatment were applied. for each cultivar and gs about 600 g of leaves were used for quality evaluation at harvest, whereas about 1.2 kg were used for the quality evaluations during storage. thus, leaves were put in open polyethylene bags (about 200 g each bag), and stored at 8°c in dark conditions. for each cultivar, 12 bags (3 replicates ´ 2 gs, s or sl, ´ 2 storage periods, 7 and 13 days) were prepared. fig. 1 climatic parameters (a= temperature; b= air relative humidity; c= photosynthetically active radiation) measured in the greenhouse during the experiment, from seedling transplantation to harvest days. the blue and red perpendicular lines indicate the harvest day for lettuce cultivated in soilless and soil condition, respectively. mineral composition soil composition soilless nutrient solution sand 24.30% silt 31.90% clay 43.80% ph 7.6 6.5 ec (ds m-1) 2.5 2.3 cl7.39 mg2+ 1.73 na+ 4.05 k+ 5.12 ca2+ 4.74 nh 4 + 0.5 no 3 9.43 p-h2po41.61 s-so420.81 cec (cmol kg-1 dw) 31.8 organic matter (g kg-1 dw) 14 total n (g kg-1 dw) 0.95 available p (g kg-1 dw) 110 available k (g kg-1 dw) 244 caco 3 (g kg-1 dw) 0.11 table 1 soil and soilless nutrient solution composition. soil classified as clay soil (usda textural soil classification, 1987). values of nutrient solution are expressed in mmol l-1. micronutrients were supplied according to johnson et al. (1957) adv. hort. sci., 2018 32(3): 353-362 356 respiration activity and the analysis of nitrate content were performed at harvest. in addition, antioxidant activity, total phenols, ammonium content and microbiological analysis were evaluated at harvest and after 7 and 13 days of storage. respiration activity the respiration activity of each cultivar was measured using a closed system as reported by kader (2002). about 100 g of leaves for each replicate were put into 6 l sealed plastic jars (one jar for replicate) where co2 was allowed to accumulate until the value of 0.1%. the time needed to reach this value was calculated, making co2 measurement at regular time intervals. for the co2 analysis, 1 ml gas sample was taken from the head space of the plastic jars through a rubber septum and injected into the gas chromatograph (p200 micro gc, agilent, santa clara, ca) equipped with dual columns and thermal conductivity detector. co2 was analyzed with a retention time of 16 s and total run time of 120 s on a 10 m porous polymer (ppu) column at a constant temperature of 70°c. respiration activity was expressed as ml co2·kg-1·h-1. antioxidant activity, total phenol, ammonium and nitrate content to determine both antioxidant activity and total phenols content, the extraction procedure reported by cefola et al. (2012), was followed. in detail, 5 g samples were homogenized (ultraturrax t-25, ika staufen, germany) in a meoh: water (80:20) solution for 1 min, and then centrifuged at 5°c at 6440 x g for 5 min. the supernatant was therefore used for the assays. the antioxidant activity assay was performed following the procedure described by brand-williams et al. (1995) with minor modifications. briefly, the supernatant, proper diluted, was pipetted into 0.95 ml of dpph solution to start the reaction. the absorbance was read after about 30 min at 515 nm. trolox was used as a standard and the antioxidant activity was expressed in g of trolox equivalents per kg of fresh weight (g teac kg-1 fw). the total phenol content was determined according to the method of singleton and rossi (1965). each extract (100 μl), proper diluted, was mixed with 1.58 ml water, 100 μl of folin-ciocalteu reagent and 300 μl of sodium carbonate solution (200 g l-1). the absorbance was read after 2 h at 765 nm. total phenol content was calculated on the basis of the calibration curve of gallic acid and expressed as g of gallic acid per kg of fresh weight (g ga kg-1 fw). for ammonium content the method reported by weatherburn (1967) was used. in detail, 5 grams of chopped sample were homogenized (ultraturrax t25, ika staufen, germany) with 20 ml distilled water for 2 min, centrifuged at 6440 x g for 5 min, and 0.5 ml extract was used for the analysis. color development, caused by the reaction with a phenol nitroprusside reagent and alkaline hypochlorite solution, was determined after an incubation of 20 min at 37°c, by reading the absorbance at 635 nm (uv1800, shimadzu, kyoto, japan). ammonium content was expressed as µmole nh4+ per kg of fresh weight (µmole nh4+ kg-1 fw). as for nitrate content, samples (about 100 g for replicates) were dried in the oven (65°c until constant weight) and were ground to fine powder. the powder (0.5 g for each replicate) were extracted on orbital shaker for 20 minutes with 50 ml of a solution containing 3.5 mmol l-1 of sodium carbonate and 1 mmol l-1 sodium bicarbonate. analysis were carried out using a ion exchange chromatography (dionex dx 200, dionex corp, sunyvale, ca, usa) with a conductivity detector, using an ionpac ag14 precolumn and a n i o n p a c a s 4 a s e p a r a t i o n c o l u m n ( d i o n e x corporation). results were expressed in mg of nitrate per kg of fresh weight (mg no3kg-1 fw). microbiological analysis samples (30 g for replicates) were homogenized for 1 min in 0.1% sterile buffered peptone water (difco laboratories, detroit, mi, usa) (1:5 dilution) using a stomacher (seward, london, uk). total aerobic mesophilic bacteria count was evaluated using plate count agar (difco) incubated at 30°c for 48 h. yeasts and moulds were counted on sabouraud dextrose agar (difco) supplemented with chloramphenicol and chlortetracycline (both 0.05 g l-1) and incubated at 25°c for 5-7 days. total counts of enterobacteriaceae were obtained by pour-plating dilutions (1 ml) in violet red bile glucose agar (difco) and plates were incubated at 37°c for 24 h. microbiological counts were expressed as log cfu g-1 of fresh weight (log cfu g-1 fw). statistical analysis in order to study the effect of gs (s or sl), cultivars, cv (‘ezra’, ‘eztoril’ and ‘ezabel’) and their interaction (gs x cv) on quality parameters at harvest, and the effect of gs, cv, storage (0-7-13 days) and their interaction (gs x cv x storage) on quality parameters, two multifactor anova were performed (statistica software). when significant effect of factors were detected, the student newman keuls (snk) test was applied to separate means. for a visual pace et al. postharvest quality of three ready-to-use multi-leaf lettuce cultivars 357 analysis of the data, principal component analysis (pca) (princomp procedure, sas software, cary, nc, usa; biplot by xlstat, addinsoft, paris, france) was performed on mean centered and standardized (unit variance scaled) data prior to analysis. the data matrix submitted to pca was made up of 18 observations 3 cultivars (‘ezra’, ‘eztoril’ and ‘ezabel’) x 2 growing system (s and sl) x 3 storage times (0-7-13 days) and 6 quality parameters (antioxidant activity, total phenols, ammonium, mesophilic bacteria, yeasts and moulds, enterobacteriaceae). 3. results effect of growing systems and cultivars on lettuces quality traits at harvest yield response was influenced by genotypes more than gs (s or sl), since a lower fresh weight was produced by the red lettuce compared to the other two cultivars (2.7 vs 3.6 kg m-2) and only in cv. eztoril there was a higher yield in s compared to sl system (4.3 vs 3.3 kg m-2). the effect of gs, multi-leaf lettuce cv (‘ezra’, ‘eztoril’ and ‘ezabel’) and their interaction on the quality parameters measured at harvest was investigated (table 2). ammonium content, antioxidant activity, total phenols and nitrate content were significantly affected by gs and cv, while respiration activity was affected only by cv. the interaction gs x cv was statistically significant only for nitrate content (table 2). the respiration activity of cv. ezra was twofold higher than the green cultivars (‘eztoril’ and ‘ezabel’). regarding ammonium content, the values found for lettuces cultivated in sl were statistically higher respect to s and, between cultivars, red multileaf lettuce had mean values statistically higher than green cultivars (table 2). growing system affects significantly the antioxidant activity and total phenols: plants cultivated in s showed significantly higher mean content than sl samples (table 2). regarding cv, there were no differences between green multileaf lettuces in antioxidant activity and total phenols, while the red cultivar ezra had lower values of antioxidants and higher values of total phenols respect to the green cultivars (table 2). as for nitrate content was almost double in sl lettuces than s grown ones (838 vs 432 mg no3kg-1 fw). the red multi-leaf lettuce (cv. ezra) had mean values of nitrate statistically higher than cv. eztoril but not different from cv. ezabel (table 2). effect of growing systems and cultivars on lettuces’ quality traits and microbial parameters during cold storage the results of multifactor anova on antioxidant activity, total phenols and ammonium content as affected by gs, cv, storage time (0, 7 and 13 days) and their interaction were reported in table 3. growing system affected antioxidant activity and total phenols, cv affected total phenols and ammonium, while storage time affected antioxidant activity respiration activity (ml co 2 kg-1 h-1) ammonium content (µmole nh 4 + kg-1fw) antioxidant activity (g teac kg-1 fw) total phenols (g ga kg-1 fw) nitrate content (mg no 3 kg-1 fw) growing system (gs) soilless 47.44 66.7 2.33 1.32 838.12 soil 45.73 49.6 4.82 1.84 431.96 cultivar ezra 72.67 a 72.80 a 3.07 b 1.89 a 765.71 a eztoril 35.82 b 48.10 b 3.82 a 1.41 b 524.32 b ezabel 31.26 b 53.60 b 3.84 a 1.44 b 615.09 ab gs ns ** *** ** *** cultivar *** ** ** * * gs x cultivar ns ns ns ns * table 2 effect of growing system (soil and soilless) and cultivar (ezra, eztoril and ezabel) on quality parameters measured at harvest when interaction among factors was not significant, the results of the mean separation test (snk test) are reported. different letters indicate statistical difference within cultivars, respectively, for p≤0.05. ns, not significant; * p≤0.05; ** p≤0.01; *** p≤0.001. 358 adv. hort. sci., 2018 32(3): 353-362 and ammonium (table 3). considering the interaction among factors, antioxidant activity was affected by gs x storage and cv x storage; total phenols were influenced by gs x cv and by gs x cv x storage, and ammonium was affected only by gs x storage (table 3). in figure 2, changes in antioxidant activity (a), total phenols (b) and ammonium content (c) during storage of the three multi-leaf lettuce cultivars, cultivated in s and sl conditions, are reported. at harvest, lettuces cultivated in s showed values of antioxidant activity significantly higher than sl lettuce. however, during storage, antioxidant activity of lettuces cultivated in s decreased rapidly, reaching approximately the same values of samples cultivated in sl after 7 day of storage at 8°c; after it remained unchanged for cv. ezabel (about 2 g teac kg-1 fw) and slightly increased for cv. eztoril (about 3 g teac kg-1 fw). the cultivar ezra cultivated in s showed a content in antioxidant activity almost constant during storage, with a 30% reduction at the end of storage (fig. 2a). whereas, lettuce cultivated in sl showed unchanged values of antioxidant activity during time, with a slight reduction at the end of storage for cv. ezabel (about 1.3 g teac kg-1 fw) (fig. 2a). regarding the content of total phenols (fig. 2b), green multileaf lettuces cultivated in s showed a slight decrease during the first week of storage, after then values rise again until the end of storage, reaching values of about 1.6 and 1.7 g ga kg-1 fw for cv. eztoril and ezabel, respectively (fig. 2b). a specular trend for green multi-leaf lettuce cultivated in sl was observed (fig. 2b). the cv. ezra cultivated in sl showed the same behavior of green lettuces; whereas ‘ezra’ cultivated in s showed an initial total phenol content of about 2.3 g ga kg-1 fw, which increased during the first week of storage, reducing to initial values until the end of the storage (fig. 2b). as regard data of ammonium content (fig. 2c) lettuces cultivated in sl showed unchanged values during postharvest storage, starting from the initial m e a n v a l u e s o f a b o u t 7 9 . 9 ± 7 . 5 , 5 7 . 0 ± 9 . 3 a n d 63.2±9.6 mmole nh4+ kg-1 fw in cv. ezra, eztoril and ezabel, respectively. whereas, lettuces cultivated in s showed an increase in ammonium content during storage, which doubled for all cultivars, starting from initial mean values of 65.5±15.7, 39.2±3.5 and 44.0±7.0 mmole nh4+ kg-1 in cv. ezra, eztoril and ezabel, respectively. fig. 2 changes in antioxidant activity (a), total phenols (b) and ammonium content (c) of three multi-leaf lettuce cultivars (‘ezra’, ‘eztoril’ and ‘ezabel’), cultivated in soil or soilless condition, during storage at 8°c. mean ± sd. table 3 multifactor anova of antioxidant activity total phenols and ammonium content as affected by growing system (soil or soilless), cultivar (‘ezra’, ‘eztoril’ and ‘ezabel’) and storage time (0, 7 and 13 days) antioxidant activity (g teac kg-1 fw) total phenols (g ga kg-1 fw) ammonium content (µmole nh 4 + kg-1fw) growing system (gs) *** ** ns cultivar (cv) ns ** ** storage time *** ns * gs x cv ns ** ns gs x storage * ns * cv x storage *** ns ns gs x cv x storage ns * ns ns= not significant; * p≤0.05; ** p≤0.01; *** p≤0.001. 359 pace et al. postharvest quality of three ready-to-use multi-leaf lettuce cultivars as concerns microbial populations evaluated during the trial, results of multifactor anova statistical analysis of mesophilic bacteria, yeasts and moulds and enterobacteriaceae as affected by growing system (gs), cultivars (cv) and storage time are reported in table 4. all factors and their interactions affected significantly the microbial populations evaluated, with the exception of gs x cv interaction for yeasts and moulds. as concerns total mesophilic bacteria, lettuces cultivated in sl showed an increase of about 1.5 log unit during storage, starting from initial mean values of 3.7±0.05 log cfu g-1 (cv. eztoril), 4.9±0.22 log cfu g-1 (cv. ezabel) and 6.1±0.29 log cfu g-1 (cv. ezra), whereas lettuces cultivated in s growing condit i o n s h o w e d a n h i g h e r s i g n i f i c a n t i n c r e a s e i n mesophilic population during storage, of about 3 log unit, starting from a mean initial count of about 4.67±0.68 log cfu g-1 (fig. 3a). yeast and mould loads from s and sl were not found to be significantly different. however, during 13 days of cold storage their amount increased significanly for all cultivars, resulting statistically higher in multi-leaf lettuces cultivated in s respect to the sl ones (fig. 3b). also in the case of enterobacteriaceae, lettuces cultivated in s showed a significant increase of about 4 log unit at the end of the storage, starting from initial values of 3.4±0.9, 2.4±0.26 and 4.10±0.10 log cfu g-1 for cv. ezra, eztoril and ezabel, respectively. in lettuces cultivated in sl conditions, the increase of about one magnitude order was found for this microbial population (fig. 3c). principal component analysis principal component analysis revealed that almost 84% of the total variability of data was explained by the first two principal components. pc1 resulted mainly and positively correlated to ammonium and the counts of the three microorganisms groups. each of them contributed to pc1 for 20-25% of the total variability (fig. 4). on the other hand, antioxidant activity and total phenols contributed to pc2 for more than 45% each. among observations, all s growing system samples after 13d storage showed a stronger and positive correlations with pc1, ammonium and the microbiological counts. at a proximate position collocated samples of the red cultivar ezra collected from sl and stored for 7 and 13 days. on the contrary, all cultivars grown on the s system, at fig. 3 changes in total mesophilic bacteria (a), yeasts and moulds (b) and enterobacteriaceae (c) of three multileaf lettuce cultivars (‘ezra’, ‘eztoril’ and ‘ezabel’), cultivated in soil or soilless condition, during storage at 8°c. mean ± sd. mesophilic bacteria yeasts and moulds (log cfu g-1 fw) enterobacteriaceae growing system (gs) ** * * cultivar (cv) *** * *** storage time *** *** *** gs x cv *** ns *** gs x storage *** *** *** cv x storage *** ** *** gs x cv x storage *** * *** table 4 multifactor anova of mesophilic bacteria, yeasts and moulds and enterobacteriaceae as affected by growing system (soil or soilless), cultivar (‘ezra’, ‘eztoril’ and ‘ezabel’) and storage time (0, 7 and 13 days) ns= not significant; * p≤0.05; ** p≤0.01; *** p≤0.001. 360 adv. hort. sci., 2018 32(3): 353-362 harvest, were negatively correlated to pc1, on the opposite site of ammonium and microbial parameters. it seems that no observations were strongly correlated to antioxidant activity and total phenols, with the exception of soil-grown ezra leaves sampled at harvest and at 7 days storage, followed by the other two cultivars coming from s at harvest and soilgrown ezra at 13 days storage (fig. 4). among observations negatively correlated to pc2, one result noteworthy, the sl-grown ‘ezabel’ sampled at 13 days after storage, since it showed a sharp decrease in the antioxidant activity at the end of the storage, as described in figure 2a. 4. discussion and conclusions the three multi-leaves lettuces studied in this research showed different qualities at harvest. in particular, the green cultivars resulted very similar and suitable for postharvest processing, on the basis of respiration activity and ammonium content. at contrary, the red cultivar was considered more perishable than the green ones, due to the high respiration activity (kader, 2002) even though it showed an higher content in polyphenols than the green cultiv a r s , a s p r e v i o u s l y r e p o r t e d b y o t h e r a u t h o r s (martínez-sánchez et al., 2012; selma et al., 2012). as regards antioxidant activity and total phenols, the higher contents measured in s cultivated lettuces could be a plant response to applied stress treatments (oh et al., 2009). compared to the sl growing system, s irrigation management implies necessarily different water conditions, keeping s plants in not optimal and constant water availability all-day. as a consequence, s plants may have occasionally experienced a water stress combined to heat stress during the highest temperature hours under greenhouse, in the few days preceding harvest (fig. 1). even a timelimited stress can activate the antioxidant synthesis in the plant metabolism (oh et al., 2009). on the other hand, the more constant availability of water and nutrients in sl grown plants allowed a higher nitrogen uptake, partially accumulated as nitrate in the vacuoles at higher rate than in s cultivated lettuces. however, the nitrate content measured at harvest was generally low in both gs compared to the limits imposed by the eu regulation no. 1258/2011. in compliance with the current regulation, nitrate accumulation in lettuce grown in the spring-summer period under greenhouse should not exceed 4,000 mg kg-1 fw. this limit is in agreement with the potentially high nitrate accumulating capacity of lettuce. however, at our latitude, the optimal light conditions found by plants during the spring months allow an efficient and fast assimilation of the up-taken nitrate. during cold storage, the sl growing system resulted able to preserve the quality of lettuces since no increase in ammonium content (senescence indicator) was registered whereas multi-leaf lettuces cultivated in s that resulted more senescence-prone. ammonium accumulates in leafy vegetables during storage, as consequence of protein catabolism. thus, ammonium was used as indicator of quality and shelf-life of green vegetable. (chandra et al., 2006; pace et al., 2014; cefola et al., 2015; cefola and pace, 2015; cefola et al., 2017). data from ammonium confirms that sl could be considered a suitable growing system to preserve postharvest quality of the cultivars analysed, although genotyping characteristics of each cultivar need also to be taken into account (selma et al., 2012). during storage nitrate measurements were not carried out, since in preliminary trials performed on the same lettuce genotype (cv. ezra) no nitrate changes after storage at 8°c for 10 days w e r e d e t e c t e d . i n p a r t i c u l a r n i t r a t e r e m a i n e d unchanged at 1550 and 1800 mg kg-1 fw, in soilless and soil lettuce, respectively (data not shown). this was supported by several contributes in literature, referring about no modification of nitrate concentration in lettuce and other species during storage at temperature in a range from 1 to 10°c (siomos et al., 2002; chung et al., 2004; konstantopoulou et al., 2010). the sl cultivation showed a positive effect fig. 4 pca biplot (pc1 vs pc2) describing the spatial distribution of quality and microbiological parameters of three multi-leaf lettuce cultivars (‘ezra’, ‘eztoril’ and ‘ezabel’) grown in soil (s) and soilless (sl) system during storage; a= ezra; b= eztoril; c= ezabel. aa= antioxidant activity; phenols= total phenols; 0d – 7d – 13d: 0-7-13 days of storage. 361 pace et al. postharvest quality of three ready-to-use multi-leaf lettuce cultivars also on microbiological quality of the green cultivars during storage. similarly, results were reported by other authors on table grape (cefola et al., 2011) and on soilless growing systems (scuderi et al., 2011; selma et al., 2012). in conclusion, the three multi-leaves lettuces studied in this research showed different qualities at harvest. in particular, green cultivars resulted very similar and suitable to postharvest processing, whereas, the red one was considered more perishable. at harvest, lettuces grown in soil showed the higher content in antioxidant activity and total phenols and the lower in nitrate than soilless samples. however, the nitrate content measured at harvest was generally low in both growing systems compared to the limits imposed by the eu regulation no. 1258/2011. regarding the postharvest storage, ready-to-use lettuces cultivated in soilless showed microbiological and qualitative performance better than those grown in soil. in particular, soilless growing system improved the storability of lettuces and allowed to the production of clean raw material, particularly suited for ready to use industry. it is interesting to note, as soilless system resulted able to limit ammonium accumulation (senescence indicator), also in red cultivar, which for genotipic traits resulted more senescence prone than green lettuces. acknowledgements this research was financed by miur research projects: “high-convenience fruits and vegetables: new technologies for quality and new products,” pon01_01435. references barrett d.m., beaulieu j.c., shewfelt r., 2010 color, flavor, texture, and nutritional quality of fresh cut fruits and vegetables: desirable levels, instrumental and sensory measurement, and the effects of processing. cr. rev. food sci., 50: 369-389. brand-williams w., cuvelier m.e., berset c., 1995 use of a free radical method to evaluate antioxidant activity. lwt food sci. technol., 28: 25-30. cantwell m., 2004 evaluating the quality of fresh-cut products. in: cantwell m. 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biotechnol., 83: 71-75. weatherburn m.w., 1967 phenol-hypochlorite reaction for determination of ammonium. anal. chem., 39: 971-974. 213 adv. hort. sci., 2018 32(2): 213-219 doi: 10.13128/ahs-21365 the effect of polyamines and sics on the compatibility, fertility and yield indices of apple cv. golden delicious p. sayyad-amin, g.h. davarynejad (*), b. abedy department of horticultural sciences and landscaping, agriculture college, ferdowsi university of mashhad, p.o. box 9177948978, mashhad, iran. key words: fruit set, putrescine, spermidine, spermine. abstract: the most critical problems of temperate fruit trees are poor fruit set and low yield. to solve the problem, some major chemical compounds such as polyamines and sics (self-incompatibility control substance, mn+b) can be used. popular polyamines including putrescine (0.1 and 0.25 mm), both spermine and spermidine (0.05 and 0.25 mm), and sics (1 and 2 mg l-1) were used alone or with cotton coverage bags to cover branches in order to investigate self-incompatibility in malus domestica. results showed that spermidine (0.25 mm) led to higher yields in comparison with that of the control. sics (2 mg l-1), also, demonstrated the highest yield compared with that of the control. at june fruit set, treatment with spermidine (0.25 mm) led to the highest percentage of fruit set and also the highest index of self-incompatibility and percentage of final fruit set among treatments. 1. introduction alternate bearing and self-incompatibility are the main issues in apple. alternate bearing is vigorous in some apple cultivars like ‘golden delicious’. most apple cultivars are self-incompatible and self-unfruitful; therefore, they need another cultivar for pollination and fertilization. gametophytic self-incompatibility (gsi) occurs when the s allele of the pollen grain matches either of the s alleles of the stigma. in such a case, the pollen tube begins to develop but stops before reaching the micropyle (asatryan and tel-zur, 2013). gametophytic self-incompatibility is controlled by glycoprotein with rnase (s-rnase) activity expressed in the pistil. s-rnase (qing-qing et al., 2009; duca et al., 2010; uchida et al., 2012) encoded by the s-locus gene is named sfb (s-haplotype-specific f-box gene) in the rosaceae. to date, in rosaceae family, si has mainly been studied in japanese pear (pyrus pyrifolia) and almond (prunus dulcis) (qing-qing et al., 2009; uchida et al., 2012). however, some requirements are necessary for suitable cross pollination: having (1) compatible pollen grains with enough quantity and high quality (in fact, pollination with (*) corresponding author: davarynej@um.ac.ir citation: sayyad-amin p., davarynejad g.h., abedy b., 2018 the effect of polyamines and sics on the compatibility, fertility and yield indices of apple cv. golden delicious. adv. hort. sci., 32(2): 213219 copyright: © 2018 sayyad-amin p., davarynejad g.h., abedy b. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 7 november 2017 accepted for publication 18 april 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(2): 213-219 214 semi-compatible pollen resulted in lower fruit-set than that with fully compatible pollen) (sapir et al., 2008), (2) an overlap period of pollination between pollinizer and pollinator (pollinizer trees must be cultivated along with the pollinator trees) (3) proper time of flowering and blooming, and finally, (4) having attractive flowers in order to reduce the number of bees visiting weeds on the orchard floor (the pollen grain of fruit trees is sticky and heavy causing not to be carried by the wind) (bekey and burgett, 1981). hand pollination is a type of cross pollination that can be effective on producing crop in adverse weather conditions. when the king flower opens, it is necessary to place bee colonies in order to do cross pollination by bees and then remove them at petal fall in fruit orchards: however, this method is also timeconsuming and needs a big number of workers (bekey and burgett, 1981; maib et al., 1996). despite the presence of hives, pollen transfer limitation and subsequent seed set reductionwas observed in orchards (quinet et al., 2016). the other suitable technique applied to do pollination involves chemical methods; in fact, some chemical compounds can be used in order to achieve enough fruit set and high yield. plant bio regulators (pbrs) had significant effects on increasing pollen germination and pollen tube length in almond pollen: the action of these pbrs significantly increased the percentage of fruit set at both the bud pink and petal fall phenological stages (maita and sotomayor, 2015). as an alternative, the use of polyamines (putrescine, spermine, spermidine), that are natural compounds involved in plant growth and development process, has been s u g g e s t e d r e d u c e f l o w e r s a n d f r u i t s d r o p ; polyamines competes with ethylene synthesis with whose they share the same precursor called s-adenosyle methionine (crisosto et al., 1988; khezri et al., 2010). polyamines as plant bio regulator revealed to increase pollen tube growth and fruit set by stimulating pollen germination (crisosto et al., 1988; liu et al., 2006) and were effective on pollen tube elongation (aloisi et al., 2015). also, self-incompatibility control substance (sics), which is a mixture of manganese and boron (son et al., 2009), could be useful for increasing fruit set. polyamines and sics were effective on increasing yield and fruit set in crops such as pear (crisosto et al., 1988; son et al., 2009), apple and apricot (asadi et al., 2013), olive (costa et al., 1986), sweet cherry (grant sheard, 2008), sweet orange (saleem et al., 2008), pistachio (khezri et al., 2010), mango (malik et al., 2005), and date palm (tavakoli and rahemi, 2014). with regard to the effect of these compounds on pollen tube growth, spermidine (spd) and spermine (spm) influence the p r o m o t i o n o f t h e p o l l e n t u b e e l o n g a t i o n a t polyamines concentrations up to 50 mm in rosaceae family, whereas higher concentration of spd and spm resulted inhibitory for pollen tube elongation in rosaceae family and correlate with male sterility in actinidia deliciosa (aloisi et al., 2015). they are compounds useful in enhancing ovule longevity (crisosto et al., 1988; liu et al., 2006), without having any deleterious and toxic effects on human life (azh et al., 2014). in the initial stage of fruit development, an active cell division takes place, which possibly needs sufficient polyamines. at the later stage of fruit development, polyamine synthesis is reduced. as biosynthesis of polyamines takes place before pollen tube emergence, low level of free polyamines in cytoplasmic male sterile plants influences cell division and its enlargement, leading to abnormal development and low viability of pollens (liu et al., 2006). polyamines increase pollen tube growth and fruit set by stimulating pollen germination. as well, they play a role in carbohydrates and nitrogen regeneration, followed by increasing chlorophyll content and leaf a r e a ( b a n i n a s a b a n d r a h e m i , 2 0 0 8 ) . u s e o f polyamines (put, spm, spd) at bloom improves ovule longevity in fruit crops such as apricot and pear. higher endogenous polyamine contents have been correlated with improved ovule viability in apricot and sour cherry (grant sheard, 2008). boron increased pollination activity (nyomora et al., 1997). in boron deficiency, phenolic compounds aggregates on stigma. the accumulation of these compounds due to the activation of dehydrogenase enzyme led to the pollen grain not to be germinated. boron increases pollen grain viability by increasing the flavenoids content of pollen grain (marschener, 1995). the percentage of fruit set as a vital factor is as important as other quantitative and qualitative traits to achieve an acceptable yield in apple (malus domestica). it seems the contemporary application of polyamine and sics together can enhance their effects. although there are numerous researches on the individual application of polyamine and sics, there was not found any literature comparing the effect of polyamines and sics on yield indices, especially along with cotton coverage bags on the index of fertility (if) and the index of self-incompatibility (isi). the aim of this study was to increase yield indices in apple cultivar ‘golden delicious’, accompanying the decrease of fruit drop using polyamines and sics. sayyad-amin et al. polyamines and sics effect on compatibility, fertility and yield indices of apple cv. golden delicious 215 2. materials and methods the study was carried out on thirteen year-old apple trees cv. golden delicious in an orchard located in mashhad (latitude of 36°20’and altitude 59° 34‘). it is an area with arid and semiarid climate and annual average precipitation of 255 mm. the foliar application was done with 5 l sprayer on four selected branches between bud swollen and flower opening pheological stages at early morning. the compounds included polyamines [putrescine (put) 0.1 or 0.25 mm; spermine (spm) 0.05 or 0.25 mm; spermidine (spd) 0.05 or 0.25 mm] and sics [1 mg/l (3.5 mg boric acid, 6.8 mg manganase sulphate) and 2 mg/l (6.8 mg boric acid, 13.6 mg manganese sulphate)] (son et al., 2009) with (+) or without (-) cotton coverage bags (ccb), and control (untreated) plants. cotton coverage bags were used in order to prevent cross and open pollination. in this study, the following traits were measured: yield, percentage of fruit set [initial (2 weeks after petal fall); june (fruit drop in june); final (at harvest)] and fruit drop. index of fertility (if) was measured based on the percentage of initial fruit set ratio in each treatment compared to control. index of self-incompatibility (isi) was evaluated based on the percentage of final fruit set ratio in each treatment in comparison with control; in this regard, the ratios of 0.2, between 0.2-1, and higher than 1 represent incompatibility, semi-compatibility and full self-compatibility, respectively (zeinanlo et al., 2001; azimi et al., 2008; seifi 2008; taslimpour and aslmoshtaghi, 2013). the randomized complete block design with four replicates was applied in this study. at final, the data w e r e a n a l y z e d b y s a s s o f t w a r e v e r . 9 . 1 ( s a s institute, 2004) and the means were compared using lsd test at 0.05. fruit diameter and length were measured with non destructive method during the fruit growth period until the harvest time. this method was better than the destructive technique. non destructive method are less time consuming and no need to laboratory space, and without harvesting. according to this method, fruit length and diameter were measured every 2 weeks without harvest, whereas in destructive method, these parameters are measured in fruits harvested every 2 weeks and their length a n d d i a m e t e r w e r e m e a s u r e d . t h e r e f o r e , n o n destructive method was used in the present study (arzani et al., 1999; dehghani et al., 2012). 3. results the effects of polyamines and sics on yield and fruit set without cotton coverage bags. data showed that spd treatment (0.25 mm) led to a higher yield in comparison to the control (p<0.05). sics (2 mg l-1) demonstrated the highest yield among all of the treatments (p<0.05). it was also found that put (0.1 mm) and spm (0.05 mm) showed increase in yield compared with the control, but it was statistically non-significant at 0.05 level. sics (1 mg l-1) increased initial fruit set when compared to control. the highest june and fruit set related to spd (0.25 mm) among all of the treatments (p<0.05). with cotton coverage bags. there was significant increase in percentage of initial fruit set by application of sics (2 mg l-1), and spm (0.25 mm) (table 1) in comparison to the control. the results also demonstrated the higher percentage of initial fruit set under treatment with spd (0.05 mm) than the control, but it was statistically non-significant at 0.05 level. percentage of final fruit set showed statistically significant difference (p <0.05) in most of the treatments in comparison with the control, except for put (0.1 mm), sics (2 mg l-1), spm (0.25 mm), spm (0.05 mm) and spd (0.25 mm). the effects of polyamines and sics on if and isi the index of fertility increased in treatments with spm (0.25 mm + ccb) and sics (2 mg l-1 + ccb). all of the treatments could led to semi or full fertility and compatibility, due to the percentage of fruit set ratio in each treatment compared to the control was larger than 0.2. the effects of polyamines and sics on fruit drop in general, final fruit drop decreased significantly in treatments with put (0.1 mm) and spd (0.05, 0.25 mm). without coverage. sics (1, 2 mgl) decreased percentage of initial fruit drop in comparison with the control of 12 and 10%, respectively (p <0.05). final fruit drop decreased significantly in treatments with put (0.1 mm) and spd (0.05, 0.25 mm). with cotton coverage. spm (0.25 mm) demonstrated the significantly (p<0.05) decrease of 14% in percentage of initial fruit drop in comparison with the control. adv. hort. sci., 2018 32(2): 213-219 216 the effects of polyamines and sics on fruit growth habit based on figure 1, fruit growth totally increased in all of the treatments; in fact, fruit length and diameter increased gradually from july 9 to harvest (fig. 1 b and d) whereas spm (0.25 mm + ccb) showed a different trend. 4. discussion and conclusions the effects of polyamines and sics on yield and fruit set in the present study, application of polyamines [put (0.1, 0.25 mm), put (0.25 mm + ccb), spd (0.05, 0.25 mm) and spd (0.05 mm + ccb)] between periods of swollen buds and the start of flowering phenological stages could increase fruit set at final fruit set stage. several researchers studied about the effects of polyamines on yield indices. polyamines (put, spm, spd) enhanced ovule longevity at bloom in fruit crops such as apricot, pear and sour cherry (grant sheard, 2008). polyamine synthesis had also positive influence on development and viability of pollen and it occurred before pollen tube emergence. therefore, lower content of free polyamines caused male sterility in flowers (liu et al., 2006). exogenous application of different polyamines at full bloom had influence on increasing fruit set and total yield in apples, olive, litchi and mango. increase in fruit set and yield by polyamines was due to raising pollination, fertilization and fruit retention (costa et al., 1986). saleem et al. (2008) stated that polyamines significantly increased initial fruit set and yield and maximum fruit set was observed in spd, spm and put, respectively. put had positive effect on increasing ovule longevity, epp (effective pollination period), n and b and might raise the pollen tube growth rate in the styles of pears (crisosto et al., 1988), but their role on raising ovule longevity might be due to the improved nutritional status of the flower (grant sheard, 2008). application of put raised fruit set and yield of ‘comice’ pear at the start of flowering (crisosto et al., 1988). fruit set, crop density and yield efficiency under low fruit set conditions were improved by put application at flower opening stage in pear (crisosto et al., 1988). according to our results, the effects of polyamines o n f r u i t s e t a n d y i e l d w e r e i n a g r e e m e n t w i t h crisosto et al. (1988) on pear, malik et al. (2005) on mango, grant sheard (2008) on sweet cherry, saleem et al. (2008), khezri et al. (2010) and asadi et al. (2013) on pistachio. in present study, sics (2 mg l-1) had also a positive influence on fruit set and yield. in boron deficiency, p h e n o l i c c o m p o u n d s a g g r e g a t e o n s t i g m a . accumulation of these compounds due to activation of dehydrogenase enzyme did not lead to germination of pollen grain. boron increased pollen grain viability by increasing the flavenoids content of pollen grain (marschener, 1995). boron increased pollination activity (nyomora et al., 1997). foliar application table 1 the effect of polyamines (putrescine, spermine and spermidine) and sics on yield, fruit set and first drop means with the same letters were not significantly different according to lsd (0.05). spm= spermine, spd= spermidine, put= putrescine, if=index of fertility, isi=index of incompatibility. treatments yield (g) initial fruit set (%) june fruit set (%) final fruit set (%) if isi first drop (%) final drop (%) control 1230 cde 55.893 d-g 40.395 b-e 18.02 f 1 b-e 1 bcd 30.7 cde 68.25 a-e without cotton coverage bag put (0.1 mm) 1416.7 c 50.179 e-h 39.046 c-f 28.636 bc 0.8625 def 1.4872 b 34.091 cd 42.424 g put (0.25 mm) 1101 def 15.152 j 37.231 c-f 27.778 bc 0.5565 f 0.3715 e 31.548 cde 55.357 efg spd (0.05 mm) 728.5 ghi 34.953 hi 32.749 c-g 22.281 de 0.6352 ef 1.2683 bcd 25.417 d-g 51.25 fg spd (0.25 mm) 1990.8 b 41.692 ghi 61.722 a 34.722 a 0.7621 def 2.4166 a 18.75 gh 20.313 h spm (0.05 mm) 1274.5 cd 74.605 abc 43.367 bcd 29.592 bc 1.3588 ab 1.0409 bcd 24.107 efg 76.259 ab spm (0.25 mm) 1130 def 69.86 bcd 23.077 g 19.268 f 1.2552 abc 1.529 b 64.245 a 68.593 a-e sics (1 mg l-1) 452 ij 75.63 abc 52.241 ab 20.26 f 1.3693 ab 1.2772 bcd 19.063 fg 56.818 d-g sics (2 mg l-1) 2349 a 59.127 c-e 40.857 b-e 29.241 b 1.0773 bcd 1.4317 bc 9.43 h 58.772 c-f with cotton coverage bag put (0.1 mm) 556 ij 42.361 f-i 28.03 fg 18.374 f 0.8869 c-f 1.4954 b 35.714 c 64.249 b-e put (0.25 mm) 1116 def 33.145 i 27.083 fg 22.917 de 0.7276 def 1.2771 bcd 48.889 b 72.222 a-d spd (0.05 mm) 696 hi 60.348 cde 30.925 efg 20.049 c 1.0839 bcd 1.4104 bc 50.379 b 63.258 b-f spd (0.25 mm) 984 efg 56.25 d-g 43.75 bc 18.5 f 1.015 b-e 0.7663 de 16.193 gh 77.399 ab spm (0.05 mm) 355 j 51.667 e-h 43.089 bcd 20.365 f 0.9567 cde 0.8757 cde 56.034 ab 74.353 abc spm (0.25 mm) 599 ij 84.858 ab 31.579 efg 17.526 f 1.5371 a 1.2116 bcd 22.5 efg 55 efg sics (1 mg l-1) 900.7 fgh 57.143 defg 32.712 c-g 25.173 cd 1.054 bcd 1.3247 bcd 28.333 c-f 56.667 d-g sics (2 mg l-1) 720 ghi 87.326 a 44.355 bc 19.903 f 1.5973 a 1.1082 bcd 49.958 b 83.953 a sayyad-amin et al. polyamines and sics effect on compatibility, fertility and yield indices of apple cv. golden delicious 217 of boron increased yield (mashayekhi and atashi, 2008) and fruit set in comparison with control and fruit abscission was lower than control (khoshghalb et al., 2011). maz ardalan and savaghebi firoozabadi ( 1 9 9 7 ) r e p o r t e d t h a t f o l i a r a p p l i c a t i o n o f m n increased fruit yield. high levels of boron in floral organs such as the stigma and style, may aid pollen germination and make faster pollen tube growth down the style and into the ovary. application of sics a day before full bloom at 1 or 2 mg l-1 on three pear cultivars increased fruit set especially at 1 mg l-1 sics. furthermore, sics at 2 mg l-1 ascended the number of seeds (son et al., 2009). in addition, regarding fruit yield, foliar application of mn alone showed significant increase in fruit yield of sweet oranges due to its effect on increasing the number of fruit/tree as well as fruit average weight (hasani et al., 2012). our results for sics were in accordance with maz a r d a l a n a n d s a v a g h e b i f i r o o z a b a d i ( 1 9 9 7 ) , khoshghalb et al. (2011) on pear, mashayekhi and atashi (2008) on strawberry, son et al. (2009) on pear, hasani et al. (2012) on pomegranate. the effects of polyamines and sics on if and isi in our experiment, all of the treatments could led to semi or full fertility and compatibilty. so, this result was in line with duca et al. (2010) on pear. if the isi is lower than 0.2 means (self or cross) incompatibility, if it is between 0.2 to 1 interpret as semi (self or cross) compatibility, and if it is higher than 1, reveals full (self or cross) compatibility (zeinanlo et al., 2001; azimi et al., 2008.; seifi, 2008). duca et al. (2010) reported that, in compatible pollinated styles, the levels of put and spm were similar and higher than spd, whereas in self-incompatible pollinated styles, put was the highest. in the compatible pollinated styles, these three polyamines showed higher content when compared to selfincompatible pollinated styles (duca et al., 2010). pollen germination and pollen tube development are important for fertilization (kuruki et al., 2017). extremely low pollen germination rates may cause fruit setting failure because of ovule degradation before the pollen tube reaches the ovary (kuruki et al., 2017). full compatibility is superior to semi-compatibility for ensuring high fruit set, even when environmental conditions are favorable for growth and pollination (sapir et al., 2008). among the natural polyamines, spm showed strongest effects on tube growth (aloisi et al., 2015). the effects of polyamines and sics on fruit drop put treatments significantly reduced secondary fig. 1 effects of foliar application of polyamines and sics on fruit length without ccb (a), with ccb (b) and fruit diameter without ccb (c) and with ccb (d) of apple (cv. ‘golden delicious’). put= putrescine, spm= spermidine, spr= spermine, ccb= cotton coverage bags. adv. hort. sci., 2018 32(2): 213-219 218 fruit drop on date palm, apple, pear, mango, sweet orange and avocado (asadi et al., 2013). a reduction in secondary fruit drop was observed in date palm, apple, pear, mango, sweet orange and avocado (tavakoli and rahemi, 2014). due to preventing enzymatic conversion of 1aminocyclopropane-l-carboxylic acid (acc) to ethylene by polyamines, the ethylene production reduced and is followed by fruit drop (tavakoli and rahemi, 2014). decreased fruit drop one week before full bloom in the “on” year and increased yield per shoot two weeks before full bloom in the “off” year were observed by application of spm (0.1 and 1 mm), but spd (1 mm) just lowered fruit abscission one week before full bloom in the “on” year (khezri et al., 2010). in our study, spd (0.25 mm) was the best treatment on fruit set (june and final), yield, isi and fruit drop. asadi et al. (2013) reported that the most effective treatment on raising fruit set were spm and spd in apricot, respectively (asadi et al., 2013). the content of spd was the highest at four development stages, followed by put and spm, respectively (valero, 2010). tavakoli and rahemi (2014) stated that treatment with spd 1 mm led to the highest fruit yield. the effects of polyamines and sics on fruit growth habit regarding the effects of polyamines on fruit length and diameter, fruit growth totally increased because of increasing fruit length and diameter in all of the treatments in our study. in the case of fruit growth, malik et al. (2005) demonstrated that the amount of polyamines increased during initial fruit growth period of apple, pear, apricot and strawberry followed by gradually decrease near maturity. polyamine content of pericarp declined from fruit set to maturity. spd and spm were higher than put during initial fruit growth compared to later during fruit development (malik et al., 2005). put application had a positive effect on fruit size and weight, which might due to its role in cell division leading to improved weight and diameter of fruit (saleem et al., 2008). mn application in sics increased fruit diameter and length but only the 0.6% rate of manganese was significant on fruit diameter (hasani et al., 2012) and influenced fruit growth habit. control trees produced significantly lower fruit yield, so naturally the fruit size was greater compared to fruit from polyamines-treated trees (saleem et al., 2008). s p e r m i d i n e i n c o n c e n t r a t i o n o f 0 . 2 5 m m , with/without cotton 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biological sciences, university of sciences and technologies of houari boumediene el-alia, bp 16011 bab ezzouar, algiers, algeria. 2 laboratory of microbiology, department of cellular and molecular biology, faculty of biological sciences, university of sciences and technologies of houari boumediene el-alia, bp 16011 bab ezzouar, algiers, algeria. 3 laboratory of sciences and environment research, universitary center of amine elokkal elhadj moussa eg. akhamoukh, 11039 sersouf, tamanrasset, algeria. key words: abiotic stresses, ahaggar, plant growth promoting rhizobacteria, rhus tripartitus. abstract: plant growth promoting rhizobacteria (pgpr) associated with ucria (rhus tripartitus) represents a good alternative for including this crop in revegetation programs in arid area. in this study, 137 bacterial strains were isolated in tryptic soy agar medium (tsa) from six samples of ucria’s rhizospheric soil (ahaggar, algeria), based on colony characteristics, gram © reaction, oxidase and catalase tests. to evaluate their pgp activities and their physiological characteristics under stress environment, ten tests were made. sixty strains of 16 genera were selected for their pgpr abilities, which represent 43.79% to the total of rhizobacteria isolated. the maximum bacterial population were bacillus (35%). 71.66% of isolates were able to solubilize the phosphate, 31.66% were able to produce indole acid acetic (iaa), 58.33% were siderophore producers, 28,33% were able to produce cyanhydric acid (hcn) and 70% were able to grow without any source of nitrogen. indeed, pgpr strains have shown tolerance and/or resistance to several experimental environmental conditions. as a conclusion, the pgpr strains of ucria’s rhizosphere were shown a good potential for biofertilization and biocontrol of crops, and their tolerance to abiotic stresses is an interesting step to support their utilities. 1. introduction rhus tripartitus (called ucria, african sumac and tahounek in tamahaq) is an important medicinal plant belonging to the anacardiaceae fam(*) corresponding author: benaissa.asmaa@yahoo.fr citation: benaissa a., djebbar r., abderrhmani a., 2018 diversity of plant growth promoting rhizobacteria of rhus tripartitus in arid soil of algeria (ahaggar) and their physiological properties under abiotics stresses. adv. hort. sci., 32(4): 525-534 copyright: © 2018 benaissa a., djebbar r., abderrhmani a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 19 december 2018 accepted for publication 27 june 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(4): 525-534 526 ily. this species often grows in areas of marginal agricultural capacity. it encountered in algeria, in arid areas especially in the mountains of the ahaggar where the indigenous people (touaregs) use it to treat gastric disorders (chermat and gharzouli, 2015). this plant is also found in the north-eastern part of saudi arabia and in tunisia where it spreads in the center to the far southern part of the country. it is used in the arabian traditional medicine for centuries to treat cardiovascular and gastrointestinal disorders and inflammatory conditions (chetoui et al., 2013; shahat et al., 2016). the study of rhizosphere bacteria from medicinal plants is very important, as they are well known to have impact on plant growth and also produce industrially important metabolites and improve quality of medicinal products (bafana and lohiya, 2013). considerable numbers of studies were focused on the beneficial effects of bacterial species that colonize the rhizosphere of many plant species and proved their beneficial effects on plant growth, yield, and productivity as well as their role in the reduction of their susceptibility to diseases caused by phytopathogenic bacteria, fungi, viruses and nematodes and even against abiotic stresses. these bacteria h a v e b e e n c a l l e d « p l a n t g r o w t h p r o m o t i n g rhizobacteria» (kloepper et al., 2004; orhan et al., 2006; miransari, 2014; nadeem et al., 2014; gupta et al., 2015). these pgpr’s can enhance the plant growth by direct mechanisms such as the fixation of atmospheric nitrogen, the solubilization of minerals l i k e p h o s p h o r u s a n d i r o n , t h e p r o d u c t i o n o f siderophores and enzymes, the synthesis of phytohormones like the auxin, indole-3-acetic acid (iaa), cytokinins and gibberellins, their role in lowering of ethylene levels and the induction of systemic resistance. indirect mechanisms are used by pgpr to benefit the plant growth by the induction of the disease resistance by producing antibiotics or hydrogen cyanide, competition for nutrients, extracellular enzymes production and others (glick, 1995; vessey, 2003; adesemoye et al., 2009; saharan and nehra, 2011; saha et al., 2016). to the best of our knowledge, there are no studies conducted on the rhizospheric bacteria associated with rhus tripartitus, a medicinal plant that grows in ahaggar (algeria). we hypothesize, that this plant harbors a diverse group of rhizospheric bacteria that can help ucria to cope with harsh environmental conditions. so, the main objectives of this study were to characterize the isolated rhizobacteria associated with the rhizosphere of rhus tripartitus, their propreties as plant growth promoting bacteria and their capacities to tolerate abiotics stresses. 2. materials and methods sample collection, isolation and characterization of rhizobacteria six soil samples are taken from the rhizosphere of t h e w i l d u c r i a s h r u b s f r o m i l a m a n r e g i o n i n tamanrasset, an arid area, which is located in the national culturel parc of ahaggar in the south of algeria (22° 49’ 59’’ north, 5° 19’ 59’’ east) during march, 2017. each soil sample was collected at a depth of 15 cm, around the root and placed in a sterile container. the samples collection was transported to the laboratory in an ice box set at 4°c. tenfold serial dilution of the samples was made by mixing the soil with sterile water, and plating on a tryptic soy agar medium (tsa). the plates were inverted and incubated, at 30°c for five days. the maximum of bacterial colonies present on plates were purified and characterized. the gram reaction, oxidase reaction and catalase test were performed as per standard procedure. in vitro screening and identification of plant growth promoting rhizobacteria the collected rhizobacterial isolates associated with rhus tripartitus were tested for their capacities to produce plant growth promoting effects. the pgpr were identified by using api galeries e20, ne20, c h b , s t a p h a n d n h ( a p i , b i o m e r i e u x s a , l y o n , france). all the strains were preserved in nutrient broth added with 20% glycerol at -80°c. nitrogen fixation. the fixation of molecular nitrogen is tested on a free nitrogen medium. the bacterial isolates are inoculated on the plates and incubated at 25°c for 24-48 h. the growth on this medium after being transferred ten times in the same medium r e f l e c t s t h e a b i l i t y o f b a c t e r i a t o f i x n i t r o g e n (haahtela et al., 1983). production of hcn. the strains ability to produce hydro cyanic acid (hcn) is carried out according to the method of lorck (1948) on solid bennett agar amended with 4.4 g/l glycine is inoculated with a loop of the bacterial culture. 90 mm whatman paper are dipped in sodium picrate solution (0.5% picric acid and 2% sodium carbonate) for one minute and then placed underneath the petri plates lids. the plated were sealed with parafilm and incubated at 30°c for four days. the appearance of an orange to benaissa et al. pgpr of rhus tripartitus 527 red color indicates the production of hcn. solubilization of phosphates. qualitative phosphate solubilization activity was tested on nbrip (national botanical research institutes phosphate) medium by applying a spot of 20 μl of bacterial suspension on the surface of the agar and incubated at 30°c for 15 days (nautiyal, 1999). a clear halo zone around the colony is an indication of phosphate solubilization. the calculation of the solubilization index (s.i.) is carried out according to the formula developed by kumar and narula (1999): s.i. = diameter of the halo around the spot/diameter of the spot production of siderophores. the production of siderophores on solid medium is carried out qualitatively on chrome azurol s (cas) medium as described by schwyn and neilands (1987). the cas plates were prepared and divided into sectors and inoculated with bacterial culture spots (10 μl of 106 cfu/ml) and incubated at 25±2°c for 48-72 h. the development of an orange yellow halo around the colony was considered positive for the siderophore production. the change in color is due to the transfer of ferric ions from the cas to the siderophores. the calculation of the ratio (halo diameter/diameter of the bacterial colony) makes it possible to compare production differences between bacterial strains. production of indole acetic acid (iaa). the production of iaa was determined according to the method of holt et al. (1994). the principle is to inoculate the selected strains on the nutrient broth containing 0.1 g/l of l-tryptophan. the change of the solution color from yellow to pink or red when we add the reagent of salkowski (50 ml, 35% perchloric acid; 1 ml 0.5 fecl3) is an indication of positive result. screening of pgpr isolates for stress tolerance influence of salinity. to evaluate the strains ability of these strains to grow in salinity levels, different concentrations of nacl (1%, 5% and 8%). the salt were added to tsa medium in the liquid stage (infusion at 40°c) and deposited on the magnetic stirrer to dilute it. influence of ph and temperature. in order to test the ability of the bacteria to grow in alkaline and/or acidic environment, two media were prepared with the addition to 250 ml of tsa of 3 g of solid naoh 6.4 m and 1 ml of kcl 12 n to obtain a ph of 8.8 and 6 respectively. for studying the effect of incubation temperature on growth of the isolates, the bacterial cultures were grown on tsa medium and incubated at a variables temperatures (10, 20, 30, 40 and 50°c). tolerance of heavy metal. the sensitivity of the selected strains to copper (cu), zinc (zn), bromine (br), cyanide (cn), fluorine (f) and silicon (si) were tested. for this test, six tsa media of 250 ml were prepared with respectively three grams of solid kbr, caf2, sio2 and one gram of k3[fe(cn)6], znso4.7h2o and cuso4.5h2o. any growth on this media reflects the ability of strains to tolerate the heavy metal toxicity. antibiotics resistance. the selected strains were tested for their susceptibility to antibiotics on mullerhinton medium. it consists of bringing the germ into contact with disks of blotting paper impregnated with a given antibiotic at concentrations determined by the standardization of the antibiogram according to the clinical and laboratory standards institute (clsi), to determine the sensibility of this germ to antibiotics which allows classifying it in the category: r (resistant), s (sensible) or i (intermediate). ten different antibiotics were used: fosfomycin (fos) 50 µg, rifampin (ra) 5 µg, nalidixicid (na) 30 µg, spiramycin (sp)100 µg, 30 µg, novobiocin (nv) 30 µg, teicoplycin (tei) 30 µg, kanamycin (k) 30 µg and erythromycin (e) 15 µg (all from sigma chemical co., st. louis, mo.). 3. results in this study, we focused on the diversity of bacterial community of ucria’s rhizosphere and the evaluation of their plant growth promoting abilities, also under abiotic stresses. isolation and characterization of rhizobacteria one hundred and thirty seven (137) culturable bacteria were isolated from the rhizosphere of six healthy ucria plants using tsa medium. the rhizobacteria isolates showed a diversity of phenotypic and cultural characteristics of their colonies. infact, 54.19% of the isolated rhizobacteria were gram negative. 100% of strains showed positive test to catalase and variability to oxidase reaction (67.88% negative). screening and identification of pgpr biochimical characterization of pgpr isolates. the isolates were grouped into 16 genera of bacillus, adv. hort. sci., 2018 32(4): 525-534 528 pseudomonas, ewingella, staphylococcus, alcaligenes, micrococcus, kocuria, chryseomonas, chryseobacterium, cedecea, shigella, yersinia, providencia, acinetobacter, haemophilus and aeromonas based on cultural, morphological and biochemical characteristics. the maximum and minimum populations were bacillus (35%), and haemophilus and aeromonas with a percentage of 1.66% each (table 1). table 1 functional diversity of pgpr strains isolated from ucria’s rhizosphere sample no division/strains functions 1 gamma proteobacteria cedecea lapagei solubilization of phosphate, production of hcn and siderophores chryseomonas luteola solubilization of phosphate, production of siderophores and nitrogen fixation chryseomonas luteola solubilization of phosphate, production siderophores and nitrogen fixation firmicutes bacillus megaterium solubilization of phosphate, production of siderophores and nitrogen fixation bacillus megaterium solubilization of phosphate, production of aia and siderophores and nitrogen fixation bacillus circulans solubilization of phosphate, production of aia and siderophores, 2 gamma proteobacteria ewingella americana solubilization of phosphate, production of aia and siderophores chryseomonas luteola solubilization of phosphate, production of aia and siderophores and nitrogen fixation cedecea lapagei solubilization of phosphate, production of siderophores and nitrogen fixation cedecea lapagei solubilization of phosphate, production of aia and siderophores and nitrogen fixation firmicutes bacillus licheniformis solubilization of phosphate, production of aia and siderophores bacillus subtilis solubilization of phosphate, production of siderophores and nitrogen fixation bacillus licheniformis solubilization of phosphate, production of aia and siderophores and nitrogen fixation. bacillus licheniformis production of aia and siderophores actinobacteria kocuria varians production of aia 3 gamma proteobacteria chryseomonas luteola solubilization of phosphate, production of aia and siderophores and nitrogen fixation providencia rattgeri production of aia ewingella americana production of siderophores firmicutes bacillus subtilis production of hcn and fixation of azote bacillus non reactiv production of siderophores and fixation of azote bacillus licheniformis production of hcn and fixation of azote 4 gamma proteobacteria yersinia pestis solubilizationof phosphate, production of siderophores and nitrogen fixation haemophilus aphrophilus solubilization of phosphate, production of aia, and nitrogen fixation aeromonas salmonicida solubilization of phosphate, production of hcn and siderophores pseudomonas aeruginosa solubilization of phosphate, production of hcn and nitrogen fixation cedecea lapagei solubilization of phosphate, production of siderophores and nitrogen fixation acinetobacter colcoaticus solubilization of phosphate and production of aia, acinetobacter baumanii solubilization of phosphate, production of aia and nitrogen fixation chryseomonas luteola solubilization of phosphate, production of hcn and siderophores. acinetobacter baumanii solubilization of phosphate and nitrogen fixation firmicutes staphylococcus lentus solubilization of phosphate and production of aia. staphylococcus lentus solubilization of phosphate, production of siderophores and nitrogen fixation actinobacteria micrococcus ssp solubilization of phosphate, production of siderophores and nitrogen fixation béta proteobacteria alcaligenes faecalis production of aia and nitrogen fixation 5 gamma proteobacteria chryseomonas luteola production of siderophores and nitrogen fixation chryseomonas luteola solubilization of phosphate, production of aia and siderophores and nitrogen fixation chryseomonas luteola solubilization of phosphate, production of hcn and aia. to be continued benaissa et al. pgpr of rhus tripartitus 529 plant growth promoting traits.the isolated rhizobacteria were screened for various pgp features responsible for plant growth promotion. the pgpr isolates represent 43.79% of the total of the rhizosp h e r i c b a c t e r i a . t h e y b e l o n g t o f o u r divisions (gamma-proteobacteria, firmicutes, actino-bacteria and béta-proteobacteria). the strains mostly belonging to the gamma-proteobacteria, most of them affiliated to the enterobacteriaceae. according to the figure 1, species of gamma proteobacteria division were predominant with 45% among the isolats of pgpr of ucria behind firmicutes with 38.33%. infact, the species cedecea lapagei, chryseomonas luteola and ewingella americana were the most representative pgpr of gammaproteobacteria division. the species chryseomonas luteola found in five samples of six rhizospheric soils (table 1). different combinations of pgp effects have been found, 18.33% strains have been able to produce up to 4 pgp effects against 40% were able to produce 3 pgp effects, 25% showed 2 traits of pgp and 16.66% were able to produce only one pgp effect. in fact, the genera bacillus, ewingella, alcaligenes, the rhizobacteria were identified through six samples and five plant growth promoting treatments: solubilization of phosphate, production of cyanhydric acid, production of indol-acid-acetic, fixation of nitrogen and production of siderophores. fig. 1 classification of rhizospheric bacteria of ucria on the pgp traits. the main bacteria divisions of plant growth promoting rhizobacteria from the rhizosphere of ucria. sample no division/strains functions 5 firmicutes bacillus circulans solubilization of phosphate, production of hcn and siderophores and nitrogen fixation bacillus circulans solubilization of phosphate, production of siderophores and nitrogen fixation bacillus licheniformis solubilization of phosphate, production of siderophores and nitrogen fixation bacillus subtilis solubilization of phosphate, production of siderophores and nitrogen fixation bacillus licheniformis solubilization of phosphate, production of hcn and siderophores and nitrogen fixation bacillus licheniformis solubilization of phosphate and nitrogen fixation bacillus circulans production of hcn bacteroidetes chryseobacterium meningosepticum nitrogen fixation actinobacteria kocuria varians nitrogen fixation micrococcus ssp solubilization of phosphate, production of hcn and siderophores and nitrogen fixation 6 gamma proteobacteria ewingella americana production of aia and nitrogen fixation shigella spp solubilization of phosphate and nitrogen fixation pseudomonas aeruginosa solubilization of phosphate, production of siderophores and nitrogen fixation ewingella americana solubilization of phosphate, production of siderophores and hcn and nitrogen fixation ewingella americana solubilization of phosphate and nitrogen fixation providencia rattgeri production of aia firmicutes bacillus subtilis solubilization of phosphate and nitrogen fixation bacillus licheniformis solubilizationof phosphate, production of hcn and siderophores and nitrogen fixation bacillus subtilis solubilization of phosphate, production of hcn and nitrogen fixation bacillus non reactiv nitrogen fixation actinobacteria micrococcus ssp production of hcn and siderophores and nitrogen fixation béta proteobacteria alcaligenes faecalis solubilization of phosphatase, production of hcn and siderophores alcaligenes faecalis production of hcn table 1 functional diversity of pgpr strains isolated from ucria’s rhizosphere continued chryseomonas and cedecea have showed positive screening for all the pgp traits, according to the table 2. screening and assessment of phosphate solublizers and iaa producers. among the total isolates screened for phosphate solublization, 71.66% were able to solubilize inorganic phosphate (fig. 2) and were identified as potential phosphate solubilizing 530 adv. hort. sci., 2018 32(4): 525-534 bacteria that showed a clear halo zone around the colonies on nbrip’s agar plates amended with bromophenol blue. 20 isolates out of total rhizospheric flora had the capacity to produce were iaa in the presence of l-tryptophane. molecular nitrogen fixation. the ability of the isolates to grow on n-free medium indicated positive results for nitrogen fixation. in fact 70% of the isolates were able to grow even being transferred ten times in this medium which indicated their capacity to fix molecular nitrogen (fig. 2). siderophore and hcn production. siderophore production was registered at 58.33% of the strains based on the appearance of a halo zone of yellow orange color around the colony inoculated on casagar plates. seventeen isolates were positive for hcn production (fig. 2). physiological properties of pgpr isolates under abiotic stresses. the isolated bacteria were tested for their ability to tolerate abnormal growth conditions after incubation in a wide range of salt, ph and temperature stress condition, heavy metal toxicity and antibiotics. influence of salinity. according to the figure 3, the most percentages of strains which can grow in the 1% and 5% salinity are respectively 96.66% and 81.66%. in addition, only 16.66% of strains can tolerate % of salinity. influence of ph and temperature. the rhizobacterial isolates seems to tolerate the alkalinity better than the acidity with 66.66% and 46.66% respectively. however, these isolates could grow up to higher temperature of 50°c with 85%, which means that the temperature hadn’t a remarkable effect on bacterial growth (fig. 3). tolerance to heavy metal. depending on the metal tested and the species, the tolerance to heavy metals is different. in fact, we recorded a very good tolerance to silicon and bromine with respectively 80% and 78.33%. 55% of our strains have well tolerated fluoride followed by 30% for cyanide. the lowest percentages recorded were for copper and zinc with 13.33% and 3.33%, respectively. these results showed that heavy metals affected the bacterial growth (fig. 4). table 2 plant growth promotion activities of rhizobacteria genera isolated from ucria’s rhizosphere genera n2 fe 2 hcn aia po2 bacillus + + + + + pseudomonas + + + + ewingella + + + + + staphylococcus + + + + alcaligenes + + + + + micrococcus + + + + kocuria + + chryseomonas + + + + + chryseobacterium + cedecea + + + + + shigella + + yersinia + + + providencia + acinetobacter + + + haemophilus + + + aeromonas + + + n2 = nitrogen fixation, fe2= siderophore production, hcn production, aia and po= phosphate solubilisation. fig. 2 siderophores production, phosphate solubilization, fixation of nitrogen, hcn and aia productions of the rhizobacteria isolates of ucria (ahaggar). fig. 3 estimation of tolerance of pgpr strains to alkalinity, acidity, salinity and temperature. fig. 4 estimation of tolerance of pgpr strains to heavy metals toxicity. br= bromine, zn= zinc, si= silicon, f= fluorine, cn= cyanide, cu= copper. 531 benaissa et al. pgpr of rhus tripartitus antibiotics resistance. the collection of pgpr isolates was tested for susceptibility to 10 antimicrobials. high frequency of resistance was observed for métronidazole (mt) followed by amoxyclav (amc) with respectively 93.33% and 91.66% of strains. the minimum resistance was recorded for fosfomycin (fos) with 16.66% (fig. 5). 4. discussion and conclusions the diversity of native bacterial species in the southern algerian soils remains largely unknown, especially for the rhizosphere of rhus tripartitus. most of the rhizobacteria isolated from ucria’s rhizospheric soils have been dominated by gram-negative (54.19%) and all were catalase positif. these results are in-line with studies realized on rhizosphere of brassica campestris (poonguzhali et al., 2006) and eragrostis tef zucc. trotter (woyessa and assefa, 2011). plant growth promoting rhizobacteria (pgpr) represent a diverse range of soil bacteria that stimulate the growth of their host when grown in association. such rhizosphere microbes benefit by utilization of metabolites secreted by plant roots as a nutrient for their growth and promote plant growth through more than one mechanism, including production of phytohormones and biocontrol of plant pathogens (rana et al., 2011). this present study showed a high diversity among the isolates of these species under different combination of pgp effects. the most often genera identified was bacillus (35%) with a high diversity of species, such as bacillus megaterium, b. licheniformis, b. subtilis, b. circulans and b. non-reactiv. pgpr of the genera bacillus have been reported in many studies (trivedi and pandey 2008; zou et al., 2010; liang et al., 2011; woyessa and assefa, 2011; nadeem et al., 2012; mishra et al., 2014; susilowati et al., 2015). the second pgpr group found througho u t t h i s s t u d y w a s b e l o n g i n g t o s p e c i e s chryseomonas luteola (11.6%) and ewingella americana (8.3%). the latter was able to increase the growth of pipper and spinach (hou and oluranti, 2 0 1 3 ) . t h e t h i r d a n d l a s t p g p r g r o u p ( 4 5 . 1 % ) b e l o n g e d t o t h e g e n e r a c e d e c e a , p r o v i d e n c i a , yersinia, heomophilus, aeromonas, acinetobacter, micrococcus, alcaligenes, chryseobacterium, shigella, staphylococcus and pseudomonas. earliers studies showed that pseudomonas pgpr is a producer of hcn (castric, 1975), improving the availability of necessary nutrient (islam et al., 2014) and an agent of biocontrol (weller and thomashow, 1993). the genera acinetobacter promotes production of wheat, pea, chickpea, maize and barley through nitrogen fixation, siderophore production and mineral solubilization (gulati et al., 2009; sachdev et al., 2010). in constrat, cedecea have never shown in the litterature as pgpr potential and molecular analysis is necessary to confirm the identification. the industrialization of chemical fertilizers such phosphate and nitrogen has increased in the agricultural sector. these minerals are considered important limiting factors for many crops (ahmad et al., 2008). however, the aim of this study is also to evaluate the ability of rhizobacteria to promote the biodisponibility of n and p, in order to reduce of industrial fertilizers. in fact, most of ucria’s rhizobacteria could fix atmospheric nitrogen (70%) and 71.66% were able to solubilize inorganic phosphate in nbrip’s medium. similary, many studies shown that phosphorus-solubilizing microorganisms are ubiquitous in soils (chandra et al., 2007; tsavkelova et al., 2007; banerjee et al., 2010). pgpr can produce auxin-like compounds that increase the development of root system thus improving nutrient uptake by plants (voisard et al., 1989); however, out of total isolated rhizobacteria, 31.66% exhibited as producers of iaa in medium supplemented with l-tryptophan. the number of pgpr producers of hcn represent 28.33% of total isolates strains. these pgp traits have the capacity to enhance indirectly plant growth and protect them from phytopathogens (lugtenberg and dekker, 1999; shahat et al., 2016). rhizobacteria producing siderophores are of great importance for the p l a n t b e c a u s e t h e y m a k e b i o a v a i b l e i r o n . t h e siderophores decreases the metal bond and formation of free radicals in the roots zone, which prevented the degradation of iaa (yang et al., 2009). among the isolates of pgpr obtained in this study, 58.33% fig. 5 estimation of resistance of pgpr strains to antibiotics. adv. hort. sci., 2018 32(4): 525-534 532 are producers of siderophores. the production of siderophores in the rhizosphere increases the bacterial competition as well the root colonization (abrol et al., 1988). the rhizosphere is characterized by large environmental fluctuations, which may promote high diversity in the rhizosphere microbial community by maintaining high niche diversity. thus, microbial community diversity may be important especially in extreme condition, like high temperature, salinity or ph changes. under stress conditions, bacterial rhizosphere may promote the plant growth (cheikh and jones, 1994). in the present research, these pgpr isolates were traited under several environmental conditions. they have showed a good potential of tolerance for the previuosly conditions, which is a value, added to their beneficial effect. therefore, the aim of this work is not only to deal with the diversity of pgpr but also the selection of resistant strains at the most extreme conditions. the ucria’s rhizobacteria, were isolated from an arid area of ahaggar (algeria) and salinity is a natural feature of ecosystems in arid and semi-arid regions (curl and truelove, 1986). the 81.66% of pgpr studied exhibited as tolerant to the presence of 5% nacl and 85% could growth until 50°c. the variation on temperature is an important factor that can affect the hormonal balance of the plant (lovley, 1995). then, certain beneficial microorganisms can influence plants response to abiotic stresses like drought a n d h i g h t e m p e r a t u r e ( g r o v e r e t a l . , 2 0 1 1 ) . therefore, the necessity of discovering species able to grow under salt stress conditions and in a high temperature, are important to include them in revegetation system in arid area. the ph is one the obvious influencing factors of microbial activity and populations in soil (woyessa and assefa, 2011). most of these isolates (66.66%) can growth over alkaline ph and more than 46% of growth recorded in acidity condition, it suggests that there is a good potential to inoculate them over a range of wide ph. the growth o f r h i z o b a c t e r i a a t a c i d i c p h v a l u e s c o u l d b e explained by their adaptation at arid soil. effectively, low rainfall can probably cause an increase in acidity of soil. on the other hand, an acidic environment of roots due to co2 and organic acid can be included in soil acidity (gururani et al., 2012). moreover, the pgpr isolates of this work, demonstrated a good tolerance in-vitro for heavy metals toxicity, with an average of 43.33% for chemicals forms of six metals. the accumulation in soil of heavy metals can perturbate the growth and the diversity of bacterial communities. many studies are shown the potential of application of pgpr in resistance and uptake of heavy metal by certain plants (lovley, 1995; yang et al., 2009; gururani et al., 2012). the ability to colonize roots and antibiotic resistance are other parameters needed to detect effective pgpr strains (siddiqui, 2005). effectively, the study of the sensitivity of rhizobacteria to antibiotics adds pgp potential. indeed, our strains have showed some resistance to the majority of antibiotics tested which can be involve high microbial competition in the rhizosphere. this resistance increased the chances of survival and colonization of the rhizospheric soil. the present study reflects the preliminary work done on the rhizosphere of rhus tripartitus in arid 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university of mashhad, mashhad, iran. 2 department of horticultural science, faculty of agriculture, isfahan university of technology, isfahan, iran. key words: combining ability, diallel, heritability, light intensity, reciprocal effects, solanum lycopersicum. abstract: limitations in access to electricity in rural areas and substantial cost of supplemental lightning necessitate breeding as response to low light conditions. seven inbred lines of tomato (solanum lycopersicum l.) and their f1 hybrids, including reciprocals, developed through a 7×7 full diallel cross were evaluated under two different levels of light. mean square for light (l) effect was significant for total yield, average fruit weight and days to first flower. variation attributable to genotypes and genotype × light (g×l) interaction had significant effect on all studied traits except days to ripening for which g×l interaction was not significant. diallel analysis across two environments indicated that general (gca), specific (sca) and reciprocal combining ability (rec) were significant for all characters implying importance of additive and nonadditive gene action along with cytoplasmic effects on genetic expression of yield, yield components and earliness. ratio of sca variance to sca variance and estimates of narrow sense heritability (h2n.s) demonstrated higher weight of additive effects in inheritance of yield, fruit number and days to ripening, while indicating predominance of non-additive effects for fruit weight and early flowering. interactions gca×l and sca×l were significant for almost all studied features. a particular genotype could not be recommended for all traits, but variation among genotypes in response to ambient light was promising for feasibility of plant breeding for non-optimal light intensity and duration. 1. introduction tomato (solanum lycopersicum l.), a member of solanaceae family, is a wide cultivated vegetable used for fresh and processing market. this day neutral plant is grown in various regions of the world with different climates (mizoguchi et al., 2007; gerszberg et al., 2015). iran is among the top 10 countries producing tomato (faostat, 2014), but it performs very poorly in terms of tomato seed production and a large portion of the seeds, particularly hybrid cultivars, is imported to the country. the major obstacle to seed production in iran is the absence of breeding programs (*) corresponding author: nemati@um.ac.ir citation: emami s., nemati s.h., azizi m., mobli m., 2018 combining ability and gene action of some tomato genotypes under low light condition. adv. hort. sci., 32(4): 459-470 copyright: © 2018 emami s., nemati s.h., azizi m., mobli m. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 10 january 2018 accepted for publication 16 may 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(4): 459-470 460 for most of the vegetables including tomato; therefore, more attention to vegetables breeding in order to produce high quality seeds is required. hybrid breeding technique is one of the most important methods used for crop improvement. the information needed to develop proper f1 hybrid cultivars via hybrid breeding could be achieved through different methods including diallel analysis, a method to analyze crosses made among (n) lines in all possible combinations (griffing, 1956 a, b). the analysis is mainly adopted when dealing with limited number of parental lines and determines genetic parameters such as heterosis, general combining ability (gca), specific combining ability (sca), heritability, and nature of gene action. heterosis demonstrates the superiority of f1 progenies compared to the average of their parents. general combining ability (gca) shows the average performance of a parental line while specific combining ability (sca) refers to the best combination of crosses. general combining ability (gca) and specific combining ability (sca) are the indicators of additive and non-additive gene effects, respectively. these parameters help plant breeders in the selection of suitable parental lines and appropriate breeding method (sprague and tatum, 1942). it should be noted that despite of the advantages of hybrid cultivars over open pollinated ones, the high price of hybrid seeds developed via hybridization programs makes the use of them more economical for intensive and indoor cultivation (zengin et al., 2015). one of the major problems affecting plants growth in greenhouses is the decreased level of light received by plants due to significant loss of solar radiation caused by reflection and absorption by greenhouse covering material (baeza and lópez, 2012) and high plant density, typically executed in greenhouse cultivations (laurent et al., 2017). since light is one of the most crucial factors influencing growth rate, production quality and quantity of plant, supplying plants with adequate light intensity with suitable quality is of importance in greenhouses particularly during autumn and winter seasons (hangarter, 1997). in developing countries such as iran, most of the greenhouses do not benefit from high technology and suffer from lack of accessibility to electricity, h e n c e ; s u p p l e m e n t a l l i g h t n i n g i s n o t a p p l i e d . moreover, substantial price of energy resources and tendency toward lower energy consumption (oz and atilgan, 2015) necessitate hybrid breeding for low light conditions. to our knowledge breeding for low light condition has not been conducted in tomato, therefore, this study aimed to investigate if there existed any differences among various tomato genotypes in response to two different light conditions w h i l e c o n s i d e r i n g t h e i n t r o d u c t i o n o f s u i t a b l e parental lines and hybrids for each condition as well as determination of stable genotypes over two environments. genetic parameters including combining ability, gene action and heritability were estimated to help breeders in choosing the best approach for the improvement of tomatoes regarding increased yield and earliness. 2. materials and methods plant material s e v e n i n b r e d l i n e s o f t o m a t o c o n s i s t e d o f ‘perimoga’, ‘la1793’, ‘ac06’, ‘ct6’, ‘mc3’, ‘c20’ and ‘kingstone’ (table 1) were cultivated in a research greenhouse of ferdowsi university of mashhad, mashhad, iran under optimum conditions. the lines were crossed in all possible two-way combinations (full diallel cross system) to develop f1 hybrids with their reciprocals. experimental design the experiment was performed in research greenhouse of ferdowsi university of mashhad with computerized temperature control system. a year round was divided into two growing seasons: the first growtable 1 description of parental inbred lines crossed in 7×7 full diallel cross system plant material abbreviation origin growth habit leaf type fruti shape perimoga p1 russia determinate vulgare oval la1793 p2 usa indeterminate vulgare round ac06 p3 iran indeterminate vulgare round ct6 p4 russia semi-indeterminate grandifolium oval mc3 p5 russia indeterminate vulgare round c20 p6 russia indeterminate vulgare oval kingstone p7 italy semi-indeterminate vulgare oval emami et al. tomato genotypes under low light condition 461 ing season was from march to august including warm seasons with high light intensity and long photoperiod (more sunny hours a day) and the second season covering cold seasons with low light intensity and short photoperiod (less sunny hours per day) started in september and ended up in january. to investigate the performance of tomato plants under two different light conditions, 21 f1 progenies together with reciprocals (42 f1 hybrid progenies) developed via a 7×7 full diallel cross system were cultivated during each of the two aforementioned growing seasons. daily average of light intensity of experimental greenhouse during each month is represented in table 2. the experiment was conducted in a completely randomized design with three replications per genotype in two different time span mentioned above (split plot). measurement of characters the observations concerning the following characteristics were recorded as described below. total yield per plant (kg) it was calculated by summing up the weight of fruits obtained from all pickings during 8 weeks from each plant. average fruit weight (g) the average fruit weight was an index of fruit size. all fruits collected from each harvest were weighted and the total weight of the fruits was divided into the number of the weighted fruits. number of fruits per plant harvested fruits of each plant in a period of 8 weeks were counted. days to first flower the number of days from seeding until the formation of first flower on the plants was recorded. days to ripening the number of days from flower anthesis to fruit ripening stage was determined through the date tagging of five flowers per plant at the time of anthesis. fruit ripening was considered the time when genetically red fruits turned pink and yellow ones turned yellow (garg et al., 2008). statistical analysis obtained data in each environment were analyzed using excel software (microsoft office 2010) using griffing’s (1956, a, b) method 3, model 1 (fixed effects) formula. combined analysis of data over two seasons was performed based on modified method 3, model 1 for several environments proposed by singh (1973) as described below: where yijk = observation of trait value of parents i and j in year k; µ = population mean; g i / g j = gca effect of parent i / j; sji / sji = sca effect of the hybrid developed from parent i × parent j / parent i × parent j; rij = rec effect of the hybrid produced by parent i × parent j; lk=effect of environment k; (gl)ik / (gl)jk = interaction between gca effect of parent i / j with environment k; (sl)ijk = interaction between sca effect of cross ij with environment k; and eijk = error of observation ijk. broad-sense heritability (h2b.s) and narrow-sense heritability (h2b.s) over environments were estimated using following formula (sharifi et al., 2010): where l indicated light condition; σ2g, σ2s and σ2r stand for variance components of gca, sca and rec, respectively; σ2gl, σ2sl and σ2rl represent variance components of gca×l, sca×l and rec×l, respectively. where f1 and yij are the mean performances of hybrids and parents, respectively. the average of light intensity per day was calculated based on 24 hours a day including night hours (n= 24, one measurements every hour). table 2 average of daily light intensity for each months (foot candle intensity/24h) average light intensity first growing season with high light intensity and duration second growing season with low light intensity and duration mar. apr. may june july aug. sept. oct. nov. dec. jan. feb. 680.7 694 750.5 863.3 893 760 650.7 547.3 550 527 535.5 655.7 adv. hort. sci., 2018 32(4): 459-470 462 3. results anova analysis the variance analysis of genotypes for each season separately showed that genotypes were highly significant (table 3); therefore, the compound analysis of variance over two environments was conducted (table 4). light condition was considered as main plot; therefore, error a refers to whole plot error. some genotypes, namely 42 f1 hybrids grown under each light treatment along with the interaction between genotypes and environments, were the sub plot of the experiment and error b represent whole plot error. compound anova analysis for two seasons represented in table 4 showed that light (l) effect was highly significant for total yield per plant, average fruit weight and days number to first flower but not significant for fruit number per plant and days number for fruit ripening. genotypes and the interaction of genotypes with light (g×l) were highly significant for all studied traits except for days to ripening in which genotypes did not show any interaction with environment. estimation of genetic parameters compound analysis of variance for general combining ability (gca), specific combining ability (sca) and reciprocal combining ability (rec) over two seasons for yield, yield components and earliness varied (table 4). the results indicated that variances due to table 3 mean squares of anova analysis over two light conditions for yield, yield components and earliness of f1 hybrids with their reciprocals developed via a 7×7 full diallel cross ** significant at p<0.01 level. sources of variation degree of freedom total yield per plant (kg) average of fruit weight (g) fruit number per plant days to first flower days to ripening light (l) 1 16.69 ** 14654.49 ** 913.52 359.53 ** 5.43 error a 4 0.74 261.6 140.91 0.61 8.8 genotypes (g) 41 2.05 ** 1944.73 ** 1593.50 ** 34.74 ** 23.38 ** g×l 41 0.17 ** 146.54 ** 96.24 ** 2.50 ** 3.99 error b 164 0.07 34.01 18.33 0.97 2.81 table 4 compound analysis of variance for combining ability over two environments for yield, yield components and earliness of f1 hybrids with their reciprocals developed via a 7×7 full diallel cross and estimates of heritability in broad and narrow senses as well as heterosis * significant at p<0.05 level, ** significant at p<0.01 level. sources of variation degree of freedom total yield per plant (kg) average of fruit weight (g) fruits number per plant days to first flower days to ripening gca 6 1.28 ** 3343.91 ** 2063.32 ** 54.66 ** 23.20 ** sca 14 1.35 ** 430.82 ** 644.60 ** 9.55 ** 8.68 ** rec 21 0.07 ** 23.00 ** 17.79 ** 0.62 * 2.80 ** gca×l 6 0.14 ** 120.47 ** 70.33 ** 2.86 ** 1.95 sca×l 14 0.06 ** 53.49 ** 42.07 ** 0.94 ** 1.68 * rec×l 21 0.03 25.29 * 14.49 ** 0.19 0.92 error 164 0.02 11.34 6.11 0.32 0.94 variance components σ2g 0.06 166.63 102.86 2.72 1.11 σ2s 0.33 104.87 159.62 2.31 1.94 σ2r 0.01 2.92 2.92 0.07 0.47 σ2g/ σ2s 0.19 1.59 0.64 1.18 0.57 σ2gl 0.01 10.91 6.42 0.25 0.1 σ2sl 0.02 21.08 17.98 0.31 0.37 σ2rl 0 6.98 4.19 -0.07 -0.01 h2 broad-sense (%) 94.77 94.94 95.7 94.38 90.38 h2 narrow-sense (%) 26.02 72.22 53.89 66.25 48.34 heterosis (%) 37.75 -22.95 55.04 3.05 -6.38 emami et al. tomato genotypes under low light condition 463 gca, sca and rec were highly significant for all studied characters. the significance of both gca and sca variances implies that all studied characters are controlled by either of additive or non-additive gene action. the effect of interaction gca×l and sca×l on all evaluated features were significant but fruit ripening period, which showed non-significant variance of gca×l. the interaction of rec×l showed remarkable effect only on average fruit weight and fruits number per plant. for total yield, fruits number and days to ripening, the gca (σ2g) was less than variance component of sca (σ2s) in a way that σ2g/σ2s was less than unity, demonstrating the predominance of nonadditive gene action in controlling mentioned traits (baker, 1978). the ratio of σ2g/σ2s for fruit weight and days to first flower was higher than unity, illustrating the more important role of additive gene action for inheritance of these attributes. estimates of broad-sense heritability percentage (h2 %) over environments was high for all studied traits ranged from 90 to 95% (table 4). narrow-sense heritability percentage (h2 %) was low for total yield, fruits number and days to ripening while relatively high for fruit weight and days to flowering. total heterosis percentage was high for yield and fruit number while negative for fruit weight. unlike yield and yield components, in earliness characters negative values show superiority but heterosis for early flowering was positive. negative but low heterosis was recorded for early maturity. estimation of mean values mean values of f1 hybrids for plant yield showed that hybrid ‘ct6×mc3’ (p4×p5) had the highest rate under both light conditions, while lowest amount w a s f o r h y b r i d ‘ c 2 0 × m c 3 ’ ( p 6 × p 5 ) ( t a b l e 5 ) . ‘perimoga×kingstone’ (p1×p7) cross and its reciprocal produced heaviest fruits over two environments. lightest fruits produced under normal light were for ‘mc3×la1793’ (p5×p2), but under low light, the fruits of hybrid ‘mc3×ac06’ (p5×p3) had the least weight. the highest number of fruits per plant was produced by hybrid ‘la1793×c20’ (p2×p6) and the lowest was f o r ‘ k i n g s t o n e × p e r i m o g a ’ ( p 7 × p 1 ) . h y b r i d ‘perimoga×ct6’ (p1×p4) commenced flowering earlier than other progenies. latest flowering under adequate light intensity was for ‘mc3×c20’ (p5×p6), w h i l e u n d e r l o w l i g h t , i t w a s f o r h y b r i d ‘la1793×ac06’ (p2×p3). overall, according to pooled data over two seasons, hybrid ‘c20×mc3’ (p6×p5) took more days to flower compared with other progenies. longest fruit ripening duration was for hybrid ‘mc3×la1793’ (p5×p2) and shortest period for ripening was observed in the cross of ‘aco6×kingstone’ (p3×p7). estimation of gca, sca and rec effect for yield and yield components, positive values of gca, sca and rec indicate the superiority of genotypes while for earliness characters negative values are desired (table 6). most of the parental lines were not stable during two growing seasons and superior parents for each character differed with environmental changes. parental line of ‘ct6’ (p4) was the best combiner for achieving higher yield in both seasons, and the lowest gca in low light and high light condition was for ‘kingstone’ (p7) and ‘la1793’ (p2), respectively. in total ‘kingstone’ (p7) was the weakest genitor for yield across two environments. for fruit weight, although ‘perimoga’ (p1) had the highest gca in warm season, parental line ‘kingstone’ (p7) acted as the best combiner in both seasons. the best donors for increased fruits number during sunny and cloudy seasons were ‘la1793’ (p2) and ‘mc3’ (p5), respectively and ‘la1793’ (p2) had the best gca over both seasons. for days to first flower, parental genotype ‘perimoga’ (p1) possessed the highest negative gca and ‘mc3’ (p5) had the highest positive and significant gca under each ambient light and over both of them. the best combiner concerning days to ripening in both tested environments was ‘la1793’ (p2) and the weakest performance was for ‘kingstone’ (p7). in yield and related trait, a positive value of sca is repetitive of a successful cross between parental lines of that hybrid, while a negative value demonstrates that parental lines did not make up a good couple. for earliness, negative values of sca are indicative of prosperity .estimation of sca for each evironment and over two environments indicated t h a t t h e b e s t c o m b i n a t i o n f o r t o t a l y i e l d w a s ‘ l a 1 7 9 3 × c 2 0 ’ ( p 2 × p 6 ) a n d t h e w o r s t w a s f o r ‘mc3×c20’ (p5×p6) (table 7). for fruit weight, hybrid ‘ac06×kingstone’ (p3×p7) had the highest sca during sunny seasons, and the hybrid developed from ‘perimoga×kingstone’ (p1×p7) had the highest magnitude during cloudy seasons and over two seasons. the highest and lowest sca estimations for fruits n u m b e r w e r e s i m i l a r t o t o t a l y i e l d . t h e c r o s s between ‘mc3×c20’ (p4×p6) was the most successful cross for decreased days to flowering during sunny months. ‘la1793×mc3’ (p2×p5) not only had the highest negative sca during cold months, but also p o s s es s ed t h e b es t s c a i n t o t a l . f o r t h i s t r a i t , 464 adv. hort. sci., 2018 32(4): 459-470 table 5 means values and standard deviation for yield components and earliness of f1 hybrids developed via 7×7 diallel cross over two light conditions (part a) p1= perimoga, p2= la1793, p3= ac06, p4= ct6, p5= mc3, p6= c20, p7= kingstone. values in parenthesis represent standard errors. ** significant at p<0.01. genotypes ♀ × ♂ total yield per plant (kg) average of fruit weight (g) fruits number per plant l1 l2 pooled l1 l2 pooled l1 l2 pooled p1×p2 2.72(±0.28) 1.48(±0.44) 2.10(±0.36) 70.13(±8.70) 45.90(±9.34) 58.02(±8.83) 38.93(±3.02) 31.90(±3.92) 35.42(±3.07) p1×p3 3.54(±0.36) 2.87(±0.48) 3.20(±0.42) 85.63(±10.48) 55.17(±8.89) 70.40(±8.53) 41.33(±1.64) 52.27(±7.01) 46.80(±2.69) p1×p4 3.81(±0.14) 3.50(±0.06) 3.65(±0.10) 88.53(±2.05) 56.47(±4.97) 72.50(±1.63) 43.00(±2.55) 62.20(±5.20) 52.60(±1.96) p1×p5 3.61(±1.02) 3.18(±0.20) 3.40(±0.60) 84.73(±7.22) 57.33(±3.14) 71.03(±2.79) 42.10(±8.98) 55.70(±6.58) 48.90(±7.65) p1×p6 2.24(±0.19) 2.23(±0.36) 2.24(±0.26) 40.60(±4.06) 43.80(±4.88) 42.20(±2.08) 55.27(±2.15) 51.87(±13.90) 53.57(±5.97) p1×p7 2.47(±0.36) 2.13(±0.33) 2.30(±0.34) 114.20(±14.97) 96.80(±10.83) 105.50(±8.28) 21.80(±3.97) 21.87(±2.20) 21.83(±2.05) p2×p1 2.29(±0.18) 1.58(±0.22) 1.94(±0.20) 64.33(±16.06) 42.17(±2.35) 53.25(±7.55) 36.60(±5.97) 37.50(±6.41) 37.05(±0.52) p2×p3 2.69(±0.17) 1.60(±0.04) 2.14(±0.08) 58.53(±7.25) 33.77(±2.03) 46.15(±2.64) 46.20(±3.10) 47.27(±2.75) 46.73(±1.05) p2×p4 3.27(±0.31) 2.77(±0.23) 3.02(±0.27) 46.13(±2.06) 39.83(±4.11) 42.98(±2.72) 70.70(±5.05) 70.07(±9.36) 70.38(±7.17) p2×p5 2.81(±0.25) 2.55(±0.20) 2.68(±0.19) 36.90(±3.33) 35.03(±2.84) 35.97(±1.21) 76.10(±4.58) 72.67(±3.25) 74.38(±3.01) p2×p6 4.20(±0.33) 2.77(±0.31) 3.49(±0.32) 44.63(±1.65) 32.63(±3.39) 38.63(±2.32) 94.03(±5.54) 84.80(±3.99) 89.42(±4.13) p2×p7 3.24(±0.33) 2.74(±0.33) 2.99(±0.33) 51.80(±4.54) 56.80(±4.54) 54.30(±4.54) 62.57(±1.72) 48.17(±2.29) 55.37(±1.94) p3×p1 3.33(±0.31) 2.42(±0.31) 2.87(±0.31) 76.60(±8.52) 54.03(±11.95) 65.32(±10.03) 43.50(±3.21) 45.27(±4.35) 44.38(±2.98) p3×p2 3.14(±0.30) 1.98(±0.25) 2.56(±0.19) 61.00(±2.11) 37.07(±3.62) 49.03(±1.37) 51.33(±3.18) 53.17(±2.90) 52.25(±2.83) p3×p4 3.34(±0.24) 2.62(±0.19) 2.98(±0.21) 68.20(±8.89) 54.23(±7.73) 61.22(±8.29) 49.17(±3.73) 48.57(±3.27) 48.87(±3.48) p3×p5 2.97(±0.25) 2.44(±0.30) 2.70(±0.27) 40.07(±1.75) 33.47(±4.46) 36.77(±3.07) 73.97(±3.10) 72.83(±1.05) 73.40(±1.15) p3×p6 2.81(±0.24) 2.48(±0.12) 2.65(±0.18) 61.50(±1.71) 37.90(±3.73) 49.70(±2.66) 45.63(±3.85) 65.67(±6.37) 55.65(±4.15) p3×p7 3.03(±0.18) 2.50(±0.20) 2.77(±0.19) 99.03(±5.56) 78.47(±2.90) 88.75(±1.66) 30.63(±0.59) 31.93(±3.61) 31.28(±1.89) p4×p1 4.27(±0.34) 3.69(±0.15) 3.98(±0.16) 83.03(±12.08) 64.67(±4.61) 73.85(±3.83) 51.77(±3.88) 57.20(±3.72) 54.48(±0.33) p4×p2 2.86(±0.2) 2.66(±0.20) 2.76(±0.09) 47.97(±4.71) 38.27(±3.84) 43.12(±1.66) 59.60(±1.73) 69.50(±1.85) 64.55(±0.15) p4×p3 3.18(±0.36) 3.01(±0.22) 3.09(±0.29) 66.53(±1.46) 54.90(±6.05) 60.72(±3.02) 47.80(±6.17) 54.83(±1.93) 51.32(±2.63) p4×p5 4.57(±0.26) 3.80(±0.34) 4.19(±0.27) 63.20(±4.30) 47.50(±3.76) 55.35(±1.30) 72.40(±3.41) 80.00(±5.73) 76.20(±3.21) p4×p6 3.32(±0.32) 3.14(±0.25) 3.23(±0.24) 63.83(±3.40) 50.20(±7.27) 57.02(±3.95) 52.27(±7.31) 62.77(±4.40) 57.52(±2.45) p4×p7 3.03(±0.31) 2.75(±0.17) 2.89(±0.21) 87.20(±11.27) 62.80(±3.24) 75.00(±5.56) 34.90(±2.80) 43.77(±0.51) 39.33(±1.57) p5×p1 3.37(±0.31) 2.97(±0.35) 3.17(±0.58) 76.83(±8.81) 47.27(±5.53) 62.05(±1.91) 44.03(±3.81) 62.77(±2.74) 53.40(±3.14) p5×p2 2.52(±0.40) 2.35(±0.27) 2.44(±0.58) 30.27(±4.99) 29.53(±2.17) 29.90(±1.41) 83.33(±3.32) 79.47(±7.33) 81.40(±5.24) p5×p3 3.29(±0.21) 2.10(±0.22) 2.69(±1.53) 44.13(±4.23) 26.87(±2.87) 35.50(±0.79) 75.00(±9.37) 77.90(±3.30) 76.45(±3.36) p5×p4 4.00(±0.20) 3.60(±0.15) 3.80(±2.08) 56.17(±4.99) 45.23(±1.63) 50.70(±2.92) 71.53(±8.22) 79.47(±0.49) 75.50(±4.29) p5×p6 1.87(±0.23) 1.51(±0.18) 1.69(±0.58) 42.17(±4.32) 37.67(±2.97) 39.92(±1.66) 44.33(±1.12) 40.10(±6.67) 42.22(±3.82) p5×p7 2.34(±0.25) 1.97(±0.35) 2.16(±0.58) 54.40(±3.10) 32.77(±3.08) 43.58(±3.09) 42.87(±2.34) 59.70(±5.28) 51.28(±3.73) p6×p1 2.57(±0.29) 1.97(±0.34) 2.27(±0.58) 55.63(±3.27) 36.90(±3.24) 46.27(±3.25) 46.10(±3.57) 53.10(±5.48) 49.60(±4.45) p6×p2 3.79(±0.39) 3.06(±0.25) 3.43(±1.53) 44.53(±2.34) 37.33(±4.52) 40.93(±3.41) 84.90(±4.27) 82.13(±4.31) 83.52(±1.90) p6×p3 2.99(±0.15) 2.59(±0.18) 2.79(±1.53) 55.77(±2.89) 43.10(±4.45) 49.43(±0.78) 53.70(±5.11) 60.27(±2.29) 56.98(±1.75) p6×p4 2.96(±0.18) 2.69(±0.24) 2.82(±1.53) 51.57(±0.46) 40.37(±6.80) 45.97(±3.35) 57.23(±3.45) 67.10(±5.23) 62.17(±0.94) p6×p5 1.77(±0.16) 1.24(±0.22) 1.51(±0.58) 50.93(±2.78) 40.23(±9.32) 45.58(±5.95) 34.67(±1.35) 31.03(±1.76) 32.85(±0.88) p6×p7 3.27(±0.35) 3.17(±0.25) 3.22(±0.58) 69.43(±3.52) 54.47(±3.54) 61.95(±3.53) 46.97(±2.77) 58.43(±8.39) 52.70(±3.27) p7×p1 2.18(±0.16) 1.83(±0.20) 2.00(±0.58) 116.40(±13.25) 99.53(±8.03) 107.90(±9.74) 18.97(±3.54) 18.30(±1.01) 18.63(±2.14) p7×p2 2.94(±0.12) 2.47(±0.17) 2.70(±1.00) 48.90(±0.35) 45.97(±3.68) 47.43(±1.95) 59.97(±2.03) 53.73(±2.06) 56.85(±1.55) p7×p3 3.25(±0.33) 2.62(±0.18) 2.93(±0.58) 108.60(±11.61) 72.67(±1.29) 90.62(±6.45) 29.93(±1.27) 35.90(±2.20) 32.92(±1.70) p7×p4 2.86(±0.09) 2.60(±0.16) 2.73(±0.58) 70.83(±2.00) 73.50(±2.56) 72.17(±1.24) 40.40(±2.40) 35.27(±0.99) 37.83(±1.63) p7×p5 2.67(±0.45) 2.18(±0.21) 2.42(±0.58) 60.07(±12.53) 39.57(±1.89) 49.82(±7.03) 44.73(±3.43) 54.97(±3.85) 49.85(±0.39) p7×p6 2.82(±0.27) 2.77(±0.27) 2.80(±1.15) 65.97(±8.25) 54.10(±5.91) 60.03(±1.24) 42.77(±1.37) 51.57(±7.46) 47.17(±3.05) f 11.42 ** 16.70 ** 16.47 ** 25.61 ** 28.12 ** 45.39 ** 49.16 ** 33.15 ** 76.95 ** lsd 0.05 0.50 0.41 0.40 11.51 8.69 7.52 6.80 8.11 5.23 to be continued 465 emami et al. tomato genotypes under low light condition table 5 means values and standard deviation for yield components and earliness of f1 hybrids developed via 7×7 diallel cross over two light conditions p1= perimoga, p2= la1793, p3= ac06, p4= ct6, p5= mc3, p6= c20, p7= kingstone. values in parenthesis represent standard errors. ** significant at p<0.01. genotypes ♀ × ♂ days to first flower days to ripening l1 l2 pooled l1 l2 pooled p1×p2 56.00(±1.00) 54.33(±0.58) 55.17(±0.76) 14.00(±1.00) 15.67(±0.58) 14.83(±0.29) p1×p3 56.67(±0.58) 54.00(±1.00) 55.33(±0.58) 16.67(±2.08) 18.67(±1.53) 17.67(±1.61) p1×p4 54.00(±1.00) 50.67(±0.58) 52.33(±0.58) 18.33(±1.15) 18.33(±1.15) 18.33(±1.15) p1×p5 62.67(±0.58) 60.33(±0.58) 61.50(±0.50) 19.00(±2.65) 20.67(±1.15) 19.83(±1.76) p1×p6 58.00(±1.00) 54.67(±0.58) 56.33(±0.58) 18.00(±3.00) 18.00(±3.00) 18.00(±3.00) p1×p7 57.33(±0.58) 53.33(±1.53) 55.33(±0.76) 19.33(±2.08) 18.67(±1.53) 19.00(±1.00) p2×p1 56.67(±0.58) 54.67(±0.58) 55.67(±0.58) 19.00(±1.00) 16.67(±1.53) 17.83(±1.15) p2×p3 60.33(±1.15) 60.67(±1.53) 60.50(±0.87) 18.33(±1.53) 17.33(±1.53) 17.83(±1.44) p2×p4 60.67(±0.58) 58.67(±0.58) 59.67(±0.58) 16.33(±1.15) 15.67(±1.15) 16.00(±1.00) p2×p5 60.67(±0.58) 58.67(±0.58) 59.67(±0.58) 15.67(±2.08) 15.67(±2.08) 15.67(±2.08) p2×p6 61.67(±1.53) 60.33(±0.58) 61.00(±1.00) 17.00(±2.00) 17.00(±1.00) 17.00(±0.50) p2×p7 58.33(±0.58) 58.00(±1.00) 58.17(±0.29) 16.33(±1.15) 17.00(±2.00) 16.67(±1.53) p3×p1 57.33(±0.58) 54.67(±0.58) 56.00(±0.00) 18.33(±2.08) 18.33(±2.08) 18.33(±2.08) p3×p2 60.00(±0.00) 59.67(±0.58) 59.83(±0.29) 20.33(±2.52) 19.33(±2.31) 19.83(±2.36) p3×p4 56.00(±1.00) 55.67(±2.31) 55.83(±1.61) 16.67(±2.08) 16.67(±2.08) 16.67(±2.08) p3×p5 60.00(±1.00) 56.33(±1.53) 58.17(±1.04) 20.00(±1.73) 17.33(±2.08) 18.67(±1.53) p3×p6 60.67(±0.58) 59.00(±1.00) 59.83(±0.76) 17.00(±1.73) 17.00(±1.73) 17.00(±1.73) p3×p7 58.33(±0.58) 56.33(±0.58) 57.33(±0.58) 22.67(±2.08) 22.67(±2.08) 22.67(±2.08) p4×p1 54.67(±1.15) 51.33(±1.15) 53.00(±0.00) 17.00(±2.00) 17.00(±1.15) 17.00(±2.00) p4×p2 60.00(±1.00) 58.00(±1.00) 59.00(±1.00) 15.00(±1.00) 14.00(±1.00) 14.50(±0.87) p4×p3 58.00(±1.73) 57.00(±1.00) 57.50(±0.50) 14.67(±0.58) 15.33(±1.00) 15.00(±0.00) p4×p5 61.67(±1.15) 59.67(±1.15) 60.67(±1.15) 18.00(±2.00) 18.00(±1.15) 18.00(±2.00) p4×p6 56.33(±0.58) 54.33(±0.58) 55.33(±0.58) 20.00(±2.65) 21.00(±0.58) 20.50(±1.80) p4×p7 57.67(±0.58) 55.67(±0.58) 56.67(±0.58) 18.67(±1.53) 18.67(±0.58) 18.67(±1.53) p5×p1 62.00(±1.00) 59.33(±0.58) 60.67(±0.76) 20.33(±1.53) 19.33(±0.58) 19.83(±1.04) p5×p2 61.67(±0.58) 58.33(±0.58) 60.00(±0.00) 13.00(±1.00) 12.67(±0.58) 12.83(±1.04) p5×p3 61.00(±0.00) 57.33(±1.53) 59.17(±0.76) 19.67(±2.52) 16.00(±1.53) 17.83(±1.04) p5×p4 60.67(±2.08) 58.67(±2.08) 59.67(±2.08) 19.00(±3.00) 20.33(±2.08) 19.67(±2.08) p5×p6 63.67(±1.53) 58.67(±0.58) 61.17(±0.58) 21.00(±1.00) 20.33(±0.58) 20.67(±1.15) p5×p7 61.00(±0.00) 58.33(±0.58) 59.67(±0.29) 21.33(±1.53) 15.00(±0.58) 18.17(±0.29) p6×p1 58.33(±1.53) 55.33(±0.58) 56.83(±0.76) 17.67(±2.52) 17.67(±0.58) 17.67(±2.52) p6×p2 62.33(±1.53) 59.67(±1.53) 61.00(±1.32) 15.00(±2.00) 15.33(±1.53) 15.17(±1.76) p6×p3 59.67(±0.58) 59.33(±1.53) 59.50(±0.87) 16.00(±1.73) 19.67(±1.53) 17.83(±1.04) p6×p4 56.67(±0.58) 54.33(±1.53) 55.50(±0.87) 20.00(±2.65) 18.33(±1.53) 19.17(±2.02) p6×p5 63.33(±0.58) 60.33(±0.58) 61.83(±0.58) 18.67(±1.53) 18.67(±0.58) 18.67(±1.53) p6×p7 59.00(±1.00) 55.33(±0.58) 57.17(±0.29) 20.67(±1.53) 21.33(±0.58) 21.00(±1.50) p7×p1 58.00(±1.00) 52.67(±0.58) 55.33(±0.76) 20.67(±2.08) 20.67(±0.58) 20.67(±1.76) p7×p2 60.00(±0.00) 59.00(±1.00) 59.50(±0.50) 20.00(±1.00) 18.00(±1.00) 19.00(±0.00) p7×p3 58.33(±0.58) 56.33(±0.58) 57.33(±0.58) 20.67(±0.58) 20.67(±0.58) 20.67(±0.58) p7×p4 58.33(±0.58) 56.33(±0.58) 57.33(±0.58) 17.33(±1.15) 18.67(±0.58) 18.00(±0.00) p7×p5 62.00(±0.00) 59.33(±0.58) 60.67(±0.29) 20.00(±1.00) 17.67(±0.58) 18.83(±1.61) p7×p6 58.67(±1.15) 53.33(±1.15) 56.00(±1.00) 19.00(±1.00) 19.00(±1.15) 19.00(±1.00) f 18.69 ** 19.82 ** 28.14 ** 4.20 ** 19.82 ** 4.82 ** lsd 0.05 1.52 1.66 1.28 2.98 2.59 2.53 continued 466 adv. hort. sci., 2018 32(4): 459-470 table 6 general combining ability (gca) effects of parental lines for yield, yield components and earliness in a 7×7 diallel cross over two light conditions p1= perimoga, p2= la1793, p3= ac06, p4= ct6, p5= mc3, p6= c20, p7= kingstone. * significant at p<0.05 level, ** significant at p<0.01 level. a difference between gca of two parental lines at p<0.05 level. parental lines total yield per plant (kg) average of fruit weight (g) fruits number per plant days to first flower days to ripening l1 l2 pooled l1 l2 pooled l1 l2 pooled l1 l2 pooled l1 l2 pooled p1 -0.02 -0.06 -0.04 18.32 ** 10.97 ** 14.64 ** -13.46 ** -11.38 ** -12.42 ** -1.93 ** -2.70 ** -2.31 ** -0.06 0.42 0.18 p2 -0.02 -0.24 ** -0.13 ** -16.82 ** -11.61 ** -14.22 ** 14.63 ** 6.67 ** 10.65 ** 0.74 ** 1.77 ** 1.25 ** -1.90 ** -2.11 ** -2.00 ** p3 0.09 -0.12 ** -0.02 5.22 ** -0.87 2.17 ** -2.98 ** -1.78 * -2.38 ** -0.46 ** 0.40 * -0.03 0.2 0.36 0.28 p4 0.48 ** 0.64 ** 0.56 ** 1.98 3.76 ** 2.87 ** 3.28 ** 6.70 ** 4.99 ** -1.63 ** -1.20 ** -1.41 ** -0.80 * -0.34 -0.57 ** p5 -0.08 -0.06 -0.07 ** -13.35 ** -11.79 ** -12.57 ** 8.71 ** 10.29 ** 9.50 ** 2.94 ** 2.30 ** 2.62 ** 0.67 * -0.38 0.15 p6 -0.20 ** -0.08 -0.14 ** -12.68 ** -8.17 ** -10.42 ** 3.99 ** 4.51 ** 4.25 ** 0.74 ** 0.24 0.49 ** 0.1 0.79 ** 0.45 * p7 -0.25 ** -0.07 -0.16 ** 17.34 ** 17.71 ** 17.52 ** -14.15 ** -15.01 ** -14.58 ** -0.40 * -0.83 ** -0.61 ** 1.77 ** 1.26 ** 1.51 ** lsd 0.05 gi gj a 0.16 0.13 0.09 3.56 2.68 2.07 2.1 2.51 1.52 0.47 0.51 0.35 0.92 0.8 0.6 table 7 specific combining ability (sca) effects of f1 hybrids for yield, yield components and earliness in a 7×7 diallel cross over two light conditions p1= perimoga, p2= la1793, p3= ac06, p4= ct6, p5= mc3, p6= c20, p7= kingstone. *significant at p < 0.05 level. ** significant at p<0.01 level. a difference between two sca of two hybrids, with a common parent. b difference between two sca of two hybrids, with non-common parent. genotypes ♀ × ♂ total yield per plant (kg) average o fruit weight (g) fruits number per plant days to first flower days to ripening l1 l2 pooled l1 l2 pooled l1 l2 pooled l1 l2 pooled l1 l2 pooled p1×p2 -0.51 ** -0.70 ** -0.60 ** 1.29 -4.52 * -1.62 -14.90 ** -15.90 ** -15.40** -1.72 ** -1.43 ** -1.58 ** 0.21 -0.1 0.06 p1×p3 0.31 ** 0.29 ** 0.30 ** -6.87 ** -4.69 ** -5.78 ** 7.36 ** 6.62 ** 6.99** 0.14 -0.23 -0.04 -0.89 -0.23 -0.56 p1×p4 0.53 ** 0.48 ** 0.50 ** 1.03 -3.36 -1.16 6.07 ** 9.07 ** 7.57** -1.36 ** -1.97 ** -1.66 ** 0.28 -0.37 -0.04 p1×p5 0.55 ** 0.66 ** 0.60 ** 11.36 ** 3.93 * 7.64 ** -3.68 ** 5.01 ** 0.67 2.08 ** 3.37 ** 2.72 ** 0.81 2.00 ** 1.41 ** p1×p6 -0.42 ** -0.29 ** -0.36 ** -21.97 ** -11.65 ** -16.81 ** 8.66 ** 4.04 * 6.35** 0.11 0.6 0.36 -0.46 -1.33 * -0.89 * p1×p7 -0.45 ** -0.43 ** -0.44 ** 15.16 ** 20.30** 17.73 ** -3.51 * -8.84 ** -6.17** 0.74 * -0.33 0.21 0.04 0.03 0.04 p2×p3 -0.21 * -0.38 ** -0.30 ** 6.92 ** -1.3 2.81 * -14.38 ** -9.97 ** -12.18** 0.64 * 1.13 ** 0.89 ** 2.78 ** 2.13 ** 2.46 ** p2×p4 -0.46 ** -0.22 * -0.34 ** -2.56 -2.3 -2.43 -4.25 ** 1.11 -1.57 1.98 ** 0.90 ** 1.44 ** 0.11 -0.67 -0.28 p2×p5 -0.29 ** 0.21 * -0.04 -0.69 6.48 ** 2.90 * 4.88 ** 3.80 * 4.34** -1.76 ** -2.43 ** -2.09 ** -2.69 ** -1.30 * -1.99 ** p2×p6 1.16 ** 0.70 ** 0.93 ** 9.64 ** 5.56 ** 7.60 ** 19.35 ** 16.98 ** 18.17** 1.28 ** 1.13 ** 1.21 ** -0.46 -0.47 -0.46 p2×p7 0.31 ** 0.38 ** 0.35 ** -14.61 ** -3.91 * -9.26 ** 9.29 ** 3.99 * 6.64** -0.42 0.70 * 0.14 0.04 0.4 0.22 p3×p4 -0.37 ** -0.24 ** -0.30 ** -4.28 2.48 -0.9 -3.31 * -8.53 ** -5.92** -0.16 0.27 0.06 -1.99 ** -1.97 ** -1.98 ** p3×p5 0.07 -0.09 -0.01 -14.22 ** -6.37 ** -10.29 ** 17.26 ** 11.55 ** 14.41** -1.22 ** -2.73 ** -1.98 ** 0.71 -1.27 * -0.28 p3×p6 -0.04 0.20 * 0.08 1.65 0.34 1 -2.84 * 4.93 ** 1.05 0.64 * 1.67 ** 1.16 ** -2.06 ** -0.77 -1.41 ** p3×p7 0.25 * 0.22 * 0.23 ** 16.80 ** 9.54 ** 13.17 ** -4.09 ** -4.60 ** -4.34** -0.06 -0.1 -0.08 1.44 * 2.10 ** 1.77 ** p4×p5 0.83 ** 0.58 ** 0.71 ** 6.60 ** 5.20 ** 5.90 ** 8.48 ** 7.43 ** 7.96** 0.61 1.20 ** 0.91 ** 0.38 1.93 ** 1.16 ** p4×p6 -0.2 -0.18 * -0.19 ** 3.95 0.49 2.22 -4.01 ** -1.59 -2.80** -1.86 ** -1.57 ** -1.71 ** 2.44 ** 1.27 * 1.86 ** p4×p7 -0.34 ** -0.43 ** -0.38 ** -4.75 * -2.51 -3.63 ** -2.98 * -7.48 ** -5.23** 0.78 * 1.17 ** 0.97 ** -1.22 * -0.2 -0.71 p5×p6 -0.95 ** -1.02 ** -0.98 ** 8.13 ** 9.71 ** 8.92 ** -24.69 ** -34.54 ** -29.62 ** 0.58 0.1 0.34 0.81 1.13 0.97* p5×p7 -0.21 -0.33 ** -0.27 ** -11.20 ** -18.95 ** -15.07 ** -2.26 6.75 ** 2.24 * -0.29 0.5 0.11 -0.02 -2.50 ** -1.26 ** p6×p7 0.45 ** 0.59 ** 0.52 ** -1.4 -4.45 * -2.93 * 3.53 * 10.19** 6.86 ** -0.76 -1.93 ** -1.34 ** -0.29 0.17 -0.06 lsd 0.05 s ij − s ik a 0.29 0.23 0.17 6.44 4.87 3.75 3.81 4.54 2.75 0.85 0.93 0.63 1.67 1.45 1.08 s ij − s kl b 0.25 0.2 0.14 5.58 4.21 3.25 3.3 3.93 2.38 0.74 0.81 0.55 1.44 1.26 0.93 emami et al. tomato genotypes under low light condition 467 ‘perimoga×mc3’ (p1×p5) was the weakest combination across both environments. the most negative value of sca for ripening period under low light belonged to ‘la1793×mc3’ (p2×p5) and under high light was for ‘kingstone×mc3’ (p7×p5). pooled value of sca in this character showed that generally ‘la1793×mc3’ (p2×p5) and ‘ac06×la1793’ (p3×p2) had the highest and the lowest negative values, respectively. the results of rec indicated that the best reciprocal combinations over two environments for total yield, average of fruit weight, fruits number, days to f l o w e r i n g a n d d a y s t o r i p e n i n g w a s f o r ‘ac06×la1793’ (p3×p2), ‘kingstone×mc3’ (p7×p5), ‘mc3×la1793’ (p5×p2), ‘kingstone×c20’ (p7×p6) and ‘mc3×la1793’ (p5×p2), respectively (table 8). the lowest pooled rec in foregoing characters was for ‘ k i n g s t o n e × c 2 0 ’ ( p 7 × p 6 ) , ‘ c 2 0 × c t 6 ’ ( p 6 × p 4 ) , ‘ c 2 0 × m c 3 ’ ( p 6 × p 5 ) , ‘ m c 3 × a c 0 6 ’ ( p 4 × p 3 ) a n d ‘la1793×perimoga’ (p2×p1), respectively 4. discussion and conclusions the results indicated that total yield, average fruit weight and flowering time were influenced by the amount of received light, while fruit number and fruit ripening period were not affected. genotype effect was highly significant for all studied traits implying the feasibility of breeding. despite of simultaneous influence of light and genotype on yield, fruit weight and days to flower, a comparison between magnitude of environment and genotype effects revealed that the genotype variation played more important in the expression of studied traits. the significance of interaction genotype × light condition (g×l) for almost all characters except days to ripening revealed that there is a genotype variation in response to light intensity as regards yield, yield components and early flowering. previous studies reported genotype variation regarding reaction to environmental light in different species (stratton, table 8 reciprocal effect (rec) for yield, yield components and earliness in a 7×7 diallel cross over two light conditions p1= perimoga, p2= la1793, p3= ac06, p4= ct6, p5= mc3, p6= c20, p7= kingstone. * significant at p<0.05 level, ** significant at p<0.01 level. a difference between two rca of two hybrids. genotypes ♀ × ♂ total yield per plant (kg) average of fruit weight (g) fruits number per plant days to first flower days to ripening l1 l2 pooled l1 l2 pooled l1 l2 pooled l1 l2 pooled l1 l2 pooled p2×p1 -0.22 0.05 -0.08 -2.9 -1.87 -2.38 -1.17 2.8 0.82 0.33 0.17 0.25 2.50 ** 0.5 1.50 ** p3×p1 -0.11 -0.23 * -0.17 * -4.52 -0.57 -2.54 1.08 -3.5 -1.21 0.33 0.33 0.33 0.83 -0.17 0.33 p4×p1 0.23 0.1 0.17 * -2.75 4.1 0.67 4.38 * -2.5 0.94 0.33 0.33 0.33 -0.67 -0.67 -0.67 p5×p1 -0.12 -0.11 -0.11 -3.95 -5.03 * -4.49 ** 0.97 3.53 2.25 -0.33 -0.5 -0.42 0.67 -0.67 0 p6×p1 0.16 -0.13 0.02 7.52 * -3.45 2.03 -4.58 ** 0.62 -1.98 0.17 0.33 0.25 -0.17 -0.17 -0.17 p7×p1 -0.15 -0.15 -0.15 * 1.1 1.37 1.23 -1.42 -1.78 -1.6 0.33 -0.33 0 0.67 1 0.83 p3×p2 0.22 0.19 0.21 ** 1.23 1.65 1.44 2.57 2.95 2.76 * -0.17 -0.5 -0.33 1 1 1.00 * p4×p2 -0.21 -0.06 -0.13 0.92 -0.78 0.07 -5.55 ** -0.28 -2.92 * -0.33 -0.33 -0.33 -0.67 -0.83 -0.75 p5×p2 -0.14 -0.1 -0.12 -3.32 -2.75 -3.03 3.62 * 3.4 3.51 ** 0.5 -0.17 0.17 -1.33 -1.50 * -1.42 ** p6×p2 -0.21 0.14 -0.03 -0.05 2.35 1.15 -4.57 ** -1.33 -2.95 * 0.33 -0.33 0 -1 -0.83 -0.92 p7×p2 -0.15 -0.14 -0.15 * -1.45 -5.42 * -3.43 * -1.3 2.78 0.74 0.83 * 0.5 0.67 * 1.83 * 0.5 1.17 * p4×p3 -0.08 -0.19 0.06 -0.83 0.33 -0.25 -0.68 3.13 1.23 1.00 * 0.67 0.83 ** -1 -0.67 -0.83 p5×p3 0.16 -0.17 -0.01 2.03 -3.3 -0.63 0.52 2.53 1.52 0.5 0.5 0.5 -0.17 -0.67 -0.42 p6×p3 0.09 0.06 0.07 -2.87 2.6 -0.13 4.03 * -2.7 0.67 -0.5 0.17 -0.17 -0.5 1.33 * 0.42 p7×p3 0.11 0.06 0.08 4.77 -2.9 0.93 -0.35 1.98 0.82 0 0 0 -1 -1 -1.00 * p5×p4 -0.29 * -0.1 -0.19 ** -3.52 -1.13 -2.33 -0.43 -0.27 -0.35 -0.5 -0.5 -0.5 0.5 1.17 0.83 p6×p4 -0.18 -0.22 * -0.20 ** -6.13 * -4.92 * -5.53 ** 2.48 2.17 2.32 0.17 0 0.08 0 -1.33 * -0.67 p7×p4 -0.09 -0.08 -0.08 -8.18 ** 5.35 * -1.42 2.75 -4.25 * -0.75 0.33 0.33 0.33 -0.67 0 -0.33 p6×p5 -0.05 -0.13 -0.09 4.38 1.28 2.83 -4.83 ** -4.53 * -4.68 ** -0.17 0.83 * 0.33 -1.17 -0.83 -1.00 * p7×p5 0.17 0.1 0.13 2.83 3.4 3.12 0.93 -2.37 -0.72 0.5 0.5 0.5 -0.67 1.33 * 0.33 p7×p6 -0.23 -0.2 -0.21 ** -1.73 -0.18 -0.96 -2.1 -3.43 -2.77* -0.17 -1.00 * -0.58 * -0.83 -1.17 -1.00 * lsd 0.05 r ij − r ik a 0.31 0.25 0.18 7.04 5.32 4.1 4.16 4.96 3.01 0.93 1.02 0.69 1.82 1.59 1.18 adv. hort. sci., 2018 32(4): 459-470 468 d.a., 1998; martínez-ferri et al., 2001) promising for plant improvement regarding maintenance of high yield and earliness under lower level of light intensity. the remarkable effect of interaction g×l on studied traits except ripening period demonstrated that genotypes were not stable across two environments and should be evaluated in a range of environments. importance of genotype selection across different environments for tomato improvement concerning yield and earliness attributing traits was reported by chadha and kumar (2001), and biswas et al. (2011). according to mean performances of hybrids, superior genotypes for various characteristics differed and none of them could be considered as the best for all of the attributes. in this regard, in order to commercialize f1 hybrids, breeding programs should be conducted to collect suitable features in one plant (breseghello and coelho, 2013). either of general combing ability (gca) or specific combining ability (sca) was highly significant for all of evaluated features illustrating both additive and non-additive gene action were involved in controlling yield, yield components and earliness. our findings supports additive-dominance model reported by chishti et al. (2008) and biswas et al. (2011) for production and earliness traits. significant contribution of rec to total sum squire is indicative of inter-allelic interactions in the expression of studied traits. similarly, rec effect on fruit weight and number was reported by hannan et al., (2007 b). higher magnitude of sca variance (σ2s) in comparison with gca variance (σ2g) for total yield, fruit number and days to ripening indicated that these traits are mainly under the control of dominant effects. similar findings were reported by solieman (2009) and el-gabry et al. (2014) for fruit yield and number. the predominance of non-additive gene action over additive effects for days to ripening was in agreement with hannan et al. (2007a) but inconsistent with garg et al. (2008) who found additive gene action to be more effective on days to ripening over two environments. the ratio of σ2g/σ2s over two light conditions was greater than unity for fruit weight and days to flowering indicative of more weight of additive effects in inheritance of these features. garg et al. (2008), rai and asati (2011) and nadeem et al. (2013) also contributed the expression of fruit weight and early flowering to both additive and dominance gene actions with preponderance of additive effects. biswas et al. (2011) who examined tomato genotypes across two environments, different in terms of temperature and light intensity, reported more important role of additive gene action in control of fruit weight. the interaction gca×l was significant for all characters except for days to ripening indicating the sensibility of additive effects to light condition. the significant interaction sca×l was indicative of instability of dominance effects under different environmental light. rec×l varied for fruit weight and number demonstrating the susceptibility of cytoplasmic effects to environment in some traits and necessity of reciprocal crosses for choosing the most suitable genotypes for target environment. estimates of broad-sense heritability percentage (h2b.s%) across two different light conditions was high, demonstrating the low effect of environment and high response of studied traits to breeding programs. relatively high narrow sense heritability percentage (h2n.s%) for fruit weight and early flowering indicated that these traits are largely controlled by additive effects; while, low h2n.s% of yield, fruit number and early maturity demonstrated higher weight of non-additive effects in inheritance of these traits. these findings agree with earlier work of biswas et al. (2011) and dutta et al. (2013) who reported low narrow sense heritability for yield and fruit number over two environments. importance of both additive and non-additive gene action with predominance of additive effects in expression of fruit weight and days to flowering revealed that selection breeding programs could be an effective strategy for genetic improvement of tomato for these characters, while exploitation of hybrid vigor should not be neglected (grilli et al., 2003). yield, fruit number and early ripening were controlled by additive-dominance effects with higher weight of dominance effects implying hybrid breeding could be adopted for improvement of these characters (gul et al., 2010). abd el-maksoud et al. (2013) proposed recurrent selection program for improvement of traits controlled by both additive and nonadditive effects. for such traits, hybridization in segregating generations followed by selection for outperforming genotypes has been recommended (dutta et al., 2013; bhattarai et al., 2016). limitation in access to clean energy resources in rural areas and global interest toward lower energy consumption necessitate breeding for low energy input. in the current study, genetic variation among tomato genotypes under different light conditions was observed and some genotypes showed more stability than others. none of parental lines or f1 emami et al. tomato genotypes under low light condition 469 hybrids exhibited high performance for all studied features, therefore, a particular genotype cannot be r e c o m m e n d e d . h o w e v e r , g e n e t i c k n o w l e d g e obtained from this research could be used in planning tomato breeding programs. more important role of additive gene 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cv. dalhari (syzygium samarangense) c.k. setiawan (*), n.a. utama, b.a. pradana a g r o t e c h n o l o g y d e p a r t m e n t , f a c u l t y o f a g r i c u l t u r e , u n i v e r s i t a s muhammadiyah yogyakarta, indonesia. key words: alginate, betel essential oil, edible coating, rose apple cv. dalhari. abstract: rose apple cv. dalhari is a local fruit grown in berbah district, special region of yogyakarta. this fruit perishable, easily loses its water content and is attacked by microbes. this research aimed to determine the best combination between alginate and betel essential oil to inhibit the growth of microbes and maintain quality of rose apple cv. dalhari. alginate and betel essential oil treatments were used at three concentration levels, alone and in combinations, respectively of 2%, 2.5%, 3% and 0%, 0.1%, 0.2%. the results showed that combination treatment of 2.5% alginate and 0.1% betel essential oil was the most effective to maintain the fruit quality. the sole addition of betel essential oil was not able to inhibit microbial growth. furthermore, combination of alginate based edible coating and betel essential oil was able to maintain the quality of rose apple cv. dalhari up to nine days. 1. introduction rose apple cv. dalhari is locally cultivated at berbah district, yogyakarta. rose apple cv. dalhari is one of the most famous fruits due to its large size, slightly fresh sour taste, and high water content. while, rose apple has thin skin makes low ability to maintain its water content and also is easily growth by microbes when it is kept at room temperature (pertiwi et al., 2012). edible coating is an alternative technology to overcome the problem and its widely known as the ability to maintain the atmosphere condition around the fruit, such as control gas exchange and control water loss, maintain the fruit’s texture, and also reduce the risk of microbial attack (campos et al., 2011). edible coating formulation can be derived from polysaccharides, lipids, proteins as long as the material are tasteless, odourless, and transparent. polysaccharides include alginate that is widely used as primary ingredient for edible coating due to its ability in holding high oxygen and in resulting good quality of edible coating (quirós-sauceda et al., 2014). alginate is obtained from marine brown algae (phaeophyceae) extraction. recently, the use of edible coating in fruit preservation is combined with another (*) corresponding author: chandra_fp@.umy.ac.id citation: setiawan c.k., utama n.a., pradana b.a., 2019 combination of alginate based edible coating-betel essential oil in extending the shelf life of rose apple cv. dalhari (syzygium samarangense). adv. hort. sci., 33(1): 33-38 copyright: © 2019 setiawan c.k., utama n.a., pradana b.a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 1 january 2018 accepted for publication 18 september 2018 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(1): 33-38 34 active ingredient, such as antibrowning agent, nutrients, and antimicrobial agent to decrease microbial growth (campos et al., 2011; guerreiro et al., 2015; atarés and chiralt, 2016; hamedi et al., 2017). some essential oils have shown their effectiveness as food preservatives due to their antimicrobial activity and antioxidant content. betel essential oil contains chavicol, eugenol, acetyl eugenol as the main compounds to prevent pathogen growth in different edible commodities (prakash et al., 2010; basak and guha, 2015). however there are still limited information available regarding the use of edible coating to maintain the freshness of rose apple cv. dalhari. the only report from previous authors refers to effect of storage temperature of rose apple cv. dalhari (widoyo, 2013) , and none mentioned the use of edible coatings. this study aimed to determine the effect of betel essential oil combined with alginate-based edible coatings to prevent pathogen growth, and extend the shelf life of rose apple cv. dalhari. 2. materials and methods rose apple fruit cv. dalhari was harvested in b e r b a h d i s t r i c t , s p e c i a l r e g i o n o f y o g y a k a r t a , indonesia in the middle of april, 60 days after flowering. harvested fruit has characteristic red colour skin and average weight 125 g/fruit from 243 f r u i t s . t h e f r u i t w a s i m m e d i a t e l y b r o u g h t t o p o s t h a r v e s t l a b o r a t o r y a t t h e u n i v e r s i t a s muhammadiyah yogyakarta. the fruits were selected b a s e d o n d e f e c t a n d s i z e , t h e n s t o r e d i n a refrigerator (14°c) until treatment. food grade sodium alginate (al) (iis, indonesia) was used for coating treatment, glycerol as a plasticizer, and betel essential oil (be). preparation and application of edible coating edible coating was prepared with diluted alginate into aquadest solution and heated in 85°c. solution was added with 1.5% glycerol and betel essential oil in different concentration. alginate-betel essential oil solution were formulated into nine treatments: al 2% (w/v) (a1s0); al 2% (w/v)-be 0.1% (v/v) (a1s1); al 2% (w/v)-be 0.2% (v/v) (a1s2); al 2.5% (w/v) (a2s0); al 2.5% (w/v)-be 0.1% (v/v) (a2s1); al 2.5% (w/v)-be 0.2% (v/v) (a2s2); al 3% (w/v) (a3s0); al 3% (w/v)-be 0.1% (v/v) (a3s1); al 3% (w/v)-be 0.2% (v/v) (a3s2). each treatment was performed in several steps as f o l l o w : t h e f r u i t w a s w a s h e d u s i n g s o d i u m h y p o c h l o r i t e 2 0 0 µ l l 1 a n d d r i e d a t r o o m temperature followed by dipping in edible coating solution for 2 min. the excess of edible coating solution was dripped off for 30 s before being dipped in cacl 2% solution for 1 min. three treated fruits w e r e p a c k a g e d w i t h p o l y p r o p y l e n e t r a y s w i t h perforated cover and stored at 14°c until analysis. analysis was performed on days 0, 3, 6, 9, 12, and 15. fruit quality assessment twenty seven groups of rose apple (9 treatments x 3 replicates) were weighed during the storage time. the weight loss (wl) of rose apple was determined using the equation: wl (%)= [(w0-wt)/w0] x 100 where w0 is the initial weight and wt is the sample weight at time t. fruit firmness was measured using a fruit hardness tester fht200 (extech instruments, usa). each fruit was tested in three different sides with a 6 mm diameter probe on equatorial position a n d r e c o r d e d i n n / m m 2. t o t a l a c i d i t y ( t a ) d e t e r m i n a t i o n w a s p e r f o r m e d w i t h m e t h o d s according to iso 750-1998. five grams of rose apple slices were homogenised and diluted to 100 ml with distilled water. the mixture was filtered and was added 3-5 drops of phenolphthalein (1% in 95% ethanol) to 10 ml solution. the solution was then s t i r r e d a n d t i t r a t e d w i t h 0 . 1 n n a o h . t a w a s determined as a percent of citric acid. total soluble s o l i d ( t s s ) w a s d e t e r m i n e d u s i n g a h a n d refractometer (atago, japan). the tools determines the juice tss content after being homogenised the flesh and it was expressed as a brix (%). reducing sugar (rs) were determined according to nelsonsomogyi technique (nelson, 1944) and the result showed in percentage. the analysis was performed in 540 nm abs using spectrophotometer uvmini-1240 (shimadzu, japan). antibacteria and antiyeast activity bacteria and yeast were isolated from decayed r o s e a p p l e u s i n g p a p e r d i s c s a n d p l a t e c o u n t . suspension was obtained from diluting 1 gr samples on destilled water in seven-fold dilution series. a petri dish was prepared with poured 10 ml nutrient agar medium and allowed to solidify. the 0.1% inoculum suspension was poured and flattened on the medium. sterilized filter paper disc dipped with 0.1% betel essential oil for 15 min and then placed on the surface of a medium. the petri incubated at room temperature for 48 h. after incubation time, measurement the inhibition zones were done with a setiawan et al. shelf life of rose apple cv. dalhari 35 ruler. in the other hand, 0.1% of betel essential oil was poured in medium before inoculated with 0.1 ml i n o c u l u m . a f t e r 4 8 h i n c u b a t i o n a t r o o m temperature, the visible microbe was counted and the result was presented in log colony forming unit (cfu). after treatment was done, all samples were analyzed with microbial count for bacterial and yeasts based on mola et al. (2017). one gram of h o m o g e n i z e d s a m p l e w a s d i l u t e d u s i n g 9 m l sterilized water. sample then diluted in seven-fold dilution series before plating. a 0.1 ml solution was poured on plate count agar (pca) surface and potato dextrose agar (pda) for enumerating total bacteria and molds. microbiological analysis was performed in three replication and the results were expressed as cfu (colony forming units) per grams fresh weight. sensory analysis the sensory was analysed by 15 semi-trained panellists on the based of 5-point hedonic scales (1=very dislike; 2=dislike; 3=neither like or dislike; 4=like; 5= like very much) for the parameters of texture and appearance according to guerreiro et al. (2015) with some modification. the panellists were recruited from staff and students of the department. they were trained in the initial test to be familiar with the fruit. all samples were analyzed on 0, 3, 6, 9, 12, 15 days after treatment. statistical analysis the experiment was carried out in randomised experimental design. the experiment data were analysed using sas statistical software package 9.4 f o r w i n d o w s a n d d u n c a n m u l t i p l e r a n g e t e s t (p<0.05) was performed for mean comparisons. 3. results w e i g h t l o s s i s o n e o f t h e m o s t i m p o r t a n t freshness indicators in fruit. as shown in figure 1, the weight loss increases in all fruit treatment during 15 days storage. however, a combination of alginate 2.5% and betel essential oil 0.1% can significantly (p<0.05) prevent the fruit weight loss, while the treatment of alginate 2% has shown the highest weight loss. weight loss of rose apple has no relation to its firmness. it can be shown in figure 2. although it shows that the value of firmness is not constant, overall the trend tends to show the similar pattern during 15 days of storage. alginate can maintain the fruit firmness’ decline even there is no significant effect between all treatment (p>0.05). a similar trend was also shown in pineapples (azarakhsh et al., 2012), and apple (rojas-graü et al., 2007 a) (omsoliu et al., 2008). t h e t a , t s s a n d r s c o n t e n t s h o w a r e g u l a r decreasing during 15 days of storage time. the ta d e c r e a s e s a f t e r t h r e e d a y s s t o r a g e t i m e i n a l l treatment. however, as can be seen in figure 3a, the fruit treated with 3% alginate shows slower ta reduction. the value of tss slowly decreases on all treatments during 15 days storage (fig. 3b) despite no significant effect (p>0.05) is observed. rose apple fruit being non-climacteric fruit, tends to maintain similar tss content during storage. concerning rs content, there is no significant effect (p>0.05) in all treatments. rs value decreased after three days storage as showed in figure 3c. the effect of the edible coating containing a different concentration of betel essential oil on microbial growth is shown in figure 4a and 4b. as fig. 1 effect of alginate based edible coating-betel essential oil on weight loss of rose-apple cv. dalhari. fig. 2 effect of alginate based edible coating-betel essential oil on fruit firmness of rose-apple cv. dalhari. adv. hort. sci., 2019 33(1): 33-38 36 shown in the figure, edible coating can inhibit both bacteria and yeast in rose apple cv. dalhari. the increase in the population of both bacteria and yeast is observed during 15 days of storage time. the addition of betel essential oil can inhibit the yeast growth until nine days, while bacteria can be shown in no essential oil addition treatment after six days storage (fig. 4a). the sensory quality evaluation is not significantly different (p>0.05) during 15 days of storage time based on both treatments. the result indicates that different changes happen in the treatment during storage time although panellists did not apprehend those differences. the result also showed that rose apple is not suitable for consumption at 9 d after storage since most panellists give a score under minimum acceptable value in overall score (fig. 5). 4. discussion and conclusions reducing effect from essential oil to weight loss can be described with conjunction of betel essential oil with polysaccharides based edible coating. this relation make the edible coating more stable and permeable. similar result was described in the experiment using lemongrass (azarakhsh et al., 2014), cinnamon and palmarosa (raybaudi-massilia et al., 2008), oregano and vanillin (rojas-graü et al., 2007 a). the use of alginate as edible coating is ex p ec t ed c a n d ec rea s e t h e w ei g h t l o s s . i n t h e present study, the results show that increasing fig. 3 effect of alginate based edible coating-betel essential oil on ta (a), tss (b) and rs (c) of rose-apple cv. dalhari. fig. 4 effect of alginate based edible coating-betel essential oil on bacteria (a) and yeast (b) population of rose-apple. fig. 5 effect of alginate based edible coating-betel essential oil on panelist preferences of rose-apple cv. dalhari. setiawan et al. shelf life of rose apple cv. dalhari 37 alginate concentration determine an increase in weight loss. in a previous study, this phenomenon has been reported and weight loss was explained as coming from edible coating itself (guerreiro et al., 2015). addition of betel essential oil does not affect the firmness (fig. 3). in a previous study, the use of lemongrass essential oil on alginate based edible coating decreases the fruit firmness due to its lower ph that triggers pectic acid hydrolysis (rojas-graü et al., 2007 a). fresh cut pineapple which are treated with alginate-lemongrass essential oil edible coating shows a decrease in firmness value during storage (azarakhsh et al., 2014). in ta observation, the result show that high concentration of alginate can decrease oxygen permeability which led reduction on the respiratory process (campos et al., 2011). ta changes are related to organic acid production and an acidity reduction maybe represent as a metabolic changes result in f r u i t o r c a u s e d b y t h e u s e o f o r g a n i c a c i d a s respiratory substance (selcuk and erkan, 2015). tss content is a critical attribute which has contribution to quality and consumer acceptability. our result demonstrate that alginate based edible coating shows ability to maintain the tss content of rose apple. this findings are in line with the previous study in sweet cherry (díaz-mula et al., 2012), guava (nair et al., 2018), cantaloupe (zhang et al., 2015), blueberry (mannozzi et al., 2017) and ber fruit (ramana rao et al., 2016). rs value is an essential indicator for determining respiratory process in the fruit. betel essential oil addition does not affect the rs value. however, many studies showed alginate can maintain rs value which is reported in carambola (gol et al., 2015), cherry (díaz-mula et al., 2012) fresh fruit has high nutrient and high water content which allow microbial to multiply (rojasgraü et al., 2007 a). betel essential oil can inhibit the microbiological growth in food preservation (prakash et al., 2010). in a previous study, the result showed that there is no fungi that can grow in media which have been added with betel essential oil more than 0.6% (v/v) (basak and guha, 2015). however, our previous study in preferences (unpublished data) on alginate-betel essential oil edible coating showed that most panellists accepted to consume rose apple with the addition of essential oil less than 0.2%. a d d i t i o n b e t e l e s s e n t i a l o i l s h o w s p r o p e r appreciation up to 9 d in appearance value. this result is related to low microbial spoilage compared to no addition of essential oil. similarly, changes are shown in rose apple cv. dalhari texture preference. the panellist show acceptable score up to 9 d. this overall result is consistent with in previous study where it is no differences between fruit with low concentration essential oil addition with control in s e n s o r y t e s t ( r a y b a u d i m a s s i l i a e t a l . , 2 0 0 8 ; azarakhsh et al., 2014; bustos et al., 2016; guerreiro et al., 2016). in conclusion, edible coating treatment with alginate 2.5% and betel essential oil 0.1 % maintained the quality of rose apple cv. dalhari by reducing weight loss, fruit firmness, microbial growth and appearance. in a study of microbial growth inhibition using paper disc test, betel essential oil has shown to inhibit both yeast and bacteria. however, in this experiment, a combination of alginate-betel essential oil has not shown the different effect to microbial growth on rose apple cv. dalhari. the main limitation of this study is the lack of information in the 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(in indonesian). zhang y., ma q., critzer f., davidson p.m., zhong q., 2015 effect of alginate coatings with cinnamon bark oil and soybean oil on quality and microbiological safety of cantaloupe. int. j. food microbiol., 215: 25-30. impaginato 263 adv. hort. sci., 2019 33(2): 263-270 doi: 10.13128/ahs-24219 effects of crop system and genotype on yield, quality, antioxidants and chemical composition of organically grown leek n.a. golubkina 1 (*), t.m. seredin 1, m.s. antoshkina 1, h.v. baranova 1, v. stoleru 2, g.c. teliban 2, g. caruso 3 1 agrochemical research center, federal scientific center of vegetable production, 143072 moscow region, odintsovo district, vniissok, selectsionnaya 14, russia. 2 department of horticulture technology, university of agriculture sciences and veterinary medicine, 3m sadoveanu, 700490 iasi, romania. 3 dipartimento di agraria, università degli studi di napoli federico ii, 80055 portici, napoli, italy. key words: allium porrum l., ascorbic acid, greenhouse, mineral elements, polyphenols, sugars. abstract: the research was carried out in order to assess the effects of nine cultivars in factorial combination with open field or greenhouse growing on yield, quality indicators, antioxidants and elemental composition of leek in moscow region. greenhouse management resulted in higher yield compared to open field cultivation, due to higher mean pseudo-stem weight, and cultivar giraffe gave the highest production. pseudo-stem dry matter was better affected by greenhouse cultivation, whereas the content of monosaccharides, total sugars, nitrates, ascorbic acid and polyphenols was enhanced by open field growing. the cultivars vesta and summer breeze showed the highest dry matter and total sugar content, whereas goliath had the highest antioxidant, selenium and potassium concentration. among the mineral elements, k and mg in pseudostems were better affected by greenhouse conditions, whereas ca attained a higher concentration under open field growing. the antioxidant system of allium porrum was characterized by significant positive correlations between se, polyphenols, ascorbic acid and potassium. 1. introduction leek (allium porrum l.) is a major crop among allium species and it is mainly grown in indonesia, turkey and, within europe, in france and belgium for producing edible pseudo-stems. the latter have high nutritional value, also due to the high content of potassium and iron (koca and tasci, 2016), and high biological activities connected with the remarkable concentration of antioxidants comparable with that of allium cepa (*) corresponding author: segolubkina45@gmail.com citation: golubkina n.a., seredin t.m., antoshkina m.s., baranova h.v., stoleru v., teliban g.c., caruso g., 2019 effects of crop system and genotype on yield, quality, antioxidants and chemical composition of organically grown leek. adv. hort. sci., 33(2): 263-270 copyright: © 2019 golubkina n.a., seredin t.m., antoshkina m.s., baranova h.v., stoleru v., teliban g.c., caruso g. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 24 september 2018 accepted for publication 25 february 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(2): 263-270 264 (sekara et al., 2017), such as polyphenols (ben arfa et al., 2015), glucosinolates, s-alkenyl-l-cysteine sulfoxides and pectic polysaccharides (ozgur et al., 2011). accordingly, leek shows antimicrobial, cardio-protect i v e , h y p o c h o l e s t e r e m i c , h y p o g l y c e m i c , a n t i rheumatic, hypotensive, antianemia, and anticancer action, improves liver, gastro-intestinal and brain e f f i c i e n c y , d e c r e a s e s b l o o d p r e s s u r e , i n h i b i t s platelets aggregation and prevents neural tube defects as well as prostate diseases (radovanović et al., 2015). protected cultivation may be appropriate to organic horticulture which is more susceptible to the environmental unbalances due to the milder farming practices and is usually more profitable than the conventional management (caruso et al., 2012; conti et al., 2015). within the crop system, cultivar assessment in terms of content of antioxidants as well as macroand micro-elements in leek pseudo-stems raises the interest of establishing the relations between the mentioned substances and, accordingly, identifying the most interesting genotypes, also based on their yield. due to the fragmented investigations relevant to varietal differences in biologically active compounds (bernaert et al., 2012) and elemental composition (koca and tasci, 2016), we carried out research aiming to evaluate the effect of both crop system and cultivar on yield, quality, antioxidant content and elemental composition of a. porrum grown either in greenhouse or in open field. 2. materials and methods plant material and growth conditions research was carried out on leek (a. porrum l.) grown in greenhouse at the experimental fields of federal scientific center of vegetable production, in odintsovo (moscow, russia, 55°40’ n, 37°12’ e) in 2015 and 2016 on a clay-loam soil, with рн 6.8, 2.1% organic matter, 108 mg kg-1 n, 450 mg kg-1 p2o5, 357 mg kg-1 k2o, exchangeable bases sum as much as 95.2%. mean temperature values from may to october were: 13.0, 16.1, 19.8, 18.6, 12.3, 6.4°c in open field; 20.4, 21.4, 23.7, 20.0, 14.5, 8.3 in greenhouse. the experimental protocol was based on the factorial combination between two crop systems (open field, greenhouse) and nine cultivars (goliath, summer breeze, premier, casimir, kalambus, camus, vesta, giraffe, bandit), using a split-plot design with three replicates. the sowing was performed on 5 december in 8 x 8 cm trays and the plantlets were transplanted in the field on 14 may, spaced 15 cm along the rows, the latter being 40 cm apart. leek crops were preceded by organically grown vegetables in the previous four years, such as carrot, bean, rape and pea. prior to planting, plough at 30 cm depth, hoeing at 15 cm and fertilization with 180 kg ha-1 n, 80 p2o5 and 120 k2o were practiced; during the growing period, 40 kg ha-1 n were supplied in three times at two-week intervals, starting at bulbification stage, and just in the last n application 7 kg ha-1 of p2o5 and of k2o were also provided. drip irrigation was activated at 80% soil available water. the organic farming practice complied with ec regulation 834/2007 and 889/2008. plant protection was achieved by applying copper oxychloride against rust and azadirachtin against aphids. harvests of ripe plants were performed from 5 to 10 october in greenhouse and from 12 to 19 october in open field, when the pseudo-stems had reached their maximum growth, and the leaf blades were trimmed at 15 cm length for obtaining the marketable product. in each plot, determinations were made of the marketable product weight (pseudostems with 15 cm long leaf blades) and the mean pseudo-stem (with 15 cm long leaf blades) weight on twenty-plant samples. further plant samples were collected, gently washed with water to remove surface contaminants and dried with filter paper. pseudo-stems and leaves were separated, cut with plastic knife, dried to constant weight and homogenized; the resulting powders were subjected to laboratory analysis. dry matter the dry matter content in leaves and pseudostems of a. porrum was assessed after dehydration of the fresh samples in an oven at 70°c, until they reached constant weight. sugars monosaccharides were determined using ferricyanide colorimetric method, based on the reaction of monosaccharides with potassium ferricyanide (swamy, 2008). total sugars were determined after a c i d i c h y d r o l y s i s o f w a t e r e x t r a c t s w i t h 2 0 % hydrochloric acid (swamy, 2008). fructose was used as an external standard. polyphenols the concentrations of the total polyphenols in each sample of leaves and pseudo-stems were determined in 70% ethanol extract (1 hour at 80oc) using the folin-ciocalteu colorimetric method, according to golubkina et al. crop system and genotype effects in allium porrum 265 golubkina et al. (2018 b) by unico 2804 uv (usa) spectrophotometer. the polyphenol content was expressed as milligrams of gallic acid equivalents per 100 grams of dry weight (mg gae 100 g-1 d.w.). ascorbic acid the ascorbic acid content in leek leaves and pseudo-stems was assessed by visual titration of fresh plant extracts in 6% trichloracetic acid with tillmans reagent (caruso et al., 2009; aoac, 2012). antioxidant activity the antioxidant activity of leek leaves and pseudo-stems was assessed using redox titration method (maximova et al., 2001; golubkina et al., 2018 b), via titration of 0.01 n kmno 4 solution with ethanolic extracts of leaves and pseudo-stems. the values were expressed in mg gae 100 g-1 d.w. nitrates the nitrate content was assessed in fresh pseudostems using ion selective electrode on ionomer expert-001 (econix, russia). elemental composition the content of al, as, b, ca, cd, co, cr, cu, fe, i, k, li, mg, mn, na, ni, p, pb, se, si, sr, v and zn in leek pseudo-stems was assessed using icp-ms on quadruple mass-spectrometer nexion 300d (perkin elmer inc., shelton, ct 06484, usa) in the biotic medicine center in moscow (golubkina et al., 2017). statistical analysis data were processed by analysis of variance and mean separations were performed through the duncan multiple range test, with reference to 0.05 probability level, using spss software version 21. the data expressed as a percentage were subjected to angular transformation before processing. as the year of research had no significant effect on the yield, quality, antioxidant and elemental composition variables examined, both as main factor or in interaction with the experimental factors “crop system” and “cultivar”, the results are reported as average values of the two years of investigation. 3. results and discussion growth, yield and quality indicators of pseudo-stems the crop system showed significant effects on leek plant biomass, pseudo-stem yield and mean weight, as these variables attained higher values in greenhouse compared to open field (table 1); as reported in the previous section, the plant commercial ripeness was anticipated by 8 days on average in the protected environment. these trends are consistent with those recorded in previous research (conti et al., 2015). differences between the varieties were recorded with regard to: biomass, which was highest in cultivar summer breeze and lowest in premier; mean pseudo-stem weight and, accordingly, yield which ranged from 22.0 to 36.1 mg ha-1, with the cultivar giraffe showing the best performance, premier and kalambus the worst. the greenhouse growing resulted in higher concentration of dry matter, ash and nitrates, but lower monosaccharides and total sugars in pseudo-stems, compared to those detected in open field (table 1), similarly to previous reports (conti et al., 2015). notably, the values of dry matter recorded in our table 1 growth and yield indicators, and content of dry matter, sugars and nitrates in a. porrum pseudo-stems * significant at p≤0.05. within each column, means followed by different letters are significantly different according to duncan test at p≤0.05. treatment plant biomass (kg m-2 d.w.) marketable pseudo-stems yield (mg ha-1) mean weight (g) dry matter (%) monosaccharides (g 100 g-1 d.w.) total sugars (g 100 g-1 d.w.) ash (%) nitrates (mg kg-1 f.w.) crop system open field 11.0 25.3 162.8 16.7 3.81 12.0 4.8 44.7 greenhouse 15.8 30.8 185.7 19.7 3.41 10.7 5.2 66.3 * * * * * * * * cultivar goliath 8.7 ef 27.6 de 177.0 de 12.1 e 5.11 a 7.5 e 8.5 b 48.0 d premier 8.3 f 22.0 g 132.6 g 14.6 d 4.62 ab 10.6 c 12.4 a 45.9 d bandit 12.0 d 31.0 bc 192.8 bc 14.9 d 3.73 c 8.9 d 4.7 c 65.3 a kalambus 9.9 e 22.3 g 134.6 g 17.1 c 4.13 bc 10.5 c 2.8 e 43.1 d cazimir 11.6 d 24.3 f 150.1 f 18.4 c 2.98 d 11.0 bc 4.1 cd 57.5 bc giraffe 18.6 b 36.1 a 225.4 a 19.8 b 3.58 c 11.2 bc 3.1 e 54.7 c camus 16.0 c 29.9 cd 188.0 cd 20.6 b 2.62 d 12.3 b 3.2 e 64.9 a vesta 15.4 c 26.3 ef 163.8 ef 22.6 a 2.74 d 14.6 a 2.9 e 60.1 ab summer breeze 20.3 a 33.1 b 204.5 b 23.7 a 2.97 d 15.5 a 3.4 de 60.5 ab adv. hort. sci., 2019 33(2): 263-270 266 research fell within the 12.1 to 23.7 % range (table 1) which is much wider than that relevant to a. porrum grown in czech republic (9-11%) (lundegardh et al., 2008). moreover, the cultivars with high dry matter content (summer breeze and vesta) have a long shelf-life and are even suitable as dry spice source, w h er ea s t h e v a r i et i es s h o w i n g l o w d r y m a t t er (goliath, premier and bandit) better fit the salad industry target. significant varietal differences in ash content were recorded (table 2), with the ratio between leaf and pseudo-stem related to this variable decreasing as follows: summer breeze > cazimir > vesta > giraffe > bandit > kalambus > camus > premier > goliath. the higher nitrate accumulation in pseudo-stems grown in greenhouse is connected with the lower light intensity occurring in the protected environment compared to open field conditions, which limits the nitrate reductase activity; however, it was much lower (105 mg kg-1 f.w.) than that relevant to the topa c c u m u l a t o r s p e c i e s ( c a r u s o e t a l . , 2 0 1 1 ) a n d referred to previous reports (santamaria, 2006). antioxidants ascorbic acid and polyphenols highly affect plant antioxidant activity (proteggente et al., 2002); in our research, the open field conditions resulted in higher content of both antioxidant compounds in leek pseudo-stems compared to greenhouse (table 2). the crop system did not affect the selenium concentration either in pseudo-stems or leaves of a. porrum. the high average content of ascorbic acid recorded in our research presumably makes the product safe and healthy, as ascorbic acid participates in producing essential nitrogen oxide for human organism, thus preventing nitrosamine formation from nitrate a c c u m u l a t i n g i n p l a n t s ( s a n t a m a r i a , 2 0 0 6 ) . moreover, wide varietal differences were found in ascorbic acid concentration, unlike the polyphenol content which was characterized by higher stability in pseudo-stems and even more in leaves (table 2). among the cultivars examined, goliath showed the highest content of ascorbic acid, polyphenols and selenium in pseudo-stems (table 2). notably, these cultivars from domestic selection are characterized by lower levels of polyphenols compared to literature references, which may be connected with the different crop cycle and harvest time (biesiada et al., 2007). bernaert et al. (2012) also reported a higher polyphenol concentration in pseudo-stems of thirty leek cultivars grown in belgium (7.3 to 11.3 mg ga g-1 d.w.) compared to our values (3.3 to 6.3), but a lower content of ascorbic acid ranging between 90 to 350 mg 100 g-1 d.w. in the latter research no correlation was recorded between ascorbic acid and polyphenol concentration in leek pseudo-stems, whereas a significant positive relationship has been found in our investigation (r = 0.94 at p<0.01). the lack of correlation relevant to the thirty leek cultivars grown in belgium (bernaert et al., 2012) presumably depends on varietal heterons not significant; * significant at p≤0.05. within each column, means followed by different letters are significantly different according to duncan test at p≤0.05. table 2 concentrations of ascorbic acid, polyphenols and selenium in leek treatment ascorbic acid in pseudo-stems (mg 100 g-1 f.w.) polyphenols (mg ga 100 g-1 d.w.) selenium (µg kg-1 d.w.) pseudo-stems leaves pseudo-stems leaves crop system open field 57.6 466.5 887.1 71.9 61.2 greenhouse 47.2 376.8 699.5 76.2 64.4 * * * ns ns cultivar goliath 169.3 a 626.1 a 827.8 a 106.4a 14.1 e premier 72 b 497.6 b 751.8 b 79.1 b 64.8 c bandit 50.6 c 455.6 bc 739.8 b 74.1 bc 47.2 d kalambus 32.1 de 368.6 de 858.5 а 70.6 bc 74.8 b cazimir 37 d 334.1 e 843.1 а 59.7 e 76.9 ab giraffe 31.3 ef 386.1 d 760.8 аb 72.9 bc 48.4 d camus 27.1 fg 401.6 cd 786.3 ab 63.1 de 81.4 ab vesta 24.3 g 345.3 de 855.5 a 68.1 cd 85.4 a summer breeze 28.0 fg 379.7 de 716.3 b 72.1 bc 72.3 bc golubkina et al. crop system and genotype effects in allium porrum 267 tion between selenium and polyphenol content was found in wheat (lachman et al., 2011) and a negative correlation between quercetin and selenium was recorded in a. cepa (golubkina et al., 2016). however, moderate doses of selenium are deemed to enhance the content of antioxidants such as polyphenols, flavonoids and carotenoids (malagoli et al., 2015). in our research, the nine leek genotypes examined showed significant correlations between the components of the antioxidant system, i.e. selenium, ascorbic acid and polyphenols: se and ascorbic acid (r= 0.93 at p≤0.01); se and polyphenols (r= 0.92 at p≤0.01); ascorbic acid and polyphenols (r = 0.94 at p≤0.01). the latter correlations relevant to leek pseudo-stems may be significantly useful in leek selection based on high antioxidant content. elemental composition the beneficial effect of many mineral elements to human health has arisen a remarkable interest to the chemical composition of vegetable crops, such as leek (koca and tasci, 2016). investigations of element content in a. porrum plants have disclosed this species ability to accumulate high concentrations of minerals, but so far assessments of the leek whole profile relevant to mineral elements and to the varietal features connected to their accumulation have been lacking. the analysis of twenty-five element content in leek pseudo-stems (tables 3-5) has allowed to assess the varietal differences in elemental profile. the concentration of calcium was higher in pseudo-stems grown in greenhouse, whereas potassium and magnesium attained higher levels in open field. sodium, geneity, consequent to genotype selection based on morphological types (light-green summer type, darkgreen winter type and intermediate autumn type). interestingly, in our research the concentration of polyphenols in leek leaves has always been higher than the pseudo-stem one, raising the issue of possible crop waste valorization as a source of these antioxidants. among the components of plant antioxidant system, selenium also plays a significant role. indeed, though it is not an essential element for plants, selenium is able to provide a powerful antioxidant defense to plants against drought, salinity, frost, flooding, uv light and herbivore (malagoli et al., 2015). notably, allium species belong to the seco n d a r y s e l e n i u m a c c u m u l a t o r s , w h i c h s h o w a remarkable tolerance to high concentration and consequent accumulation of this element due to se ability to substitute sulfur in natural compounds, as previously reported in leek (koca and tasci, 2016). in our research, a. porrum grown in moscow region showed a se accumulation range from 60 to 107 µg kg1 d.w., which is much lower than the values recorded in turkey (koca and tasci, 2016). this suggests the significant effect of selenium status in the environment on plant ability to concentrate this microelement. the adverse correlation between selenium content in leaves and pseudo-stems (r= 0.95 at p≤0.01), similar to that recorded for polyphenols, entails a rather stable level of selenium accumulation in plant. reports relevant to selenium in plant secondary metabolites, as well as to polyphenols particularly in absence of selenium uptake are rather scarce and often controversial. in this respect, a positive correlatable 3 macroelement concentration in a. porrum pseudo-stems (g kg-1 d.w.) ns not significant; * significant at p≤0.05. within each column, means followed by different letters are significantly different according to duncan test at p≤0.05. treatment calcium potassium magnesium sodium phosphorus crop system open field 4.2 22.5 1.0 0.27 2.83 greenhouse 4.8 18.9 0.8 0.28 2.73 * * * ns ns cultivar goliath 3.7 bc 58.7 a 0.8 c 0.32 bc 3.12 b premier 3.1 cd 5.4 e 0.8 c 0.36 b 2.74 bc bandit 10.4 a 26.3 b 2.1 a 0.75 a 2.53 cd kalambus 4.0 b 15.2 c 0.6 d 0.16 e 2.43 cd cazimir 4.3 b 9.0 d 0.6 d 0.15 e 2.08 d giraffe 2.6 d 17.9 c 0.7 cd 0.12 e 2.49 cd camus 4.4 b 19.6 c 1.1 b 0.16 e 4.07 a vesta 3.9 bc 16.9 c 0.7 cd 0.18 de 2.77 bc summer breeze 4.4 b 17.7 c 0.8 c 0.26 cd 2.80 bc 268 adv. hort. sci., 2019 33(2): 263-270 phosphorus and all the microelements and heavy metals analyzed were not significantly affected by the crop system (tables 3-5). moreover, the potassium concentration was positively correlated with the ash content (r= 0.78 at p≤0.01). from the comparison between the nine leek cultivars it arose that the three cultivars premier, goliath and cazimir had contrasting features from each other. indeed, goliath was characterized by the highest content of k, fe, b, zn and se, and the lowest of cd. premier accumulated preferably со, i, al, as, cd, ni, pb and sr, but poorly cu and zn. cazimir showed the highest concentration of na and zn, but the lowest of k, i, se, cr and ni. among the minerals examined, the highest correlation coefficients were recorded between al and as, pb, v, со and li. indeed, the physiological role of al in plants has not been completely understood so far, though this element is supposed to both activate at low doses some enzymes and control membrane permeability (ahn and matsumoto, 2006). lithium also showed wide varietal differences, consistently with previous reports (kabata-pendias and pendias, 2010). the correlations recorded in our research are in agreement with those found in five species grown both in ecological unpolluted and in oil-polluted areas of nigeria (essiett et al., 2010). as for selenium, though the leek varietal differences are rather low compared to other elements, the significant correlation recorded between se and k is a remarkable characteristic of this allium species and it has been very scarcely investigated so far. in spinach, the fertilization with sodium selenate increased the potassium content in the female plants but not in the male ones (golubkina et al., 2017), whereas in other research (põldma et al., 2011) garlic biofortification led to selenium antagonistic activity towards k. taking into account that potassium participates in plant protection against all forms of biotic and abiotic stress along with selenium and other antioxidant compounds (wang et al., 2013), the close relationship between the two minerals in leek suggests intensive interactions between all components of the defense system. indeed, potassium was predominant in leek elemental composition, showing significant correlations with both ash (r= 0.78 at p≤0.01) and polyphenols (r= 0.96 at p≤0.01). the known ability of potassium to decrease the activity of polyphenol oxidase in plants and enhance polyphenol accumulation (mudau et al., 2007) may be a good explanation of the positive correlation between polyphenols and potassium in leek plants. the active table 4 microelements concentration in a. porrum pseudo-stems (mg kg-1 d.w.) table 5 heavy metal concentration in a. porrum pseudo-stems (mg kg-1 d.w.) within each row, means followed by different letters are significantly different according to duncan test at p≤0.05. within each row, means followed by different letters are significantly different according to duncan test at p≤0.05. element goliath cazimir premier vesta kalambus summer breeze bandit giraffe camus b 20.5 a 14.8 bc 16.3 b 9.5 de 8.5 e 9.4 de 9.4 de 12.3 cd 10.7 d co 0.08 b 0.05 d 0.10 b 0.03 d 0.29 a 0.04 d 0.04 d 0.04 d 0.10 b cu 4.7 df 4.4 ef 3.4 g 5.7 bc 6.4 ab 5.0 ce 6.8 a 4.0 fg 5.3 cd fe 206 a 110 c 168 b 97 bd 74 e 80 de 93 ce 100 cd 215 a i 0.06 bc 0.04 c 0.31 a 0.04 c 0.06 bc 0.06 bc 0.04 c 0.08 b 0.08 b li 0.11 b 0.04 c 0.15 a 0.03 c 0.01 c 0.03 c 0.02 c 0.03 c 0.11 b mn 12.2 c 12.0 c 22.6 a 9.8 c 6.3 d 9.5 c 10.6 c 19.2 b 21.7 ab si 14.1c 10.5 d 27.3 a 9.2 e 12.8 cd 13.1 cd 11.0 de 18.8 b 15.4 c sn 0.15 c 0.22 b 0.02 d 0.16 c 0.48 a 0.18 bc 0.51 a 0.22 b 0.23 b zn 23.5 ab 26.7 a 11.8 f 18.1 de 16.0 e 19.2 ce 21.5 bc 22.2 bc 21.2 bd element goliath cazimir premier vesta kalambus summer breeze bandit giraffe camus al 78.0 c 29.5 d 126.0 a 21.0 df 7.7 g 24.1 de 12.2 fg 19.3 ef 88.4 b as 0.03 b 0.02 bc 0.05 a 0.02 bc 0.01 c 0.02 bc 0.01 c 0.02 bc 0.06 a cd 0.08 d 0.10 bc 0.18 a 0.11 b 0.08 d 0.06 d 0.11 b 0.17 a 0.11 b cr 0.13 c 0.08 g 0.48 a 0.10 df 0.10 eg 0.12 cd 0.09 fg 0.15 b 0.11 ce ni 1.05 a 0.46 c 0.96 ab 0.98 ab 0.56 c 0.57 c 0.83 b 0.60 c 1.08 a pb 0.34 b 0.28 bc 0.83 a 0.10 e 0.10 e 0.13 de 0.20 cd 0.12 e 0.83 a sr 28.3 ab 25.3 c 30.4 a 24.6 c 28.2 ab 17.3 d 28.8 ab 26.4 bc 28.5 ab v 0.21 b 0.07 cd 0.28 a 0.07 cd 0.04 d 0.09 c 0.07 cd 0.07 cd 0.30 a golubkina et al. crop system and genotype effects in allium porrum 269 participation of potassium in the antioxidant defense system of this allium species is also characterized by positive correlation of the element with the ascorbic acid content (r= 0.95 at p≤0.01). in this respect, the results of the present work reveal the close relationship between the main components of the leek antioxidant system, including polyphenols, ascorbic acid, selenium and potassium. in our research, the lowest negative correlation coefficients were recorded between selenium, chromium and iodine (table 6). se is known as an antagonist of cr and its protective role towards cr has been previously reported (qing et al., 2015). the interaction between se and i is more complex; both the elements are not essential for plants, but at low concentrations they may improve plant growth, development and protection from biotic and abiotic stresses (pilon-smits, 2015). separate plant fortification with se and i showed the possibility of mutual stimulation by the two elements (golubkina et al., 2018 a). the selective accumulation of selenium in the spinach male plants and of iodine in the female ones suggests the participation of phytohormones in the interactions between selenium and iodine (golubkina et al., 2017). with regard to heavy metals, highly significant correlations were found between v and al, as, со, pb and fe (table 6). 4. conclusions from research carried out in moscow region with the aim to assess the effects of open field or greenhouse conditions on yield and quality performances of nine leek (a. porrum) cultivars under organic farming, useful remarks have been drawn. the genotypes examined had a uniform behavior with both the crop systems, showing higher yield and dry matter when grown in the protected environment, but better quality 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(2014) reported that treating cultivars of gerbera stems by sucrose in combination with an antimicrobial compound (hqc, chlorine) resulted in less bending than the same concentration of the antimicrobial compounds alone. perik et al. (2014) noted that other factors might also be involved in bending and showed that a mixture of chemicals delayed the time to bending in six tested cultivars of gerbera. sodium nitroprusside (snp), the inorganic nitrous compound (nitroferricyanide) with the formula na2[fe(cn)5no].2h2o, is an important signaling molecule. this molecule has diverse physiological functions for plants such as inducing tolerance to adverse environmental factors (shi et al., 2016; kumar rai et al., 2018). application of snp in vase solution resulted in extending the vase life of gerbera cut flowers (shabanian et al., 2018); however, there is very limited information regarding the positive effects of exogenously applied snp in extending the vase life. ortho-hydroxybenzoic acid or salicylic acid is an endogenous plant growth regulator. exogenous application of salicylic acid (sa) can affect the antioxidant capacity of plant cells and prolong vase life of cut flowers, such as rose (alaey et al., 2011) and anthurium (promyou et al., 2012). however, little work has been reported on the role of sa on cut flower vase-life improvement and physio-chemical attributes related to senescence. methyl jasmonate (mj) has been found as a naturally occurring substance in higher plants. in this context, the application of mj induced antioxidant system activity can suppress fungal infection and enhance stress resistance (kanani and nazarideljou, 2017). to our knowledge, mj effects on specific physiological and biochemical processes in gerbera cut flower have not been studied yet. as a derivative of 8-hydroxyquinolines, 8-hydroxyquinoline sulfate is widely used as antibacterial since the beginning of the 1950s. this compound is a c t i v e a g a i n s t g r a m n e g a t i v e b a c t e r i a o f t h e enterobacteriaceae family, fungi of the candida genus, and mycoplasma (chupakhina et al., 2012). in previous studies, 8-hydroxyquinoline sulfate (8-hqs) remarkably increased vase life of rose cut flowers (ichimura et al., 1999). in the present study, the role of three plant growth regulators (i.e., snp, mj, and sa) in regulating the activities of the antioxidant enzyme, relative water content (rwc), water uptake, the contents of malondialdehyde (mda), protein, pigments and soluble sugar in the petals, and time of stem bending of gerbera cut flowers were investigated. the objective of the present study is to provide a theoretical basis for the application and optimization dosage of plant growth regulators in combination with an antimicrobial compound, i.e., 8-hqs, in improving the vase life of gerbera cut flowers during the vase-holding period. 2. materials and methods plant material and treatment ‘sun spot’ cut-gerberas (gerbera jamesonii), harvested at normal harvest maturity, were obtained from a commercial grower (dezfol, khuzestan, iran). the length of the stem varied between 65 and 70 cm. the harvested flowers were packed into parchment paper and transported to the laboratory within 1-2 h. then, stems were re-cut to a uniform length of 55, under distilled water to avoid air embolism. each sixflower sample was placed randomly in 250 ml of various concentrations (50, 100, and 200 µm) of holdsolutions containing 8-hqs, sa, mj, and snp. hold solutions were used alone or in combination with 100 µm 8-hqs for 24 h. distilled water was used as control and sucrose (4%, w/v) was added to all solutions. to maintain the proper concentrations of holdsolutions, the mouths of the vases were covered with plastic wrap (around the stem) to minimize evaporation and to prevent contamination. then, flowers were individually sited in glass bottles of 25 cm height, approximately 150 ml of distilled water in each bottle, under laboratory condition. the laboratory was maintained at 22°c, 60±5% relative humidity, and 16 mmol m-2 s-1 photons irradiance using cool fluorescent lamps for a 12 h photoperiod (07:0019:00 h). measurements time of stem bending was determined as described by perik et al. (2012). relative fresh weight (rfw) was calculated by the hemati et al. plant growth regulators as hold solutions of cut-flower gerbera 189 following formula: rfw (%) = [(fw t=10 -fw t=0 )/ fw t=0 ] ×100 where fwt=10 is the fresh weight of flower (g) at 10th day and fwt=0 is the fresh weight of the same flower (g) at first day (he et al., 2006). water uptake (ml) was calculated by subtracting in the weight of the remaining water at the end of the experiment from the initial weight. soluble carbohydrate content in petals was measured by the anthrone colorimetric method according to the method of xue (1985). total anthocyanin content of petal was measured by the ph differential method of yang et al. (2009). total carotenoid content of gerbera petal tissue was estimated using the method of wellburn (1994). flavonoid petal tissue was measured according to the method of markham (1982). protein content in petal was estimated by the method of bradford (1976) using bovine serum albumin as the protein standard. malondialdehyde (mda) content in petals was measured by the thiobarbituric acid reaction following the procedure of hodges et al. (1999). peroxidase (pox) activity was evaluated by oxidation of guaiacol, as a substrate, according to chance and maehly (1955). statistical analysis data were analyzed using analysis of variance (anova) in sas software. means were compared by one-way anova and duncan’s multiple range test at the 5% level of significance. 3. results and discussion vase life as shown in figure 1, hold-solutions affected senescence of gerbera jamesonii in a dose-dependent manner. compared with control, different concentrations of sa, with or without 8-hqs, all prolonged the senescence of gerbera as shown by the tighter stem and more showy flowers. compared with other concentrations of sa, 50 µm sa+ 100 µm 8-hqs markedly prolonged the longevity of the cut flowers. as shown in figure 1, different concentrations of snp, with or without 8-hqs, all prolonged the length of vase life of gerbera cut flower, compared with control. compared with control, 50 µm, 100 µm, and 200 µm snp noticeably increased the length of vase life of gerbera jamesonii cut flower by 65%, 78%, and 43%, respectively. however, there was no significant difference in the stem bending of flowers among different concentrations of snp. among various concentrations of 8-hqs, stem bending of cut flowers treated by 100 μm 8-hqs was significantly lower than those treated by other concentrations. compared with control, 50, 100, and 200 μm 8-hqs increased the vase life by 69%, 100%, and 78%, respectively. different concentrations of mj all significantly increased vase life, compared with the control (fig. 1). however, mj treatments were less effective on vase life compared with 8-hqs, sa, and snp treatments. shabanian et al. (2018) showed that snp extended the vase life of gerbera cut flowers as compared with their respective control treated with water alone. in agreement with this finding, treatments with snp extended the vase life of other cut flowers, e.g., carnation (zeng et al., 2011), chrysanthemums (mansouri, 2012), gladiolus (dwivedi et al., 2016), and rose (liao et al., 2013). the mechanism of sa action, as a hold solution, in vase life of cut flowers has not been clarified; however, published data suggest some association with ethylene production. zhang et al. (2003) showed that application of sa resulted in suppression acc synthase and acc oxidase activities and biosynthesis of ethylene in kiwifruit. in gladiolus, the maximum vase-life was obtained once flowers treated with a solution containing 100-ppm 5-sulfosalicylic acid + 4% sucrose (ezhilmathi et al., 2007). 8-hqs is a subclass of quinolones with a wide variety of biological effects. the 8-hydroxyquinoline derivatives emerged as a hold-solutions being widely explored for several biological functions such as antifungal effects (oliveri and vecchio, 2016) and antimicrobial (abouelhassan et al., 2017). according to van doorn (1997), the bending of gerbera cut flowers was caused by low turgescence of the flower scape when facing water uptake problems. in addition, he notified that bactefig. 1 effect of the different preservative solutions (µm) on time of stem bending in gerbera cut flowers. columns followed by different letters are significantly different at p=0.05. adv. hort. sci., 2019 33(2): 187-195 190 ria in the vase water were the most common cause o f x y l e m b l o c k a g e a f f e c t i n g w a t e r u p t a k e . accordingly, antimicrobial compounds such as 8hydroxyquinoline citrate (elhindi, 2012) and essential oils (salehi salmi et al., 2018) were applied to improve vase-life of cut flowers. the effect of the application of mj on vase life of cut flowers varies widely among species and cultivars. these reports indicated that the ethylene production rate might change with the kind of genes, which were stimulated by mj (salimi et al., 2016). the results of the present study indicated that the decline in the vase life was significantly less in cut flowers in mj-treated, compared with other treatments. water loss and water uptake comparing the results of the different hold-solutions revealed statistically significant differences such that the maximum and minimum amounts of water loss, in the 10th day, occurred on cut gerbera hold in 100 µm 8-hqs+ 200 µm mj and 100 µm 8-hqs+ 100 µm snp, respectively (fig. 2). from figure 2 the maximum water loss occurred also on cut gerbera hold in mj 200 µm alone (together with 100 µm 8-hqs+ 200 µm mj). also, data showed that cut flowers treated with 50 and 100 µm 8-hqs; 200 µm sa; 50 and 100 µm snp; 50, 100, and 200 sa+ 100 µm 8-hqs; 50 µm mj+ 100 µm 8-hqs; 50, 100, and 200 µm snp + 100 µm 8-hqs hold solutions lost lower water the control (fig. 2). the lowest amount of water uptake appeared on 10th day in the cut flowers treated with 100 µm 8hqs+ 200 µm mj (fig. 3). however, there was no significant difference between this treatment and 50 µm mj, 200 µm mj, and 200 µm snp control treatments. in other treatments, water uptake was increased over 10 days of postharvest life in comparison with control. however, water uptake amounts showed significant differences among hold-solutions such that the maximum amount of it was observed in a cut flower treated with 100 µm 8-hqs+ 100 µm snp (fig. 3). cut flower senescence is closely associated with water uptake stem and rwc of petals, whereas, these characteristics are closely related with the contents of osmoregulation substances such as soluble sugars and soluble proteins (hou et al., 2018). soluble carbohydrates of petal changes of sugars content of gerbera petals are shown in figure 4. maintenance of elevated total soluble carbohydrates content exhibited by the flowers under hold-solutions treatments can be correlated with the delay in senescence and the increase in vase life of gerbera flowers. the results indicate that treatment with hold-solutions, except the high concentration of mj, with or without 8-hqs, caused a significant decrease in reducing sugars compared with the control. reducing carbohydrate starvation or its symptoms led to unwanted color changes and eventually increased susceptibility to microorganisms. postharvest treatments can reduce carbohyd r a t e s t a r v a t i o n d u r i n g t h e v a s e l i f e p h a s e . postharvest treatments like sugar feeding often havefig. 2 relative water loss of cut flower of gerbera in various hold solutions with different concentrations (µm). the indicator was determined on 10st day. columns followed by different letters are significantly different at p=0.05. fig. 3 effects of different concentrations (µm) of various hold solutions on the water uptake. the indicator was determined during 10 days. columns followed by different letters are significantly different at p=0.05. fig. 4 total soluble carbohydrates in petals of hold-solutionstreated and untreated gerbera cut flowers stored at 22°c for 10 days. hemati et al. plant growth regulators as hold solutions of cut-flower gerbera 191 est amount of carotenoid content among all treatments at the end of the experiment. figure 7 depicts the contents (expressed as mg/g fresh weight) of flavonoid obtained from petals of gerbera cut flowers untreated and treated with hold solution at different concentrations. by comparing untreated-control samples, hold solutions differences could be clearly established (fig. 7). in particular, the concentrations of petal flavonoid were significantly (p<0.05) higher in treated cut flowers with snp, sa or mj; except 50 µm sa, 200 µm mj, 50 µm snp treatments; than control. as can be seen, cut flowers treated with 100 and 200 µm snp+ 100 µm 8-hqs exhibit higher concentrations of flavonoid than the other treatments. despite the results of this study, most investigations on vase life of cut flowers do not present data on the changes in pigmentation and those that use subjective color grades for evaluation. browning and discoloration are important factors in determining display quality of cut flowers and in many cases are the major reason for the termination of vase life (elhindi, 2012; khalaj et al., 2017; salehi salmi et al., 2018). petal coloration is caused by the accumulation of pigment, including carotenoids, flavonoids, and betacyanins, within epidermal cells. anthocyanins are synthesized via the phenylpropanoid and flavonoid pathways (tanaka et al., 2008). carbon metabolite levels, directly and indirectly, affect almost every metabolic process in a plant life. anthocyanin and carotenoid biosynthesis occur concomitantly with sugar accumulation in plant tissue (hara et al., 2003; zhang et al., 2015). similarly, in our study, some vase solutions promoted soluble carbohydrates contents in the petals of gerbera cut flowers (fig. 4), accompanying higher anthocyanin contents and presenting better ornamental quality of petal color (figs. 5, 6, and 7). it is reported that application of mj enhanced a positive effect on vase life in general. it seems that 8-hqs, by preventing vascular blockage, caused sugars directly to reach flowers in the transpiration stream via xylem. increased sugar caused by exogenous sucrose is well known from earlier studies (ichimura et al., 1999; promyou et al., 2012). han et al. (2018) have illustrated that snp treatment inhibited significantly the degradation of sucrose of peach fruit at the end of storage. they suggested sugars were significantly affected by snp treatment probably due to the activities of sucrose metabolism enzymes. yu et al. (2016) found that mj treatment could increase the encoding level and enzyme activity of sucrose phosphate synthase, which resulted in the enhancement of sucrose content. petal pigments the highest concentration of anthocyanin in gerbera florets was in the sa+ 8-hqs treatments, followed by the 8-hqs and sa treatments. the mj, with or without 8-hqs, treatments did not result in a significant increase in anthocyanin concentration compared with that of the control (fig. 5). carotenoid content of petals were increased under treatment with all concentrations of sa with or without 8-hqs, all concentrations of snp with or without 8-hqs, high concentrations of 8-hqs, and all concentrations of mj with 8-hqs (fig. 6) while the control had the lowfig. 5 effects of different concentrations (µm) of various hold solutions on anthocyanin content. the indicator was determined during 10 days. columns followed by different letters are significantly different at p=0.05. fig. 6 effects of different concentrations (µm) of various hold solutions on amount of carotenoid. the indicator was determined during 10 days. columns followed by different letters are significantly different at p=0.05. fig. 7 effects of different concentrations (µm) of various hold solutions on flavonoid. the indicator was determined during 10 days. columns followed by different letters are significantly different at p=0.05. 192 adv. hort. sci., 2019 33(2): 187-195 accumulation of flavonoids in daucus carota (sircar et al., 2012). pox activity the pox activity in the petal of gerbera flowers that were treated with different concentrations of all hold-solutions, except 100 µm mj+ 100 µm 8-hqs, slightly increased during vase life (fig. 8). the highest activity of pox was observed in cut flowers hold in 50 µm 8-hqs, 100 µm sa, and 200 µm 8-snp solutions. the enzymatic antioxidant system can work against the accumulation of reactive oxygen species (ros). regulation of the antioxidant status and ros production by snp in plant cells subjected to either biotic or abiotic stressors is well established (vidal et al., 2018). previously, it has been shown that snp provides protection in broccoli florets against rapid yellowing after harvest (shi et al., 2016). this study was carried out to provide evidence for the ability of snp to regulate flower senescence through regulation of the antioxidant status of gerbera petal cells. salicylic acid can also act as a protector against several stressful impacts, scavenge free oxygen radicals, and counteract oxidative damage by regulating cellular redox balance and accelerating the transformation of superoxide anion and enhancing the activities of antioxidant enzymes (zhang et al., 2003). sa treatment reduced chilling injury in anthurium via improving the activities of sod, cat, and pox (promyou et al., 2012). kumar rai et al. (2018) reported that sa and snp enhanced tolerance to heat stress in lablab purpureus, by elevating antioxidant enzyme activity of pox, sod, and cat and thus alleviating heatinduced oxidative damage. the present study, for the first time, indicated that sa and snp treatment delayed the senescence of gerbera flowers via improving the activity of antioxidant enzymes of pox. mj was reported to stimulate pox activity in banana plants and reduce the level of o2 and h2 o2 (sun et al., 2013). in this regard, fan et al. (2016) reported inducing resistance responses in eggplant fruit by increasing the expression of pox genes. protein there was a significant difference among the type of hold-solution treatments on protein in the 10th day, although the protein content showed no significant differences among concentrations of a holdsolution. protein content for sa treatments, with or without 8-hqs, increased compared with the control; however, it was not significantly different from other treatments to control (fig. 9). the increase observed in the protein content, through treatment with sa, was likely the result of less protein degradation (alaey et al., 2011) or an increase of protein synthesis (ezhilmathi et al., 2007). under flower senescence, the stimulation of protein synthesis leads to protein accumulation that may involve in the enhanced activity of enzymes as a defense mechanism (promyou et al., 2012). to support the accumulation of proteins due to sa treatment, it was reported that sa results in a pronounced increase in total protein content and the formation of new proteins in roses (alaey et al., 2011). it seems that mj by increasing antioxidant defense enzymes leads to maintaining carbohydrate at high levels, as antioxidant inhibits the oxidation of cell biomolecules like proteins and carbohydrates (kanani and nazarideljou, 2017). in the present study, different concentrations of snp and sa, with or without 8-hqs, could markedly increase the contents of soluble sugars and soluble proteins, which increased the rwc of petals and water uptake. these increases were helpful in increasing the water retaining capacity and also played an important role in increasing vase life of gerbera cut flowers. in addition, schouten et al. (2018) suggested that snp enhances flow through xylem vessels by increasing the ionic strength of the vase water. fig. 8 effects of different concentrations (µm) of various hold solutions on activity of pox. the indicator was determined during 10 days. columns followed by different letters are significantly different at p=0.05. fig. 9 petal proteins of gerbera cut flower in various hold solutions with different concentrations (µm). the indicator was determined on day 10. columns followed by different letters are significantly different at p=0.05. hemati et al. plant growth regulators as hold solutions of cut-flower gerbera 193 mda in the present experiment, a noticeable decrease in the mda in control treated cut flowers compared with the other vase-solutions (fig. 10). accumulation of elevated amounts mda in control treated cut flower was recorded. this accumulation indicates the presence of oxidative stress in gerbera petals. zhang et al. (2015) reported over-reduction of the electron transport chain in mitochondria as the main source of o2 production under specific stress conditions. production of h2o2 can occur during lipid catabolism as a side-product of fatty acid oxidation. ross are also involved in the detoxifying reactions catalyzed by cytochromes in both the cytoplasm and the endoplasmic reticulum (kumar rai et al., 2018). obviously, the observed high activity of pox in saand snptreated cut flowers are as a protective mechanism against senescence. it has been revealed that mj mitigates the ros effects in maize seedlings subjected to oxidative stress (ahmadi et al., 2018). 4. conclusions treatment with sa+8-hqs extends the vase life of gerbera cut flowers at relatively low sa concentrations and leads to generating the maximum costeffectiveness. in conclusion, our results demonstrated that 8-hqs improves the vase life through increasing water uptake and consequently increases total soluble carbohydrates. also, 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sciences, university of tennessee, usa. key words: bio-fertilizer, chemical fertilizer, dpph test, inoculation, organic fertilizer, perilla, rosmarinic acid. abstract: medicinal plants are one of the main natural resources of iran from ancient times. perilla is one of the most important medicinal plants of the mint family lamiaceae, since there is no study about adaptability of perilla in iran climate conditions and different fertilizer systems, this experiment was conducted in two experimental sites. the experiment was conducted as split-plot factorial based on a randomized complete block design with three replications at two experimental regions. the main factor was three chemical fertilizer levels (control, 50, 100, 200 kg/ha) and subplots were different kinds of organic fertilizer (control, humic acid, and compost application) and inoculation with piriform osporaindica (inoculation and without). among levels of chemical treatments, 50 and 100 kg/ha lead to a better result. also, humic acid allows to achieve the highest amount of measured traits between different treatments of organic fertilizer. the highest plant yield (147.2 g/m2) and rosmarinic acid yield per area (3.432 g/m2) was achieved in 100 kg/ha at chemical fertilizer with humic acid and biological fertilizer application and the lowest plant yield (89.86 g/m2) and rosmarinic acid yield per area (1.253 g/m2) was observed in control. also, the highest stomatal conductance was obtained with application of compost fertilizer (67.67 mmol h2o m -2s-1). integrated application of the studied fertilizers showed the more positive effect on yield and quality of perilla than individual application of those fertilizers. 1. introduction the perilla is a medicinal plant belonging to the labiatae family and is widely cultivated in southeast asian countries (igarashi and miyazaki, 2013). a number of studies have shown that advantages of perilla are related to the metabolites contained therein (ghimire et al., 2017). to date, very limited information exists regarding the adaptability and management of chemical, organic and bio-fertilizer of perilla in iran. organic matter effects on physicochemical properties and health of soil. it also affects the efficiency of fertilizer application, pesticides and herbicides. one of the most effective organic fertilizers on the growth of (*) corresponding author: mmohamm9@utk.edu citation: ghane m., mohammadi m., pirdashti h., 2019 yield and physiological response of perilla (perilla frutescens) under different soil fertility treatments. adv. hort. sci., 33(2): 205-214 copyright: © 2019 ghane m., mohammadi m., pirdashti h. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 26 august 2018 accepted for publication 18 february 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(2): 205-214 206 plants and improvement of soil status is humic acid. humic acid can be obtained from any material such as organic matter, coal, or well-decomposed compost. this material plays an important role in increasing soil moisture, absorbing micronutrients and contributing to carbon sequestration (spaccini et al., 2002). another organic material that plays an important role in soil fertility is compost and it comes from p l a n t a n d f o o d r e s i d u e s . s o m e e x p e r i m e n t a l researches have shown the important role of compost in crop production (adugna, 2016). compost retains moisture in the soil, slow release nutrients to crops and finally increase the crop yield. application of compost obtained from plant remains leads to increased fertility, soil nutrients and increases water retention in the soil. zemanek (2011) also confirmed that application of 50 t/ha and 100 t/ha compost has a positive effect on soil moisture retention. moreover, biologic fertilizers assists well in mineralization and channelization of nutrients leading to enhanced plant productivity (fischer et al., 2007; ansari et al., 2017). biologic fertilizers adopt various possible ways to accelerate the rate of crop production (rizvi et al., 2015; ansari et al., 2017). they increase and improve plant growth by increasing access to nutrients in the root rhizosphere. these fertilizers provide nutrients through biological processes such as biological nitrogen fixation, phosphorus solubilization, and plant growth stimulation. also, they help to natural nutrient cycle and build soil organic matter (kapoor et al., 2015). use of biologic fertilizers ensures healthy plants growth, while enhancing the sustainability and the vigor of the soil. these biologic fertilizers play a special role in increasing plant nutrition and fertility of soils (vessey, 2003). a number of studies have shown that the chemical composition of secondary metabolites in the perilla plant is influenced by various factors such as soil conditions, temperature, growth season (kiazolu et al., 2016), geographic region (ruberto et al., 2002), and phenological stages (saeb and gholamrezaee, 2012). therefore, this study was carried out to evaluate the yield and physiological traits of perilla cultivars under different fertilizer treatments in two regions. based on the work, attempt was also made to provide amicable solutions to address the challenges of organic farming with the help of perilla cultivation in two different locations in dry region of iran. 2. materials and methods the field experiments were conducted at two locations in esfahan province: 1-mashhad ardehal (latitude 34° north, longitude 51° east, altitude 1800 m above mean sea level) 2sensen, iran (latitude 33°, longitude 51°, altitude 945 m above mean sea level) during 2015. mashhad ardehal is located in warm and dry condition and sensen is located in the mountainous and dry region. the meteorological data recorded during the period of plant cultivation are given in table 1. in order to determine the physical and chemical characteristics of experimental fields, two weeks b e f o r e p l a n t i n g , t h e s o i l s a m p l e s w e r e t a k e n . physicochemical properties of experimental field soil are presented in table 2. this experiment was conducted as split-plot factorial based on a randomized complete block design with three replications at two experimental sites in the year 2015. the main factor was three chemical fertilizer levels (50, 100, 200 kg/ha) plus control, and sub plots were different kinds of organic fertilizer (humic acid, and compost application), plus control; inoculation with piriformospora indica (inoculation and without inoculation) was also evaluated. table 1 weather characteristics in mashhad ardehal and sensen year temperature mean (°c) precipitation mean (mm) humidity mean (%) sensen mashhad ardehal sensen mashhad ardehal sensen mashhad ardehal 2015 19.8 18.4 89.3 90.8 42 49 average of 5 years 20.86 19.88 143.36 133.42 40.6 46.6 parameter location sensen mashhad ardehal total nitrogen (%) 0.09 0.28 phosphorous availability (mg/kg) 15.56 12.18 potassium availability (mg/kg) 245.6 209.7 ph 7.93 7.83 ec (ds.m-1) 2.82 0.89 organic carbon (%) 0.53 1.63 clay (%) 14.3 10.3 silt (%) 33.3 43.4 sandy (%) 52.4 46.3 table 2 soil analysis of the experimental site ghane et al. yield and physiological response of perilla 207 f o l i a r a p p l i e d h u m i c a c i d ( 9 5 % p u r i t y ) w a s obtained from humic strong company (70% w/w, ph 5.17, ec: 4.80 ms/cm) and added to the plots at 4 stages of the plant growth. compost fertilizer was prepared from barij essence company (including 1.5% nitrogen, 1.1% phosphorous, 0.9% potassium, 50% organic matter). compost fertilizer was used as a strip one week after plantation. perilla seeds were purchased from barij essence company, isfahan, iran. first of all, the seeds were germinated in the laboratory and then transplanted to the pots with 10 cm diameter and 15 cm height at t h e 4 l e a f s t a g e . t h e m y c o r r h i z a l l i k e f u n g u s piriformospora indica was prepared in mycology laboratory of sari university, iran and then prepared biologic fertilizer was placed two weeks in incubator with 20-25˚c and 50 rpm under dark condition. before planting of perilla plants in the field, half of the plants in pots were inoculated with biologic fertilizers and then translocated to the field. each plot was 2×2 meter and had 4 furrows with 25 plant per plot. the recommended dose of chemical fertilizer (050-100 and 200 kg/ha) in the form of urea, triple super phosphate and sulfate potassium was applied to grow the crop. nitrogen was applied in three splits, the first along with phosphorus and potassium fertilizer at the time of soil preparation while the second part at the time of transplanting of plants and the third part at the flowering of crops. date of transplanting of both experimental sites was 20 of march. after plant growth, different traits were studied at the suitable stage. a furrow irrigation system was applied for both experimental sites. hand weeding of the experimental area was performed as required. in both experimental sites, after flowering five plants from each plot were harvested from two central rows and then plant height, fresh weight and dry weight of plants were measured. samples dried in an oven at 80°c for 24 hours and the mean of dry weight for each treatment at each replicate was determined. rosmarinic acid determination by hplc the dried seeds of perilla were pulverized (60 mesh) for 3 min using an hr 2860 coffee grinder (philips, drachten, netherlands), and each sample (1.0 g) extracted in 30 ml of 80% methanol for 6 h at room temperature in a shaking incubator. the supernatant was centrifuged at 3000g for 3 min and then filtered through a 0.45 lm syringe filter (whatman inc., maidstone, uk) prior to hplc analysis. for quantification, the peak areas of the isolated compounds were integrated from the hplc chromatogram at 330 nm using the dionex software. the stock solutions were prepared by dissolving in methanol to obtain a 1 mg/ml concentration. calibration curves were obtained with methanol at eight different concentrations (0.5, 1, 2, 5, 10, 25, 50, and 100 g/ml). all calibration curves had coefficients of linear correlation r2>0.998. stomatal conductance twenty days after plantation stomatal conductance was measured in the shade-enclosure with saturated light using promoter (model kr1301, korea tech) stomatal conductance (mmol h2o m-2 s-1) was measured at 12:00-14:00 hours on a clear, cloud-less day in fully expanded, healthy, turgid, three flat and uniform in color and size leaves of each samples (barbieri et al., 2012). dpph radical-scavenging activity this test was used for the determination of the free radical-scavenging activity of the extracts (ebrahimzadeh et al., 2008). dpph test was perf o r m e d f o l l o w i n g t h e m e t h o d p r o p o s e d b y ebrahimzadeh et al. (2008). three young fully developed leaves were selected from each replication. polyphenoloxidase (ppo) activity crude extract was prepared by homogenization of frozen plant sample in buffer medium. leaves of perilla plant which were stored at -20°c was used for the enzyme extraction. 10 g of the sample were cut quickly into thin slices and homogenized in 50 ml of 100 mm sodium phosphate buffer (ph 7.0) containing 1 mm ascorbic acid and 0.5% (w/v) polyvinylpyrrolidone for 5 min at 4°c. the homogenate was filtered through three layers of cheesecloth and then the filtrate was centrifuged at 5,000 x g for 15 min, and the supernatant was collected. enzymatic activity was assayed by determining the rate of increase in absorbance at 420 nm and 25˚c in a perkin-elmer lambda 15 uv/vis spectrophotometer (shimadzu corp., tokyo, japan). the reaction mixture contained 3.0 ml of catechol substrate, the solution freshly prepared in 0.05 m sodium phosphate buffer at ph 6.5 and a fixed quantity of ppo. the reference cuvette contained only the catechol substrate solution. the reaction was conducted at 25°c. the ppo activity was defined as a change of 0.001 in absorbance at the conditions of the assay (pizzocaro et al., 1993). catalase activity assay cat activity was measured by monitoring the h2o2 decomposition at 240 nm in 3 ml of reaction adv. hort. sci., 2019 33(2): 205-214 208 mixture containing 50 mmol/l phosphate buffer (ph 7.0), 15 mmol/l h2o2, 100 ml enzyme extract and 0.1% (v/v) triton x-100 (aebi, 1984). the activity was e x p r e s s e d i n t e r m s o f m m o l h 2o 2 r e d u c e d min/mg/protein. statistical analysis the data were tested for homogeneity and normality of residuals using the bartlett tests and kolmogorov-smirnov, respectively. a combined anova was used to compare treatments for 2 location using proc glm of sas 9.1 software. means were separated by application of lsd test when the f test proved significant at p≤0.05 and 0.01. 3. results and discussion combined statistical analysis of two studied location is presented in table 3. also, the analysis of variance of mashhad ardehal and sensen locations are shown separately in table 4 and 5. in mashhad ardehal location, the maximum plant yield (157.6 g/m2) was achieved in 100 kg/ha chemical fertilizer plus humic acid treatment and the minimum amount (90.68 g/m2) was produced in zero levels of chemical fertilizer and without application of organic treatment (fig. 1, table 3). while in sensen location, the maximum plant yield (126.9 g/m2) was achieved with the application of 50 kg/ha chemical fertilizer plus humic acid (fig. 1). between treatments of chemical × organic × biologic fertilizer interaction, the maximum plant yield (147.2 g/m2) was produced in 100 kg/ha along with humic acid and application of biologic fertilizer (table 6). differences between plant yield (147.2 g/m2 and 89.86 g/m2) showed the importance of fertilizer application in improving yield in this plant. in all of the chemical fertilizer treatments, the plant yield was increased with application of organic and biologic fertilizer, especially with organic fertilizer. so, the integrated fertilizer application was very positive. in the present investigation, application of organic fertilizer especially humic acid significantly increased plant fig. 1 effect of chemical and organic fertilizers on plant yield of perilla in mashhad ardehal and sensen regions. different letters in each column denote a significant difference at p≤0.05 (lsd test). *, ** significant at 5% and 1% probability levels, respectively. table 3 anova of some morphologic, yield and essence traits of perilla under different fertilizer systems in sensen and mashhad ardehal in kashan regions s.o.v. d.f. means squares plant yield rosmarinic acid content rosmarinic acid yield stomatal conductance dpph catalase activity polyphenol oxidase location (l) 1 14012.64 ** 13.80 ** 8.87 ** 248.11 ** 224.60 ** 0.00357 * 0.000935 ** replication (location) 4 56.75 0.70 0.01 4.02 18.56 0.00047 0.000021 chemical fertilizer (c) 3 10188.25 ** 244.44 ** 13.92** 1504.81 ** 99.75 ** 0.03396 ** 0.010834 ** l×c 3 1899.70 ** 27.75 ** 2.53** 184.57 ** 19.74 0.01396 ** 0.000478 ** ea 12 28.48 1.18 0.04 5.74 44.51 0.00063 0.000072 organic fertilizer (o) 2 1435.58 ** 27.93 ** 1.69** 76.66 ** 75.18 * 0.00178 0.000454** biologic fertilizer (b) 1 308.17 ** 8.41 ** 0.53** 213.84** 0.24 0.00003 0.000146 * c × o 6 73.04 4.75 ** 0.06* 8.46 47.80 * 0.00170 * 0.000217 ** c × b 3 130.19 * 1.05 0.06 2.01 2.82 0.00257 ** 0.000022 o × b 2 236.97 ** 1.31 0.09* 2.47 19.55 0.00014 0.000008 l × o 2 71.26 4.79 ** 0.17** 13.49 2.83 0.00253 * 0.000275 ** l × b 1 88.64 4.42 * 0.22** 1.92 22.75 0.00026 0.000003 l × c × o 6 83.62 * 2.89 ** 0.03 10.55 5.98 0.00244 ** 0.000180 ** l × c × b 3 18.09 0.16 0.001 3.44 0.20 0.00178 * 0.000009 l × o × b 2 95.96 0.77 0.11* 9.61 0.13 0.00075 0.000010 c × o × b 6 191.38 ** 2.38 * 0.11 ** 2.76 18.59 0.00068 0.000004 l × c × o × b 6 66.33 1.42 0.04 3.05 6.50 0.00009 0.000006 eb 80 37.44 0.79 0.02 5.84 19.15 0.00059 0.0000224 cv (%) 5.11 4.69 6.66 3.64 2.42 5.48 3.06 ghane et al. yield and physiological response of perilla 209 tion in perilla. the highest rosmarinic acid (23.48 mg.g dm) was acheved in 100 kg/ha chemical fertilizer plus humic acid and without application of biologic fertilizer (table 6). similarly, with plant yield, rosmarinic acid concentration was affected by fertilizer application. these results are in agreement with hendawy et al. (2015) on mentha piperita. they reported foliar application of humic acid increased growth characteristics and finally possessed the best oil percentage and yield in mint plant. zaghloul et al. (2009) reported also the application of humic acid increased oil content of thuja orientalis. at both of studied location and with/without application of biologic fertilizer, the maximum rosmarinic acid yield was achieved by using humic acid. the maximum rosmarinic acid yield (2.967 g/m2) was observed in mashhad ardehal and application of yield in all levels of chemical fertilizer. however, there was not clear trend about application of biologic fertilizer. previously, researchers (ciarkowska et al., 2017) had highlighted the important role of humic acid, especially in root formation. rosmarinic acid concentrations in perilla were significantly affected by location, chemical and organic fertilization (table 3) and, at both locations and all chemical fertilization levels, the humic acid application increased rosmarinic acid content in perilla. this fact exhibited the importance of humic acid on rosmarinic acid content in perilla. the maximum rosm a r i n i c a c i d ( 2 5 . 0 1 m g . g d m ) w a s a c h i e v e d i n mashhad ardehal along with using 100 kg/ha chemical fertilizer and application of humic acid (fig. 2). application of organic fertilizer especially humic acid with/without biologic fertilizer in all chemical fertilizer levels increased rosmarinic acid concentras.o.v. d.f. means squares plant yield rosmarinic acid content rosmarinic acid yield stomatal conductance dpph catalase activity polyphenol oxidase replication 2 61.95 0.46 0.01 5.24 24.06 0.0006 0.000040 chemical fertilizer (c) 3 10082.10 ** 195.60 ** 13.42** 1256.35 ** 540.52 ** 0.0411 ** 0.00749 ** ea 6 10.21 1.61 0.04 5.48 58.46 ** 0.0003 0.00013 ** organic fertilizer(o) 2 1049.35 ** 27.60 ** 1.46** 42.86 ** 581.28 ** 0.0012 0.00071** biologic fertilizer (b) 1 363.69 ** 12.52 ** 0.71** 87.60 ** 13.86 0.0002 0.00005 c × o 6 50.03 * 3.84 ** 0.07** 13.03 ** 11.17 0.0006 0.00037 ** c × b 3 42.09 0.63 0.03 2.73 1.39 0.0027 ** 0.00001 o × b 2 240.40 ** 1.60 0.18** 1.28 8.78 0.0006 0.00001 c × o × b 6 89.83 ** 1.67 0.08 ** 5.60 5.38 0.0009 * 0.000005 eb 40 21.24 0.86 0.02 3.16 14.71 0.0004 0.00003 cv (%) 3.56 4.79 5.65 2.63 2.32 4.48 3.64 s.o.v. d.f. means squares plant yield rosmarinic acid content rosmarinic acid yield stomatal conductance dpph catalase activity polyphenol oxidase replication 2 51.56 0.96 0.01 2.80 13.07 0.00030 0.000003 chemical fertilizer (c) 3 2005.86 ** 76.59 ** 3.03** 433.02 ** 326.18 ** 0.00677 ** 0.003819 ** ea 6 46.75 0.76 0.04 5.99 30.56 0.00090 0.000009 organic fertilizer(o) 2 457.49 ** 5.13 ** 0.39** 47.28 ** 521.70 ** 0.00307 * 0.000019 biologic fertilizer (b) 1 33.13 0.31 0.03 128.16** 9.14 0.00005 0.000095 ** c × o 6 106.63 3.80 ** 0.01 5.98 36.55 0.00351 ** 0.000025 c × b 3 106.20 0.58 0.02 2.72 1.62 0.00156 0.000019 o × b 2 92.53 0.48 0.02 10.79 10.91 0.00030 0.000006 c × o × b 6 167.87 * 2.15 * 0.06 ** 0.21 19.72 0.00065 0.000006 eb 40 53.64 0.74 0.02 8.51 23.59 0.00077 0.000012 cv (%) 6.67 4.59 7.88 4.48 2.98 6.37 2.28 table 4 anova of some morphologic, yield and essence traits of perilla under different fertilizer systems in mashhad ardehal in kashan regions *, ** significant at 5% and 1% probability levels, respectively. *, ** significant at 5% and 1% probability levels, respectively. table 5 anova of some morphologic, yield and essence traits of perilla under different fertilizer systems in sensen in kashan regions 210 adv. hort. sci., 2019 33(2): 205-214 humic acid beyond biologic fertilizer (fig. 3). also, between chemical × organic × biologic fertilizer treatments, the maximum rosmarinic acid yield (3.432 g/m2) was observed in 100 kg/ha along with humic acid and biologic fertilizer application (table 6). integrated application of chemical, organic and biological fertilizers improved soil physical and chemical properties and resulted in the increase in availability of nutrients and ultimately the yield and quality of plants. darzi et al. (2007) reported the application of mycorrhiza and vermicompost and phosphate solubilizing biologic fertilizers determined increased essential oil yield in fennel. stomatal conductance stomatal conductance is one of the most import a n t f a c t o r i n d e t e r m i n i n g t h e p h o t o s y n t h e s i s amount. the study of stomatal conductance between location and chemical fertilizer treatments showed the highest stomatal conductance (75.88 mmol h2o m-2 s-1) in mashhad ardehal with application of 100 kg/ha chemical fertilizer (fig. 4). at both of studied location especially in mashhad ardehal, application of chemical fertilizer increased the stomatal conductable 6 means comparison of some morphologic, yield and essence traits of perilla under different fertilizer systems in sensen and mashhad ardehal in kashan regions chemical fertilizer organic fertilizer biologic fertilizer (inoculation) plant yield (g/m2) rosmarinic acid content (mg/g d.m.) rosmarinic acid yield (g/m2) 0 kg/ha control (-) 93.12 lm 14.40 lm 1.34 l (+) 89.86 m 13.94 m 1.25 l compost (-) 99.40 kl 15.31 l 1.52 k (+) 103.40 jk 17.29 jk 1.67 ij humic acid (-) 99.91 kl 16.44 k 1.64 jk (+) 95.17 lm 17.31 jk 1.65 jk 50 kg/ha control (-) 115.70 gh 19.52 def 2.25 gh (+) 128.40 de 19.38 defg 2.54 ef compost (-) 129.50 de 19.56 def 2.52 ef (+) 126.90 de 20.11 ced 2.54 e humic acid (-) 127.90 de 18.92 fgh 2.42 efg (+) 141.20 ab 20.37 cd 2.89 bc 100 kg/ha control (-) 131.40 cd 21.05 bc 2.79 d (+) 126.70 de 21.58 b 2.75 d compost (-) 138.00 bc 21.7 b 3.02 bc (+) 137.70 bc 21.63 b 3.00 bc humic acid (-) 131.70 cd 23.48 a 3.11 b (+) 147.20 a 23.02 a 3.43 a 200 kg/ha control (-) 107.20 ij 17.52 ij 1.88 i (+) 113.50 hi 18.4 ghi 2.09 h compost (-) 126.20 def 18.2 hij 2.30 g (+) 119.40 fgh 19.13 egh 2.29 g humic acid (-) 116.90 gh 19.33 efg 2.27 gh (+) 122.70 efg 19.07 fgh 2.35 fg different letters in each column denote a significant differences at p≤0.05 (lsd test). fig. 2 effect of chemical and organic fertilizer on rosmarinic acid content of perilla in mashhad ardehal and sensen regions. different letters in each column denote a significant difference at p≤0.05 (lsd test). fig. 3 effect of organic and biologic fertilizer on rosmarinic acid yield of perilla in mashhad ardehal and sensen regions. different letters in each column denote a significant difference at p≤0.05 (lsd test). ghane et al. yield and physiological response of perilla 211 tance of perilla leaves. increasing of stomatal conductance increases the photosynthesis activities and finally improved the plant yield (atteya, 2003). the results of stomatal conductance are agreement with result of plant yield. this result showed the increasing of stomatal conductance can be useful to increasing of plant yield especially with 100 kg/ha application of chemical fertilizer. the highest stomatal conductance was obtained with application of compost fertilizer (67.67 mmol h2o m -2 s-1) (table 7). organic fertilizers can provide better moisture conditions by increasing the absorption and preservation of water and the availability of water and food, and this can contribute to the favorable conditions for photosynthesis in the plant. if there is enough stomatal conductance, co2 gas will enter the stomata more easily and sufficient photosynthesis will be performed. also means comparison showed inoculation with mycorrhiza improved stomatal conductance as compared to without inoculation (table 7). the physiological effects of mycorrhizal fungi symbiosis include aboveground modifications of water relations and physiological status in terms of leaf water potential, relative water content, stomatal conductance, co2 assimilation, and efficiency of photosystem ii as compared to non-mycorrhizal plants (barzana et al., 2012). mycorrhizal fungi increase the contact surface with soil and moisture around the plant roots by 10 to 1,000 times, thus increasing the plant’s ability to use the resources in its surroundings (sharma, 2002). dpph test many researchers have used dpph test to express the antioxidant status of plants (dasgupta and de, 2007; sahu et al., 2013). the results showed the antioxidant activity of perilla plant in mashhad ardehal (165.29 ug/ml) was higher than sensen (162.79 ug/ml) location (table 7). perilla is very sensitive to free radicals productions. in a study in korea on different species of perilla, different species for dpph test showed a significant difference (choi et al., 2002). increasing levels of chemical fertilizer reduced the plant’s efficiency in inhibiting free radicals. the highest amount of dpph test was obtained (169.47 μg/ml) under control condition and the lowest (55.85 μg/ml) was obtained from the highest level of fertilizer application (200 kg/ha) (table 7). omar et al., (2012) reported the antioxidant activity determined using the dpph was high with the application of organic fertilizer compared to chemical fertilizer in cassava tubers. organic fertilizers, especially compost (277.17 μg/ml), increased the antioxidant activity and inhibited free radicals (table 7). uthairatanakij et al. (2017) reported an increase in antioxidant activity and inhibition of free radicals due to the application of organic fertilizers. organic fertilizers with plant sources activated defense mechanisms against pests, diseases and other tensions (brandt and molgaard, 2001). catalase activity t h e h i g h e s t c a t a l a s e a c t i v i t y ( 0 . 5 3 3 2 n m o l h2o2/min) was observed in mashhad ardehal region with the application of 100 kg/ha fertilizer and withfig. 4 effect of chemical fertilizer on stomatal conductance of p e r i l l a i n m a s h h a d a r d e h a l a n d s e n s e n r e g i o n s . different letters in each column denote a significant difference at p≤0.05 (lsd test). treatments stomatal conductance (mmol h 2 o m−2s−1) dpph (ug/ml) polyphenol oxidase (μmol/min) location mashhad ardehal 67.61 a 165.29 a 0.156 a sensen 64.98 b 162.79 b 0.152 b chemical fertilizer 0 kg/ha 57. 36 d 169.48 a 0.130 c 50 kg/ha 65.99 c 161.73 c 0.152 b 100 kg/ha 72.05 a 166.41 b 0.167 a 200 kg/ha 69.80 b 158.55 d 0.168 a organic fertilizer control 66.03 b 158.54 b 0.152 b compost 67.68 a 167.27 a 0.158 a humic acid 65.19 b 166.31 a 0.153 b biologic fertilizer (inoculation) (-) 65.08 a 164.08 a 0.153 b (+) 67.52 b 164.00 a 0.155 a table 1 weather characteristics in mashhad ardehal and sensen different letters in each column denote a significant differences at p≤0.05 (lsd test). adv. hort. sci., 2019 33(2): 205-214 212 out application of biologic fertilizer. the lowest amount was obtained from mashhad ardeal area without using of chemical and biologic fertilizer. also, in the sansen location, the highest catalase activity was obtained from 200 kg/ha chemical fertilizer and biologic fertilizer application (fig. 5). among the chemical and organic fertilizer treatments in two studied locations, the highest catalase activity was obtained from 100 kg/ha of chemical fertilizer with compost application in mashhad ardehal region (0.5182 nmol h2o2/ min) and the lowest amount (0.338 nmol h2o2/ min) was obtained without using of chemical and organic fertilizer control in mashhad ardehal location (fig. 6). logan et al. (1999) stated that nitrogen had a significant effect on the activity of enzymes involved in photosynthesis, such as ribulose-1,5-bisphosphate. in another study the researchers reported increased nitrogen levels caused to production of antioxidant enzymes like apx, sod, cat, and pod in the populus yunnanensis plants (lin et al., 2012). our result indicated that sources of fertilizer had a significant influence on the level of catalase activity in field grown perilla. polyphenol oxidase activity among the treatments of chemical and organic fertilizer in different locations, similar to catalase activity, the highest polyphenol oxidase activity was obtained from 100 kg/ha with compost application in mashhad ardeal location (0.193 μmol/min) and the lowest amount (0.129 μmol/min) was obtained from without application of chemical and organic fertilizer in mashhad ardehal area (fig. 7). at all levels of chemical fertilizer in both locations, application of organic fertilizers increased the activity of polyphenol oxidase activity, and in most cases, organic matter compost was superior to humic acid. also, the application of biologic fertilizer led to an increase in polyphenol oxidase activity (table 7). similar to our results, the application of biologic fertilizer increased the amount of polyphenol oxidase activity in triticale (kheirizadeh-arough et al., 2016). 4. conclusions in the present experiment, the results showed that proper nutritional management of perilla medicinal plant has a special role in improving quantitative and qualitative traits. among individual fertilizer treatments, 50 and 100 kg/ha of fertilizer and among organic fertilizers, humic acid played a more effective role in improving the studied indices, but the application of biologic fertilizer separately did not have a very significant effect on this plant. integration with other fertilizers has been shown to be more effective. mashhad ardehal area has better conditions for cultivating perilla due to its soil characteristics and climatic characteristics. also, the combined application of different fertilizer sources in comparison with the single application of each of them in both studied regions significantly improved the growth characteristics, as well as essential oil yield and biochemical fig. 5 effect of chemical and biologic fertilizer on catalase activity of perilla in mashhad ardehal and sensen regions. different letters in each column denote a significant difference at p≤0.05 (lsd test). fig. 6 effect of chemical and organic fertilizer on catalase activity of perilla in mashhad ardehal and sensen regions. different letters in each column denote a significant difference at p≤0.05 (lsd test). fig. 7 effect of chemical and organic fertilizer on polyphenol oxidase activity of perilla in mashhad ardehal and sensen regions. different letters in each column denote a significant difference at p≤0.05 (lsd test). ghane et al. yield and physiological response of perilla 213 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center, horticultural scinces research institue, areeo, karaj, iran. 3 horticulture crops research department, west azarbaijan agricultural and natural resources research and education center, areeo, urmia, iran 2 soil and water research department, isfahan agricultural and natural resources research and education center, areeo, isfahan, iran. key words: drought stress, genotypes, growth vigor, pyrus spp., seedlings. abstract: one of the important products in iran and isfahan province is the pear that its cultivation has been limited by drought stress and global warming in recent years. the use of drought-tolerant rootstocks is one of the available solutions for pear orchards in semi-arid regions. in addition, the lack of dwarf or semi-dwarf rootstocks, which are appropriate and compatible with iran climatic conditions, limited high density pear orchards. in order to obtain drought tolerant rootstocks, in this research fruit of pyrus glabra, pyrus syriaca, and pyrus salicifolia, along with p. communis cv. spadona, dargazi, as well as khoj n. 1 and n. 2 species were collected from different regions of iran in august and september of 2016. the seeds were separated from the flesh and dried at room temperature. the seeds were cultivated in uniform and light texture of soil in november in the field condition to break the seed dormancy. seedlings were irrigated regularly for three months in order to establish in the soil; drought stress began in july. in order to apply drought stress, irrigation time was considered based on 80% of allowed water depletion. morphological traits of seedlings were recorded before stress and at the end of the stress period (late september). the viability percentage of seedlings after drought stress was between 14.28% (p. communis cv. dargazi progenies) to 82.55% for p. salicifolia. comparison of the means and cluster analysis, among populations showed that the three populations of p. salicifola, p. glabra and p. communis cv. khoj n. 2 had the lowest height and were placed in the same group. after studying single genotypes in these three populations, genotypes no. 31, 32, 41, 57 and 12 from p. salicifolia, genotypes 10, 11, 7, 3 and 9 of p. glabra and genotype 4 of p. communis cv. khoj n. 2 populations were selected as drought tolerant and dwarf genotypes and were taken to the propagation phase for future evaluation. 1. introduction pear is from the rosaceae family and the pyrus genus. this species has been cultivated in iran since ancient times, and iran is one of the earliest (*) corresponding author: mtatari1@gmail.com citation: tatari m., 2019 selection of open pollination progenies in some pear species in order to achieve dwarf and drought tolerant rootstocks. adv. hort. sci., 33(2): 245-255 copyright: © 2019 tatari m. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 24 september 2018 accepted for publication 25 february 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(2): 245-255 246 areas of pear distribution and variation in the world (abdollahi, 2011). drought stress is the most important environmental stress that occurs annually with extreme damage to crops, especially in arid and semi-arid regions (xoconostle-cazares et al., 2010). in dry and semi-arid regions of iran, the amount of annual evaporation is higher than precipitation. in recent years, the cultivation of fruit trees, such as pears, has been limited due to climate change and reduce rainfall. considering that the drought tolerance of the rootstocks is transmitted to the scion (landsberg and jones, 1981), the choice of drought tolerant rootstocks with a low water requirement is one of the solutions to drought problem of pear orchards in arid and semi-arid regions such as iran (cheruth et al., 2009). another problem in the cultivation of pears in iran is the lack of suitable dwarf or semi-dwarf rootstocks that are compatible with cultivars and climatic conditions of the country for high density orchards. the pear and quince are used as rootstocks for pear trees. the quince rootstocks are very dwarf (em-c) and semi-dwarf (ba29), but these rootstocks could not overcome the problem of graft incompatibility with some pear cultivars. these rootstocks are not also tolerant to winter frosts and chlorosis caused by iron deficiency in the calcareous soils (harotko, 2007). in recent years, a number of clonal rootstocks have been introduced into iran such as pyrodwarf, fox-11 and some of the american rootstocks of the old home × farmingdale series (abdollahi, 2011). the use of native pear species as a seed or clonal rootstocks can be a solution for access to proper rootstocks with good adaptability to the climatic conditions of the country. some of these species are dwarf and have a high tolerance to unfavorable environmental conditions that can be used to produce clonal rootstocks (abdollahi et al., 2012). in the pear genus, 22 species have been identified in the world that are mainly native to europe, africa and asia. of the 22 identified species, there are 12 species in iran (sabeti, 1995). in some parts of the world, these species are used as the rootstock for pear commercial cultivars. for example, a large number of pear cultivars in turkey are grafted on p. elaeagrifolia, in syria and lebanon on p. syriaca, in ancient yugoslavia, turkey and greece on p. amygdaliformis, in the south of russia on p. salicifolia and in algeria and morocco are grafted on p. longipes (henareh, 2015). w i l d g e r m p l a s m h a s e v o l v e d i n n a t u r a l d r y ecosystems for tolerance of stress conditions such as h i g h t e m p e r a t u r e s , d r o u g h t a n d s a l i n i t y . identification of wild pear germplasm is very important to use as a rootstock in semi-arid regions (zarafshar et al., 2014). according to this, three wild pear genotypes from p. syriaca were exposed to four irrigation treatments. the results showed that the coile wild genotype (p. syriaca) was more tolerant to drought conditions compared with other genotypes due to its high relative water content during drought stress and non-decreasing dry weight. drought stress reduced leaf photosynthesis, stomatal conductance and transpiration in a number of pear species (javadi and bahramnejad, 2011). in another study, drought tolerance was evaluated in three populations of wild pear germplasm (p. boisseriana) in greenhouse conditions. among the three populations studied, the collected population from semi-arid regions showed higher drought tolerance than the other two populations that were collected from semi-humid areas, and recovered more rapidly after irrigation. these seedlings were introduced as promising sources for use as rootstock for commercial pear cultivars in drought conditions (zarafshar et al., 2014). in a research carried out by ghasemi et al. (2014), chlorophyll index was significantly different between studied pistachio rootstocks. in these rootstocks, chlorophyll index in stress conditions was lower than drought conditions. so, the reduction of chlorophyll content can be caused by chlorophyll degradation under drought stress conditions, which leads to reduced pure photosynthesis. the rootstocks of european pear species are from very vigor to relatively dwarf. these rootstocks are compatible with all the pear tree cultivars and have considerable tolerance to the adverse conditions of soil and fire blight disease. other species of pyrus genus, such as p. syriaca boiss is compatible with commercial pear cultivars and is currently used in some countries (radnia, 1996). seed cultivation after cross or open pollination is one of the breeding methods that is frequently used in breeding programs. in this way, it is possible to achieve a wide variation for choice of new rootstocks and cultivars. for example, the manon cultivar was obtained from open pollination of beurre bosc cultivar. the pib-bu3 dwarf pear rootstock was obtained from open pollination of p. longipes, while pi-bu4 and pi-bu7 rootstocks were obtained from open pollination of the p. pyrifolia species (mohan jain and priyadarshan, 2009). currently, most pear cultivars in iran are grafted on different seedling rootstocks of the p. communis tatari selection of drought tolerant pear rootstocks 247 species. considering that wild pear genotypes grow on rocks and dry or low moisture soils, as well as some of them have a small growth in the form of shrubs, so some of them can be used as drought tolerant and dwarf rootstocks for commercial pear cultivars (ashraf and karimi, 1991; henareh, 2015). the aim of this research was an evaluation of drought stress tolerance in some wild pear species, and then selection of the dwarf genotypes among the drought tolerant genotypes. 2. materials and methods plant materials in this research, seeds from open pollination of pyrus syriaca boiss., pyrus salicifolia pall. and pyrus. glabra boiss. along with p. communis l. cv. spadona and dargazi as well khoj including large fruit (khoj no. 1) and small fruit (khoj no. 2) were evaluated. p. syriaca boiss. is distributed from west azarbaijan to fars in the zagros mountains and northwest of iran (abdollahi, 2011). this species has shown compatibility with spadona and kochia pear cultivars and have improved the growth of the scion in calcareous soils. it is currently used as a rootstock in some countries (fallouh et al., 2008). p. glabra species is known as anchuchek in iran, and has spread mainly in the zagros mountains. the seeds of this species are large a n d a r e c o n s u m e d a s s n a c k s i n f a r s p r o v i n c e (abdollahi, 2011). p. communis is more commonly known as khoj and is scattered in the forests of the north, west azarbaijan, sardasht and baneh in kordestan province. the fruit of this species is very diverse. two types of native khoj including large fruit (khoj n. 1) and small fruit (khoj n. 2) were studied in this research (moazedi et al., 2014). p. communis cv. dargazi is native commercial cultivar of iran that has been introduced as tolerant rootstock (mansouryar et al., 2017). p. communis cv. spadona is high yielding and has high resistance to chlorosis due to iron deficiency. it is also relatively tolerant to psylla and fire blight. p. salicifolia species spread in the northwest of iran, including west and east azerbaijan provinces (abdollahi, 2011). cultivation of seeds fruits of studied species were collected in august and september of 2016 from different regions of west azarbaijan and isfahan provinces as well as northern regions of iran and transferred to the laboratory. the seeds were separated from the flesh and kept in a cool and dry place in paper bags after washing and drying, in order to eliminate the chilling requirement, seeds were cultivated in separate rows in the nursery soil with a sandy loam texture at isfahan agricultural and natural resources research center, in december. from each of these species, 500 seeds were cultivated at intervals of 2-3 cm in the nursery and each produced seedlings were studied as a genotype. during chilling period, adequate moisture was provided and prevented from drying of the culture bed. irrigation of seedlings after emergence of seeds, the seedlings were irrigated for three months in order to establish in the nursery soil. determination of irrigation time based on allowed water depletion of pear trees is after a 5 0 % d e c r e a s e i n h u m i d i t y , b u t b e c a u s e o f t h e seedling roots had not yet been sufficiently developed, and needed enough moisture for better growth, so allowed water depletion was considered 35%. in the irrigation intervals, soil moisture at various depths of the soil was measured up to a depth of 100 cm by the time domain reflectometry device (tdr, trase6050x1) (doorenbos and pruitt, 1977; alizadeh, 2006). to obtain the amount of irrigation (irrigation volume), first, net irrigation depth was calculated according to formula 1. in= ∑ [(θfci θbli)×di] (1) in this formula, in is the net irrigation depth (mm), θfci is the moisture content of the field capacity for each layer, θbli is the soil moisture before irrigation for each layer, di is the root development depth (mm), and i is the number of each soil layer. then, according to formula 2, gross irrigation depth was calculated. ig = in/(1-lr)×ei (2) in this formula, ig is the gross irrigation depth (mm), in is the net irrigation depth (mm), (1-lr) is the amount of leaching and ei is the irrigation efficiency (usually 80-90% for drip irrigation). irrigation volume was obtained from the multiplication of gross irrigation depth in the irrigation plot area. this volume was controlled by the installed counter on the pipe before irrigation plot. for irrigation of seedlings leaked tubes with drippler at 10 cm intervals were used. the outlet flow of each drippler was measured in one liter per hour at an appropriate pressure. in this study, the efficiency of the drip system and the leaching requirement was considered 90% and 10% respectively. adv. hort. sci., 2019 33(2): 245-255 248 drought stress drought stress began in july, which coincided with the beginning of the stress period in isfahan region. irrigation frequency was changed in order to apply drought stress, and irrigation time was considered based on 80% of allowed water depletion. table 1 shows the volume of pure and impure irrigation water and number of irrigation per month. evaluated traits the morphological traits of seedlings were recorded separately for each genotype at the end of the stress period (late september). these traits included height and diameter of seedlings, number and length of internode, crown width, number of suckers and branches, chlorophyll index and leaf dimensions. before applying stress, seedling height and diameter at 5 cm above the soil surface were recorded. the difference in both seedling height and diameter b e f o r e a n d a f t e r s t r e s s w e r e a l s o c a l c u l a t e d . qualitative traits, including leaf chlorosis and trichome, as well as seedling growth vigor were recorded after stress using national guideline for distinctness, uniformity and stability in pear (dus guileline) in pears. the abbreviation and measurement method of evaluated traits are given in table 2. data analysis mean comparison was calculated with sas software (version 9.1). descriptive statistics including mean, minimum, maximum and coefficient of variation and also cluster analysis were performed by ward method based on squared euclidean distance with spss software (version 15). 3. results viability percent viability percent of seedlings of the studied pear species after the stress was shown in table 3. according to the results, p. salicifolia showed the highest survival in drought stress conditions. after that, p. communis cv. khoj n. 1 and 2 were placed in the next rank. the lowest percentage of seedling viatable 2 symbol and measurement method for recorded traits (based on dus guideline) table 1 number of irrigation, net and gross volume of irrigation water irrigation february march april may june july august september october november total number of irrigation 3 3 5 7 8 1 1 1 1 1 32 net volume of irrigation water (m3.ha-1) 540 540 900 1260 1440 180 180 180 180 180 5580 gross volume of irrigation water (m3.ha-1) 666.6 666.6 1111.1 1555.5 1777.7 222.2 222.2 222.2 222.2 222.2 6888.2 characteristic symbol unit measurement method leaf chlorosis lc code no chlorosis (1), low chlorosis (3), medium chlorosis (5), high chlorosis (7), very high chlorosis (9) leaf trichome lt code no trichome (1), low trichome (3), medium trichome (5), high trichome (7), very high trichome (9) seedling growth vigor gv code very low (1), low (3), medium (5), high (7), very high (9) seedling height se cm ruler seedling diameter sd mm caliper height difference hd cm calculation diameter difference dd cm calculation internode number in number counting internode length il cm ruler (average of internodes in branches) crown width cw cm meter number of secondary branches nsb number counting number of suckers ns number counting chlorophyll index ci chlorophyll meter (spad) leaf length ll cm ruler (average of 10 leaves) leaf width lw cm ruler (average of 10 leaves) table 3 seedlings viability percent of pear cultivars and species after drought stress species viability percent pyrus communis cv. spadona 14.77 pyrus communis cv. dargazi 14.28 pyrus communis cv. khoj n. 1 55.17 pyrus communis cv. khoj n. 2 43.79 pyrus syriaca 20.33 pyrus salicifolia 82.55 pyrus glabra 17.74 tatari selection of drought tolerant pear rootstocks 249 were lower than other species. similarly, p. communis cv. spadona showed the highest height difference before and after applying stress. t h e s e e d l i n g d i a m e t e r o f p . c o m m u n i s c v . spadona, khoj n. 1, dargazi and p. syriaca was more than the other seedling diameter, so that p. glabra and p. communis cv. khoj n. 2 had the lowest seedling diameter. no significant difference was observed among the seedling diameter of the species before and after drought stress. p. communis cv. spadona had the highest number of internode after the end of drought stress. the lowest number of internode belonged to p. glabra and p. communis cv. khoj n. 2. p. salicifolia produced the crown with the widest (24.71 cm) width, compared to other species. it should be noted that p. communis cv. spadona, khoj n. 1 and p. syriaca did not show any significant difference with pyrus salicifolia. p. communis cv. khoj n. 2 had the lowest crown width (4.75 cm). p. salicifolia did not produce a secondary branch. there were no significant differences in the number of secondary branches among the other species. p. salicifolia showed the highest chlorophyll index, but did not show significant differences with p. communis cv. spadona, khoj n. 2, p. syriaca and p. glabra. the lowest chlorophyll index belonged to p. communis cv. dargazi (table 5). p. communis cv. spadona , with a length of 6.06 cm and a width of 2.7 cm had the highest leaf length and width compared with other species. after p. communis cv. spadona, p. communis cv. khoj n. 1 had the highest leaf length and width. the lowest leaf length was related to p. communis cv. khoj n. 2 with an average of 2.01 cm. p. syriaca also produced the leaves with the lowest width (1.37 cm). p. communis cv. spadona and dargazi had the longest internode, but did not show significant differences with p. communis cv. khoj n. 1 and p. syriaca. bility during drought stress belonged to p. communis cv. spadona and dargazi. traits before applying stress the mean comparison of recorded traits among populations before applying drought stress is presented in table 4. according to the results, p. communis cv. spadona had the highest seedling height. the seedling height in the wild species of p. glabra, p. salicifolia and p. communis cv. khoj n. 2 was the lowest. p. communis cv. khoj n. 2 and p. glabra produced seedlings with the lowest diameter. the largest seedling diameter belonged to p. communis cv. spadona and p. syriaca (table 4). generally, without drought stress conditions, growth of p. glabra, p. salicifolia and p. communis cv. khoj n. 2 were lower than p. communis cv. spadona, khoj n. 1, dargazi and p. syriaca. traits after applying stress the mean comparison of recorded traits among populations after stress is presented in table 5. p. communis cv. spadona had the highest seedling height (36.73 cm) after stress. the seedling height of p. glabra, p. salicifolia and p. communis cv. khoj n. 2 table 4 the mean comparison of the seedling height and diameter among populations of pear species before applying stress similar letters in each column indicate no significant difference (lsd). table 5 mean comparison of some traits in the seedling populations of pear species after applying stress similar letters in each column indicate no significant difference (lsd). species seedling height (cm) seedling diameter (mm) pyrus communis cv. spadona 33.52 a 3.44 a pyrus communis cv. khoj n. 1 23.38 b 3.15 ab pyrus communis cv. dargazi 24.23 b 3.15 ab pyrus syriaca 20.92 b 3.31 a pyrus salicifolia 12.80 c 2.62 bc pyrus communis cv. khoj n. 2 11.83 c 1.74 d pyrus glabra 7.54 c 2.25 cd species seedling height (cm) height difference (cm) diameter (mm) diameter difference (mm) internode number crown width (cm) number of secondary branches chlorophyll index leaf length (cm) leaf width (cm) internode length (cm) pyrus communis cv. spadona 36.73 a 3.20 a 4.34 a 0.89 a 20.23 a 10.03 ab 0.23 ab 44.99 ab 6.06 a 2.70 a 1.91 a pyrus communis cv. khoj n. 1 26.65 b 2.80 ab 4.00 a 0.84 a 14.56 b 10.62 ab 0.68 a 42.48 b 5.45ab 2.23 b 1.21 ab pyrus communis cv. dargazi 25.83 b 1.60 ab 3.81 a 0.66 a 14.66 b 7.83 b 0.66 a 42.02 b 4.73 bc 2.10 bc 1.93 a pyrus syriaca 21.60 b 1.41 ab 4.02 a 0.70 a 12.91 bc 9.16 ab 0.25 ab 49.77 ab 4.50 c 1.37 d 1.34 ab pyrus salicifolia 14.21 c 0.67 b 3.56 ab 0.94 a 10.05 cd 11.24 a 0.00 b 53.83 a 4.30 c 1.73 c 0.94 b pyrus communis cv. khoj n. 2 13.67 c 0.84 b 2.63 c 0.88 a 8.31 de 4.75 c 0.21 ab 46.53 ab 2.01 d 1.97 bc 0.71 b pyrus glabra 10.22 c 0.68 b 2.92 bc 0.66 a 5.27 e 8.31 b 0.36 ab 48.85 ab 3.98 c 2.10 bc 0.73 b 250 adv. hort. sci., 2019 33(2): 245-255 the lowest internode length belonged to p. salicifolia, p. glabra, and p. communis cv. khoj n. 2 (table 5). cluster analysis of populations according to the results of cluster analysis, species were classified into three groups at five s q u a r e d e u c l i d e a n d i s t a n c e b a s i s o f t h e w a r d method (fig. 1). p. communis cv. spadona and dargazi were placed in the first group. p. syriaca and p. communis cv. khoj n. 1 in the second group, while p. communis cv. khoj n. 2, p. salicifolia and p. glabra species in the third group. study of single genotypes the mean and the range of traits for each of the examined genotypes, as well as the coefficient of variation in each trait are presented in table 6. each genotype was identified by a number and genotypes with minimum and maximum values were presented. internode length (66.12%), seedling height before stress (60.45%) and seedling height after stress (56.85%) had the highest and chlorophyll index (14.34%) had the lowest coefficient of variation. in order to achieve dwarfing genotypes, the trait of seedling height was considered. the three pear populations, including p. salicifolia, p. glabra and p. communis cv. khoj n. 2 had a lower mean seedling height than other populations, so the height of single genotypes of them after stress was shown in figures 2, 3 and 4, respectively. in p. salicifolia, genotypes no. 31 (4.5 cm), 32 (5 cm), 41 (5.5 cm), 57 (6 cm) and 12 (6 cm) had the lowest seedling height. genotypes fig. 2 height of single genotypes in p. salicifolia after stress. table 6 mean, range and coefficient of variation in studied traits fig. 1 grouping of pear species based on measured characteristics by ward method. characteristics cv (%) standard deviation mean minimum maximum genotype (number of genotypes) rate genotype (number of genotypes) rate seedling height before stress 60.45 9.83 16.26 p. glabra (42) 1.4 p. communis cv. spadona (23) 53.9 seedling diameter before stress 28.35 0.76 2.68 py. communis cv. khoj n. 2 (33) 0.75 p. communis cv. spadona (64) 6.3 seedling height after stress 56.85 10.28 18.08 p. glabra (10 & 11) 3 p. communis cv. spadona (2) 55 seedling diameter after stress 26.47 0.94 3.55 p. communis cv. khoj n. 2 (8) 1.6 p. salicifolia (8) 7.28 height difference 55.32 2.19 1.82 p. salicifolia (24 & 28) 0 p. communis cv. khoj n. 1 (9) & p. syriaca (3, 4,5,6 & 7) 19 diameter difference 55.17 0.48 0.87 p. salicifolia (47) 0.04 p. salicifolia (66) 3.06 internode number 44.65 5.00 11.20 p. glabra (7,10 & 11) 3 p. communis cv. khoj n. 1 (14) 26 crown width 30.67 3.11 10.14 p. communis cv. khoj n. 1 (5) & p. glabra (10) 4 p. communis cv. khoj n. 1 (8) 30 number of secondary branches 37.22 0.67 0.18 many genotypes 0 p. communis cv. khoj n. 2 (4) 4 chlorophyll index 14.34 7.81 54.46 p. communis cv. khoj n. 1 (8) 23.2 p. salicifolia (36) 69.8 leaf length 31.93 1.37 4.29 p. communis cv. khoj n. 2 (15) 1 p. communis cv. spadona (13) 7.2 leaf width 26.70 0.51 1.91 p. syriaca (5) 1 p. communis cv. spadona (2) 3.3 internode length 66.12 0.82 1.24 p.salicifolia (30) 0.3 p. communis cv. spadona (1) 10 tatari selection of drought tolerant pear rootstocks 251 no. 10 (3 cm), 11 (3 cm), 3 (6 cm), 9 (6 cm) and 7 (6 cm) showed the lowest seedling height in the p. glabra species. genotype no. 4 had the lowest height from p. communis cv. khoj n. 2 population. cluster analysis of single genotypes based on all studied traits cluster analysis among the single genotypes of the p. salicifolia, p. glabra and p. communis cv. khoj n. 2 populations was shown in figures 5, 6 and 7. genotypes no. 12, 31, 32, 41 and 57 of p. salicifolia (fig. 5) and genotypes no. 10, 11, 3, 9 and 7 of p. glabra (fig. 6) were placed in the groups close to each other. qualitative traits for selected genotypes were presented in table 7. all genotypes had a small trichome and had low or very low growth potentials. leaf chlorosis was not observed in genotypes except for the genotype no. 31 from p. salicifolia and genotype n o . 3 f r o m p . g l a b r a t h a t h a d l o w c h l o r o s i s . genotypes no. 12 and 32 of p. salicifolia were green and very green, respectively. genotypes no. 9, 7, 10 and 11 of p. glabra and genotype no. 4 of the p. communis cv. khoj n. 2 preserved their green color after applying drought stress. 4. discussion and conclusions viability percent after the drought stress period, 35.51% of the genotypes survived, and the rest of them were dried. most surviving genotypes belonged to p. salicifolia species. p. communis cv. spadona and dargazi fig. 3 height of single genotypes in p. glabra after stress. fig. 4 height of single genotypes in p. communis cv. khoj n. 2 after stress. fig. 5 grouping of drought tolerant genotypes in p. salicifolia species based on measured traits by ward method. fig. 6 grouping of drought tolerant genotypes in p. glabra species based on traits measured by ward method. adv. hort. sci., 2019 33(2): 245-255 252 showed the lowest percentage of survival (table 3). for a long time, wild pear genotypes have been considered in iran’s plateau due to the tolerance to biotic and abiotic stresses (javadi et al., 2005). the adaptation of wild pears with rocky areas and dry or low moisture soils can lead to more tolerance of them under drought stress conditions compared with domestic and commercial rootstocks (henareh, 2015). traits before and after applying stress the analysis of variance showed that the investigated species had a significant difference in most of the studied traits, which is due to the diversity among populations, so it is possible to select species for different values of a trait. according to table 4, in normal conditions, seedling height and diameter of p. glabra, p. salicifolia and p. communis cv. khoj n. 2 were less than p. communis cv. spadona, khoj n. 1, dargazi and p. syriaca. similarly, after applying drought stress, the seedling height and diameter of p. glabra, p. salicifolia and p. communis cv. khoj n. 2 species were lower than other populations (table 5). morphological adaptations in plants can be one of the adaptive mechanisms under drought stress (pire et al., 2007). the first reaction of plants against drought stress is a reduction in their vegetative growth. drought stress affects the vegetative characteristics of trees, including their height (higgs and jones, 1990). due to height difference among populations before and after stress, it seems that the effect of drought stress on the seedling height trend of these populations before and after applying the stress is almost same. before drought stress, the seedling diameter of p. communis cv. spadona, khoj n. 1, dargazi and p. syriaca was higher than the seedling diameter of p. salicifolia, p. glabra and p. communis cv. khoj n. 2, while the diameter difference before and after drought stress among compared species did not show a significant difference, therefore, it can be concluded that the effect of drought on the seedling diameter among populations was not the same. other results also showed that the negative effect of drought stress on seedling diameter was less than its effect on seedling height (haghighatnia et al., 2013). growth of branch and internode length is an appropriate index for detecting the effect of drought stress on the plants, so that the occurrence of drought stress can be observed even before the change in the water potential of the leaves (grimplet et al., 2007). among the remaining genotypes after the stress, p. communis cv. spadona had the highest and p. glabra and p. communis cv. khoj n. 2 had the lowest number and length of internode. the chlorophyll meter indicates the relative fig. 7 grouping of drought tolerant genotypes in p. communis cv. khoj n. 2 based on the measured traits by the ward method. table 7 growth vigor, trichome and chlorosis of leaf in selected genotypes species no. genotype leaf chlorosis leaf trichome seedling growth vigor pyrus salicifolia 12 no chlorosis low trichome low 31 low chlorosis low trichome low 32 no chlorosis low trichome low 41 no chlorosis low trichome low 57 no chlorosis low trichome low pyrus glabra 3 low chlorosis low trichome low 9 no chlorosis low trichome low 7 no chlorosis low trichome low 10 no chlorosis low trichome very low 11 no chlorosis low trichome very low pyrus communis cv. khoj n. 2 4 no chlorosis low trichome low tatari selection of drought tolerant pear rootstocks 253 chlorophyll concentration, based on the difference between the light transmittance in two red and infrared wavelengths, which correlates with the chlorophyll content of the leaves (hoel and solhaug, 1998). in the present study, p. salicifolia and p. communis cv. dargazi showed the highest and the lowest chlorophyll index, respectively. preservation and not decomposition of chlorophyll in p. salicifolia during drought stress indicates the tolerance of that species to this stress (tarahomi et al., 2010). cluster analysis of populations drought tolerance in plants has a direct or indirect relationship with a complex of traits, therefore, all the traits should be considered for selection of tolerant plants. for this reason, in this research cluster analysis was used to facilitate the selection of drought tolerant species. cluster analysis classified species into three groups at five squared euclidean distance (fig. 1). p. communis cv. spadona and dargazi were placed in the first group that had the lowest survival rate after drought stress. the highest seedling diameter belonged to the plants of this group. in the second group, p. syriaca and p. communis cv. khoj n. 1 were placed, which showed low to moderate survival rate after drought stress. in total, the most crown width, primary and secondary seedling height and diameter, as well as the highest number of internode were observed in the species of the first and second groups. p. communis cv. khoj n. 2, p. salicifolia and p. glabra species formed the third group. these populations had the lowest seedling diameter and height. the lowest seedling growth vigor was also found in these species; therefore, selection of dwarf and drought tolerant genotypes in this group was more possible than other groups. it seemed that traits such as seedling height and diameter were the most effective grouping traits. in the research carried out by aran et al. (2012), seedling h e i g h t w a s a l s o o n e o f t h e i m p o r t a n t t r a i t s i n seedling grouping. of course, different values of some traits were seen in this group. for example, the crown width and viability percentage were observed at low, medium and high levels in this group. study of single genotypes the mean, the range of changes, and the coefficient of variation of each trait were shown in table 6. coefficient of variation shows the extent of variability in relation to the mean of the population. in the traits with a high coefficient of variation has provided a higher selection range. genetic variation helps the plant to overcome environmental changes and also provides more chance for selection of new cultivars (liu, 2006). in this research, high variation was observed for internode length and seedling height before and after stress. the variability of some traits in 15 cultivars of vitis vinifera l. was previously investigated by mousazadeh et al. (2014). they reported that leaf traits had the highest diversity among the studied traits. doulati baneh et al. (2013) and tahzibihagh et al. (2012) also observed a high variation in the morphological characteristics of the leaves of grape and pear cultivars. in the current research, the coefficient of variation in leaf dimensions was not high compared to other traits, because of less scatter in relation to the mean of the population. the high diversity coefficient for internode length and seedling height before and after stress indicates the high range of variation in these traits among the studied seedlings, so it is possible to select genotypes based on these two traits. survived genotypes after drought stress were selected in order to dwarfing. for this purpose, seedling height of each genotype was investigated separately. considering that the average height of three populations including p. salicifolia, p. glabra and p. communis cv. khoj n. 2 were lower than other populations, therefore selection was carried out among genotypes of these populations. the highest number of genotypes was selected from p. salicifolia. the least seedlings height was belonged to p. glabra. only a dwarf genotype was selected from the p. communis cv. khoj n. 2 population (figs. 2, 3 and 4). finally, 11 dwarf genotypes were selected from these species. cluster analysis of single genotypes cluster analysis was performed for three populations with a lower seedling height. according to this, five selected genotypes of p. salicifolia (fig. 5), five genotypes of p. glabra (fig. 6) and a genotype of the p. communis cv. khoj n. 2 population (fig. 7) were classified in the same subgroups. these genotypes had lower seedling height and internode number. leaf width was also lower in these genotypes. q u a l i t a t i v e t r a i t s o f 1 1 s e l e c t e d g e n o t y p e s showed that all genotypes had low trichome. the survival and tolerance of these genotypes to drought stress was not along with the increase of leaf trichome. the growth vigor of genotypes was low, and two genotypes 10 and 11 from p. glabra had very low growth vigor. these genotypes also had the lowest seedling height. in fact, their growth vigor was correlated with the seedling height. this positive correlation was observed between seedling height and adv. hort. sci., 2019 33(2): 245-255 254 growth vigor in bitter cherry seedlings (mahlab) ( g a n j i m o g h a d a m a n d k h a l i g h i , 2 0 0 6 ) . u n d e r drought stress, studied genotypes did not show chlorosis, only genotypes 3 of p. salicifolia and 31 of p. glabra were greenish-yellow after drought stress. selected genotypes were transferred to a propagation phase through cutting and layering. after studying single genotypes in these three populations, genotypes no. 31, 32, 41, 57 and 12 from p. salicifolia, genotypes 10, 11, 7, 3 and 9 of p. glabra and genotype no. 4 of p. communis cv. khoj n. 2 populations were selected as drought tolerant and dwarf genotypes and were taken to the propagation phase for future evaluation. references abdollahi h., 2011 pear, butany, cultivars and 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f.a., flores-elenes l., ruiz-medrano r., 2010 drought tolerance in crop plants. am. j. plant physiol., 5: 241256. zarafshar m., akbarinia m., askari h., hosseini s.m., r a h a i e m . , s t r u v e d . , s t r i k e r g . g . , 2 0 1 4 morphological, physiological and biochemical responses to soil water deficit in seedlings of three populations of wild pear tree (pyrus boisseriana). biotechnol. agron. soc. environ., 18: 353-366. impaginato 433 adv. hort. sci., 2018 32(3): 433-441 doi: 10.13128/ahs-22932 effect of wounding intensity on physiological and quality changes of strawberry fruit m.t. solomon, f. piazzolla, m.l.v. de chiara, m.l. amodio (*), g. colelli dipartimento di scienze agrarie, degli alimenti e dell’ambiente, università degli studi di foggia, via napoli, 25, 71122 foggia, italy. key words: ascorbic and dehydroascorbic acid, ‘candonga’, cutting degree, fresh-cut, respiration rate, total phenolic content. abstract: wounding makes fresh-cut product more perishable than whole fruit. the effect of wounding intensity on respiration rate and nutritional quality of fresh-cut ‘candonga’ strawberries was investigated. fruit were submitted to six levels of cutting intensity whole fruit (who), 4, 16, 64, and 128 pieces and chopped (cho) samples. respiration rate, and the main nutritional parameters were evaluated at the processing day and after 2 days of storage at 5°c. results showed that wounding intensity significantly influenced respiration rate, ascorbic and dehydroascorbic acids, total phenolic content, and antioxidant capacity. respiration rate increased with wounding intensity up to the level of 64 pieces (10.01 µg kg-1 s-1) compared to who (5.5 µg kg-1 s-1) and then decreased in the cho samples (2.81 µg kg-1 s-1). at day 2, the stress caused by the high intensity of cutting (64 pieces and cho) induced a higher degradation of ascorbic acid, phenolic compounds, and antioxidant capacity. stress-related changes decrease when the wounding damage was so high that it completely compromises the functionality of the cells (from 64 pieces up). these results should be considered for processing and packaging optimization of minimally processed strawberries-based products. 1. introduction the continuous physiological activity of living plant tissues induces severe compositional and structural variations, also associated to ripening and senescence of fresh produce during postharvest life. tissue responses cannot be blocked but it is possible to delay them within certain limits in order to prolong fruit shelf life (el-ramady et al., 2015). the physiological stresses due to physical damage and wounding occurring during minimal processing, make fresh-cut products more perishable than whole fruit (nicola and fontana, 2014). immediate response of plant cells start from a wound signal formed in adjacent and distant tissues, which gives rise to a wide range of different physiological and biochemical reactions. common are respiration rate and ethylene production increase, variation in product quality, synthesis and/or loss of phytochemicals with consequent decrease of nutritional content, stimulation of enzymatic activity (*) corresponding author: marialuisa.amodio@unifg.it citation: solomon m.t., piazzolla f., de chiara m.l.v., amodio m.l., colelli g., 2018 effect of wounding intensity on physiological and quality changes of strawberry fruit. adv. hort. sci., 32(3): 433-441 copyright: © 2018 solom m.t., piazzolla f., de chiara m.l.v., amodio m.l., colelli g. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 22 march 2018 accepted for publication 28 september 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(3): 433-441 434 and bacterial spoilage (brecht, 1995; surjadinata and cisneros-zevallos, 2003). the complex interrelationship among the different effects of wounding on physiological processes of fresh-cut products, are comprehensively described by saltveit (1997). wound responses could vary depending on different factors: species and cultivar of the product, maturity stage, temperature of processing and storage, cutting-type and sharpness of the blades, but also on process temperature (el-ramady et al., 2015), o2 and co2 levels, and water vapor pressure (brecht, 1995). the basis for the wound-induced changes include altered genes expression and changes in enzyme activities involved in an effort to heal the damaged tissues providing defense mechanisms of the plant aimed to prevent further and more serious damages (chung et al., 2006). during storage time the increase in respiration rate is usually responsible for the aging of the products due to consumption of reserve energy during redox process. as a consequence, the higher the respiration rate, the shorter is the storage life and therefore the faster is the quality deterioration. respiration is a function of the climacteric or non-climacteric behavior of the product and of the physiological age of climacteric fruit (gunes and lee, 1997). as already reported wounding induces an increase in enzyme activity, in particular higher activities of phenylalanine ammonia-lyase (pal), peroxidase (pod) and polyphenol oxidase (ppo) are observed. (saltveit et al., 2005 b). the main effect of the postcutting interaction of substrates with enzymes, such as ascorbate oxidase, ppo, and pod, is the degradation of phytonutrients. phenols oxidation and the resulting browning may induce a reduction in nutrient content resulting often in degradation of color, texture and flavor of fresh-cut products (saltveit, 1997; francis et al., 2012). it is possible to reduce wound-induced browning with the application of antioxidant or calcium-based active compounds and treatments that interfere with the synthesis or oxidation mechanisms of the phenolic compound precursors (brecht, 1995; saltveit, 1997; saltveit et al., 2005 a). however, according to francis et al. (2012), the induced synthesis of phenolic compounds after cutting caused an increase in nutritional value for lettuce, celery, carrot, parsnips, and sweet potato, while in the same study a decrease of phenols was observed in cut zucchini, radish, potato, and red cabbage, pointing out the influence of product on the wound-induced response type. amodio et al. (2014) described the consecutive reaction mechanism that regulates the phenolic content in fresh-cut produce during storage and how their variation is related to cut intensity. they observed, for example, that there was an increase in k1 values (the rate constant for the de novo synthesis of the phenols), when fresh-cut lemons were cut as half-slices rather than slices. this result was also in agreement with the increase in pal activity resulting from the higher level of wounding on vegetable tissues. at the same time, the cut intensity did not affect the rate of phenolic oxidation. the authors stated that one of the most important factor affecting the phenolic content and synthesis is the biological variability; in fact, each product shows a particular combination of factors that can contribute to the amount and composition of wound-induced phenolics (francis et al., 2012). fernando reyes et al. (2007) stated that the final concentration of phenolic compound in cut products is strongly affected also by the type of tissue and the initial level of reduced ascorbic acid. strawberry-based product attracted in the recent years the food industry due to the high amount of bioactive compounds (vitamin c, anthocyanins and flavonols). for this reason strawberries are one of the r i c h e s t f r u i t s i n t e r m o f a n t i o x i d a n t c a p a c i t y (cordenunsi et al., 2002; pertuzatti and barcia, 2015). understanding the stress-induced changes is important in order to develop reliable approaches to control the stress responses, and improve the quality of minimally processed fresh products, particularly when the fruit is subjected to a pronounced mechanical damage. this makes very interesting the investigation on the effect of wounding on soft and fresh fruit like strawberries. therefore, the main objective of the present study was to determine the effect of wounding intensity, on physiological and quality changes of fresh strawberry fruit, with a particular focus on the respiration rate and nutritional compounds. 2. materials and methods sample preparation ‘candonga’ strawberries (fragaria x ananassa duch.) were purchased from local stores in foggia (south italy) and stored at 5°c overnight. in the next morning, fruit with uniform color and size, free of physical defects and decay, were divided into six groups (treatments), each one corresponding to one different level of wounding intensity: whole fruit (no cutting), cutting into 4, 16, 64, 128, pieces and chopped defined as who, p4, p16, p64, p128 and solomon et al. effect of wounding intensity on quality of strawberry fruit 435 cho, respectively. about 12-15 fruit were cut (300 g) for each replicate and 150 g of product were used for initial determinations, whereas the remaining samples were stored at 5°c under a continuous flow of humidified air for 2 days. three replicates were used for each cutting intensity treatment and for each quality parameter determination. vitamin c, total phenolic and anthocyanin content, antioxidant capacity, soluble solid content, ph value, titratable acidity and sugar/acid ratio were evaluated at the processing day (day 0) and after storage (day 2). separate samples were used for respiration rate measurement. respiration rate respiration rate of fresh-cut strawberries was measured in static conditions as described in kader (2002). respiration rate (expressed as µg kg-1 s-1 of co 2) was determined at 120 min after cutting. separate samples (about 300 g each) of strawberries were placed in 5 l sealed glass jars with a plastic septum for sampling gas; jars were closed after an equilibration time of about 1 hours. from each jar, a gas sample (0.5 ml) was collected after the required time to accumulate co2 in the headspace up to a concentration of 0.1-0.2%, and injected into a gas chromatog r a p h ( s h i m a d z u , m o d e l 1 7 a , k y o t o , j a p a n ) , equipped with a thermal conductivity detector ( 2 0 0 ° c ) . s e p a r a t i o n o f c o 2 w a s a c h i e v e d o n a carboxen 1006 plot (30 m × 0.53 mm, supelco, bellefonte, pa, usa), with a column flow of 7 ml min−1, and an oven temperature of 180°c. compositional attributes vitamin c content was measured in 5 grams of fresh homogenized strawberry tissue as l-ascorbic acid (aa) and l-dehydroascorbic acid (dha) contents expressed as g of aa, dha or total vitamin c (aa + dha) per 1 kg of fresh weight (g kg-1) following the procedure by zapata and dufour (1992) with slight modifications. total phenolic content (tpc) was analyzed using the folin-ciocalteau method of singleton and rossi (1965), with some modifications where five grams of fresh tissue were homogenized in an ultraturrax (ika t18 basic, wilmington, nc, usa) with 10 ml of extraction buffer containing 200 ml of distilled water, 800 ml of methanol and 2 mm (84 mg l-1) of sodium fluoride (naf). the absorbance was read at 725 nm compared with a blank (prepared in the same way, replacing the sample with 100 µl of distilled water) using a spectrophotometer (uv-1700 shimadzu, jiangsu, china). tpc was calculated based on the calibration curve of gallic acid and results were expressed as g gallic acid equivalents per 1 kg fresh weight (g kg-1). total anthocyanin content (tac) was determined following the protocol described by cordenunsi et al. (2002), with small modifications using hydrochloric acid/methanol mixture as extraction medium. 700 µl of extract plus 300 µl of 1% hcl-meoh solution were put in cuvettes and absorbance was read immediately in a spectrophotometer at 510 nm. results were expressed g of pelargonidin-3-glucoside (pg-3-glu) equivalents per 1 kg of fresh weight (g kg -1). the antioxidant capacity (ac) assay was conducted on the same extract made for tpc, following the method of brand-williams et al. (1995), with slight modifications. fifty µl of extract were mixed with 950 µl of dpph (2, 2-diphenylpicrylhydrazyl) solution and absorbance was read at 515 nm after 24 h. trolox (6-hydroxy-2, 5, 7, 8-tetramethlychromane-2carbxylic acid) was used as a standard and results were expressed in g of trolox equivalents per 1 kg of fresh weight (g kg-1). total soluble solids (tss) were obtained by measuring the refractive index of fresh strawberry juice using a digital refractometer (atago rx-7000cx; atago co. ltd., japan) at 25°c and expressed as percentage. one gram of sample was used to determine the ph and titratable acidity (ta), with an automatic t i t r a t o r ( t 5 0 m t e r m i n a l , m e t t l e r t o l e d o , switzerland) against a volume of 0.1 n naoh until reaches the final ph of 8.2. ta was expressed as g of citric acid equivalent per 1 kg of product (g kg-1). tss/ta ratio was also calculated. statistical analysis data were subjected to a 2-way anova (for treatment and sampling time); treatment means were separated by tukey’s test at p<0.05 using stat graphics centurion xvi.i (stat point technologies, inc., warrenton, va usa) software. 3. results wounding intensity had a significant effect on all parameters except ph; the same for storage time with exception of total phenolic content, while the interaction between the two factors showed a significant effect on respiration rate, ascorbic and dehydroascorbic acids, total vitamin c, total phenolic content and antioxidant capacity but no significant effect adv. hort. sci., 2018 32(3): 433-441 436 on tac, tss, ph, ta and tss/ta ratio. following, these effects are described in detail. the effect of w o u n d i n g i n t e n s i t y o n t h e r e s p i r a t i o n r a t e o f ‘candonga’ strawberry fruits after 120 min post-cutting at 5°c is shown in figure 1. wounding induced a significant raise in respiration of strawberries tissues that resulted to be increasing with cutting intensity up to a certain point from 5.5 (who) to 10.01 µg kg-1 s -1 of co 2 (p64). further increase of wounding beyond p128 did not stimulate respiration rate: a significant decrease up to a minimal level of 2.81 µg kg-1 s-1 was observed for chopped samples. the effect of wounding intensity on aa, dha and total vitamin c content (aa+dha) of fresh-cut ‘candonga’ strawberries is shown in figure 2. at day 0, aa and total vitamin c were not significantly affected by wounding intensity, with almost similar values referred to ascorbic acid among treatments ranging from 0.37 (p64) to 0.44 g kg-1 (p4). on the other hand, an increase in dha content could be observed already at day 0 with a significant difference among treatments ranging between 0.067 g kg-1 in the who and 0.22 g kg-1 in the treatment p128. the mean values of total vitamin c content at day 0 ranged from 0.51 (who) to 0.61 (p128) g kg-1. p128 and cho samples showed in fact significantly lower values of ascorbic acid (0.27 and 0.17 g kg-1, respectively) than other treatments, with 64-piece sample showing an intermediate behavior (0.41 g kg-1). moreover a significant increase of dha with the increase of wounding intensity was observed for all samples starting from p16. the lowest and highest dha level at day 2 were found with the who and cho samples (0.12 and 0.55 g kg-1, respectively). the highest level of wounding intensity (cho) induced about a 5-fold increase in dha content. the effect of wounding intensity on tpc, tac, and ac of fresh-cut ‘candonga’ strawberries is shown in table 1. the different cutting stress significantly affected the tpc of fresh-cut strawberries. at day 0, p128 had significantly higher (p<0.05) total phenolic content compared to other treatments with the exception of p64, with p16 showing the lowest amount (2.27 g kg-1 of gallic acid equivalent) and, together with p4 (2.37 g kg-1), resulting in significantly lower content of phenolic compounds than whole fruit. there was no significant difference in tac both at day 0 and 2 although the mean tac value of sample p4 showed slight increase during storage time. the mean value of tac ranged from 0.19 (p4) to 0.22 (cho) g kg-1 of pg-3-glu (table 1). wounding intensity also significantly affected antioxidant capacity of fresh ‘candonga’ strawberries. at day 0, samples cut into 16 (6.35 g kg-1 of trolox equivalent) and 64 (6.59 g kg-1) pieces had significantly higher ac than the cho (5.41 g kg-1) and who (5.20 g kg-1) samples. a similar trend was also observed after 2 days from cutting when p16 sample showed significantly higher ac value when compared to the cho samples, although this difference was not significant if compared to the rest of the treatments. a particular behavior was observed for cho strawberries (table 1): no significant effect of storage time was observed on the tpc, tac, and ac values of this sample indifig. 2 effect of wounding intensity on ascorbic acid (aa, black bars, g kg-1), dehydroascorbic acid (dha, grey bars, g kg-1) and total vitamin c content (sum of aa and dha, g kg-1) of fresh-cut ‘candonga’ strawberries at day 0 and day 2 of storage at 5°c. who, p4, p16, p64, p128 and cho stay for whole fruit, cut into 4, 16, 64, 128 pieces and chopped. mean value (n=3) is reported. different lowercase and uppercase letters, indicate significant differences among treatments and storage times, respectively, according to tukey’s test (𝑃≤0.05). different italics letters indicate significant differences among treatments for total vitamin c content for each storage time. fig. 1 effect of wounding intensity on respiration rate at 5°c of ‘candonga’ strawberry fruit. who, p4, p16, p64, p128 and cho stay for whole fruit, cut into 4, 16, 64, 128 pieces and chopped. error bar represent st. dev of mean values (n=3). different lowercase letters indicate significant difference among treatment according to tukey’s test (𝑃≤0.05). solomon et al. effect of wounding intensity on quality of strawberry fruit 437 respiration rate gives an immediate overview of the metabolism of a commodity (fig. 1), where higher respiration is an indicator of accelerated metabolism which is usually inversely related to shelf-life. strawberry is among the commodities with highest respiration rate (saltveit, 2002), that can be further i n c r e a s e d a s a c u t c o n s e q u e n c e . a c c o r d i n g t o surjadinata and cisneros-zevallos (2003), an increase in respiration may occur due to simultaneous enzyme synthesis and decrease. the newly synthesized enzymes could in fact be degradated by an inactivation system. it is possible that, after a certain cutting degree, the very high tissue damage compromised the cell functionality. the transition from respiring to non-respiring tissues after wounding is probably related to the damage of the membrane system or mitochondria and consequent disruption of oxidative phosphorylation. changes in ammonium dihydrogen phosphate (adp) and ammonium transferase phosphate (atp) concentrations in wounded tissue indicate that oxidative phosphorylation failed to keep place with atp utilization in injured tissues (lafta and fugate, 2011). according to these authors, a 41% reduction in atp concentration and a simultaneous increase in adp (31%) were observed between day 1 and day 4 after incremental injury of sugar beet root. costa et al. (2011), reported that respiration rates of fresh-cut strawberries was higher than whole fruit and that low storage temperature significantly influenced this parameter. this is possibly due to the fact that cutting increases the surface exposed to the air, and as a consequence, oxygen is able to diffuse into the internal cells more rapidly. moreover, injured cells show an increased metabolic activity (nilsson and hedenqvist, 2011; saltveit, 1997). thus, respiration is stimulated by physical damages given to the cating that these compounds were quite preserved during cold storage, and did not show after two days of storage great variations if compared with other samples. in addition, this sample showed also the lowest respiration and no significant difference in total vitamin c if compared to the who samples. these characteristics may be therefore exploited to maintain the quality of fresh-blended products. tss, ph value, ta and tss/ta ratio of fresh-cut strawberry subjected to different wounding intensity and stored for two days at 5°c were significantly affected by wounding intensity (with exception of ph) and storage time, but not by their interaction (data not shown). the mean values of tss ranged from 7.6 (who at day 0) to 8.6% for who, p16 and cho samples at day 2. almost similar ph values (3.9 to 4.0) were determined for all treatments and sampling times. ta ranging from 0.07 to 0.08 g kg-1 of citric acid showed almost no differences among treatments and sampling time except for p64 which had a slightly lowest value after two days. similarly, almost no differences in the tss/ta ratio were recorded. the highest and lowest values were 9.4 (p4 at day 0) and 12.1 (p16 at day 2). 4. discussion and conclusions the effect of wounding intensity on respiration rate and compositional values of fresh ‘candonga’ strawberry fruit was clearly determined. in general, high degree of wounding intensity (p128 and cho) caused a significant decrease in respiration rate. in addition, the chopped sample did not show any significant difference in tpc, tac and ac during storage time. days at 5 °c wounding intensity who p4 p16 p64 p128 cho total phenolic content 0 2.88±0.07 ab 2.37±0.16 bc 2.27±0.04 bc 2.95±0.02 aab 3.23±0.08 aa 2.94±0.05 ab 2 2.62±0.12 bb 3.19±0.18 aa 3.25±0.17 aa 2.52±0.15 bb 2.87±0.10 bab 2.58±0.07 ab total anthocyanin content 0 0.19±0.004 ns 0.19±0.007 ans 0.20±0.004 ns 0.21±0.018 ns 0.20±0.019 ns 0.20±0.010 ns 2 0.21±0.015 ns 0.21±0.002 bns 0.21±0.014 ns 0.22±0.002 ns 0.22±0.021 ns 0.22±0.012 ns antioxidant capacity 0 5.89±0.03 abc 6.02±0.05 aabc 6.35±0.12 aa 6.59±0.09 aa 6.27±0.12 aab 5.41±0.06 ac 2 5.20±0.22 bab 5.98±0.44 bab 6.16±0.02 ba 5.44±0.03 bab 5.67±0.09 aab 4.62±0.10 ab table 1 effect of wounding intensity on total phenolic ontent (tpc, in g kg-1 of gallic acid equivalent), total anthocyanin content (tac, in g kg-1 of pelargonidin-3-glucoside), and antioxidant capacity (ac, in g kg-1 of trolox equivalent) of fresh-cut ‘candonga’ strawberries at day 0 and day 2 of storage at 5°c iwho, p4, p16, p64, p128 and cho stay for whole fruit, cut into 4, 16, 64, 128 pieces and chopped; mean values (n=3) ± standard deviations are reported. different lowercase and uppercase letters, indicate significant differences among treatments and storage times, respectively, according to tukey’s test (𝑃≤0.05). 438 adv. hort. sci., 2018 32(3): 433-441 fruits: the more the severity of damage, the more the degree of respiration rate increase (kader, 1987; zhu et al., 2001). the main consequence of an increase in co2 and ethylene production as a response to cutting process (saltveit, 1997) could be a reduction of the fresh-cut product shelf life. moreover, moisture in the cut surface may impede gas diffusion, and this, together with increased respiration, could possibly lead to anaerobiosis, causing further deterioration of the tissues (saltveit, 1997; surjadinata and cisneroszevallos, 2003). however, wound-induced respiration depends on the type of tissue, temperature, controlled atmospheres and degree of cutting (zhu et al. 2001). as described by surjadinata and cisneroszevallos (2003) respiration rate of carrot tissues after wounding showed a typical increase (resulting in a m a x i m u m p e a k ) a n d t h e n a d e c r e a s e r e a c h i n g steady-state respiration values similar to that of whole carrot. in some plant tissues, such as potato, this behavior may be related to the oxidation of fatty acids and carbon dioxide, being these reactions responsible for increased respiration after wounding (gunes and lee, 1997). according to surjadinata and cisneros-zevallos (2003), wounding stimulates enzym a t i c a c t i v i t y o f p h o s p h o f r u c t o k i n a s e a n d cytochrome oxidase from the respiration pathway, which catalyse the phosphorylation of fructose-6phosphate to fructose-1,6-bisphosphate (a key regulatory step in the glycolytic pathway) and the electrons transfer to oxygen, respectively. a higher enzymatic activity could be due to activation of already present enzyme or to de novo synthesis, as suggested by surjadinata and cisneros-zevallos (2003). their higher activity results in an increase of respiration rate in wounded tissues (lafta and fugate, 2011). on the processing day total vitamin c content did not vary among the treatments, most probably because ascorbic acid was oxidized to dehydroascorbic. the same vitamin c content trend showed in figure 2 was observed by costa et al. (2011), and may be ascribed to the fact that tissue had only slightly responded to the stress. after 2 days from cutting a slight increase in aa was observed for samples p16 and p64 while a noticeable reduction was found with the increase of cutting intensity. as a results of enzymatic and non enzymatic oxidation of ascorbic acid, prolonged storage period, mechanical damages or t h e r m a l t r e a t m e n t , d h a a m o u n t i s k n o w n t o increase. the oxidized form thus represents the majority of vitamin c (davey et al., 2000; lee and k a d e r , 2 0 0 0 ) . i n t h e p r e s e n t s t u d y d i f f e r e n t behaviors were observed: as for p128 and cho samples the decrease in aa amount due to oxidation was accompanied by an increase in dha amount ending in a rise of total vitamin c content of the products. s i n c e t h i s i n c r e a s e w a s p r o p o r t i o n a l w i t h t h e decrease in aa concentration, it can be supposed that no further oxidation of dha into 2,3-diketogulonic acid occurred. regarding p16 and p64 samples at day 2, higher aa amounts were detected, suggesting the occurrence of new synthesis or the presence of different sources of ascorbic acid. cordenunsi et al. (2005), reported that ascorbic acid synthesis in strawberries occurs during the storage period, and that temperature may affect it. as for sweet pepper fruit, it was observed that wounding stress activated both biosynthesis and metabolism reduction of ascorbic acid, leaving unaffected the level of aa in t h e p r o d u c t ( i m a h o r i e t a l . , 1 9 9 7 ) . m o r e o v e r , wolucka and van montagu (2003) proposed a new vitamin c biosynthesis pathway in which l-gulose and l-gulono-1,4-lactone act as direct precursors of ascorbic acid in plant tissue during storage. as for the present work it was possible to suppose that new synthesized ascorbic acid replaced the amount which was oxidized. as a result, ascorbic acid content did not decrease over time while a huge increment in d h a o c c u r r e d f o r p 6 4 s a m p l e . s o m e a u t h o r s observed in different strawberry cultivars that dha evolution with storage time, when associated with ascorbic acid retention, can be considered an evidence of a redox system (aa/dha) triggered during cold storage, reported to be a cultivar-specific more than fruit-specific process (cordenunsi et al., 2005). moreover, to date, many of the products deriving from dha degradation are still unclear, although 2,3diketogulonic acid, threose and oxalic acid, glyoxal, methyl glyoxal and diacetyl have been identified or hypothesized as byproduct of dehydroascorbic acid decomposition (fayle et al., 2000). ascorbate, its product of oxidation (dha) and consequently dha metabolism play significant roles in the apoplast (lin and varner, 1991). for this reason dha results to be a key-factor in ascorbate catabolism, and it could be oxidized to oxalate or hydrolyzed to 2,3-diketogulonate and downstream carboxypentonates. the prevalence of one of the two reactions (oxidation or hydrolysis) is dependent on the status of the reactive oxygen species (parsons et al., 2011). in general, dha/aa ratio tends to rise during storage time although the oxidized form is unstable and is easily decomposed, this leading to a decrease in its biological activity. the changes in the form of ascorbic acid result to be important from a technological and a 439 solomon et al. effect of wounding intensity on quality of strawberry fruit nutritional point of view (lee and kader, 2000; cordenunsi et al., 2005). a possible explanation for what is showed in table 1 may be found in the prevalence of phenolics de novo synthesis with respect to their oxidation as response to cutting stress. as described by kang and saltveit (2002), the cutting related to processing of fresh-cut fruit and vegetables may induce an increase in their antioxidant capacity, enhancing synthesis and accumulation of phenols. in fact, wounding stimulates the activity of phenylalanine ammonia-lyase, which is responsible for the catalyzation of the first step of phenylpropanoid metabolism due to which tartaric acid is converted into chlorogenic acid. an increase in its activity leads to accumulation of phenols, enzymatic oxidation and tissue discoloration (saltveit, 2000; adams and brown, 2007). in contrast, however, wounding also induces antioxidant degradation, resulting in oxidation of active compounds such as ascorbic acid and phenolic compounds. their final content is the balance result between production and oxidation rates, being these rates affected by storage temperature or cutting intensity as modeled by amodio et al. (2014). however, in the case of a more serious damage, as for cho samples, tpc degradation was supposed to be faster than their production, ending in the lowest value of tpc. at day 2 however, tpc increased more clearly in p4 and p16 samples, which showed significantly higher values than other treatments, including the who. this is supported by literature, torres-contreras et al. (2014) report that phenolic content in white potato tubers that were subjected to different wounding stresses showed an accumulation of 100% and 65% tpc for slices and pieces, respectively, whereas shredded potatoes stored at 10°c for 96 hours showed 40% lower phenolic content if compared to the starting product. similarly, an accumulation of wounding-induced tpc (approximately 60%) and an increase in the activity of pal enzyme were found in purple-flesh sliced potato tissues stored at 15°c for 4 8 h o u r s ( r e y e s a n d c i s n e r o s z e v a l l o s , 2 0 0 3 ) . according to surjadinata and cisneros-zevallos (2003), phenolic antioxidant accumulation is dependent on the level of wounding intensity. the phenolic content increased with wounding intensity by 97, 76, and 252% when cut as slices, pieces and shreds, respectively compared to non-wounded carrots (0.45-0.52 g kg-1). moreover, the same behavior was observed by hu et al. (2014) on fresh-cut lotus root. after 7 days of storage pal activity of fresh-cut lotus root slices was 68% higher compared to control. at the same time 130% increase in phenolic content was detected. antioxidant activity also increased due to total phenol accumulation; however, wounding resulted in a significantly higher browning by increasing the ppo activity of the slices. phenols represent the main structural and defense-related functions in p l a n t c e l l s v i a t h e p h e n y l p r o p a n o i d p a t h w a y . wounding of fruit and vegetables tissues obviously causes rupture of the cell membrane and this induce several physiological responses causing the combination of phenolics with the oxidative enzymes and/or the synthesis of different classes of phenolics to repair the wounding damage. in strawberries, tac was reported to be in the range between 0.15 to 0.80 g kg-1 (padmanabhan et al., 2016) and cold storage may induce their biosynthesis and accumulation (holcroft and kader, 1999). accordingly, about 60% accumulation of tac in sliced potatoes was described by reyes and cisneros-zevallos (2003), and a similar increase was reported for anthocyanin content in pomegranates stored at low temperatures (arendse et al., 2014). the high amount of vitamin c, phenolics including anthocyanins and therefore antioxidant activity in strawberries, even after severe cutting stress, is very important for beneficial effects on consumer health (giampieri et al., 2012). the relatively higher values of ac determined for cho sample could probably be due to the higher values of total vitamin c and phenolics. antioxidant capacity of fruit is, in fact, strictly related to the presence of vitamin c and phenolic compounds, being these effective systems to scavenge oxygen radical, ac level is influenced by the occurrence of different active phytochemical compounds (giampieri et al., 2012). as reported by tulipani et al. (2008), vitamin c in strawberries is the greatest contributor (>30%) to the total antioxidant capacity followed by anthocyanins (contributing for 25-40%). the remaining part was composed mainly of ellagic acid derivatives and flavonols (padmanabhan et al., 2016). antioxidant activity is therefore an expression of total vitamin c and total phenolic content of the product, including anthocyanin and ellagitannins groups (giampieri et al., 2012). as for ph, tss and ta, no clear trend and very slight differences were observed, as also reported by li et al. (2017) studying the effect of different cutting styles on postharvest quality of pitaya fruit. in this case, fresh-cut processing also showed little effect on the contents of vitamin c, tss and ta. this behavior could be interesting since the nutritional quality of the products do not greatly change after treatments, 440 adv. hort. sci., 2018 32(3): 433-441 allowing to maintain their fresh-like state. the results of the present study demonstrated that the application of wounding intensities could be used as simple emerging technology to induce the accumulation of tpc in plants (torres-contreras et al., 2014) and selection of appropriate wounding intensity and/or abiotic stress can enhance the nutritional and functional and health-related values of fresh produce (reyes and cisneros-zevallos, 2003; hu et al., 2014). these results should be taken into consideration for processing and packaging optimization of minimally processed products from fresh strawberries, although further investigations extending shelf life period would be necessary in order to make these results useful for industrial application. references adams j.b., brown h.m., 2007 discoloration in raw and processed fruit and vegetables. crit. rev. food sci., 47: 319-333. amodio m.l., derossi a., colelli g., 2014 modeling phenolic content during storage of cut fruit and vegetables: a consecutive reaction mechanism. j. food eng., 140: 1-8. arendse e., fawole o.a., opara u.l., 2014 effects of postharvest storage conditions on phytochemical and radical-scavenging activity of pomegranate fruit (cv. wonderful). sci. hortic. amsterdam., 169: 125-129. brand-williams w., cuvelier m.e., berset c., 1995 use of a free radical method to evaluate antioxidant activity. lwt food sci. technol., 28: 25-30. brecht j.k., 1995 physiology of lightly processed fruits and vegetables. hortsci., 30: 18-22. chung t.t., west g., tucker g.a., 2006 effect of wounding on cell wall hydrolase activity in tomato fruit. postharvest biol. tec., 40: 250-255. cordenunsi b.r., genovese m.i., oliveira do nascimento j.r., aymoto hassimotto n.m., josé dos santos r., lajolo f.m., 2005 effects of temperature on the chemical composition and antioxidant activity of three strawberry cultivars. food chem., 91: 113-121. cordenunsi b.r., oliveira do nascimento j.r., genovese m.i., lajolo f.m., 2002 influence of cultivar on quality parameters and chemical composition of strawberry fruits grown in brazil. j. agr. food chem., 50: 2581-2586. costa f.b., duarte p.s., puschmann r., finger f.l., 2011 quality of fresh-cut strawberry. hortic. bras., 29: 477-484. davey m.w., montagu m.v., inzé d., sanmartin m., kanellis a., smirnoff n., benzie i.j.j., strain j.j., favell d., fletcher j., 2000 plant l-ascorbic acid: chemistry, function, metabolism, bioavailability and effects of processing. j. sci. food agric., 80(7): 825860. el-ramady h., domokos-szabolcsy é., abdalla n., taha h., fári m., 2015 postharvest management of fruits and vegetables storage pp. 65-152. in: lichtfouse, e. 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and these treatment enhanced total phenol, anthocyanin and antioxidant activity in the three cultivars. in both years of experiment, rdi remarkably enhanced anthocyanin, flavonoids, antioxidant activity and phenolic concentration. overall, the results indicated that 80% etc might be sufficient to gain adequate yield in ‘keshmeshi’, ‘sahebi’ and ‘sharabi’ without undermining the quality of fruit. 1. introduction water deficiency is one of limiting factors influencing grape production throughout the world (rabiei et al., 2003). in order to improve water use efficiency (wue), it is necessary to recognize factors restricting wue and then introduce appropriate approaches for mitigating the harmful effects of such factors (mccarthy et al., 2002). in this respect, balancing (*) corresponding author: ghasemnezhad@guilan.ac.ir citation: ashori m., ghasemnezhad m., biglouei m.h., 2019 influence of post-véraison water deficit on berries yield and quality of three table grape cultivars. adv. hort. sci., 33(1): 67-75 copyright: © 2019 ashori m., ghasemnezhad m., biglouei m.h. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 10 april 2018 accepted for publication 17 october 2018 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(1): 67-75 68 between vegetative and reproductive phases in plants in favor of hindering excess vegetative growth is one of these approaches being used to improve not only wue but also fruit quality of grapevine, because excess growth of shoots may have adverse impact on fruit quality (chalmers et al., 1981). reduction in vegetative growth in favor of improving wue can be implemented by many approaches. one of such approaches is to constrict the amount of water required for plant growth through reducing the number of irrigation or decline in amount of water in each irrigation (chalmers et al., 1981; goodwin and jerie, 1992). for achieving this purpose, regulated deficit irrigation (rdi) was introduced (santesteban and mirandam royo, 2011). as more than 82% of iran territory is located in arid and semiarid zone with drought climate and extreme temperature changes in the world (amiri and eslamian, 2010), this approach may pave the way for planting crop in a vast majority of abandoned farmland deprived of adequate water distribution, although employing this approach may reduce crop production because of reduction in water use (chalmers et al., 1981). under drought condition, rdi abridges vegetative growth in favor of enhancing reproductive growth and this could improve yield (santesteban and mirandam royo, 2011). at critical stages of plant growth and development, change in water status certainly influences fruit quality by affecting vegetative growth and fruit development (van leeuwen et al., 2004; ezzhaouani et al., 2007). therefore, rdi improves cluster quality through reduction in berry drop (ojeda et al., 2002). under a mild drought stress, acevedo-opazo et al. (2010) proposed an appropriate threshold for managing rdi schedule to keep quality of grape fruits. the positive rdi effect on phenolic compounds, tss, and anthocyanins has previously been documented (ojeda et al., 2002; van leeuwen et al., 2004). the highest yield (48 kg a tree) with high fruit quality was observed in seedless grape cultivars experiencing rdi under drought condition (faci et al., 2014). according to the results obtained by zabihi and azarpajouh (2004) and dolatibaneh and norjo (2012), rdi probably decrease photosynthetic capability in leaves of grape; and as a result, it reduces (fruit) yield. in other research, it was shown that rdi increased berry quality and increase water use efficiency in grape trees pinillos et al. (2016). rabiei et al. (2003) pointed out that using rdi at the end of growth season significantly increased phenolic compounds in merlot cultivar. with respect to mentioned issues, the current experiment was designed to investigate effects of different rdi treatments on yield and fruit quality of the three grape cultivars (i.e. keshmeshi, sahebi and sharabi) during the onset of véraison to harvest under climate of khorramabad province, iran. 2. materials and methods vineyard site and experimental design this experiment was conducted during the 2015 and 2016 growing seasons at semiarid climate in lorestan province, iran (47°e, 51°n, 1400 m). ownrooted vitis vinifera l. cvs. keshmeshi, sahebi and sharabi were planted in 2007 by spacing of 3×2 m in n-s oriented row. vines were trained to spur-pruned bilateral cordon with 6, 4 and 4 nodes per vine in ‘keshmeshi’, ‘sahebi’ and ‘sharabi’, respectively according to flowering type. the vineyard was dripirrigated using pressure-compensated emitters (flow rate 4 lh-1). the vines were fertigated from june with a nutrient compound containing 150 kg ha-1 n, 25 kg ha-1 k, 6 l ha-1 s and 4 kg ha-1 fe chelate. three rdi strategies were compared with the full irrigation practice (control). specifically, the control received full irrigation over fruit growth and development (etc 100), whereas rdi treatments were: 80 % of etc, 60% of etc and 40% of etc. rdi treatments were applied at the onset of berries color change (véraison) to harvest time. daily metrological data were obtained from a database of khoramabad’s meteorology station located closely to the experimental vineyard and water requirement for each vine was calculated according to cropwat 8.0 software. furthermore, irrigation scheduling was based on soil water holding capacity, daily crop water requirement, effective depth of root, percentage of wetted soil surface according to defined treatments (100%, 80%, 60% and 40% etc) using the following relations. where in= net irrigation water content (mm), fc= field capacity (%), pwp= permanent wilting point (%), pb= soil bulk density (gr.cm-3), dr= effective depth of root (120 cm) i= irrigation interval (day), k= operation coefficient of rdi treatments, etc= plant ashori et al. post-veraison water deficit on table grape cultivars 69 water requirement. all vines were equally irrigated based on their water requirement before starting rdi treatment. in this study, the drip irrigation system was equipped with two droplets adjusted manually for 4 lh-1 per vine. in order to access required pressure, a floating pump was used and the amount of water allocated to each irrigating turn was measured by water counter with 0.1 l accuracy. directing water into each plot containing three grapevines was performed through stopcock water equipped in drip irrigation. for each irrigation treatment, three replications were established according to a randomized block design. each replication consisted of five lines of 60 vines each, and the experimental measurements were performed on 20 homogeneous vines from the 3 central lines chosen at the beginning of the study according to their trunk cross sectional area (tcsa). sampling and analysis the berries were harvesting from 10 vines per each treatment and three samples for each vine, based on minimum maturity index of sugar content and berry color change. the mean total yield, clusters weight, number of berry per cluster and berry weight was measured from 10 vines for each treatment and three samples for vines. soluble solid content (ssc) was measured by a digital refractometer (model eurromex rd). titratable acidity (ta) was measured with a digital titrator in presence of 0.1 naoh and ph was determined by a digital ph meter (model sclto tt). total phenolic was determined by using the folinciocalteau method (brand-williams et al., 1995) with slight modification in a uv-vis spectrophotometer (t80+, pg instruments ltd). briefly, 0.5 g of extract was mixed with 3 ml of methanol 85%, and then centrifuged for 10 min by 10000 per min. then, 300 µl of diluted extract was mixed with 1.5 ml of diluted folin-ciocalteau reagent (1:10 in distilled water). after vortexing, 1.2 ml of 7.5% sodium carbonate was added and allowed to stand for 90 min at room temperature. the absorption at 760 nm was measured and results were expressed as gallic acid equivalents (gae) per 100 g fresh weight. total flavonoids content were determined spectrophotometerically according to the zarrouk et al. ( 2 0 1 2 ) . c a t e c h i n w a s u s e d a s a s t a n d a r d . t h e flavonoids content were expressed as mg catechin equivalents (re) per 100 g fw. total antioxidant capacity (tac) was measured using the 2, 2 diphenyl-1-pic-rylhydrazyl (dpph) radical scavenging method described by brand-williams et al. (1995) with slight modification. the amount of 75 μl of fruit extract was mixed with 2925 μl 0.1 mol l-1 dpph in methanol. after incubating at room temperature for 30 min in the dark, the absorbance of the mixture was measured at 517 nm using uv-vis spectrophotometer. for each sample, three separate determinations were recorded. antioxidant activity w a s e x p r e s s e d a s t h e p e r c e n t a g e d e c l i n e i n absorbance in comparison with the control, corresponding to the percentage of dpph scavenged (% dpphsc), which was calculated as: %dpphsc = [(acontrol-asample)/acontrol]× 100 data analysis the results obtained for each irrigation treatment were compared using anova and when significant differences were found, duncan’s test was used to identify differences in mean values. data from each year were analyzed separately; all analyses were performed with sas 17.0. 3. results and discussion fruit yield the results of anova showed that simple effect of rdi on total yield at first year was significant (p<0.01). compared to controls, 80% etc did not show a significant effect on yield at first year, whereas applying 40% and 60% etc reduced fruit yield by 33% and 48%, respectively. also, it was found that sahebi in comparison to sharabi gained higher fruit yield at first year (table 1). the results of this experiment showed that the interaction effect of cultivar × rdi had a significant effect on yield in second year of experiment (fig. 1). in ‘keshmeshi’ and ‘sharabi’, using 40% etc significantly resulted in a yield reduction, while 40% and 60% etc decreased fruit yield in sahebi. santesteban and mirandam royo (2011) and di vaio et al. (2001) showed that rdi brought about a reduction in grape yield, although they proposed rdi as a useful approach to enhance quality and quantity of fruits in the grapes grown especially in subtropical climate. on the contrary, dolatibane and norjo (2012) and zabihi and azarpajouh (2004) stated that rdi could reduce grape yield. the negative effect of rdi on fruit yield is associated with its harmful effect on photosynthesis activities and consequently supplying required assimilates for berry’s formation adv. hort. sci., 2019 33(1): 67-75 70 (conesa et al., 2016). also, a remarkable increase in transpiration rate of berries under severe drought stress can be accounted for losing berries weight and lowering grape yield. conesa et al. (2016) reported that rdi had not a significant effect on grape yield and rdi-experiencing plants had the same yield as controls. in this study, we also didn’t find any significant differences between 80% rdi and control during two consecutive growing seasons. berry weight and cluster weight the results of anova showed that berry weight was significantly affected by simple effects of cultivar and rdi (p<0.01). the interactional effect of cultivar × rdi had a significant effect on cluster weight in both years of experiment (p<0.01). in general, the weight of grape berry and cluster was significantly decreased as the percentage of rdi was gradually decreased (from 100% to 40% etc) in both experimental years. comparing to controls, although applying 40% and 60% etc reduced cluster weight of ‘keshmeshi’ at first year, all rdi treatments did not render a reduction in cluster weight of keshmeshi in second year of experiment. in ‘sahebi’, cluster weight was diminished by applying 40% and 60% etc at first year, whereas by just 40% etc in second year of experiment. in this regard, all rdi treatments reduced berry and cluster weight of sharabi while 80% etc did not significantly reduce cluster weight in sharabi only in second year (fig. 2). the results of this research revealed that effect of rdi on berry and cluster weight is strongly correlated to cultivars. according to coombe (1992), change in berry’s water status under drought is directly related with change in berry weight. rabiei et al. (2003) figured out that rdi may be responsible for a reduction in berry weight in merlot cultivar. in this regard, dolatibaneh and norjo (2012) stated that the highest berry weight was obtained at full irrigation; and, in spite of finding non-significant difference between 50% and 75% etc treatments, they concluded that berry weight is often depressed under rdi performance. as compared to controls, the cell size of fruit experienced different rdi regimes reduced due to reduction in vegetative growth, leaf area, and photosynfig. 1 effect of regulated deficit irrigation (rdi) on fruit yield of three grape cultivars in the second year. bars represent standard error of three replicates. values with the different letters are significantly different according to duncan’s multiple range test at p<0.05. season treatment levels yield (kg vine-1) berry weight (g) ssc (%) ta (%) ph 2015 rdi control 23.70 a 1.11 a 17.55 a 0.68 a 3.51 a 80% etc 21.75 a 1.08 a 17.18 a 0.69 a 3.40 a 60% etc 15.90 b 0.89 ab 17.24 a 0.72 a 3.48 a 40% etc 12.24 d 0.72 b 17.12 a 0.66 a 3.48 a significance ** ** ns ns ns cultivar keshmeshi 19.47 ab 0.60 b 19.84 a 0.78 a 3.35 b sahebi 22.77 a 1.28 a 14.15 b 0.6 2b 3.59 a sharabi 15.20 b 0.98 a 17.83 a 0.67 b 3.46 ab significance ** ** ** ** ** 2016 rdi control 24.40 a 0.96 a 20.25 a 0.66 a 3.50 a 80% etc 23.56 ab 0.92 a 21.31 a 0.64 a 3.47 a 60% etc 21.82 b 0.78 b 20.33 a 0.68 a 3.45 a 40% etc 18.71 c 0.62 c 21.67 a 0.67 a 3.42 a significance ** ** ns ns ns cultivar keshmeshi 17.31 b 0.57 b 24.50 a 0.75 a 3.37 b sahebi 29.02 a 0.99 a 17.95 c 0.59 b 3.59 a sharabi 20.04 b 0.90 a 20.22 b 0.65 ab 3.41 b significance ** ** ** ** ** table 1 the effects of regulated deficit irrigation (rdi) on fruit yield and quality of grape cultivars ns= not significant, ** significant at p<0.01, * significant at p<0.05. ashori et al. post-veraison water deficit on table grape cultivars 71 thesis products, and this probably led to reduction in berry and cluster weight (ezzhaouani et al., 2007). in current research, the grape cultivars had different responses to rdi especially at 40% and 60% etc regimes. regarding these results, different responses to rdi treatments suggests the sensitivity of different cultivars to drought stresses. berry length and diameter the data presented in figure 3 showed a significant interaction effect of cultivar × rdi on traits of berry length and diameter. in both years of experiment, 40% etc reduced berry length and diameter, whereas 80% etc did not have a significant effect on both of them. accordingly, the cultivar response to rdi was found different (fig. 3). under rdi performance, the cell size was lower than that of at full irrigation. due to reduction in the vegetative growth and leaf area of rdi-subjected plants, the size of berry and photosynthetic products were reduced (deluc et al., 2009). zabihi and azarpajouh (2004) reported that water deficiency resulted in reduction in the size of berry, which is in agreement with our findings. total soluble solid the results of this research revealed that only simple effect of cultivar on tss was significant in both years of experiment, and the highest and lowest tss were found in ‘keshmeshi’ and ‘sahebi’, respectively (table 1). contrary to dolatibaneh and norjo (2012) who stated that rdi had a positive effect on tss of grape fruit, lanari et al. (2014) figured out a non-significant effect of rdi of tss in grape fruit. according to previous research, mild drought may stimulate aba production in favor of increasing some sugar production through different ways in order to cope w i t h s t r e s s f u l c o n d i t i o n . i n t h i s r e s p e c t , s o m e researchers pointed out that water deficiency could reduce photosynthesis activities and consequently constrict sugar production, but whenever water deficiency is exacerbated even fruits may not ripe properly and this probably affects tss status in fruits (dolatibaneh and norjo, 2012). titratabe acidity and ph the results of this experiment demonstrated that only cultivar treatment had a significant effect on ta at first year of experiment, and additionally sahebi and sharabi obtained the lower ta as compared to keshmeshi cultivar. contrary to the results obtained at first year of experiment (table 1), an interaction effect of cultivar × rdi had a significant effect on ta in second year of experiment. although, rdi did not have effect on ta in ‘keshmeshi’, rdi at 60% etc significantly increased ta in sahebi cultivar. in addition, applying 40% and 60% etc caused a reduction in ta of sharabi cultivar (fig. 4). in both years of experiment, ph was only affected by cultivar; and highest ph was observed in sahebi as compared to the two other cultivars. dolatibaneh and norjo (2012) pointed out that rdi could decline the rate of organic acid of grape fruit. during cluster ripping, coinciding with rising environmental temperature, drought stress is able to decrease acidity of berry and accordingly paves the way for minimizing the rate of ta in grape berries. lanari et al. (2014) reported that rdi did not have a significant effect on fig. 3 effect of regulated deficit irrigation (rdi) on rachis weight of three grape cultivars in two growing seasons. bars represent standard error of three replicates. values with the different letters are significantly different according to duncan’s multiple range test at p<0.05. fig. 2 effect of regulated deficit irrigation (rdi) on berry weight of three grape cultivars in the second year. bars represent standard error of three replicates. values with the different letters are significantly different according to duncan’s multiple range test at p<0.05. 72 adv. hort. sci., 2019 33(1): 67-75 ta, whereas findings of zabihi and azarpajouh (2004) showed water availability increased organic acids in grape berries. total phenol the results of this experiment revealed that the interaction effect of cultivar × rdi had a significant impact on total phenol (fig. 5). comparing to controls, applying only 80% etc increased total phenol in ‘keshmeshi’ while applying all rdi treatments in sahebi enhanced total phenol in both years of experiment. in sahebi, the highest amount of phenol was found by employing 40% (at first year) and 80% (in s e c o n d y e a r ) e t c t r e a t m e n t s . t h e r e s u l t s a l s o revealed that using 40% and 60% etc (at first year) as well as 60% and 80% etc (in second year) had significantly affected total phenol in sharabi. rabiei et al. (2003) reported that applying rdi at the end of growth season significantly increased phenol content of merlot comparing to controls, which is in agreement with our findings. a prolonged drought stress was found to significantly decrease total phenol and other phenolic compounds such as frolic acid, coumaric acid, and caffeic acid (krol et al., 2014). by investigating effects of different rdi regimes on phenolic compounds in shirazi cultivar, ojeda et al. (2002) demonstrated that rdi, dependent on type of phenolic compounds, may change the amount of phenol in grape berries. in this regard, some factors including type of cultivar, climate, soil, media culture, maturely period, and yield are effective on the amount of phenols and other biocompounds in grapes (deluc et al., 2009; bindon et al., 2011; zarrouk et al., 2012). in our research, rdi treatments had different impacts on phenol; and the results of this research, dependent on type of cultivar, suggest that an appropriate rdi can increase total phenol in grape berries. flavonoids t h e r e s u l t s o f t h i s r e s e a r c h r e v e a l e d t h a t flavonoid in berries was influenced by rdi treatments. in general, applying 60% and 80% etc significantly increased flavonoid in all the three cultivars. as compared to other cultivars, ‘keshmeshi’ was lowly affected by rdi, in a way that 60% etc at first year and 80% etc in second year of experiment significantly enhanced flavonoid content in berries. in contrast, applying 80% and 60% etc at first year and 40%, 60%, and 80% etc in second year significantly increased flavonoid content in sahebi. as compared t o t h e s e c o n d y e a r o f e x p e r i m e n t , t h e r a t e o f flavonoid in sharabi was significantly elevated by all rdi treatments at first year of experiment relative to controls (fig. 6). in grape berries, flavonoids are the abundant secondary metabolites influencing wine’s physical features (especially color and astringency) (brillante et al., 2017). the biosynthesis of such secondary metabolites is highly affected by environmental stresses (kuhn et al., 2013). the previous research has demonstrated that regulating vineyard irrigation is efficiently able to increase these metabolites (kennedy et al., 2002). similarly, findings of zarrouk et al. (2012) revealed that rdi significantly increased flavonoid compounds in grape’s aragonez cultivar in fig. 4 effect of regulated deficit irrigation (rdi) on titratable acidity percentage of three grape cultivars in second year. bars represent standard error of three replicates. values with the different letters are significantly different according to duncan’s multiple range test at p<0.05. fig. 5 effect of regulated deficit irrigation (rdi) on total phenol of three grape cultivars during two seasons. bars represent standard error of three replicates. values with the different letters are significantly different according to duncan’s multiple range test at p<0.05. ashori et al. post-veraison water deficit on table grape cultivars 73 the two subsequent years, which are in consistent with ours. deluc et al. (2009) reported that effect of r d i o n f l a v o n o i d c o n t e n t o f c h a r d o n n a y a n d cabernet sauvignon cultivars was different, in a way that its content in former cultivar increased while in latter one decreased. in current research, drought stress at 60% and 80% etc raised flavonoid content in the all grape cultivars and this manifests the positive effect of rdi on increasing grape quality. total antioxidant capacity the results of anova revealed that interactional effect of cultivar × rdi had a significant effect on total antioxidant capacity (tac) in both years of experiment (table 1). as compared to full irrigation, applying 60% and 80% etc at first year of experiment significantly increased tac in ‘keshmeshi’ and ‘sahebi’. in second year of experiment, all rdi treatments interestingly enhanced tac in sharabi relative to controls. moreover, tac was increased by using 60% and 80% etc in keshmeshi, as well as 60% and 80% etc in sahebi, and just 80% etc in sharabi (fig. 7). in a similar way, soukhtesaraee et al. (2017) figured out that drought stress significantly increased tac in chefta and yaghoti cultivars, but did not affect bidanehsefid. tangolar et al. (2015) showed that rdi increased tac a t d i f f e r e n t g r o w t h s t a g e s o f r a z a k i c u l t i v a r . comparing to full irrigation, rdi depending upon some factors such as time of applying rdi and type of cultivar diminished tac in leaf and root of kiszmisz cultivar (krol et al., 2014). plant materials possessing antioxidant properties surely contain different phenolic compounds. the antioxidant properties of these materials, due to their redox ability and chemical structures are able to neutralize free radicals through forming a complex with metal ions (krol et al., 2014). in many previous research, the strong correlation between tac and phenolic content were reported (tangolar et al., 2015). in our research, a positive relationship was found between phenol and tac. anthocyanin the results of this research showed that the interaction effect of cultivar × rdi had a significant effect on anthocyanin content in both years of experiment (table 1). comparing to controls, all the three rdi t r e a t m e n t s i n c r e a s e d a n t h o c y a n i n c o n t e n t i n keshmeshi cultivar. in other words, applying 40%, 60%, and 80% etc at first year and 60% and 80% etc in second year increased anthocyanin contents in sahebi (fig. 8). anthocyanins are a class of phenolic compounds responsible for generating black and red colors in grape epidermis. these compounds possess powerful antioxidant property and also play a significant role in physiological process. according to previfig. 6 effect of regulated deficit irrigation (rdi) on flavonoid content of three grape cultivars on both seasons. bars represent standard error of three replicates. values with the different letters are significantly different according to duncan’s multiple range test at p<0.05. fig. 7 effect of regulated deficit irrigation (rdi) on antioxidant capacity of three grape cultivars on two seasons. bars represent standard error of three replicates. values with the different letters are significantly different according to duncan’s multiple range test at p<0.05. adv. hort. sci., 2019 33(1): 67-75 74 ous study, water shortage can strongly influence anthocyanin accumulation in grape cultivars (ozden et al., 2010; kyraleou et al., 2016). also, bindon et al. (2011) reported that an increase in time of irrigation could remarkably hinder anthocyanin biosynthesis. so far, the effect of drought stress on reduction in canopy’s density in favor of entrance more light radiation into tree canopy has been reported. through this, the light quantitatively required to anthocyanin biosynthesis is supplied (tangolar et al., 2015). previous research showed that rdi, through influencing physiological and hormonal responses, changed the pathway of anthocyanin biosynthesis (shellie, 2011; nelson et al., 2015). in present research, it was found that rdi significantly increased anthocyanin accumulation of iranian grape cultivars in both years of experiment, although the cultivar responses towards rdi were various. 4. conclusions overall, the results of this research revealed the positive effect of rdi (especially 80% etc) on yield and some fruit qualities in keshmeshi, sahebi, and sharabi cultivars. in this regard, rdi significantly increased not only yield but also some fruits’ qualitative features like anthocyanin, phenol, flavonoid, and total antioxidant capacity in comparison to controls. therefore, 80% etc can be advised to maintain and increase fruit qualities of keshmeshi, sahebi, and sharabi cultivars under regional conditions. references acevedo-opazo c., ortega-farias s., fuentes s., 2010 effects of grapevine (vitis vinifera l.) water status on water consumption, vegetative growth and grape quality: an irrigation scheduling application to achieve regulated deficit irrigation. agric. water manag., 97(7): 956-964. amiri m.j., eslamian s.s., 2010 investigation of climate change in iran. j. environ. sci. eng., 3(4): 208-216. bindon k., myburgh p., oberholster a., roux k., du toit c., 2011 response of grape and wine phenolic composition in vitis vinifera l. cv. merlot to variation in grapevine water status. afr. j. enol. vitiv., 32(1): 7188. brand-williams w., cuvelier m.e., berset c.l., 1995 use of a free radical method to evaluate antioxidant activity. lwt food sci. technol., 28(1): 25-30. brillante l., martinez-luscher j., yu r., plank c.m., s a n c h e z l . , b a t e s t . l . , k u r t u r a l s . k . , 2 0 1 7 assessing spatial variability of grape skin flavonoids at the vineyard scale based on plant water status mapping. j. agric. food chem., 65(26): 5255-5265. chalmers d.j., mitchell p.d., van heek l., 1981 control of peach tree growth and productivity by regulated water supply, tree density, and summer pruning (trickle irrigation). j. am. soc. hortic. sci., 106: 307-312. conesa m.r., falagán n., josé m., aguayo e., domingo r., pastor a.p., 2016 post-veraison deficit irrigation regimes enhance berry coloration and health-promoting bioactive compounds in ‘crimson seedless’ table grapes. agric. water manag., 163: 9-18. coombe b.g., 1992 research on development and ripening of the grape berry. am. j. enology vitic., 3(1): 101110. deluc l.g., quilici d.r., decendit a., grimplet j., wheatley m.d., schlauch k.a., cramer g.r., 2009 water deficit alters differentially metabolic pathways affecting important flavor and quality traits in grape berries of cabernet sauvignon and chardonnay. bmc genomics, 10(1): 212. di vaio c., cirillo c., boselli m., masi e., 2001 dry matter accumulation and partitioning of cabernet sauvignon pot-grown vines under different water regimes. adv. hort. sci., 15(1-4): 25-30. dolatibaneh h., norjo a., 2012 effect of deficit irrigation on quantitative and quality traits of fruit and water productivity of three grapevine cultivars. seed plant prod. j., 27(4): 435-450. 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(in farsi). tangolar s.g., tangolar s., tarim g., kelebek h., topcu s., 2015 the effects of bud load and applied water amounts on the biochemical composition of the ‘narince’ grape variety. not. bot. hort. agrobot. cluj napoca, 43(2): 380-392. van leeuwen c., friant p., jaeck m.e., kuhn s., lavialle o., 2004 hierarchy of the role of climate, soil and cultivar in terroir effect can largely be explained by vine water status. joint int. conf. on viticultural zoning, pp. 433-439. zabihi h.r., azarpajouh e., 2004 grape response to different soil moisture regimes. j. soil water sci., 18(10): 34-39. z a r r o u k o . , f r a n c i s c o r . , p i n t o m a r i j u a n m . , brossa r., santos r.r., pinheiro c., chaves m.m., 2012 impact of irrigation regime on berry development and flavonoids composition in aragonez (syn. tempranillo) grapevine. agric. water manag., 114: 18-29. impaginato 13 adv. hort. sci., 2019 33(1): 13-21 doi: 10.13128/ahs-22679 eco-physiological and biochemical characterization of rhus tripartita (ucria) grande growing in algerian sahara under arid climate a. benaissa 1, 2 (*), r. djebbar 1, l. boucelha 1 1 department of biology and physiology of organisms, laboratory of plant physiology, faculty of biological sciences, usthb bab ezzouar bp 16011 algiers, algeria. 2 laboratory of science and environment research, universitary center o f a m i n e e l o k k a l e l h a d j m o u s s a e g . a k h a m o u k h , b p 1 1 0 3 9 tamanrasset, algeria. key words: ahaggar, aridity, biochemistry, ecophysiology, rhus tripartita. abstract: rhus tripartita (ucria) grande, is an anacardiaceae autochthonous shrub of the algerian sahara. its ecological, pastoral and therapeutics interests prompted us to carry out an eco-physiological and biochemical behavior in relation to aridity. therefore, relative water content of shrub leaves were found on average 81.55% and the maximum of electrolyte leakage recorded was 14.29. the biochemical determination of proteins and sugars shows that leaves are a true source of protein (33.76 mg/g fm) and sugars (938.93 μg/g fm) while the proline assay gave a value of 824.40 μg/g. the quantitative study of flavonoids in the leaves gave a result of 36.53 mg/g. the analysis of photosynthetic pigments content showed respectively results of 28.1 μg/g, 31.24 μg/g, 56.47 μg/g and 11.23 μg/g for chlorophyll a, chlorophyll b, total chlorophyll and carotenoids. the total antioxidant capacity was evaluated and gave result of 95.5 mg gae/g. therefore, rhus tripartita was found to accumulate high proportions of primary and secondary metabolites which showed a good adaptation to its arid environment. in conclusion, the plant can be considered as a xeromorphic plant, that is, a desert-adapted plant that is not limited by the water availability. 1. introduction rhus tripartita (ucria) grande is synonymous with searsia tripartita (ucria) moffett, an anacariaceae forage plant; it is traditionally used by the tuaregs (local inhabitants) of ahaggar (algeria). this saharomediterranean shrub is widespread from north africa to egypt (sahki and sahki, 2004). ferchichi (1999) has described the shrub as a very droughtresistant species characterized by abundant foliage throughout the year despite the soil’s moisture status, and it can be planted successfully on poor and marginal lands. on the geomorphological level of the algerian (*) corresponding author: benaissa.asmaa@yahoo.fr citation: benaissa a., djebbar r., boucelha l., 2019 eco-physiological and biochemical characterization of rhus tripartita (ucria) grande growing in algerian sahara under arid climate. adv. hort. sci., 33(1): 13-21 copyright: © 2019 benaissa a., djebbar r., boucelha l. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 15 february 2018 accepted for publication 7 september 2018 ahs advances in horticultural science adv. hort. sci., 2019 33(1): 13-21 14 saharan environment, this plant belongs to the grouping of the ahaggar mountainous massifs where it grows on shallow soils. plant growth is strongly influenced by many biotic and abiotic factors. ecologists and physiologists have long been interested in the effects of environmental factors that lead to plant adaptation and distribution. therefore, phyto-ecophysiological studies are important to understand the species response in extreme environments and especially their valuation in these areas. by staying in extreme environments, the sahara is one of the driest and hottest deserts in the world (dutil, 1971). the desert fact is explained by extreme values of climatic parameters which constitute the essential factors of vegetation’s distribution and the grounds evolution (daoud and halitim, 1994). moreover, aridity is a natural selection force that influences plant adaptive strategies to water stress (sayed, 1998). hence different adaptive strategies of plants to aridity have been developed through considerable survival mechanisms. these strategies are divided into three general categories: drought escape, drought avoidance and dehydration tolerance (levitt, 1972; turner, 1979). drought escape is employed by plants under conditions where water limitation late in the growing season is likely, and it ensures that the plants can complete their life-cycle quickly during the brief period of favorable conditions. drought avoidance is a strategy for avoiding lower water status during drought by maintaining relatively high tissue water content due to reduced evaporatory water loss and an efficient water uptake (levitt, 1972). the third category, drought tolerance, is the ability of plants to withstand water deficit and maintain metabolism at low tissue water content (valliyodan and nguyen, 2008). in this strategy, osmotic adjustment, osmoprotection, antioxidation and scavenging defense system have been the most important physiochemical and biochemical bases responsible for drought tolerance. cell tissue and water conservation, antioxidant defense, cell membrane stability, compatible solutes and plant growth regulators mainly contributes in above said physiochemical and biochemical mechanisms. the three strategies are not mutually exclusive, and the same plant may use more than one strategy in order to adapt to periods of drought stress (nilsen and orcutt, 1996). therefore, to adapt with these natural constraints, plants have had to produce primary metabol i t e s s u c h a s p r o t e i n s a n d s o l u b l e s u g a r s . t h i s includes photosynthetic mechanisms, osmoregulation and antioxidant enzymes (liu et al., 2011; guo and gan, 2012). furthermore, water stress has a positive effect on the production and accumulation of secondary plant metabolites (ncib et al., 2018). their biosynthesis is often stimulated in response to biotic or abiotic stress (naczk and shahidi, 2004). the aim of this work is to study the adaptive physiological response of rhus tripartita to its naturally arid ecosystem (ahaggar, algeria). for this purpose, we will content to discuss our results in relation to water deficit and oxidative stress. 2. materials and methods presentation of study area localisation. the native plants of rhus tripartita (fig. 1) are located in the ilamane region (100 km away from tamanrasset city) which is situated in the ahaggar national and cultural park (22°49’59” north, 5°19’59” east). this park with an area of 4.500.000 ha, was created by the decree n. 87-231 of 03 november 1987. it is located in the mountainous center part of the volcanic ahaggar in the extreme algerian south (fig. 1). fig. 1 location of rhus tripartita growing in the rocky mountains of ilamane in the ahaggar national and cultural park. benaissa and djebbar rhus tripartita (ucria) grande under arid climate 15 features edapho-climatic. before looking more specifically at plant physiology that interests us, it is necessary to present the main characteristics of the ahaggar. the bioclimate is arid, with very varied winter and summer temperatures and very low rainfall. the rains occur from may to september and usually i n c r e a s e i n a u g u s t . t h e a n n u a l r a i n f a l l v a r i e s between 180 mm and 250 mm. the thermal regime varies between the two very short winter and longer summer seasons. dubief (1959) noted the maximum average on the ground, in july, at 52°c towards 2700 m of altitude (assekrem) and estimates at 60°c this value at 1376 m (tamanrasset). these high temperatures condition the biology of plants in this environment. on the edaphic plane, soil encountered in this area is very little evolved because of a low rainfall. however, this park is marked by a geomorphological distinction (mountainous mass, wadis, hammadas, gueltates, plains and rocky ravines); this has resulted in a high heterogeneity in the soil composition (sandy, sandy-loamy, sandy-clayey, shallow and rocky). therefore, in the ilamane region, edaphic variability is a result of shallow skeletal soils that characterize this area. determination of physiological parameters in order to study the rhus tripartita physiology and biochemistry characteristics in relation to its arid environment, different measurements of primary and secondary metabolites are performed. all analyzes were performed on three replicates of six shrubs naturally growing in this area. the sampling was conducted during december 2017 (winter season). therefore, we consider that 6 individuals are largely sufficient given the physiognomic homogeneity of the population. relative water content (rwc) the rwc measurement is an old method (slatyer, 1967) which is currently widely used to estimate the plant water status and in particular water deficit. a 12 mm leaf disc taken from the second leaf by means of a punch is weighed directly (weight of the fresh vegetable material, wfvm). it is then placed in the refrigerator in a test tube containing distilled water for 24 hours and weighed to obtain the turgor weight (tw). the fragments are finally placed in an oven at 80°c for 48 hours and then weighed to obtain the weight of dry vegetable matter (wdvm). the rwc is calculated according to the following equation: rwc (%) = (wfvm wdvm) x 100/(tw wdvm) metabolites compositions for the analysis of sugars, proteins and proline, 100 mg of fresh material from each repeat was used. total hydro-soluble protein. soluble proteins are assayed according to bradford method (bradford, 1976). this method is a colorimetric assay based on the color change of coomassie blue after binding to aromatic amino acids (tryptophan, tyrosine and phenylalanine) and the hydrophobic residues of amino acids present in proteins. proline free foliar. the technique used for proline determination is that of troll and lindsley (1955) modified by magné and larher (1992). it is based on the ability of proline to react in acidic and hot conditions with ninhydrin (revealing aromatic amino acids including proline) to give a pink compound soluble in organic solvents such as toluene. ethanol soluble sugars. the soluble sugars assay method is based on the technique of mccready et al. (1950). in the presence of 91% sulfuric acid (h2so4) and hot conditions, the oses produce furfural derivatives that react with the anthrone to give a bluegreen compound. photosynthetic pigments. the assay was performed by colorimetric method; the pigments being naturally colored. to do this, read the od of the samples using the spectrophotometer previously calibrated with 80% acetone (corresponding to od= 0). the od readings were carried out at wavelengths 647 a n d 6 6 3 n m f o r c h l o r o p h y l l s a n d 4 7 0 n m f o r carotenoids. the concentrations were determined according to lichtenthaler (1987). antioxidant system total anti-oxidant capacity (cat). the total antioxidant capacity of rhus tripartita leaf was estimated using the method described by prieto et al. (1999). the amount of 0.1 ml of methanolic extract (2 g of dry matter in 10 ml of methanol) was mixed with 1 ml of molybdate reagent (0.6 m sulphuric acid, 28 mm sodium phosphate and 4 mm ammonium molybdate). the absorbance was measured at 695 nm after incubation in boiling water bath about 90 min. the total antioxidant capacity was expressed as the milligramme gallic acid equivalent per gram of dry matter (mg gae/g dm). lipid peroxidation. lipid peroxidation is estimated according to the method of cakmak and horst (1991) which consists in grinding 100 mg of fresh plant material in 1 ml of 1% trichloroacetic acid (tca) foladv. hort. sci., 2019 33(1): 13-21 16 lowed by centrifugation at 12000 rpm/20 min. then, 1 ml of thiobarbituric acid (tba) at 0.5% (prepared in 20% tca) is added to 500 μl of extract and incubated with marie-bath at 95°c/30 min. after cooling, the optical densities (od) reading is carried out at 532 and 600 nm. the mda measurement content is calculated using its molar extinction coefficient (ƹ= 155 mmol/l cm). electrolyte leakage. electrolyte leakage (el) was estimated by measuring the electrolytic conductivity according to modified method of pike et al. (1998). this technique consists in placing ten foliar disks 12 mm in diameter in 20 ml of distilled water. a first measurement of the conductivity (ec) is carried out after stirring for 3 hours. then, a second measurement of the conductivity (et) is conducted, after marie-bath heating (95°c) for one hour. electrolyte leakage (%) is determined by the ratio (ec/et). total flavonoid content. total flavonoid content w a s e s t i m a t e d u s i n g t h e m e t h o d r e p o r t e d b y dewanto et al. (2002). it consists to mix 250 μl of vegetable methanolic extract with 25 μl of 5% nano2, added with 150 μl of alcl3 (2%). after 5 min, 0.5 ml of 1m naoh is added to the solution. after 10 min of incubation, the absorbance was measured at 510 nm. statistical analysis the data were subjected to statistical analysis using the microsoft excel 2010 program. all values of biochemical compounds and secondary metabolites are the mean ± se (standard error) of three replicates of a single sample. unidirectional analysis of variance (anova) was used and differences between individual means were considered significant at p<0.05. 3. results and discussion aridity depends on several climatic factors (temperature, wind, radiations) and above all on evaporation, an essential factor to calculate the water deficit. therefore, a rocky environment such as that of our study site, expresses accentuated drought effects because the soil scarcity. moreover, relatively rapid soil drought after rains causes water stress even during the rainy season (pimienta-barrios et al., 2001). to this end, we will discuss our results in relation to the influence of hydric and oxidative stress as the main parameters of aridity. therefore, plant eco-physiological study depends on several criteria, depending on environmental changes and their adaptation to them. indeed, plant survival in arid environments depends on different coping mechanisms. in this work, we have analyzed the variation of physiological parameters of rhus tripartita in relation to the arid climate of ahaggar. these parameters are often measured to study the different plants responses to abiotic stress. relative water content the low water availability can cause tissue dehydration, for this purpose, plant can control its hydric potential to cope with these high temperatures of arid zones. therefore, the relative water content of the plant gave a high value ranging from 54.79% to 81.55% (fig. 2). the highest content is recorded in shrub n. 5 with a significant difference compared to the same species shrubs. poole and miller (1975) reported that species of the same genus: rhus ovata, rhus laurina and rhus interguifolia; showed high water potential in water-deficit conditions. metabolite content in extreme temperatures, the plant can produce metabolites to protect itself from harmful and damaging effects. the total protein assay of r. tripartita showed levels that ranged from 20.03 mg/g in shrub n.3 to 33.76 mg/g in shrub n. 1 (fig. 3), which repres e n t f a i r l y h i g h l e v e l s c o m p a r e d t o p e r i p l o c a angustifolia labill, a shrub growing in tunisian arid areas with a rate of 5.204 mg/g dm (dghim et al., 2015). however, this amount is twice as high as that of the same species collected in libya (10.1 mg/g dm) (le houérou, 1991) and four times higher than that found in other species of the same genus such as rhus lancea (7.79 mg/g ms) (aganga and mosase, fig. 2 relative water content (rwc%) of rhus tripartita leaves according to climatic aridity conditions. benaissa and djebbar rhus tripartita (ucria) grande under arid climate 17 2001) but it is one and a half times higher in other forage species: medicago sativa (19.4 mg/g ms) (le houérou, 1991). on the other hand, proline is an amino acid whose rate increases proportionally more rapidly than other amino acids in plants under water stress. it has been suggested as a study parameter for the selection of drought-resistant plants (bates et al., 1973). the proline level recorded in r.tripartita showed a significant difference (p<0.05) between the six shrubs of the same species with an interval of 212.45 µg/g 851.28 µg/g (fig. 3). plants tolerant to stress, have relative stability or low accumulation of proline compared to sensitive plants (lemzeri, 2006). according to dix and pearce (1981), the proline accumulation is not an adaptation reaction to stress, but rather a sign of metabolic disturbance. alternatively, proline may confer a protective effect on the plant by induction of stress-protecting proteins (vinocur and altman, 2005). however, osmotic stress can produce harmful effects in the plant’s cell compartments. a wide range of metabolites can intervene to avoid these effects including a variety of sugars and alcoholic sugars such as mannitol and trehalose (vinocur and altman, 2005). the study of sugars compound in rhus tripartita leaves was relatively high (938.93 µg/g) (fig. 3). several studies have investigated the soluble sugars accumulation in several stressful species (garg et al., 2002; penna, 2003; silva and arrabac, 2004). ahaggar is a region known for its continuous sunshine all year round. in the presence of strong radiation, several plants use protective mechanisms to reduce the absorption of solar radiation (harrison et al., 2010). the study of rhus tripartita photosynthetic pigments leaves showed results of 28.1 µg/g, 31.24 µg/g and 53.56 µg/g for chlorophyll a, b and total chlorophyll respectively (table 1). similar to those recorded in rhus typhina under water stress conditions (liping, 2007), these results indicate that dry conditions do not have significant effects on the degradation of photosynthetic pigments, which e x p l a i n s t h e d a r k g r e e n c o l o u r o f t h e l e a v e s . nevertheless, photosynthesis of plants in arid zones that believe under permanent conditions of water d e f i c i e n c y ; a r e s u b j e c t t o p h o t o i n h i b i t i o n r i s k (voronin et al., 2003). furthermore, the amount of photosynthetic pigments in the leaves of these plants has been shown to be relatively low (valladares and sanchez-gomez, 2006) which contradicts the findings of this study. the water stress is considered one of the environmental factors limiting photosynthesis, therefore plant growth (ozturk et al., 2010). fig. 3 soluble sugar content (μg/g), total proteins content (mg/g) and proline content (μg/g) in rhus tripartita leaves according to climatic aridity conditions. shrub chlorophyll a chlorophyll b total chlorophyll carotenoids shrub n° 1 17.367 ± 0.284 24.800 ± 0.527 41.300 ± 0.584 8.480 ± 0,006 shrub n° 2 23.600 ± 0.156 29.967 ± 0.295 53.560 ± 0.383 8.022 ± 0.025 shrub n° 3 24.133 ± 0.157 18.880 ± 0.271 43.067 ± 0.415 5.797 ± 0.017 shrub n° 4 24.967 ± 0.151 31.243 ± 0.564 56.473 ± 0.378 11.237 ± 0.079 shrub n° 5 24.033 ± 0.390 20.783 ± 0.533 44.577 ± 0.443 6.619 ± 0.089 shrub n° 6 28.100 ± 0.340 21.203 ± 0.363 49.503 ± 0.257 6.841 ± 0.039 table 1 content of photosynthetic pigments (µg/g) in the fresh leaves of rhus tripartitus (chlorophyll a, chlorophyll b, total chlorophyll and carotenoids) 18 adv. hort. sci., 2019 33(1): 13-21 antioxidant system in this study, the oxidative stress effect is evaluated through the quantification of mda and the electrolyte leakage since secondary aldehyde products from lipid peroxidation are generally considered to result from oxidative stress (del rio et al., 2005). the results presented in figure n. 10 showed that the mda content in the shrub is significantly higher (7.007 nmol/g) than other shrubs [5.165-5.673 nmol/g]. the electrolyte leakage was studied using conductivity measurement. it varies between 12.39 and 14.293 in the six shrubs of rhus tripartita as shown in figure n. 4. similary, in rhus typhina, high levels of mda have been recorded in water stress conditions (liping, 2007). therefore, mda is a degradation product of lipid peroxidation reactions that are formed during the attack of polyunsaturated lipids by reactive oxygen species (ros). however, this is the most widely used assay to characterize oxidative damage in plants (shulaev and oliver, 2006). moreover, high temperatures can produce metabolic disturbances based on reactive oxygen species and antioxidant systems. however, the present work showed a variability in total anti-oxidant capacity from one shrub to another and is within a range of 19 mg/g 100 mg/g (fig. 4). therefore, it is known that tac is mainly due to phenolic compound (tlili et al., 2014). on the other hand, the flavonoids constitute the main group of polyphenols, ubiquitous in plants, t h e y a r e f o r m e d f r o m a r o m a t i c a m i n o a c i d s (hernandez et al., 2009). they act as antioxidant molecules that ensure the binding of ros produced during stress and thus neutralize their effects before the manifestation of oxidative damage at the cellular level (lovdal et al., 2010). the determination of total flavonoids contents in rhus tripartita leaves gave result of 35.89 mg/g (fig. 4). this level is relatively similar to the same species under water stress conditions (39.2 µg/mg) (ncib et al., 2018) but higher than in other species of the same family such as rhus punjabensis (30.50 µg/mg) (tabassum et al., 2017). several studies have demonstrated the richness of rhus tripartita in flavonoids (mahjoub et al., 2007, 2010; tlili et al., 2014). therefore, flavonoid is considered as a phytochemical adaptation to the biotic and abiotic environment (dixon and paiva, 1995). in the same perspective, the carotenoids are soluble antioxidant lipids that play a very important role in abiotic stress tolerance (sieferman-harms, 1987; gill and tuteja, 2010). the carotenoids content found in our shrub leaves is relatively low (11.63 µg/g) (table 1) compared to another shrub of tunisian arid zones: periploca angustifolia labill (dghim et al., 2015). it is known that carotenoids have a protective role against photooxidation (ladygin et al., 2008) which may explain the adaptive power of this shrub. the relationship between aridity and the results found is certainly evident. it can be explained by the fig. 4 malondialdehyde content (nmol/g), total antioxidant capacity (mg/g), electrolyte leakage and flavonoid content (mg/g), of rhus tripartita leaves in relation to climatic aridity conditions. benaissa and djebbar rhus tripartita (ucria) grande under arid climate 19 fact that the plant increases in both primary and secondary metabolite production under drought conditions. it should be noted that a difference in the metabolites composition has been recorded among different individuals of the same species. that said, not all shrubs had the same orientation towards the sunshine and some were grouped with the species myrtis nivellei and periploca laevigata. these two parameters can explain the difference between the results of the six individuals. 4. conclusions in summary, we presented a simple preliminary work on the physiological information of rhus tripartita in arid environments. therefore, our results have shown that this shrub presents an interesting adaptation to its climatic and edaphic environment. the approaches developed in this study certainly contribute to the understanding of shrub ecophysiology and can be improved plant productivity in arid regions. in conclusion, and according to classification of jenks and hasegawa (2005) of plants from desert and semi-arid zones, rhus tripartita can be considered as a xeromorphic plant, a species adapted to the desert that is not limited by water availability. references aganga a.a., mosase k.w., 2001 tannin content, nutritive value and dry matter digestibility of lonchocarpus capassa, zizyphus mucronata, sclerocarya birrea, kirkia acuminata and rhus lancea seeds. anim. feed sci. tech., 91: 107-113. bates l.s., waldren r.p., teare i.d., 1973 rapid determination of free proline for water-stress studies. plant soil, 39(1): 205-207. bradford m.m., 1976 a rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein dye binding. anal. biochem., 72: 248-254. cakmak i., horst w.j., 1991 effect of aluminium on lipid peroxidation, superoxide dismutase, catalase, and peroxidase activities in root tips of soybean (glycine max). physiol. plant., 83(3): 463-468. daoud y., halitim a., 1994 irrigation et salinisation au sahara algérien. institut national de la recherché agronomique d’algérie (inraa), sécheresse, 5(3): 151160. del rio d., stewart a.j., pellegrini n., 2005 a review of recent studies on malondialdehyde as toxic molecule and biological marker of oxidative stress. nutr. metab. cardiovasc. dis., 15(4): 316-328. dewanto v., wu x., adom k.k., liu r.h., 2002 thermal processing enhances the nutritional value of tomatoes by increasing total antioxidant activity. j. agricul. food chem., 50(10): 3010-3014. dghim f., bouaziz m., mezghani i., boukhris m., neffati m., 2015 laticifers identification and natural rubber characterization from the latex of periploca angustifolia labill. 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( e d . ) p l a n t genetics/genomics. volume 2. genetics and genomics of soybean. springer, new york, usa, pp. 406. vinocur b., altman a., 2005 recent advances in engineering plant tolerance to abiotic stress: achievements and limitations. curr. opin. biotechnol., 16(2): 123132. voronin p.y., ivanova l.a., ronzhina d.a., ivanov l.a., anenkhonov o.a., black c.c., p’yankov v.i., 2003 structural and functional changes in the leaves of plants from steppe communities as affected by aridization of the eurasian climate. russian j. plant physiol., 50(5): 604-611. impaginato 49 adv. hort. sci., 2019 33(1): 49-56 doi: 10.13128/ahs-22863 ameliorate the cadmium toxicity in solanum tuberosum l. plants with selenium and silicon application a.o.s. dorneles 1 (*), a. soares pereira 2, g. possebom 3, c. peligrinotti tarouco 4, l.v. rossato 4, l. almeri tabaldi 4 1 federal university of pelotas, postgraduate program plant physiology, 96010-900 pelotas, rio grande do sul, brazil. 2 federal university of pelotas, postgraduate program family farm production systems, 96010-900 pelotas, rio grande do sul, brazil. 3 federal university of santa maria, postgraduate program agricultural engineering, 97105-900 santa maria, rio grande do sul, brazil. 4 federal university of santa maria, biology department, 97105-900 santa maria, rio grande do sul, brazil. key words: elements beneficial, potato, toxic metals, toxicity. abstract: the present study aimed to prove the efficiency of se or si as relievers of the cd toxicity in potato plants. solanum tuberosum plants, asterix genotype, from in vitro propagation were placed in pots with sand and irrigated with complete nutrient solution for 60 days under six treatments: t1: control (nutrient solution); t2: 2.5 µm se; t3: 2.5 mm si; t4: 50 µm cd; t5: 50 µm cd + 2.5 µm se; t6: 50µm cd + 2.5 mm si. the treatments were arranged in completely randomized design, with four replicates for each treatment and six plants per replicate. the plants were collected at 30 and 60 days after application of the treatments. cadmium was highly toxic in all parameters (dry and fresh weight, plant height, leaf number, leaf area, root and photosynthetic parameters), in both assessments. however, se and si were effective in mitigating cd toxicity in all parameters, although si has been shown to be more efficient than se in dry weight and plant height parameters. thus, from data obtained in this study, it is clear that the beneficial elements tested have power to ameliorate cd toxicity. 1. introduction plants differ in their ability to absorb, accumulate and tolerate heavy metals, including cadmium (cd), and toxic levels of heavy metals affect a variety of plant processes (gupta et al., 2013). cadmium is one of most toxic heavy metals, having a high mobility in environment (tang et al., 2015), being absorbed by roots and transported to shoot of many plant species (shi et al., 2005). although cd has no known biological function in plants (pence et al., 2000; pereira et al., 2016), it can be easily absorbed and transported by xylem (lux et al., 2011), since it has an electronic con(*) corresponding author: athos_odin@hotmail.com citation: dorneles a.o.s., soares pereira a., possebom g., peligrinotti tarouco c., rossato l.v., almeri tabaldi l., 2019 ameliorate the cadmium toxicity in solanum tuberosum l. plants with selenium and silicon application. adv. hort. sci., 33(1):49-56 copyright: © 2019 dorneles a.o.s., soares pereira a., possebom g., peligrinotti tarouco c., rossato l.v., almeri tabaldi l. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 9 march 2018 accepted for publication 4 october 2018 ahs advances in horticultural science adv. hort. sci., 2019 33(1): 49-56 50 figuration and state of zinc-like valence (nan et al., 2002). this heavy metal is toxic even at low concentrations, inducing stress responses in plants from 510 μg g-1 soil (while and brown, 2010). several studies have reported significant reductions in biomass accumulation in plants exposed to cd (farooq et al., 2013; said et al., 2014; vaculík et al., 2015), and this inhibition in biomass production can occur in short time of exposure to this heavy metal (han et al., 2015). in contrast to others toxic heavy metals, cd present in soil is easily absorbed by plant roots, particularly in acidic soils (guimarães et al., 2008). the plant development stage and time of exposure to heavy metal affect the absorption and distribution of cd in different parts of plant (gonçalves et al., 2009 a). thus, it is necessary to develop strategies that result in lesser absorption of these toxic elements present in soil by plants, optimizing the use of natural resources and production of safe food, especially when it comes to a plant used in food such as potatoes. potato (solanum tuberosum l.) is one of main foods for mankind, consumed by more than one billion people worldwide, due to its composition, gastronomic and technological versatility, as well as the low market price of tubers (coelho et al., 1999; dorneles et al., 2016). according to birch et al. (2012), potatoes are third most important crop in world, behind only rice and wheat. the potato is susceptible to cd, and this sensitivity can be accentuated by level of cd in soil, time of exposure and cultivar (gonçalves et al., 2009 b). one of options sought to solve this problem with cd in plant growth is use of beneficial elements, which when used in low concentrations can alleviate the damaging cd effects. in this sense, selenium (se) and silicon (si) are recognized as beneficial elements for growth of some plants, and can increase the tolerance of plants to abiotic stresses. these elements are recognized as being capable of mitigating metal toxicity in plants (wu et al., 2017). however, it is necessary to evaluate the potential use of these mitigating elements in the presence of cd, since some studies show that these may not be effective to reduce the toxicity of this element in some species (khattab, 2004; liu et al., 2013). however, si has been shown to be effective in alleviating al toxicity in potato plants (dorneles et al., 2016) and cd in species such as peanuts (shi et al., 2010), chinese cabbage (song et al., 2009), rice (tripathi et al., 2013), maize (lukacová et al., 2013), wheat (khan et al., 2015) and sunflower (said et al., 2014). selenium, however, has stress-alleviating properties that are more focused on biochemical mechanisms activation (kumar et al., 2012; feng et al., 2013; tamaoki and maruyamanakashita, 2017). however, it already has great potential in use to relieve stresses by metals (he et al., 2004; pezzarossa et al., 2012). however, this element, to date, has not been tested as potential to relieve the stress caused by cd toxicity in potato plants. thus, the aim of this work was to test the possibility of using se or si as reliever of cd toxicity in potato plants. 2. materials and methods plants of solanum tuberosum l., asterix genotype, were used for the experiment, which were propagated in vitro from nodal segments for 25 days in ms culture medium (murashige and skoog, 1962). after this period, plants were transferred to plastic vessels containing 1 plant each and 5 kg of sand, being watered daily with complete nutritive solution. this solution had the following composition (mg l-1): 85.31 n; 7.54 p; 11.54 s; 97.64 ca; 23.68 mg; 104.75 k; 176.76 cl; 0.27 de b; 0.05 mo; 0.01 ni; 0.13 zn; 0.03 cu; 0.11 mn and 2.68 fe (feso4/na-edta). after five-day of acclimation period, treatments were applied, which consisted of following combinations: treatment 1: complete nutritional solution and absence of cd, se and si; treatment 2: complete nutritional solution + 2.5 μm se; treatment 3: complete nutritional solution + 2.5 mm si; treatment 4 : c o m p l e t e n u t r i t i o n a l s o l u t i o n + 5 0 μ m c d ; treatment 5: complete nutritional solution + 50 μm cd + 2.5 μm se; treatment 6: complete nutritional solution + 50 μm cd + 2.5 mm si. these solutions were applied daily maintaining 80% of the vessel capacity, which was monitored by daily determining the weight of the vessel. the treatments were arranged in a completely randomized design, with four replicates for each treatment and six plants per replicate. the ph of the solutions was adjusted daily (4.5±0.1). two collects were performed, the first collect being at 30 days of exposure to treatments and the second at 60 days. in both collections, the following evaluations were carried out: photosynthetic parameters were evaluated in fourth fully expanded leaf of four plants per replicate: the photosynthetic rate (a μmol co2 m-2 s-1), the stomatal conductance of water vapors (gs mol h2o m-2 s-1), internal co2 concentration (ci μmol dorneles et al. selenium and silicon ameliorate the cadmium toxicity in potato 51 m-2), water use efficiency (wue mol co2 mol h2o1), and the transpiration rate (tr mmol mmol h2o m-2 s-1) obtained by the ratio between the amount of co2 fixed by photosynthesis and the internal co2 concentration. the evaluations were carried out in p e r i o d b e t w e e n 8 a n d 1 1 h w i t h u s e o f i r g a portable meter, brand li-cor, model li-6400xt. growth parameters: fresh and dry biomass, height, leaf area, number of leaves, and morphological parameters of the root system (length, diameter, volume and number of root branches), according to methodology described by dorneles et al. (2016). for statistical analysis of data, it was verified the normality of error distribution through andersondarling test and homogeneity of error variances through the bartlett test (estatcamp, 2012) for all variables of experiment. the averages were submitted to analysis of variance and compared by the scott-knott test, with 5% significance, using sisvar (ferreira, 2011). the graphic program used was sigmaplot 12.5. 3. results for experimental conditions tested, cadmium (cd) presented great toxicity to potato plants both at 30 and 60 days of growth. this is evidenced by averages of fresh weight accumulation obtained by plants exposed to cd, which did not increase after 30 days of cultivation, remaining unchanged up to 60 days (fig. 1) both being statistically smaller than in the control treatment. besides, in general, when applied in isolation, si and se did not promote greater accumulation of fresh and dry weight in comparison to control plants (figs. 1, 2). however, both elements tested proved to be effective in mitigating cd toxicity from first collect, inducing a higher accumulation of fresh weight in both tissues, compared to the treatment where only cd was applied. the enhancing effects of se and si are more evident in shoot, where plants exposed to cd combined with se or si maintained some growth in second collect (fig. 1 a). the roots of potato plants (fig. 1 b) also show a significant improvement in presence of both beneficial elements. this probably contributed to growth of shoot, considering that roots are the first tissue to come into contact with cd. for dry weight, potato plants showed similar behavior to that observed for fresh weight in both tissues (fig. 2). cadmium showed to be toxic to both tissues for dry weight, inhibiting this accumulation until last collect. however, se and si induced biomass accumulation similar to control plants, even when applied together with cd. silicon induced greater accumulation of shoot dry weight when compared to se (fig. 2 a). in roots, cd prevented the dry weight increase, while plants treated with se and si presented increase in root dry weight in second collect (fig. 2 b), that is, se and si alleviated the toxic effects of cadmium. in addition to affecting the production of fresh and dry biomass, cd inhibited the production and expansion of leaves, as well as the growth in height of potato plants. however, plants that received si and se together with cd obtained higher means even in comparison to control plants (figs. 3 a, b). in addition, the number of leaves and height of plants exposed to cd were higher when applied si, in comparison to plants treated with se as amendment. both se and si were effective in reducing the toxicity of cadmium from first collect, inducing higher height, number of leaves and leaf area than plants with only fig. 1 effect of selenium (2.5 μm) or silicon (2.5 mm) on shoot (a) and roots fresh weight (b) of potato plants grown in presence of cadmium (50 μm) at 30 and 60 days after application of the treatments. different lowercase letters indicate significant differences between treatments in same collect. different uppercase letters indicate significant differences between collects for same treatment. adv. hort. sci., 2019 33(1): 49-56 52 cd in nutrient solution. there was no statistical difference between treatments for root length and root diameter at 30 days, but plants treated with cd presented root length 45% lower than the control (table 1). at 60 days, plants exposed to cd showed root length 16% and root different lowercase letters indicate significant differences between treatments in same collect. different upper case letters indicate significant differences between collects for same treatment. fig. 2 effect of selenium (2.5 μm) or silicon (2.5 mm) on shoot (a) and roots dry weight (b) of potato plants grown in cadmium presence (50 μm) at 30 and 60 days after application of the treatments. different lowercase letters indicate significant differences between treatments in same collect. different uppercase letters indicate significant differences between collects for same treatment. fig. 3 effect of selenium (2.5 μm) or silicon (2.5 mm) on leaves number (a), plants height (b) and leaf area (c) of potato plants grown in cadmium presence (50 μm) at 30 and 60 days after application of the treatments. different lowercase letters indicate significant differences between treatments in same collect. different upper case letters indicate significant differences between collects for same treatment. table 1 length, diameter, volume and number of branch roots of solanum tuberosum plants grown in presence of selenium (2.5 μm), silicon (2.5 mm) and cadmium (50 μm) at 30 and 60 days after application of the treatments collect treatments root length (cm) root diameter (mm) root volume (cm3) branch number 30 days control 798±0.24 aa 0.35±0.02 ba 1.64±0.49 aa 715±68 aa se 776±18.3 aa 0.38±0.04 ba 0.91±0.25 ab 394±18 bb si 743±9.24 aa 0.35±0.02 ba 0.84±0.19 ab 401±20 bb cd 438±3.76 ba 0.34±0.02 aa 0.37±0.07 ac 201±13 ad se + cd 650±4.62 aa 0.36±0.00 ba 0.65±0.05 ab 323±35 bc si + cd 711±3.43 aa 0.41±0.03 aa 0.83±0.08 ab 318±38 bc 60 days control 998±0.57 aa 0.45±0.02 aa 1.83±0.21 aa 790±48 aa se 884±21.6 aa 0.45±0.01 aa 0.85±0.03 ab 794±58 aa si 862±25.2 aa 0.45±0.02 aa 0.76±0.08 ab 790±62 aa cd 839±10.11 ab 0.34±0.02 ab 0.34±0.02 ac 253±24 ac se + cd 795±5.50 aa 0.46±0.00 aa 0.75±0.04 ab 423±21 ab si + cd 791±6.11 aa 0.44±0.02 aa 0.85±0.07 ab 452±22 ab dorneles et al. selenium and silicon ameliorate the cadmium toxicity in potato 53 diameter 25% lower, compared to the control. on the other hand, all treatments presented lower root volume compared to control, but this effect was more significant in the treatment containing only cd in the growth medium, both at 30 and 60 days of cultivation. this same behavior was observed for the number of branches at 30 days. at 60 days, branch numbers was lower in the treatments containing cd, se + cd and si + cd, and this effect was more significant in the treatment containing only cd in the growth medium. cadmium showed to be highly toxic, reducing volume of roots and ramifications to less than half of values presented by control plants. however, si and se presented an amendment effect on all root variables, mainly for total length and diameter, both at 30 and 60 days of cultivation. in these variables, plants exposed to cd treated with both si and se were statistically the same as control plants. plants exposed to cd had photosynthetic rate (a) reduced by 37% compared to control plants at first collect, and 42% at second collect (table 2). besides, cd caused significant reductions in stomatal conductance (gs), internal co2 concentration (ci) and transpiratory rate (trmmol) in both collects. the water use efficiency (wue) in both collects was higher only for the treatment with se + cd. however, in both collects si and se were effective in mitigation of cd toxicity for all variables except for wue (table 2). 4. discussion and conclusions in the present study, cd promoted a significant reduction in biomass production in potato plants (fig. 1). the reduction in biomass accumulation caused by cd may be due, in part, to its effect on the inhibition of nutrient uptake by roots (cao et al., 2014; li et al., 2016). the effects of cd on absorption of nutrients may be due to damage caused by this element in the roots. since root tissues are first to come into contact with the cd present in solution, they are also the most affected. several studies have already reported negative effects of cd on cellular level of root tissues (benavides et al., 2005; lux et al., 2011; martinka et al., 2014). the data presented in this work show the significant reduction in root parameters caused by cd. this behavior may be due to effect of cd on degradation of membranes and nucleotides of root cells (han et al., 2015). this degradation of membranes and organelles can be explained by the increase in concentration of reactive oxygen species (ros) induced by cd (chou et al., 2012; farooq et al., 2013; said et al., 2014; han et al., 2015). it is possible that this effect of cd on increase of ros, reported in these studies, may inhibit cell division of roots (said et al., 2014) in addition, the cd has affinity for phosphates and some amino acids components of enzymes and table 2 effect of selenium (2.5 μm) or silicon (2.5 mm) on photosynthetic rate (a-μmol co2 m-2 s-1), stomatal conductance (gs mol h2o m-2 s-1), internal co2 concentration (ci μmol m-2 s-1), transpiration rate (trmmol h2o mmol m-2 s-1),and water use efficiency (wue co2 mol h2o mol-1) of solanum tuberosum plants grown in cadmium presence (50 μm) at 30 and 60 days after application of the treatments collect treatment a gs ci trmmol wue 30 days control 9.47±0.13 ba 0.20±0.07 aa 288±0.48 aa 9.47±0.13 ba 192±4.59 bb se 8.39±0.32 bb 0.16±0.04 aa 295±11.8 ba 8.39±0.32 bb 178±11.8 bb si 8.92±0.31 bb 0.04±0.00 ac 272±2.44 bb 8.92±0.31 bb 200±20.3 bb cd 5.92±0.27 bc 0.10±0.00 ab 257±8.54 ac 5.92±0.27 bc 228±8.63 ab se + cd 8.73±0.07 bb 0.06±0.03 ac 297±5.57 aa 8.73±0.07 bb 308±3.05 aa si + cd 8.26±0.03 bb 0.10±0.00 ab 255±9.29 ac 8.26±0.03 bb 236±12.6 ab 60 days control 12.5±0.14 aa 0.20±0.03 aa 289±0.24 aa 12.5±0.14 aa 205±0.25 ab se 10.3±0.32 ab 0.14±0.01 aa 268±14.6 ab 10.3±0.32 ab 215±0.24 ab si 10.1±0.06 ab 0.06±0.00 ab 298±5.53 aa 10.1±0.06 ab 236±0.02 ab cd 7.41±0.31 ad 0.04±0.00 bc 269±8.13 ab 7.41±0.31 ad 220±0.26 ab se + cd 9.08±0.72 ac 0.08±0.00 ab 257±8.54 bc 9.08±0.72 ac 298±0.14 aa si + cd 8.98±0.61 ac 0.10±0.00 ab 255±9.29 ac 8.98±0.61 ac 245±0.22 ab different lowercase letters indicate significant differences between treatments in same collect. different upper case letters indicate significant differences between collects for same treatment. 54 adv. hort. sci., 2019 33(1): 49-56 proteins, which leads, in addition to damage to membranes, genetic damage and can disrupt oxidative phosphorylation in exposed tissues (hasanuzzaman and fujita, 2012; nahar et al., 2016). the present study also shows the effect of cd under photosynthetic parameters, in which there was a significant reduction in plants exposed to this heavy metal. this reduction in photosynthetic parameters may be a consequence of the degradation of chlorophylls caused by cd (lópez-millán et al., 2009). furthermore, cd causes disorder in arrangement of grana and thylakoids, which limits the efficiency of most photosynthetic parameters (han et al., 2015; bayçu et al., 2016). the potato plants used in present study, treated with si or se as amendments of cd toxicity, showed higher biomass production than plants exposed to only cd. in addition, se and si significantly reduced the damage caused by cd in photosynthetic parameters. these results of biomass and photosynthetic rate are related, since the beneficial effects of si and si on the cd toxicity in photosynthetic parameters are expressed, to a greater or lesser degree, in higher production of biomass by plants. each element used as amendment in this work (se or si) act in different ways in the plant. silicon have been proven to be effective in easing stress by cd in different species such as cucumber (feng et al., 2010), corn (malčovská et al., 2014; vaculík et al., 2015) and cotton (farooq et al., 2016). this effect of si may be due to its deposition on cell wall which increases its plasticity and elasticity (vaculík et al., 2009). the si deposited on cell wall of the plants may increase cd retention in apoplast (lukačová et al., 2013; vaculík et al., 2015), which may reduce the availability and translocation of cd. due to these cell wall modulations, si can inhibit cd uptake (liu et al., 2013). these effects of si can explain the more evident amelioration of cd in shoot than in roots for dry weight in potato plants (fig. 2). in addition, silicon has photoprotective properties when deposited on leaves (tripathi et al., 2017), which may explain the mitigation of cd toxicity in gas exchange parameters. there is a chance that se also has the property of forming complexes with toxic or heavy metals, but were only found complexes of se-mercury (hg) in plants (said et al., 2014). however, there are reports that selenium reduces cd accumulation in tissues (lactuca sativa l.) (he et al., 2004). while si is recognized for increasing the enzymatic antioxidants activity (debona et al., 2017), se has the property of inducing resistance by activating routes of synthesis of hormones linked to stress response and antioxidant activities (freeman et al., 2010; feng et al., 2013; tamaoki and maruyama-nakashita, 2017). thus, the mechanism of mitigation of se may be more directly related to antioxidant system. many studies have reported this effect of se under plant antioxidant enzymes, as well as its effect on direct removal of ros (cartes et al., 2010; zembala et al., 2010). in present study, the cd toxicity was reduced in most of evaluated parameters, mainly under photosynthetic parameters. the possible effect of se under enzyme activity may help to explain its most evident effect under these parameters. selenium was more effective in reducing the effects of cd on photosynthetic parameters than for other parameters evaluated. for potato plants under experimental conditions used in this study, si and se elements proved to be effective in mitigating cd toxicity. thus, these elements have potential for use in fertilizers applied to contaminated soils. future biochemical, histological and molecular analyzes may complement the elucidation of action mechanisms these elements. acknowledgements t h e a u t h o r s t h a n k t h e c o o r d e n a ç ã o e aperfeiçoamento de pessoal de nível superior, conselho nacional de desenvolvimento científico e tecnológico, and fundação de amparo à pesquisa de estado do rio grande do sul for the research fellowships. references bayçu g., gevrek-kürüm n., moustaka j., csatári i., rognes s.e., moustakas m., 2016 cadmium-zinc accumulation and photosystem ii responses of noccaea caerulescens to cd and zn exposure. environ. sci. pollut. res., 24: 2840-2850. benavides m.p., gallego s.m., tomaro m.l., 2005 cadmium toxicity in plants. j. plant physol., 17: 21-34. birch p.r.j., bryan g., fenton b., gilroy e.m., hein i., jones j.t., prashar a., taylor m.a., torrance l., toth i.k., 2012 crops that feed the world 8: potato: are the trends of increased global production sustainable? 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effects of foliar application of organic chelate fertilizers on french bean production under field conditions in a calcareous soil m.k. souri (*), m. aslani department of horticultural science, tarbiat modares university, tehran, iran. key words: aminochelate, amino acid, calcareous soil, nutrient uptake, pod yield, quality. abstract: aminochelate are organic-based chelate fertilizers with higher efficiency for agricultural applications. in the present study, foliar application of three organic-based chelate fertilizers, a macro-micro mixture and soil applied npk were evaluated on french bean growth characteristics under open field in a calcareous soil. the results showed that plant growth, pod yield (79%) and pod quality were improved by application of chelate fertilizers. growth parameters as plant height, number of leaves and lateral shoots, shoot dry weight, pod number and pod length were significantly increased by foliar application of the chelate fertilizers. the concentrations of nitrogen, potassium and iron in pods and above all in leaves were increased by foliar application of chelate fertilizers compared to control and soil applied npk. pod ph and tss were not influenced by treatments; however, foliar application of the chelate fertilizers resulted in higher titratable acidity (40%), vitamin c (112%) and protein (35%) content of pods. the results indicate that organic-based chelate fertilizers can be effective safer alternatives for simple chemical salts in calcareous soils. 1. introduction calcareous or lime soils are the dominant type of soil in many parts of the world, and are characterized by high ph as well as high levels of carbonates and bicarbonates. nutrients and particularly iron uptake in such soil conditions is restricted. in many cases lime-induced chlorosis refers to iron deficiency (souri, 2015). the leaf chlorosis is mainly due to high ph of soil solution and sap solution induced by high concentrations of carbonates and bicarbonates, which results in precipitation of nutrients in soil and cell apoplast (mengel, 1994; nikolic and römheld, 2002). plant cultivation in calcareous soils requires especial management techniques and strategies particularly regarding micronutrients supplementation (souri, 2015). various fertilizers, as well as different forms of nutrient elements do not have the same uptake efficiency (jeppsen, 1991; fernández and (*) corresponding author: mk.souri@modares.ac.ir citation: souri m.k., aslani m., 2018 beneficial effects of foliar application of organic chelate fertilizers on french bean production under field conditions in a calcareous soil. adv. hort. sci., 32(2): 265272 copyright: © 2018 souri m.k., aslani m. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 3 november 2017 accepted for publication 20 march 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(2): 265-272 266 ebert, 2005). since decades chelate fertilizers have been used due to their better suitability and efficiency to meet plant’s need of nutrient elements particularly under calcareous soil conditions. simple nutrient salts are in inorganic form, which for utilization by plant roots must be restructured to ionic form in soil solution. this makes them very vulnerable to various inactivation processes, resulting in their low efficiency rate (mengel, 1994; nikolic and römheld, 2002; souri, 2016). on the other hand, high price of commercial synthetic chelate fertilizers such as edta or eddha restricts their application by many farmers. in addition, there is also great doubt on their impacts on plant, environment and ecosystem health issues (souri, 2015). aminochelate fertilizers are claimed as suitable alternatives for simple salt fertilizers. for nutritional purpose the safety of fertilizer products is v e r y i m p o r t a n t . o r g a n i c c h e l a t e s s u c h a s aminochelate fertilizers are formulated mainly for foliar application, even if in various studies their soil application also resulted in higher growth compared to soil applied npk (souri and yarahmadi, 2016) or a more complete fertilizer (salwa, 2011; garcia et al., 2011). the method of fertilizer application also plays an important role in uptake efficiency by plant roots (fernández and ebert, 2005). there are generally two methods of soil and foliar application for most fertilizers. most of micronutrients and nitrogen fertilizers including amino acids can simply be applied to leaves with acceptable uptake efficiency (jeppsen, 1991; marschner, 2011). nevertheless, foliar application cannot fully replace soil application of fertilizers in agriculture (souri, 2015; dehnavard et al., 2017). aminochelates are composed of amino acids and a single or several nutrient elements (metals) and represent a more suitable form of fertilizers for sustainable production than routine fertilizers (souri, 2015). in cultivation systems, application of nitrogen and micronutrients needs a precise and intelligent management, in which various organic chelates can play important role. however, general chemical properties of various aminochelate fertilizers have not been yet studied in soil or within the plant tissues. in recent studies, it was shown that application of amino acid chelates of nutrients in nutrient solution or on plant foliage, significantly increases plant growth and biomass production (zeid, 2009; ghasemi et al., 2014; souri et al., 2017), leaf number, leaf area, leaf spad index (garcia et al., 2011; souri and yarahmadi, 2016), fruit yield (naseri et al., 2013; pourebrahimi, et al., 2013; fahimi et al., 2016), fruit quality (machado et al., 2008; souri et al., 2017) and composition of plants (zhou et al., 2007; garcia et al., 2011; ghasemi et al., 2014). moreover, application of organic-based fertilizers can also result in higher soil microbial activity and fertility, whereas chemical forms of fertilizers with higher salt effects generally reduce soil microbial activity and soil fertility (salwa, 2011; souri, 2015). it was shown that soil application of amino acids has suppressive effect on some soil pathogens such as meloidogyne incognita (saeed et al., 2005). green bean is a relatively sensitive plant to lime soil, showing dwarf growth and low yield due to restriction of flower development and fruit set. in the present study effects of foliar application of three organicbased chelate fertilizers were investigated on french bean growth characteristics and pod quality factors under calcareous soil conditions. 2. materials and methods experimental site t h i s s t u d y w a s p e r f o r m e d a t f a c u l t y o f agriculture, tarbiat modares uni., tehran-iran during 2013. the field in which the experiment was conducted had a calcareous soil. the soil sample was collected from three parts of the field (0-30 cm depth), mixed together and analyzed for some physicochemical characteristics that are presented in table 1. application of treatments the experiment was arranged in complete randomized blocks using six treatments and four replications. treatments were: 1) control (without any fertilizer application), 2) soil application of npk, 3) foliar application of biomin (amino acid based fertilizer; arbico-co, texas, usa), 4) foliar application of humifolin (humic acid based fertilizer; tradecrop co., spain), 5) foliar application of delfanplus (only amino acid; tradecrop co., spain) and 6) foliar application of a mixture of macro-micro solution. each replicate physico-chemical caracteristics data texture loam-clay ph 7.8 ec ds/m 2.6 total c (%) 0.77 total n (%) 0.09 caco3 (%) 5.5 extractable p (mg kg-1) 15.2 exchangeable k (mg kg-1) 250 table 1 some properties of the soil used in the experiment souri and aslani foliar application of organic chelate fertilizers on green bean 267 was a plot of 0.7×1m consisting of 12 plants. green bean seeds (phaseolus vulgaris l) were directly sown in the soil, and after germination they were thinned to 12 plants per plot. throughout the growing period all plots and plants were treated the same regarding irrigation, and pest-disease control. in npk treatment, a final amount of 6 g per plant from a 20:10:20 formulation was used in three split applications that were incorporated into the soil just near the root systems using 200 ml water. the first application was before sowing and the rest was applied in one week interval after second week of emergence. foliar application of organic-based and mix fertilizers were done five times during growth period, in a constant concentration of 0.2%. plants were foliar sprayed at 6-7 o’clock in the morning using a portable sprayer, by which the upper and the lower surface of leaves were treated. the first spray was done at four leaf stage, and the remaining foliar sprays were applied at one-week intervals. all the organic-based commercial fertilizers used in present study were in liquid form, consisting of one or several nutrient elements. composition of fertilizers the composition of various fertilizers is presented as follows: biomin: a liquid fertilizer containing 2% n, 2.5% zn, 1.5% mn, 1% fe, 0.4% mg, 0.4% cu, and 0.02% mo; humifolin, a liquid fertilizer containing 42% organic compounds including 37% fulvic and humic acid, 5% various vitamins, 0.5% phosphorus, 0.28% fe, 0.041% zn, 0.0035% mn, 0.0023% cu, 0.0012% mg, and 0.0012% b; delfanplus, a liquid fertilizer consisting of 24% free amino acids, 9% total nitrogen, 5% n-protein and 43% organic carbon. macro-micro mixture consisted of 5% n, 2.5% zn, 2.5% fe, 2% mn, 0.5% mg, and 0.5% cu. plant sampling and measurements french bean plants were grown for ten weeks, and various growth traits were measured during growth period, as well as at harvest time. plant pods were harvested several times during their growth period. cumulated yield of plants during 2-3 harvests (in each replicate) was recorded and the average was p r e s e n t e d a s p l a n t f i n a l y i e l d o f f r e s h p o d s . accordingly, total number of pods per plant was recorded. the average number of leaves and lateral shoots of plants were counted per replicate (plot) and calculated per plant. chlorophyll index of leaves was recorded using a portable spad meter (model 502 plus, illinois, usa) with 10 reading per plant and at least 120 readings for each replicate, by which the average was recorded in the results. plant stem diameter was measured by caliper (model mitutoyo japan). average shoot dry weight per plant was calculated after drying four randomly chosen plants from each replicate at 65°c for 24 hours. total soluble solid (tss) as brix index was determined with a refractometer using a drop of pod extracted juice. for determination of pod ph and titratable acidity, 10 g of fresh pods (from each replicate) was cut, crushed and centrifuged at 9000 rpm. ten ml of supernatant was titrated using 0.1 m naoh until a final ph of 8.2, and the pod acidity based on citric acid in 100 g fresh pods was calculated using the following formula: ta= 100 ×m×n×v/s×n where ta is the amount of pod acidity (mg/100 g fw), m the molecular weight of dominant pod acid (citric acid= 64 g) n: valence of dominant acid, n: normality of naoh, s: weight of pod sample (g) and v the volume of consumed naoh. for measurement of pod vitamin c, 10 g of fresh pods from final harvest was gently washed, cut in small pieces and then crushed in a mortar in presence of 10 ml of 2% metaphosphoric acid. the mixture was immediately centrifugated at 9000 rpm (eppendorf centrifuge 5810r, hamburg, germany) for 5 minutes at 4°c. the supernatant was used for titration by 2,6 dichloro indophenols, and the amount of vitamin c for 100 g fresh pod was calculated in relation to records of a standard curve of l-ascorbic acid concentration of 0, 25, 50, 100 and 200 mg l-1. determination of pod protein content was done using coomassie brilliant blue g250 dye according to bradford’s method (1976). the nutrient concentration of n, k and fe were determined in plant leaves and green pods using kjeldahl, flame photometer and atomic absorption spectrophotometer methods. statistical analysis data were analyzed using spss 16 and differences among treatments were determined at 5% level by duncan’s test. graphs were prepared using excel microsoft. 3. results plant vegetative growth vegetative growth characteristics of plants are presented in table 2. application of various fertilizers had significantly improved plant growth. all the most important growth factors were improved especially adv. hort. sci., 2018 32(2): 265-272 268 by foliar application of organic-based chelate fertilizers (table 2). in particular, plant height was increased by application of three organic chelates, while there was no difference among control plants and those plants treated with soil applied npk or by foliar application of macro-micro mixture. stem diameter was significantly higher in plants that received foliar application of organic chelates (table 2). number of leaves was maximum in plants treated with biomin and humifolin; however they had no significant difference with delfanplus treated plants. the least number of leaves was in control plants. number of lateral shoots was increased by foliar application of biomin and humifoline; however, there was no significant difference among all organic fertilizers. there was no significant improvement of lateral shoots by soil applied npk or foliar application of macro-micro mixture (table 2). plants treated with delfanplus produced longest internodes that showed no difference with biomin or humifolin treated plants (table 2). application of all fertilizer treatments, except foliar application of macro-micro mixture, resulted in significantly higher spad values compared to control plants (table 2). determination of shoot dry weight revealed that plant growth and biomass production was significantly improved by foliar application of the organic fertilizers, as well as by soil applied npk treatment. on the other hand, the maximum shoot dry weight was recorded for biomin treatment, which showed significant difference with control and all other fertilization treatments (table 2). plant yield and nutrient status application of all fertilizers increased the pod yield compared to control plants (table 3). plants p r o d u c e d s i g n i f i c a n t l y h i g h e r y i e l d w h e n t h e y received foliar application of organic chelate fertilizers (table 3). number of seeds per pod and number of pods per plant had a similar trend, in which foliar application of three organic chelate fertilizers recorded higher values. regarding pod length, plants treated with foliar application of three organic chelates had the longest pods compared to all other treatments. all fertilizer treatments increased the pod dry weight. plants treated with biomin produced significantly higher pod dry weight (table 3), although application of all the organic chelates significantly increased pod dry weight compared to soil applied npk or foliar application of macro-micro mixture. nutrient profile of plant leaves and pods were significantly increased by foliar application of organic fertilizers, only for n and k, and also by npk soil table 2 effects of various fertilization treatments on vegetative growth traits of french bean plants in a calcareous soil under field conditions data are mean of four replicates. comparison of means was done using duncan’s test at 5% level. treatments plant height (cm) stem diameter (cm) number of leaves number of lateral shoots length of internode (cm) spad index shoot dry weight (g) control 26 b 0.62 b 14 c 4 b 5.6 b 36 b 7.2 d npk 31 ab 0.79 b 19 b 5 b 5.5 b 40 a 11.3 c biomin 39 a 1:05 24 a 8:00 6.7 ab 41 a 22.1 a humifolin 39 a 0.96 a 24 a 7:00 6.1 ab 41 a 15.6 b delfanplus 38 a 0.98 a 22 ab 6 ab 7:02 42 a 17.5 b macro-micro mixture 29ab 0.67 b 17 bc 4 b 5.1 b 38 ab 8.5 d table 3 effects of various fertilization treatments on yield and fruit characteristics of french bean plants grown in a calcareous soil under field conditions data are mean of four replicates. comparison of means was done using duncan’s test at 5% level. treatments plant pod yield (g) number of seeds/pod number of pods per plant pod length (cm) pod dry weight (g) pod ph pod tss control 31.4 c 2.9 c 17 c 5.5 b 8.3 d 6.20 a 1.7 a npk 38.3 b 3.8 b 28 b 6.5 b 10.8 c 6.30 a 1.8 a biomin 59.1 a 5.1 a 49 a 9.1 a 21.1 a 6.21 a 2.4 a humifolin 53.5 a 4.7 a 43 a 9.3 a 16.2 b 6.22 a 2.1 a delfan plus 56.6 a 4.8 a 45 a 9.6 a 16.6 b 6.23 a 2.0 a macro-micro mixture 36.7 b 3.9 b 27 b 6.5 ab 11.4 c 6.24 a 2.1 a souri and aslani foliar application of organic chelate fertilizers on green bean 269 application (table 4). foliar application of biomin and delfanplus resulted in significantly higher k and fe concentration in leaves, and k concentration of pods, compared to humifolin. the lowest nutrient concentrations were recorded in control and macro-micro mixture treatments. determination of nutrients in green pods (table 4) showed that the significant highest amount of n was in plants sprayed with biomin aminochelate, and then by delfanplus, humifolin and soil applied npk, respectively. potassium concentration was significantly higher in foliar spray of biomin and delfanplus, while the lowest concentration was in pods treated with macro-micro mixture and in control plants. iron concentration of green pods was significantly increased by organic fertilizers. pod quality there was no significant difference in pod ph and pod total soluble solids (tss) among treatments (table 3). however, determination of titratable acidity (fig. 1) revealed that plants treated with humifolin resulted in significantly higher pod acidity (but not different from biomin and delfanplus), while the least titratable acidity was measured in soil applied npk, control and macro-micro treatments, that showed no difference with biomin and delfanplus treatments (fig. 1). foliar application of three organic fertilizers resulted in significantly higher vitamin c content of pods compared to control plants (fig. 2). pod protein content was higher in plants treated with humifolin and biomin, and lower in control and plants treated by foliar application of macro-micro mixture. pod protein content was intermediate for soil applied npk and foliar application of delfanplus (fig. 3). 4. discussion and conclusions in the present study plant growth was improved by foliar application of three organic chelate fertilizers. many parameters of vegetative growth as well as plant yield, nutrient content and fruit quality were improved by application of these three commercial fig. 1 effects of various fertilization treatments on titratable acidity of french bean pods. data are mean of four replicates ± sd. comparison of means was done using duncan’s test at 5% level. treatments leaf pod n (%) k (%) fe (mg kg-1 dw) n (%) k (%) fe (mg kg-1 dw) control 2.1 c 1.7 c 55.3 c 3.7 c 2.8 c 77.2 b npk 2.7 b 2.6 a 64.1 c 5.2 b 4.6 b 85.1 b biomin 3.5 a 2.8 a 126.3 a 6.5 a 5.3 a 108.3 a humifolin 2.9 b 2.3 b 97.7 b 5.6 b 4.7 b 96.6 a delfanplus 3.2 ab 2.6 a 118.5 a 5.7 b 5.1 a 99.0 a macro-micro mixture 2.3 c 1.8 c 66.1 c 3.7 c 2.7 c 82.4 b table 4 effects of various fertilization treatments on some nutrients in leaves and pods of french bean grown in field with calcareous soil conditions data are mean of four replicates. comparison of means was done using duncan’s test at 5% level. fig. 2 effects of various fertilization treatments on vitamin c content of french bean pods. data are mean of four replicates ± sd. comparison of means was done using duncan’s test at 5% level. fig. 3 effects of various fertilization treatments on protein content of french bean pods. data are mean of four replicates ± sd. comparison of means was done using duncan’s test at 5% level. adv. hort. sci., 2018 32(2): 265-272 270 and nutrient uptake has been shown (david et al., 1994; el-ghamry et al., 2009; salwa, 2011; canellas et al., 2015). the biostimulant effects of humic substances are characterized by both structural and physiological changes in roots and shoots related to nutrient uptake, assimilation and distribution (nutrient use efficiency traits). in addition, they can induce shifts in plant primary and secondary metabolism related to abiotic stress tolerance which collectively modulate plant growth as well as promoting fitness (canellas et al., 2015). in the present study, improved growth and plant performance of french bean might be also due to the higher nitrogen and micronutrients content of plant leaves. the n, k and fe concentrations in plant leaves were significantly improved by foliar applications of organic chelate fertilizers. metal ions such as fe, zn, mn and cu are essential for healthy plant growth, being required for various metabolism reactions (marschner, 2011). they have direct and distinct effects on plant performance, as well as on yield and quality parameters. however, uptake of micronutrients such as iron by roots from soil could be limited due to low chemical stability and precipitation of t h e s e e l e m e n t s p a r t i c u l a r l y i n c a l c a r e o u s s o i l (fernández and ebert, 2005; souri, 2015). in calcareous soils, with high ph and carbonate-bicarbonates levels, plants are prone to iron and other micronutrient deficiencies. lime-induced chlorosis is one of the most important nutritional disorders, affecting many plant yield and quality traits. aminochelate fertilizers represent an excellent n source for plant, in both foliar and soil applications. in present study, n concentration of leaves and pods were significantly improved by organic fertilizers. higher nitrogen content of plant was also reported for radishes (liu et al., 2008) and marigold (souri and yarahmadi, 2016), when plants were treated by foliar application of aminochelate fertilizers. improvement in nutrient elements profile of tomato was observed when amino acids were applied in nutrient solution, which finally improved plant growth and nutrient concentrations, particularly n status of plants (garcia et al., 2011). nitrogen has an important role in growth, yield and quality of crops and must be usually applied to meet the plant needs. french bean can fix atmospheric n2, so it may need less n fertilization; however, in some parts of iran farmers use also high rates of urea to enhance plant growth. on the other hand, application of high amount of n fertilizers could lead to significant reduction in yield and quality, as well as organic fertilizers. increasing in plant growth and various yield traits were also reported in other studies (machado et al., 2008; garcia et al., 2011; naseri et al., 2013; salwa, 2011; ghasemi et al., 2014; sadak et al., 2015; souri and yarahmadi, 2016). in a recent study it was shown that foliar application and to lesse r e x t e n t s o i l a p p l i c a t i o n o f a c o m m e r c i a l aminochelate fertilizer increased the growth, yield and quality of tomato, cucumber and bean plants (souri et al., 2017). foliar application of a mixture of amino acid on bean plants significantly improved the tolerance to seawater salinity stress (sadak et al., 2015). similarly, application of amino acids significantly improved growth parameters of shoots and fresh weight as well as pod yield of soybean plants under pathogen infection (saeed et al., 2005). vegetative growth of plant height and dry weight of potato plants were increased by foliar application of amino acids (el-zohiri and asfour, 2009). three foliar application at 6-7 leaves stage and two more sprays in two weeks intervals using fe and zn aminochelates with different concentrations up to 0.3%, resulted in 1 5 3 5 % h i g h e r p o t a t o t u b e r y i e l d p e r h e c t a r e (pourebrahimi et al., 2013). stimulatory effects of amino acids on plant growth characteristics have been well documented, particularly under adverse climatic conditions such as salt and drought stresses (rai, 2002; zhou et al., 2007; garcia et al., 2011; salwa, 2011; sadak et al., 2015). amino acids are key important player in plant metabolism. they are the intermediate compounds in nitrogen assimilation, and represent the main form by which nitrogen is translocated within the plant through phloem (marschner, 2011). various amino acids and peptides are precursor of physiologically i m p o r t a n t p h y t o h o r m o n e s ( c o b b e t t a n d goldsbrough, 2002; marschner, 2011) or they are involved in detoxification of different toxins within the plant cells (cobbett and goldsbrough, 2002; souri, 2015). in addition to the amino and carboxyl groups, amino acids have a side chain or r group that is attached to the α-carbon. each amino acid has unique characteristics arising from the size, shape, solubility and ionization properties of its r group. nevertheless, the side chain of amino acids exerts a deep effect on their biological activity as well as on the structure and activity of proteins. by far, glycine is the main and widespread used amino acid in manufacturing aminochelate fertilizers, despite frequently a mixture of amino acids may be included (souri, 2015). similarly, the stimulating effect of foliar or soil application of humic acid on increased plant growth souri and aslani foliar application of organic chelate fertilizers on green bean 271 various pollutions. leaf greenness, as the best health indicator of plants, depends on chlorophyll biosynthesis and concentration, which in part is affected by n and micronutrients. aminochelates generally contain all these effective nutrient elements. application of aminochelates, as a source of n fertilizer, can also result in lower nitrate accumulation in plant tissues. high nitrate content of plant tissues is a negative factor, particularly in leafy vegetables that are fresh consumed. application of reduced form of nitrogen (such as ammonium or amino acids) instead of oxidized form (nitrate) can lead to less nitrate accumulation in plant tissues (marschner, 2011; souri et al., 2017). on the other hand, plant might have different response to various fertilizers of a given nutrient (souri, 2015). however, there are always distinct clear responses of vegetative and reproductive growth, as well as quality parameters to nitrogen fertilizers (marschner, 2011; souri, 2016). the higher efficiency of aminochelate fertilizers can be due to the chelating effects of amino acids or organic acids on nutrient elements. the chelating effects of amino acids on nutrients has been commercially used for improving nutritional status of animals and human for more than 6 decades, and for plants in recent years (souri, 2016). as it is well known, amino acids are “zwitterions” in biological systems, and have distinct different behavior in acidic and basic solutions, to maintain the ph of the system. the structure of an amino acid allows it to act both as an acid and a base, depending on solution ph. this behavior is quite important in plant nutrition, as ph is one of the main factors responsible for nutrient use efficiency, and frequently high ph (in calcareous soils) and low ph (in acidic soils) restrict nutrients solubility and bioavailability (souri, 2015). nevertheless, in present study organic chelate fertilizers had various composition of one or several components of amino acid or nutrients. conducting scientific research with such amino or organic chelates due to their mix nature of various components is quite difficult (souri, 2016). in conclusion, foliar application of organic chelate fertilizers resulted in higher plant growth under calcareous soil conditions. aminochelates are composite fertilizers of amino acids and various nutrient elements particularly iron and zinc and separating the effects of each component is relatively difficult. the effect of organic chelate fertilizers particularly aminochelates on many physiological and molecular responses of plants has not been well studied. application of organic chelate fertilizers can avoid all negative effects of routine chemical salt fertilizers such as urea or ammonium nitrate, including leaching, volatilization and nitrate accumulation in vegetable tissues. therefore, they represent modern and suitable alternatives to simple salts, and even to commercial synthetic chelates such as edta due to their cheaper price. nevertheless, these claims need to be evaluated in 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acta physiol. plant, 31: 65-70. zhou z., zhou j., li r., wang h., wang j., 2007 effect of exogenous amino acids on cu uptake and translocation in maize seedlings. plant soil, 292: 105-117. impaginato 43 adv. hort. sci., 2020 34(1): 43­48 doi: 10.13128/ahsc­8300 cefixime manages internal bacterial contamination during tissue culture operation a. ameri 1, g.h. davarynejad 1 (*), a. tehranifar 1, n. moshtaghi 2 1 department of horticultural science and landscape, ferdowsi university of mashhad, mashhad, iran. 2 department of biotechnology and plant breeding, ferdowsi university of mashhad, mashhad, iran. key words: antibiotic, in vitro culture, growth, pyrus communis. abstract: large­scale propagation of pyrus communis, which is a difficult­to­ root species, is dependent on tissue culture technique. however, internal bac­ terial contaminations are an obstacle in tissue culture of fruit tree species. for this purpose, this investigation was conducted with several experiments to manage bacterial contamination. first, gram test for bacterial contamination related to pyrus shoots proliferating was conducted. gram test revealed that bacteria contaminating pear shoots were negative gram. then, we investigated the application of cefixime (0, 100, 300 and 500 mg l­1) or ampicillin (0, 100, 300 and 500 mg l­1) for managing bacterial contaminations. it was found that the contaminated area on medium containing 500 mg l­1 cefixime (63.585 mm2) was lower than other treatments (803.84 mm2). therefore, cefixime at 500 mg l­1 was selected to control the bacterial contamination. next, different proce­ dures were used included shaking with (1: sterile distilled water, 2: 500 mg l­1 cefixime and culturing in media with 500 mg l­1 cefixime, 3: 500 mg l­1 cefixime, culturing and subculturing in media with 500 mg l­1 cefixime 4: disinfection). the third procedure was known the best due to the low bacterial contamina­ tion percentage and rate also the healthy growth of plants. finally, the effect of gibberellic acid at 0 and 1 mg l­1 was investigated to compensate for shoot growth reducing in the presence of cefixime. 1 mg l­1 gibberellic acid improved the growth indices in the presence of cefixime. 1. introduction fire blight, the most devastating disease of pear, leads to the death of the whole pear tree through the systematical infection in all underground and aerial parts of the tree (vanneste, 2000; evrenosoğlu et al., 2019). from the horticultural science perspective, the revival of the pear orchards is dependent on large­scale propagation. the majority of culti­ vated pear is pyrus communis (morgan et al., 1994) and p. communis cv. williams is sensitive to fire blight (abdollahi et al., 2010). however, pyrus communis is difficult­to­root (zhu et al., 2003; sun et al., 2011); therefore, the tissue­culture technique can support large­scale propagation of pear. (*) corresponding author: davarynej@um.ac.ir citation: ameri a., davarynejad g.h., tehranifar a., moshtaghi n., 2020 ­ cefixime manages inter‐ nal bacterial contamination during tissue culture operation ­ adv. hort. sci., 34(1): 43­48 copyright: © 2020 ameri a., davarynejad g.h., tehranifar a., moshtaghi n.. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 20 october 2018 accepted for publication 6 march 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(1): 43­48 44 one of the most important factors in tissue culture is t h e c o n t r o l o f m i c r o b i a l c o n t a m i n a ti o n . p l a n t immune system acts against pathogens. there are three steps of plant defense responses included the response in the step of entry, establishment, and spread of pathogens. overall, plant immune systems can be classified into cell wall reinforcement and pro­ grammed cell death. in pathogen entry condition, cell wall reinforcement is efficient and in pathogen estab­ lishment and spread conditions programmed cell death can be restrictive (abramovitch and martin, 2004). when pathogens suppress the plant defenses responses, the plant was invaded by pathogens and contaminated. one of the phytopathogens is bacte­ ria; they cause serious troubles in vitro conditions. some of the modes of bacteria actions to suppress the host defenses refer to the use of type iii effector proteins and toxins (abramovitch and martin, 2004) as well as, type iv secretion systems to inject effector proteins into cells (angot et al., 2007). tissue culture technique is very sensitive to special pathogenic fac­ tors and all of the microbes in air condition and equipment. in other words, all of the microbes infect cultures with the aim of nutrition (nadha et al., 2012). internal infections in plant cultures had often harmful effects for shoot proliferation, shoot rooting and quality of plant growth (nadha et al., 2012). in tissue culture, the selection of the appropriate antibi­ otic is important. there are several reports about the use of antibiotics to manage the bacterial contamina­ tion (phillips et al., 1981; falkiner, 1990; kneifel and leonhardt, 1992; barrett and cassells, 1994; falkiner, 1997; nadha et al., 2012). cefixime is an antibiotic belonging to cephalosporin class. the mode of action of cephalosporins is related to inhibiting the cell wall biosynthesis so that this class arrests the formation of peptide bonds (kohanski et al., 2010). affect sites of various antibiotics are different; therefore the efficiency of different antibiotics is dif­ ferent in the removal of bacteria. there are some observations for different antibiotics affect sites in the previous studies, such as, inhibiting of cell wall synthesis that is related to benzylpenicillin and phos­ phomycin, inhibiting protein synthesis related to chloramphenicol and streptomycin, inhibiting of rna and dna synthesis related to rifampicin and nalidixic acid (phillips et al., 1981). one of the side effects of antibiotic application in plant tissue culture is the reduction of growth. however, in control of bacterial infection in guadua angustifolia, streptomycin sul­ fate decreased growth shoot, but kanamycin caused intensive growth with high­quality; therefore, the effects of antibiotics are different on plant growth (nadha et al., 2012). the management of contamina­ tion in tissue culture leads to the prevention of waste of time and energy. in this investigation, we examine the use of antibiotics to manage the bacterial conta­ minations coupled with the use of ga3 to compen­ sate for the poor growth of the plant in the presence of antibiotic in the media. 2. materials and methods plant materials three months old proliferating micro­shoots of pyrus communis cv. williams, as the most common cultivar in the world, exhibited bacterial contamina­ tion. contaminated micro­shoots were picked to investigate the experiments of bacterial control for the large­scale production of p. communis. these m i c r o ­ s h o o t s w e r e m a i n t a i n e d i n m s m e d i u m (murashige and skoog, 1962) supplemented with 1.5 mg l­1 ba, 0.1 mg l­1 naa and 3% sucrose. gram test of bacteria two drops, approximately 50 µl, of a 3% (w/v) solution of potassium hydroxide were placed on a clean glass slide as outlined by ryu (1940). bacterial cells were transferred from culture media aseptically with a flat wooden toothpick and placed into the drop of koh with rapid, circular agitation. after 5­8 sec, the toothpick was alternately raised and lowered just off the slid surface to detect a stringing effect. it was considered gram­negative bacteria if drop vis­ cosity increased within 15 sec (suslow et al., 1982; schaad et al., 2001). antibiotic selection test contaminated shoots were cultured on ms medi­ um (murashige and skoog, 1962) supplemented with either ampicillin 0, 100, 300 and 500 mg l ­1 or cefixime at 0, 100, 300 and 500 mg l­1. vessel cul­ tures with 32 mm inner diameter were maintained at a constant temperature of 25±1°c and in 16/8 h light/dark photoperiod (45 µmol m­2 s­1) using cool white fluorescent lamps (sylvania, germany). after a week, the contaminated area was measured in each treatment. bacterial contamination removing in the before step, we selected the proper antibi­ otic (cefixime at 500 mg l­1). then, we used four dif­ ferent procedures using cefixime at 500 mg l­1 to control any eventual bacterial contamination. for each procedure, 3 micro­shoots were cultured in a ameri et al. ‐ in vitro plant rescue by cefixime 45 candle jar as a replicate. 1) shaking of contaminated shoots with sterile dis­ tilled water (control); 2) the first, shaking of contaminated shoots with cefixime at 500 mg l­1 then, cultured in media with cefixime at 500 mg l­1. finally, sub­culturing in free antibiotic media; 3) the first, shaking of contaminated shoots with cefixime at 500 mg l­1, then, cultured in media with cefixime at 500 mg l­1. finally, sub­culturing in media with cefixime at 500 mg l­1; 4) disinfection of contaminated shoots (immersing in 1% hypochlorite sodium for 5 min then rinsed with sterile water three times). ms medium (murashige and skoog, 1962) supple­ mented with 1.5 mg l­1 ba, 0.1 mg l­1 naa and 3% sucrose were used for each procedure. cultures were maintained at a constant temperature of 25±1°c and in 16/8 h light/dark photoperiod (45 µmol m­2 s­1) u s i n g c o o l w h i t e fl u o r e s c e n t l a m p s ( s y l v a n i a , germany). after 30 days, several traits were evaluat­ ed: percentage of fungal contamination and bacterial contamination (bc), bacterial contamination rate (bcr) and general health. based on the following equation (e1) bacterial contamination rate was counted per each micro­ shoot in each candle jar: bcr =∑ ni di w h e r e b c r = b a c t e r i a l c o n t a m i n a ti o n r a t e , n i = number of the contaminated shoot in each day, di = day number. rescued shoot improvement micro­shoots related to the best procedure were transferred to ms medium (murashige and skoog, 1962) supplemented with 1.5 mg l­1 ba, 0.1 mg l­1 naa, 3% sucrose, 500 mg l­1 cefixime and gibberellic acid (ga3) treatments. the concentrations of ga3 were 0 and 1 mg l­1. ph was adjusted at 5.8 with naoh prior to autoclaving at 98 kpa and 121°c, and the media were solidified using 0.8% agar. cefixime antibiotic and ga3 added to the media after autoclav­ ing by filtering. related traits of this experiment were included: the percentage of new growth, the per­ centage of proliferation, the average number of bud and leaf, as well as, the average shoot length. the evaluation of declined antibiotic dose after the six months using cefixime at 500 mg l­1, proliferated shoots were divided into two groups. each group of plants was cultured in media with cefixime at either 500 mg l­1 or 250 mg l­1. ph was adjusted at 5.8 with naoh prior to autoclaving at 98 kpa and 121°c, and the media were solidified using 0.8% agar. cefixime antibiotic and ga3 were added to the media after autoclaving with the syringe filter (pore size: 0.22 µm). after 10 days, the percentage of bacterial contaminations and bacterial contamination rate were measured per each micro­shoot in each candle jar based on (e1). rooting micro‐shoots after six months, micro­shoots were transferred to ½­strength ql (quoirin and lepoivre, 1977) medi­ um supplemented with 1.5 mg l­1 naphthaleneacetic acid (naa) and 500 mg l­1 cefixime. cultures were maintained a week in dark conditions, then, trans­ ferred to 16/8 h (light/dark) photoperiod and light intensity of approximately 45 µmol m­2 s­1 photosyn­ thetic photon flux density (ppfd) emitted by cool­ white fluorescent tubes in 35% relative humidity. statistical analysis these experiments were arranged as a completely randomized design with three replications. the data were statistically analyzed using a one way anova test and means were compared with the duncan test at the 5% level of confidence. all of the statistical tests were performed using sas (statistical analysis system) software v9.1. all of the percentage data were transformed to arcsin √x. 3. results and discussion the result of the gram type detection showed contamination of this investigation is related to gram­negative bacteria. in the antibiotic selection step, we compared ampicillin and cefixime. we used 0, 100, 300 and 500 mg l­1concentrations of each antibiotic. ampicillin is a common antibiotic in tissue culture, and its activity spectrum is related to gram­ positive and gram­negative bacteria, whereas the cefixime is antibiotic acting against gram­negative bacteria. in antibiotic selection test, results revealed 500 mg l­1cefixime could overcome contamination better than other treatments. the contaminated area on medium containing 500 mg l­1cefixime (63.585 mm2) was lower than other treatments (803.84 mm2). therefore, we selected 500 mg l­1 cefixime to control the contamination for later experiments. anova revealed that the difference between the four procedures was significant (p≤0.01) for bacterial contamination percentage and the rate of bacterial adv. hort. sci., 2020 34(1): 43­48 46 contamination (table 1). four procedures were used for the survival and rescuing of shoot from bacterial contamination. the third procedure (the first, shak­ ing of contaminated shoots with 500 mg l­1 cefixime; then, cultured in media with 500 mg l­1 cefixime. finally, sub­culturing in media with 500 mg l ­1 cefixime) was known the best due to the low bacteri­ al contamination percentage, rate and finally the healthy and fresh growth of plants. these indices in other procedures were not desirable; as the highest percentage of bacterial contamination was observed in the first procedure (shaking with sterile distilled water). all of the procedures led to necrotic plants except for procedure 3 (table 2). the presence of antibiotic in media effected on plant growth and weakened their growth; therefore, we used ga3 and its effect evaluated on plants growth. ga3 application in media containing 500 mg l­1 cefixime had a signifi­ cant effect on the percentage of new growth, the average of bud number, the average shoot length and the average of leaf number (p<0.05) (table 3). without the application of ga3 were not observed any proliferation and new bud formation; while in media containing ga3, 22.53% proliferation and 1.46 the average number of bud were observed (table 4). as well as, the results showed the decrease in the antibiotic dose to 250 mg l­1 cefixime increased bcp table 4 ­ evaluation of gibberellic acid in ms medium along with pgrs and 500 mg/l cefixime on secondary growth traits values are mean ± standard error. table 1 ­ analysis of variance traits under study table 2 ­ evaluation of different procedure to come over bacterial contamination during the culture ns= no significant; **= significant at the 0.01 level of probability according to duncan test. **= significant at the 0.01 level of probability according to duncan test. *= significant at the 0.05 level of probability according to duncan test. source of variance df means square bacterial contamination rate of bacterial contamination treatment 3 2963.807 ** 45.48649 ** error 8 7.350.308 0.5436 procedure fungal contamination (%) bacterial contamination (%) bacterial contamination rate general health after 30 days 1 0 90 ± 0 9.47 ± 0.77 necrotic leaves 2 0 74.55 ± 2.42 4.93 ± 0.119 necrotic leaves 3 0 17.01 ± 1.22 0.45 ± 0.053 green leaves and healthy 4 0 57.85 ± 1.56 7.47 ± 0.323 necrotic leaves 1= shaking with sterile water. 2= the first, shaking of contaminated shoots with cefixime (500 mg/l) then, culturing in media with antibiotic. finally, sub­culturing in free antibiotic media. 3= the first, shaking of contaminated shoots with cefixime (500 mg/l), then, culturing in media with antibiotic. finally, sub­culturing in with antibiotic media. 4= disinfection (immersing in 1% hypochlorite sodium for 5 min then shaking with sterile water for three times. values are mean ± standard error. table 3 ­ analysis of variance of traits under study source of variance df means square percentage of new growth percentage of proliferation average of bud number average shoot height average of leaf number treatment 1 514.20 ** 761.53 ** 3.22 ** 60.16 * 54.0 * error 4 6.022 4.53 0.0066 7.33 0.33 concentration (mg/l) new growth (%) proliferation (%) average bud number average shoot height (mm) average leaf number 1 43.93 ± 1.23 22.53 ± 1.74 1.46 ± 0.07 9.66 ± 2.03 10.33 ± 0.33 0 25.41 ± 1.58 0 ± 0 0 ± 0 3.33 ± 0.88 4.20 ± 0.33 ameri et al. ‐ in vitro plant rescue by cefixime 47 leifert and cassells (2001) mentioned alternatives for the antibiotic in their review. these alternatives included medium acidification and autotrophic cul­ ture (e.g. culture without carbohydrate) (leifert and cassells, 2001). in other literature were noted to acti­ vating of endogenous bacteria as a result of sub­cul­ t u r i n g i n m e d i a w i t h c y t o k i n i n s ( k n e i f e l a n d leonhardt, 1992). however, plant tissue culture with­ out cytokinins, carbohydrates and with the modifica­ tion in media acidity is impossible. in this regards, this investigation showed with the presence of cefixime in media containing cytokinins, carbohy­ drates could manage the bacterial contamination. based on the results of this investigation, cefixime at 500 mg l­1 had not any toxicity effect on growth and proliferation. cefixime is an antibiotic belonging to cephalosporins class. the cephalosporin antibiotics have been introduced as the appropriate antibiotic plant tissue culture since they have low eukaryote toxicity (mathias and boyd, 1986) which our results emphasize this point. 4. conclusions bacterial contamination incidence is common and unavoidable during the in vitro propagation of fruit tree species. this investigation presented a proce­ dure to manage the bacterial contamination of p. communis cv. williams during the in vitro culture. based on the results of this investigation, cefixime at 500 mg l­1 could control the bacterial contamination. the use of antibiotic in a medium is associated with a decrease in the growth of plants. this side effect of antibiotic was managed with the application of ga3 at 1 mg l­1. therefore, we suggest cefixime at 500 mg l­1 for in vitro propagation of fruit trees. references abdollahi h., tahzibi f., ghahremani z., 2010 ­ correlation between fire blight resistance and morpho‐ logical characteristics of pear (pyrus communis l.). ­ acta horticulturae, 896: 339­345 abramovitch r.b., martin g.b., 2004 ‐ strategies used by bacterial pathogens to suppress plant defenses. ­ curr. opin. plant biol., 7(4): 356­364. angot a., annette v., stéphane g., nemo p., 2007 ­ exploitation of eukaryotic ubiquitin signaling pathways by effectors translocated by bacterial type iii and type iv secretion systems. ­ plos pathog., 3(1): e3. barrett c., cassells a.c., 1994 ‐ an evaluation of antibi‐ to 41% and bcr to 1.2 (table 5). therefore, using the antibiotics at 500 mg l­1 should continue because the plants grow without bacterial contamination only in the presence of 500 mg l­1 cefixime (fig. 1). nadha et al. (2012) stated the removal of kanamycin from the medium did not result in resumption contamination after 10 days (nadha et al., 2012); while other litera­ ture mentioned that the usage of antibiotics for inhibiting the bacteria growth has impermanent impact and removal of antibiotics has accompanied by resumption contamination (falkiner, 1990; barrett and cassells, 1994; falkiner, 1997; leifert and cassells, 2001) confirming the results of this experi­ ment. in the consumption of antibiotic, resistant­bac­ teria theory is undeniable. despite long­term using of cefixime, about six months, it could not only remove bacterial contamination, but also act without any resistant­bacteria. finally, rescued shoots were able to produce healthy roots. table 5 ­ evaluation of decreasing of cefixime on bcr and bcp after ten days fig. 1 ­ contaminated shoots in vitro culture (a). contaminated shoot after sub­culturing on media without cefixime (b). new growth after using ga3 treatment (c, d) different letters in columns indicate significant difference between treatments at 5% level. traits cefixime concentration (mg/l) 250 500 bacterial contamination rate 41 a 0 b bacterial contamination percentage 1.2 a 0 b 48 adv. hort. sci., 2020 34(1): 43­48 otics for the elimination of xanthomonas campestris pv. pelargonii (brown) from pelargonium x domesticum cv. ‘grand slam’explants in vitro. ­ plant cell tissue organ cult., 36(2): 169­175. evrenosoğlu y., mertoğlu k., bilgin n.a., misirli a., özsoy a.n., 2019 ­ inheritance pattern of fire blight resistance in pear. ­ sci. hortic., 246: 887­892. falkiner f.r., 1990 ­ the criteria for choosing an antibiot‐ ic for control of bacteriain plant tissue culture. ‐ int. soc. plant tiss. cult. newsletter, 60:13­23. falkiner f.r., 1997 ­ antibiotics in plant tissue culture and micropropagation. ‐ what are we aiming at?, pp. 155­ 160. ­ in: cassells a.c. (ed.) pathogen and microbial contamination management in micropropagation. kluwer academic publishers, springer, dordrecht, the netherlands, pp. 371. kneifel w., leonhardt w., 1992 ­ testing of different antibiotics against gram‐positive and gram‐negative bacteria isolated from plant tissue culture. ‐ plant cell tissue organ cult., 29(2): 139­144. kohanski m.a., dwyer d.j., collins j.j., 2010 ­ how antibiotics kill bacteria: from targets to networks. ‐ nat. rev. microbiol., 8(6): 423­435. leifert c., cassells a.c., 2001 ­ microbial hazards in plant tissue and cell cultures. ­ in vitro cell. dev. biol., plant, 37(2): 133­138. mathias r.j., boyd l.a., 1986 ­ cefotaxime stimulates cal‐ lus growth, embryogenesis and regeneration in hexa‐ ploid bread wheat (triticum aestivum l em. thell). ‐ plant sci., 46(3): 217­223. morgan d.r., soltis d.e., robertson k.r., 1994 ‐ systematic and evolutionary implications of rbcl sequence variation in rosaceae. ‐ am. j. bot., 81: 890­ 903. murashige t., 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edition. ­ am. phytopathol. soc. press (aps), st. paul, mn, usa, pp. 373. sun q., sun h., bell r.l., li h., xin l., 2011 ‐ variation of phenotype, ploidy level and organogenic potential of in vitro regenerated polyploids of pyrus communis. ‐ plant cell tissue organ cult., 107: 131­140. s u s l o w t . v . , s c h r o t h m . n . , i s a k a m . , 1 9 8 2 ­ application of a rapid method for gram differentiation of plant pathogenic and saprophytic bacteria without staining. ­ phytopathology, 72(7): 917­918. vanneste j.l., 2000 ­ fire blight: the disease and its causative agent, erwinia amylovora. ­ cabi publishing, wallingford, uk, pp. 370. zhu l.h., li x.y., ahlman a., welander m., 2003 ‐ the rooting ability of the dwarfing pear rootstock bp10030 (pyrus communis) was significantly increased by intro‐ duction of the rolb gene. ‐ plant sci., 165: 829­835. impaginato 27 adv. hort. sci., 2020 34(1s): 27­33 doi: 10.13128/ahsc­7855 short­term low temperature treat­ ments of harvested wine grapes (cv. vermentino) affect the volatile organic compound profile of the berries m. modesti (*), r. shmulevitz, s. brizzolara, p. tonutti life sciences institute, scuola superiore sant’anna, piazza martiri della libertà, 33, 56124 pisa, italy. key words: aroma, post­harvest, temperature conditioning, terpenoids, vitis vinifera. abstract: in the recent years, due to the climate change and the effects of greenhouse gases average temperatures are increasing. grapes cultivated in mediterranean areas are exposed to high temperatures especially during the late growing season and at harvest. this may induce undesirable biochemical processes (e.g. aroma losses and oxidative reactions) with negative effects on the berry composition and specific quality traits of the resulting wine. in the present study the effects of short­term low temperature treatments on har­ vested grapes before vinification have been evaluated. bunches of wine grapes cv. vermentino have been hand­harvested and then refrigerated at 4°c and 10°c for 24 and 48 hours, while 22°c has been applied as control temperature. grapes were analysed in terms of technological parameters (weight loss, total soluble solids, titratable acidity, ph and total polyphenols) and volatile organic compound profile by hs­spme gc­ms. low­temperature post­harvest treat­ ments affect total polyphenols content of the berries and appear to reduce the heat­related aroma loss, increase the content of four volatile terpenoids and decrease the accumulation of ethyl acetate. 1. introduction several challenges characterize the wine industry and have a marked impact on the production chain, final quality of the wines and consumer acceptance. one major problem, in particular in warm­temperate cli­ mates, is represented by the increase of the average temperatures, main­ ly due to the accumulation of greenhouse gases, that often leads to differ­ ent pheno/physiological processes in grape berry and strongly affects berry development. high temperatures induce anticipated and unbal­ anced ripening and, at harvest, undesirable biochemical changes such as aroma losses and oxidative processes (ribéreau­gayon et al., 2006). this negatively affects grape composition and wine quality. hence, it is crucial to find and develop effective strategies for mitigating these negative effects. one option is represented by the application of postharvest cool­ (*) corresponding author: margherita.modesti@santannapisa.it citation: modesti m., shmulevitz r., brizzolara s., tonutti p., 2020 ­ short‐term low temperature treatments of harvested wine grapes (cv. vermentino) affect the volatile organic compound profile of the berries. ­ adv. hort. sci., 34(1s): 27­ 33. copyright: © 2020 modesti m., shmulevitz r., brizzolara s., tonutti p. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 15 january 2020 accepted for publication 4 may 2020 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(1s): 27­33 28 ing treatments of the bunches. postharvest protocols based on controlling (low­ ering) temperature are used for the management and storage of fresh horticultural crops with the main goal of prolonging commercial life and freshness (tonutti, 2013). the effects of low temperature on harvested fruits are diverse and depend on a number of factors including the fruit type, pre­harvest fac­ tors, ripening stage, the applied temperature and the duration of the treatment (kader, 1999). both prima­ r y a n d s e c o n d a r y m e t a b o l i s m s a r e a ff e c t e d (brizzolara et al., 2020), with changes in the composi­ tion and quality parameters, including those related to polyphenols and volatile organic compounds (vocs) (valenzuela et al., 2017, brizzolara et al., 2018). this is also the case of table grapes that, when stored at 0°c to prolong commercial life, show changes in the voc profiles and related­metabolic pathways, resulting in altered overall flavours (maoz et al., 2019). with other goals, post­harvest treatments can also be applied on specific crops undergoing process­ ing. this is the case of wine grapes on which tech­ niques such as controlled dehydration, high carbon dioxide, ozone, ethylene and pre­cooling treatments have been applied or studied to modulate the com­ position of the harvested berries and the style of the resulting wines (mencarelli and tonutti, 2013; becatti et al., 2014; mencarelli and bellincontro, 2018). maintaining harvested wine grapes at low tempera­ ture is a practice that is already applied in certain production areas and for specific enological purpos­ es. low temperature treatments prior to vinification appear to have a positive effect on the aromatic pro­ file of the wines, especially when white­skinned berries are processed. this empirical approach has, so far, very little scientific evidence and, differently from table grapes, just few studies report the effects of such treatments on technological parameters and secondary metabolism (including aroma compounds) of wine grapes. marais (2003) showed that keeping grapes (cv. pinotage) overnight at 10°c and then maintaining the same temperature during skin con­ tact with the must prior to fermentation resulted in the production of the most typical and highest quali­ ty pinotage wines, compared to the same treatments carried out at 15°c. this effect appears to be related to changes in ester metabolism occurring in the berries. mencarelli and bellincontro (2018) reported that following a 10°c treatment applied on wine grapes during post­harvest partial dehydration (a practice used to produce special wines, such as the “passiti”) an up regulation of genes involved in the phenylpropanoid pathway occurs together with a s l i g h t i n c r e a s e o f s ti l b e n e s a n d a d e c r e a s e o f polyphenol oxidase activity. the present study aimed at evaluating the effect of a short­term low tempera­ ture conditioning on harvested wine grapes cv. vermentino in terms of technological parameters and vocs profile. 2. materials and methods grapes samples and cooling treatments bunches of white­skinned wine grapes (vitis vinifera l.) cv. vermentino were hand harvested in 2018 in correspondence of an average total soluble solid (tss) value of 21°brix. the grapes were collect­ ed from a commercial vineyard (lodolina) located in the hills of candia (massa province, tuscany, italy. 44°02’197.6” n, 10°11’265.9” e). the vines are trained at simple guyot, and all agronomic practices f o l l o w t h e d i s c i p l i n a r y o f p r o d u c ti o n f o r t h e appellation of controlled origin (doc) candia dei colli apuani. after harvest, grapes were immediately transported to the laboratory and selected based on absence of evident defects or diseases. grapes were randomly distributed into six lots (of 5 kg each) and subjected to post­harvest low temperature treat­ ments as follow: two lots were cooled at 4°c (±0.5) for 24 (4°c 24 h) and 48 (4°c 48 h) h; two other lots were cooled at 10°c (±0.5) for 24 (10°c 24 h) and 48 (48°c 48 h) h. the last two lots were used as a control and kept at 22°c (±0.5) for 24 (22°c 24 h) and 48 (22°c 48 h) h. immediately after harvest (t0) and at the end of each treatment,30 berries per biological replicate (three biological replicates per lot) were col­ lected and immediately analyzed for technological parameters. for vocs analysis 30 berries per biologi­ cal replicate (five biological replicates for each treat­ ment) were homogenized and a nacl buffer solution (1 m) has been added (1:1) by using an ultraturrax (mod. t25, ika),immediately frozen in liquid nitrogen and stored at ­80°c. technological parameters the weight loss (wl) of 5 bunches from each lot was measured by using a technical balance. these 5 bunches were tagged and weighed at t0 and at the end of each treatment. for each of the three biologi­ cal replicates a total of 30 berries were manually pressed and the obtained must was centrifugated modesti et al. ‐ low‐temperature conditioning of harvested wine grapes 29 (8,000 rpm, 5 min, 22°c), filtered with syringe filters (0.22 µm pore size, 33 mm diameter, sigma­aldrich, italy) and used for the following analyses: ph, using a ph meter (ph­metro glp21; crison instruments); tss employing an optical refractometer; titratable acidity (ta), titrating 7.5 ml of filtered must with 0.1 n sodi­ um hydroxide (naoh), expressed in g/l of tartaric acid equivalent. for each of the three biological repli­ cates, 30 berries were powdered with liquid nitrogen and total polyphenols were then extracted from 250 mg of berries powder with 1.25 ml of 80 per cent methanol and then centrifugated at 4°c, 10,000 rpm for 15 min. the total polyphenols content (tpc) was then measured using the folin­ciocalteau method (singleton and rossi, 1965), expressed as mg of gallic acid equivalents (gae) x 100 g−1 fresh weight. hs‐spme gc‐ms analysis the pre­homogenized (as described above) sam­ ples were thawed and 10 g were weighed in a 20 ml glass crimpvial for headspace analysis (cat. no. su860049, sigma­aldrich, italy) sealed with silicone septa for spme (cat. no. 27362, sigma­aldrich, italy). the grape samples were incubated under agitation for 30 minutes at 40°c. vocs were sampled at the same temperature for 30 min using an spme fiber (50/30 μm, dvb/car/pdms, 1 cm long; supelco, bellefonte, pa, usa). the fiber was desorbed into the injector of the gc set at 250°c for 5 min (splitless mode). a clarus 680 gas chromatograph equipped w i t h a s p l i t / s p l i t l e s s i n j e c t o r ( p e r k i n e l m e r ® , waltham, massachusetts) was used for the analysis. volatiles were separated on a fused­silica capillary column (db­wax, 60 m, 0.32 mm id, 0.25 μm film thickness; restek, bellefonte, pa). helium was used as carrier gas with a flow rate of 1 ml min­1. the gc­ ms settings employed were the same adopted by genova and montanaro (2012). for the identification of the compounds, a mass spectrometer (clarus 500 m a s s s p e c t r o m e t e r , p e r k i n e l m e r ® , w a l t h a m , massachusetts) coupled to the gc was used. each chromatogram was deconvoluted using amdis soft­ ware (national institute of standards, gaithersburg, md, usa). each peak was identified by comparing the experimental spectra with those of the national institute for standards and technology (nist98, version 2.0, usa) data bank including only com­ pounds with 75 per cent of identity or more. the peaks were quantified using turbomass software (turbomass®, version 5.4.2 perkinelmer inc., usa, 2008), by integration of the peak’s areas. the area of each peak was normalized on the sum of the areas of all peaks detected in the same chromatogram to eliminate variations in fiber adsorption. the efficien­ cy of the fiber was monitored by running on daily bases a quality check (qc) sample, calculating the percent of variance in the total area of the qc chro­ matograms. for each sampling time and treatment five biological replicates were analyzed. statistical analysis each set of replicates was tested to detect out­ liers performing principal component analysis (pca) employing metaboanalyst online tool (chong et al., 2019). one­way anova was performed on technological parameter and gc­ms data following a post hoc tukey’s honestly significant difference (hsd) test (with p= ≤ 0.05) for multiple comparation using graphpad prism version 7 (graphpad software, la jolla california usa).vocs revealing statistically sig­ nificant differences between treatments were then analyzed by means of partial least square discrimi­ nant analysis (pls­da) using metaboanalyst online too (chong et al., 2019). 3. results considering technological parameters, as expect­ ed all samples lost weight after 24 and 48 h, follow­ ing both cooling treatments (4 and 10°c) and control conditions (22°c) (table 1). the wl percentage was higher in the control, which showed the highest value after 48 h. the lowest wl value was recorded for grapes cooled at 4°c for 24 h. the wl of grapes cooled at 10°c for 24 h was not significantly different from samples kept at 4 and 22°c for the same time. both cooled samples at 48h showed significantly l o w e r w l v a l u e s t h a n t h e r e s p e c ti v e c o n t r o l . compared to t0 samples the ph values were slightly lower in all samples except for grapes kept at 22°c for 48 h, while ta values significantly decreased only in berries kept for 24h at 4°c and in the 48 h control sample (table 1). with the exception of this latter sample, compared to t0 a general reduction of tss values was observed in comparison with t0 sample. tpc was significantly lower in comparison to t0 in control berries kept at 22°c for 24 and 48 h (table 1). low temperature treatments induced variable effects on this parameter with increases in 10°c 24 h and decreases in 10°c 48 h samples. the grapes vocs profile was acquired by hs­spme gc­ms. a total of 35 vocs has been detected. among adv. hort. sci., 2020 34(1s): 27­33 30 ment is reported in figure 2. the model explains 55.3 per cent of the variability with still an overlapping of the treatments (fig. 2a). fig. 2b reports the vip scores for the employed features. the highest score is attributed to isoledene, which is markedly accumu­ lating in berries kept at 10°c. furthermore, cooled grapes showed again a higher content of terpenoids comparing with the t0. as far as ethyl acetate is con­ cerned, this compound shows the highest level in the control grapes and the lowest in t0 samples. 4. discussion and conclusions in detached fruits, wl progresses with time and is dependent on the vapour pressure deficit, the evapo­ them, 14 terpenes, 7 esters, 4 alcohols, 3 aldehydes, 2 alkanes, 2 benzene derivates, 1 ether, 1 alkane and 1 phenol were identified. one­way anova test was run on the whole vocs dataset: a total of 11 com­ pounds resulted significantly different (p≤0.05) between treatments (data not shown). among the 11 statistically significant vocs, 5 compounds of inter­ est, known for their impact on grapes and wine aroma, were present and so used for a pls­da analy­ sis. these compounds were three sesquiterpenes ( c a d i n e n e , c u b e b e n e a n d i s o l e d e n e ) a n d t h e monoterpene dihydro­citronellol, which are generally associated with floral and spicy notes, and ethyl acetate, which is considered an off­flavor and associ­ ated with the anaerobic metabolism. the pls­da was carried out separately for the two sampling times. cadinene, cubebene and isoledene, dihydro­citronel­ lol and ethyl acetate levels were used as predictor variables, while the different treatments and t0 were used as response variable. the effect of the 24 h treatment is reported in figure 1. after 24 h of treat­ ment, the model explained 51.6 per cent of the vari­ ability present in the dataset and in this projection the different treatments and t0 samples partially overlaps (fig. 1a). figure 1b reports the vip scores for the employed features. the highest score is attributed to cubebene, which seems to be strongly accumulated in berries held at 10°c. noticeably, the level of all the terpenoids considered is higher in the cooled grapes, regardless the temperature, with the only exception of the monoterpene dihydro­citronel­ lol which showed the lowest level in 4°c sample. interestingly, the most marked variation for the three sesquiterpenes (cubebene, cadinene and isoledene) is observed when comparing cooled with t0 samples. on the other hand, control grapes kept at 22°c are characterized by an accumulation of ethyl acetate. a slight accumulation of this compound is found also in grapes held at 10°c (fig. 1b).the effect of 48 h treat­ table 1 ­ technological parameters in vermentino wine grapes at harvest (t0) and after post­harvest treatments at 4°c for 24 h (4°c 24 h), 10°c for 24 h (10°c 24 h), 4°c for 48 h (4°c 48 h) and 10°c for 48 h (10°c 48 h). 22°c is the temperature of the control sam­ ples different letters indicate statistically significant differences at p≤0.05 according to the results of the tukey’s hsd test. values are the mean of three biological replicates +/­ sd. technological parameters t0 4°c 24 h 10°c 24 h 22°c 24 h 4°c 48 h 10°c 48 h 22°c 48 h weight loss (%) ­ 1.4±1.5 c 2.3±0.6 bc 4.5±1.9 b 2.8±1.5 bc 4.5±1.5 b 9.3±2.1 a ph 3.46±0.02 b 3.40±0.0 c 3.33±0.0 d 3.34±0.0 d 3.39±0.0 c 3.36±0.01 d 3.50±0.0 a titratable acidity (g/l­1) 4.6±0.2 ab 4.2±0.0 c 5±0.1 a 4.5±0.0 b 5±0.1 a 4.7±0.0 a 3.7±0.0 c total soluble solid (° brix) 21±0.0 a 19±0.0 b 16.5±0.4 d 19±0.0 b 17.6±0.3 c 18±0.0 c 20.4±0.2 a total polyphenols content (gae/100 gr fw) 613.6±60.4 b 550±47.3 bc 722.7±84.5 a 560.4±9 c 555.6±83 bc 492.1±77.2 c 363.3±46.7 d fig. 1 ­ a) partial least squares discriminant analysis (pls­da) performed on vocs detected in vermentino grapes fol­ lowing cooling treatment at 4 and 10°c for 24 h and con­ trol treatment at 22°c. cadinene, cubebene, dihydro­ citronellol, isoledene and ethyl acetate levels were used as predictor variables while the different treatments and t0 were used as response variables. each color repre­ sents different treatment with five replicates. 95% confi­ dent intervals are presented in ellipses. b) the variable importance in projection scores of pls­da (vip scores). the coloured boxes on the right indicate the relative con­ centrations of the corresponding metabolite in each group under study. modesti et al. ‐ low‐temperature conditioning of harvested wine grapes 31 eral genes involved in the phenylpropanoid pathway and to the accumulation of stilbenes and flavonoids. maintaining harvested berries at 4­10°c can be con­ sidered as mild stress: it is well known that posthar­ vest cold stress induces changes in fruit secondary metabolic pathways and compounds, including phenylpropanoids (dixon and paiva, 1995; ruiz­ garcía and gómez­plaza, 2013; mencarelli and bellincontro, 2018). concerning the volatile compounds, our results indicate that, as general effect, low temperature con­ ditioning of vermentino grapes has an impact on the volatile terpenoid content of the berries. it is well known that the presence of terpenoids significantly affects the aroma of grapes and wines (d’onofrio et al., 2017), and this is particular important for wines vinificated from neutral variety such as vermentino. terpenoids are classified based on the number of carbons present in the chemical structure: monoter­ penes (10 carbons), sesquiterpenes (15 carbons), diterpenes (20 carbons), triterpenes (30 carbons), and carotenes (40 carbons) (yu and utsumi, 2009; li et al., 2019). among the different classes, a signifi­ cant influence on the aroma of grapes and wine has been attributed to the monoterpene class which, in wine, is generally associated with pleasant floral notes (d’onofrio, 2011). along with monoterpenes, sesquiterpenes are another important subclass. to date, there has been limited research on sesquiter­ penes since they are considered less volatile and aroma­active than monoterpenes (may and wüst, 2012; black et al., 2015). however, sesquiterpenes h a v e b e e n r e c e n t l y c o r r e l a t e d w i t h s i g n i fi c a n t organoleptic characteristics of grapes (d’onofrio et al., 2017). indeed, their concentrations in berries can be crucial for the final wine quality (luo et al., 2019) since sesquiterpenes are more stable than monoter­ penes and once extracted from the berry they can be retained in the finished wine (dunlevy et al., 2009). it has been suggested that they provide balsamic, woody and spicy notes (slaghenaufi and ugliano, 2018). based on our preliminary results, it can be hypothesized that low temperature post­harvest treatment is effective in improving specific aromatic traits of vermentino berries and, possibly, wines. the observed increase of terpenoids could be the result of changes in specific metabolic steps of this chemi­ cal class. a key reaction is the conversion of farnesyl pyrophosphate (fpp) to sesquiterpenes, catalyzed by the different members of the terpene synthase (tps) family (tholl, 2006; muhlemann et al., 2014). it is well known that tps activity and so terpenoids rative driving force for water movement and affected by both temperature and relative humidity (cirilli et al., 2012). harvested fruits, including grape berries, already react at low wl values with metabolic changes eventually affecting grape composition (costantini et al., 2006; rizzini et al., 2009; tonutti and bonghi, 2013). in the present trial, control grapes that showed the highest wl values most likely under­ went specific water stress­related reactions more pronounced than those occurring in low temperature samples. previous studies (bellincontro et al., 2009) have demonstrated that a temperature between 5 and 10°c helps to reduce weight loss and to maintain the cellular structure of the berries, with a general reduction of metabolic events. in both control sam­ ples (kept for 24 or 48 h at 22°c), high values of tss well correlate with the loss of weight and the conse­ quent concentration of solutes. the variability pre­ sent among samples for this parameter but also for ph and ta might be the consequence of the hetero­ geneity of the samples, collected in a commercial vineyard. the effects of cooling grapes before vinification on these technological parameters appear to be more clearly defined after 48 h of treatment. this appears also true concerning tpc that increased in cooled samples after 48 h. the effect of postharvest low temperature on tpc has been reported for table grapes by maoz et al. (2019) who showed that stor­ age at 0°c for 6 weeks, led to an upregulation of sev­ fig. 2 ­ a) partial least squares discriminant analysis (pls­da) performed on vocs detected in vermentino grapes fol­ lowing cooling treatment at 4 and 10 °c for 48 h and con­ trol treatment at 22°c. cadinene, cubebene, dihydro­cit­ ronellol, isoledene and ethyl acetate levels were used as predictor variables while the different treatments and t0 were used as response variables. each color represents different treatment with five replicates. 95% confident intervals are presented in ellipses. b) the variable impor­ tance in projection scores of pls­da (vip scores). the coloured boxes on the right indicate the relative concen­ trations of the corresponding metabolite in each group under study. 32 adv. hort. sci., 2020 34(1s): 27­33 biosynthesis strongly depends on both endogenous and environmental factors (robinson et al., 2014). specific studies performed on grape berries in the field showed that high temperatures could reduce terpenoids biosynthesis, and also induce the degra­ dation of thermolabile compounds (d’onofrio, 2011). concerning specifically post­harvest, tps have been studied in toon buds by zhao et al. (2019). they found a strong increase of transcripts related to ter­ penoid biosynthesis under low temperature condi­ tion, which resulted with sesquiterpenoid accumula­ tion. additional studies are so needed to understand if the observed increase of terpenoids in vermentino berries is due to a low­temperature induced biosyn­ thesis or just a maintenance of the pre­accumulated compounds. the use of low temperature in post­harvest and food production is widespread. this preliminary study shows that cooling treatment immediately after harvest have significant metabolic effect on wine grapes. as general effect, it seems that cooling treatments are effective in improving the terpenoids­ r e l a t e d a r o m a p o o l o f t h e n e u t r a l v a r i e t y vermentino. this effect appears to be strongly dependent on the applied temperature as well as on the treatment duration: the specific effects of cooling and the interplay between these two parameters (temperature x treatment duration) 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(batsch)] cv. capdeboscq. the experimental design was completely randomized, in a 2 x 4 factorial design, four replications and five plants per plot. the treatments were the combination of two cultivation environments (on open­air benches and greenhouse bench­ es) and four doses of crf (0, 2, 4 and 8 g l­1 of substrate), in the 19­06­10 npk formulation. ninety days after their transplanting, the variables plant height, stem diameter, number of leaves per plant, shoot dry matter, root dry matter, total dry matter, plant height and stem diameter ratio were evaluated in addi­ tion to the dickson quality index. all morphological variables evaluated pre­ sented a quadratic positive response to the increase of the applied fertilizer until the dosage of maximum technical efficiency (around 6.2 g l­1). the mainte­ nance of the plants in greenhouse benches and the incorporation of 4 g l­1 crf to the substrate ensures greater efficiency in the input use, reducing the amount of time necessary for peach trees cv. capdeboscq to achieve their graft­ ing point and to be used as rootstocks. 1. introduction southern brazil is the greatest national peach producer and this region is recognized as one of the main production centers of stone fruit trees in the country, especially peach trees. however, the traditional production system of these species is mostly performed in the field (bianchi et al., 2014; ibge, 2018), strongly influenced by climatic conditions. the use of protected environments is an alternative for the production of stone fruit trees, which can ensure the survival of the plants during the most critical phase of the tree production chain. especially in rootstock production by seeds, the seedlings emergence stage and initial growth require environment control, as well as optimal nutrition and irrigation, and protection against pests and diseases in order to assure the fast growth and development of the plants (souza et al., 2017). the production of fruit plants in protected environments, such as (*) corresponding author: renata.d.menegatti@gmail.com citation: menegatti r.d., souza a.g., bianchi v.j., 2020 ­ different environments and doses of controlled‐ release fertilizer in peach rootstocks production. ‐ adv. hort. sci., 34(2): 157­166. copyright: © 2020 menegatti r.d., souza a.g., bianchi v.j. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 3 january 2020 accepted for publication 1 april 2020 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(2): 157­166 158 greenhouses, allows the maintenance of optimum conditions for cultivation throughout the year, antici­ pating and extending the grafting period, reducing production costs and increasing the plants quality standard for sale (oliveira et al., 2017). in brazil, the traditional production system of stone fruit trees requires 360­540 days to grow a plant that is suitable for trading (mayer et al., 2015). in this system, the necessary time for rootstocks to reach the grafting point is of approximately 240 days (fischer et al., 2016). in part, this long period is due to the slow growth of plants in the field during the winter and early spring. in order to reduce the time between seed germi­ nation and the production of rootstocks suitable for grafting, the cultivation in a protected environment, as well as the use of appropriate substrates and fer­ tilizers in the proper doses for the crop are alterna­ tives to optimize the initial phase of the plants growth (bianchi et al., 2014; jamal et al., 2017; menegatti et al., 2019 b). the environmental conditions of the plant pro­ duction system directly influence the plants physio­ logical processes and can directly affect plants growth (souza et al., 2017). protected environments can promote greater uniformity for the plants growth in comparison with plants produced in the field or in an unprotected environment (reis et al., 2010; fischer et al., 2016; oliveira et al., 2017). different environments can also affect the germination of seeds, and the growth and quality of the seedlings produced. the interaction between environmental conditions with the application of fertilizers may con­ tribute to optimize the space for plant production in nursery and to reduce the plant production systems impact on the environment. in addition to the use of commercial substrates, the increase of the nutrient supply is recommended b e c a u s e t h e s u b s t r a t e a l o n e d o e s n o t p r o v i d e enough nutrients for the complete development of the plants (dutra et al., 2016). among the many types of fertilizers available, the controlled­release fertilizer (crf) is the most efficient. crf promotes the slow release of nutrients and the absorption of the ideal amount throughout the plants’ growth period, a l l o w i n g t h e m t o a c h i e v e m a x i m u m s t r e n g t h (zamunér filho et al., 2012; menegatti et al., 2017 a). the concomitant use of fertilizers with the sub­ strate favors the formation of more vigorous plants in shorter time, which reduces the period in which they stay in the nursery and, consequently, the pro­ duction costs (muniz et al., 2013; menegatti et al., 2017 b). however, few are the researches that report the use of fertilization as an additional factor to the production of stone fruit trees (zhang et al., 2014; jamal et al., 2017; menegatti and bianchi, 2019). even scarcer are the studies that consider plant prop­ agation in a protected environment, such as a green­ h o u s e i n c o m p a r i s o n w i t h o p e n e n v i r o n m e n t s (picolotto et al., 2007; reis et al., 2010). the scarce information about the use of con­ trolled­release fertilizers in the production of peach rootstocks in protected environments encouraged the accomplishment of this study, whose objective was to verify the effect of different environments and doses of crf on the initial growth of peach trees cv. capdeboscq for rootstock purposes. 2. materials and methods ripe peach fruits of cv. capdeboscq were harvest­ ed in january 2017 from clonal mother plants kept in the germplasm collection of peach rootstocks at the federal university of pelotas (ufpel), brazil. the experiment was conducted between october (2017) and january (2018), at the department of botany­ ufpel, capão do leão, rs, brazil, at 21° 48’ south lati­ tude, 41° 20’ west longitude and an altitude of 11 m. after the harvest of the fruits, the post­harvest management of the pits was carried out according to picolotto et al. (2007). then the seeds were stratified, as described by souza et al. (2017). after the stratifi­ cation period (35 days at 7°c), the seeds were sown, 1.0 cm deep, in 72­cell polystyrene trays (114 cm3 per cell) containing a mixture of orchard soil + vermi­ c u l i t e + m e d i u m s a n d + c o m m e r c i a l s u b s t r a t e plantmax® (1:1:1:1) as substrate, and kept in a green­ house. when the seedlings, hereinafter referred as “ p l a n t s ” , r e a c h e d t h e t r a n s p l a n t p o i n t ( 1 5 c m between collar and apex), they were transplanted into 1­liter plastic bags containing washed sand, which was used as substrate (table 1) and whose crf (osmocote®) doses had the n­p­k formulation of 19­ 06­10 (4­6 months), which were previously incorpo­ rated into the sand. the experimental design was completely random­ ized, in a 2 x 4 factorial design, with two environ­ ments (on open air benches and on benches inside the greenhouse) and four doses of osmocote® (0, 2, 4 and 8 g l­1 substrate), with four replications and five plants per replication. “protected environment” refers to the arco menegatti et al. ‐ peach rootstocks production 159 pampeana metallic structure greenhouse model, cov­ ered with a 150­millimeter thick, low­density poly­ ethylene plastic film, arranged at the north­south direction and with the following dimensions: 10.0 m x 21.0 m and with the maximum height of 5.0 m. the benches used in the two environments were 1­meter high metallic structures, positioned at ground level. the environmental open air conditions and the ones in the greenhouse during the period of the experi­ ment are described in table 2. ninety days after transplantation, when 75% of the plants of one of the treatments reached the grafting point (at least 5 mm of stem diameter and 10 cm above the soil), the plants were evaluated for the variables stem diameter (sd), plant height (h) and number of leaves (nl). based on these data, it was possible to calculate the plant height and stem diam­ eter (h/sd) ratio. the height of the rootstocks was measured using a graduated ruler, and the stem diameter was measured with a digital caliper. the plants were dried in a forced air circulation oven at 70°c for 72 hours to obtain the shoot dry matter (sdm), root dry matter (rdm) and total dry matter (tdm) per plant. the dickson quality index (dqi) was obtained by the formula: dqi = tdm/ [(h/sd) + (sdm/rdm)], according to gomes and paiva (2011). the stem diameter increase (δsd) was obtained through the data collected every 15 days until the end of the experiment (90 days after transplanta­ tion). possible differences between treatments were verified by analysis of variance (anova). the vari­ ables that exhibited significant differences were sub­ mitted to regression analysis in order to verify the plants growth response in proportion to the crf increasing doses in both growing environments. the data analysis was performed in the statistical pack­ age sisvar (ferreira, 2011). 3. results and discussion at the end of the experiment (90 days after trans­ plantation), the survival rate of the peach rootstock plants was of 100% for all treatments. all variables exhibited interaction (p <0.05) between the environ­ ment factors and the crf (osmocote®) doses (table 3), indicating that the study of factor interaction is important to define the best condition to stimulate plant growth and development. table 1 ­ average chemical composition of the sand substrates used in the production of peach tree rootstocks *sand; **om= organic matter; v= base saturation; sb= sum of bases; cec= cation exchange capacity (cec). substrates om** % v % h+al mg dm­3 sb mg dm­3 cec mg dm­3 p mg dm­3 k μg dm­3 ca μg dm­3 mg μg dm­3 zn μg dm­3 fe μg dm­3 mn μg dm­3 sand* 0.00 67.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.44 0.00 0.00 table 2 ­ environmental conditions: temperature (t°c), relative humidity (rh%) and global radiation (w m­²) in the two cultivation environments (in open­air benches and greenhouse benches) during the cultivation period of peach trees cv. capdeboscq environment temperature (°c) rh (%) global radiation (w m­²) greenhouse 23.42 66.81 348.50 open air 21.15 78.62 477.17 table 3 ­ summary of the variance analysis for contrasts between the environmental factors of cultivation and crf (osmocote®) doses for the variables stem diameter (sd), plant height (h), number of leaves per plant (nl), shoot dry matter (sdm), root dry matter (rdm), total dry matter (tdm), plant height and stem diameter (h/sd) ratio, and the dickson quality index (dqi) of peach trees cv. capdeboscq 90 days after transplantation * significant at the probability level (p<0.01) and ** significant at the probability level (p<0.05) by the f test. source of variation df mean square sd (mm) h (cm) nl sdm (g) rdm (g) tdm (g) h/sd dqi environment (e) 1 128.2 ** 1897.8 ** 56.2 ** 603.8 ** 96.9 ** 216.2 ** 474.4 ** 42.0 ** dose of crf (d) 3 137.7 ** 549.8 ** 109.8 ** 233.8 ** 37.9 ** 84.1 ** 103.0 ** 60.7 ** e x d 3 8.0 ** 162.9 ** 11.6 ** 70.1 ** 12.6 ** 26.9 ** 31.4 ** 3.9 * mean 4.3 60.8 55.0 5.9 9.6 15.5 13.4 1.7 cv (%) 4.0 4.3 11.9 10.1 25.5 17.0 6.4 9.7 mean greenhouse 4.6 80.9 64.0 8.48 13.8 22.3 16.7 1.8 mean open air 3.9 40.7 46.0 3.3 5.3 8.6 10.0 1.5 adv. hort. sci., 2020 34(2): 157­166 160 all morphological variables exhibited a quadratic behavior in the adjustment of the regression equa­ tions (figs. 1, 2, 3 and 4), proving that the highest dose test results decreases the variables values, that is, increasing the fertilizer dosage allows the increase of the plants growth up to the maximum technical efficiency dose (mted). the mted for plant height in the protected envi­ ronment was of 6.43 g l­1, corresponding to the height of 113.8 cm, which was three times higher than the control treatment (substrate without the addition of osmocote®) in the same environment, 90 days after transplantation (fig. 1). the mted for plant height in the external environment was of 5.09 g l­1, whose plants reached the height of 52.1 cm, in contrast to the 23.9 cm high of the treatment with­ out the addition of crf (fig. 1). similar height growth was also observed with the application of osmocote® in the studies performed by silva et al. (2011) in the production of rangpur lime rootstock [citrus limonia l. (osbeck)] and by dutra et al. (2016) in the growth of canafistula [peltophorum dubium (spreng.) taub.]. however, the environments and doses tested in their studies were different of this one. the positive effects of crf application on plant growth in different species reinforce the necessity of specific studies to enable the definition of the mted for each species and cultivar, which may provide s u p e r i o r g r o w t h a n d e ffi c i e n c y i n t h e u s e a n d exploitation of fertilizers by the plants. the estimated mted for the sd variable of the plants cultivated in greenhouse was of 7.29 g l­1, cor­ r e s p o n d i n g t o a d i a m e t e r o f 5 . 5 1 m m ( f i g . 2 ) . according to the current legislation of the secretary o f a g r i c u l t u r e a n d f o o d o f r i o g r a n d e d o s u l (ordinance 302/98), the grafting must be performed when the rootstocks reach a sd over 5 mm and a height of 10 cm from the ground. thus, the mted estimated for plant grown in greenhouse allows the rootstocks to reach the minimum diameter for graft­ ing at 90 days after transplantation. on the other hand, in the external environment, the plants have not reach the minimum stem diame­ ter required for grafting during the experiment, even at the highest crf dose (fig. 2). in peach rootstock production, it is extremely important to define the environment and the fertiliz­ er mted to obtain the ideal sd for grafting in a short­ er period. the minimum diameter of 5 mm is used to make sure that the rootstocks phloem and xylem have greater competence to perform rapid vascular connection between the scion/rootstock (santarosa et al., 2016), which will allow the effective transloca­ tion of the nutrients absorbed by the roots (root­ stock) to the aerial part (scion cultivar). this condi­ tion will result in a higher percentage of graft­take and growth of the grafted plants, and it will reduce the period to obtain commercial plants. souza et al. (2013) stress the influence of the rootstock diameter to reduce the time to develop grafted plants of “ponkan” tangerine. the plants rapid growth was obtained with the use of rootstocks with larger diameter in the appropriate cultivation conditions in the greenhouse. the best plant growth in both height and stem diameter was obtained in plants grown in a green­ house with the mted. this suggests that the green­ house environment provided better conditions of temperature, humidity and luminosity for the plant growth. associated with the mted, these conditions fig. 1 ­ plant height (cm) of the peach plants cv. capdeboscq, in relation to the dosage of the controlled­release fertilizer (crf) and two cultivation environments, 90 days after transplanting. fig. 2 ­ stem diameter (mm) of peach plants cv. capdeboscq, in relation to the dosage of the controlled­release fertilizer (crf) and two cultivation environments, 90 days after transplanting. menegatti et al. ‐ peach rootstocks production 161 allowed the plants to reach the ideal point for rapid grafting, which is a desired aspect in the production system of peach tree rootstocks. paricá seedlings [schizolobium amazonicum huber ex ducke] grown in a protected environment also exhibited superior performance for plant height and stem diameter (frigotto et al., 2015). these authors concluded that greenhouse cultivation significantly increases the growth variable values in comparison with external environment cultivation. the highest mean number of leaves per plant (97) was obtained with the estimated mted (4.69 g l­1) for the plants grown in the greenhouse (fig. 3a). as for the variable number of leaves, it was found that the plants of cv. capdeboscq cultivated in an open sky achieved dmet higher than plants kept in a greenhouse (fig. 3), a fact that may be related to phytosanitary problems, such as, for example, small leaf spots and necroses detected in the leaves of this treatment, during the conduction of the plant experi­ ment. these leaf damage possibly induced damage to the leaf photosynthetic apparatus, however, this damage may have been efficiently reversed through the emission and growth of new leaves. this hypothesis can be supported by the fact that, at this moment, the plant enhances the production of photoassimilates and destines most of it, the maintenance and maximization of the aerial part, m a k i n g t h i s o r g a n t h e d r a i n o f g r e a t e r e n e r g y fig. 3 ­ number of leaves per plant (3a), shoot dry mass (3b), root dry mass (3c) and total dry mass (3d) of peach plants cv. capdeboscq, in relation to the dosage of crf and two cultivation environments. demand, both to stimulate the leaf growth maximi­ zing the capture of light, as well as to boost the thic­ kening of the stem diameter ensuring the robustness of the rootstock. it should also be noted, according to the results obtained in this work, that plants grown in the open suggest that they have prioritized the increase in the diameter of the stem at the expense of growth in height, as shown in table 4, a strategy that can increase the robustness of the plants and decrease the exposure of the aerial part, guaranteeing their survival for a longer period, as well as the maintenan­ ce of the physiological processes in this cultivation environment, which expose the plants to sudden environmental variations. the cultivation environment has a strong influen­ ce on environmental conditions, such as temperature and global radiation, parameters that are indirectly related to the efficiency of plants in terms of light absorption capacity, and later conversion to energy, as well as in the absorption and use of nutrients. a gradual production increase of shoot dry mat­ ter, root dry matter and total dry matter up to the crf mted was registered (fig. 3b, 3c and 3d), regardless of the cultivation environment. however, the plants grown in greenhouse presented higher val­ ues for total dry matter. these results corroborate the effects of osmocote® in the growth of rangpur lime rootstocks, as reported by scivittaro et al. 162 adv. hort. sci., 2020 34(2): 157­166 (2004). they found that as controlled­released fertil­ izer doses increased, the dry matter production of the rangpur lime rootstocks increased as well. the leaf area has not been quantified in this study. however, there are previous studies that sup­ port the increase of the number of leaves per plant is directly proportional to the growth of the leaf area (menegatti et al., 2017 a). the greatest leaf area of plants grown in greenhouses provides greater effi­ ciency in solar energy uptake for photosynthesis and photoassimilate production, which is directly related to the nutrient supply, including nitrogen (n), present in the crf formulation used in this study. the use of crf in the mted ensures the availabili­ ty and efficient utilization of n by the plants because the leaching level of n is reduced in comparison with conventional fertilizers (zamunér filho et al., 2012; muniz et al., 2013). n is an essential element to the components of the photosynthetic system, such as chlorophylls, carboxylase activity/oxygenase of ribu­ lose 1.5­bisphosphate and carboxylase of phospho­ enolpyruvate (bassi et al., 2018), thus maintaining satisfactory rates of carbon assimilation (taiz and zeiger, 2017) and consequently guaranteeing the production of photoassimilates that support plant growth. the crf effects on the production of rangpur lime plants for use as rootstocks were registered by serrano et al. (2006) and silva et al. (2011). they con­ cluded that the fertilizer mted increases the vari­ ables shoot dry matter, root dry matter and total dry matter, stressing the importance of fertilization for the maintenance of the photosynthetic process in order to increase the total dry matter and conse­ quently the plants growth. the relation between plant height and stem diam­ eter (fig. 4a) presents a balanced growth of the plants raised in greenhouses. the h/sd ratio is one of the parameters most used in the plant’s quality evaluation. moreover, it fig. 4 ­ ratio between height and stem diameter and dickson quality index of peach plants cv. capdeboscq, in relation to the dosage of controlled­release fertilizer and two cultivation environments. table 4 ­ mean values of the differences between greenhouse and open air for the variables stem diameter (sd), plant height (h), num­ ber of leaves per plant (nl), shoot dry matter (sdm), root dry matter (rdm), total dry matter (tdm), plant height and stem diameter (h/sd) ratio, and dickson quality index (dqi) of peach plants cv. capdeboscq 90 days after transplantation dose sd (mm) h (cm) nl sdm (g) rdm (g) tdm (g) h/sd dqi 0 0.47 10.6 4.6 0.7 1.2 1.9 1.9 0.1 2 0.35 33.4 19.8 4.1 5.8 9.9 6.3 0.2 4 0.84 51.6 38.9 7.0 11.9 19.0 8.4 0.5 8 1.11 64.9 6.4 8.7 14.9 23.7 9.7 0.5 menegatti et al. ‐ peach rootstocks production 163 reflects the accumulation of reserves and ensures greater resistance and adequate potential of the rootstock (souza et al., 2013). the index considers two parameters in a single indicator, and it can be used as a guide to determine the quality of the plants. the balance of the plants growth confirmed by the h/sd ratio may have been favored by the use of crf. the encapsulated fertilizers, such as crf, allow the slow release of nutrients through a porous struc­ ture (serrano et al., 2006), becoming available to the plants root system over time and according to their n u t r i ti o n a l n e e d , a v o i d i n g t h e l e a c h a n d l o s s observed in conventional fertilizers. in addition to the h/sd ratio, the dqi is a good indicator of plant quality. for its calculation, it con­ siders the plants robustness and biomass distribution balance. therefore, the higher the value, the better the quality standard of the plants will be (gomes and paiva, 2011). the ideal value considered for the dqi is approxi­ mately of 2.00 (gomes and paiva, 2011). in our study, the highest dqi was close to 2.2 for the estimated mted of 6.18 g l­1 in plants grown in a greenhouse. however, for the plants kept in the external environ­ ment, the highest dqi value was of 1.83 with an mted of 5.72 g l­1, which is lower than the ideal value (fig. 4b). previous research from dutra et al. (2016) and zamunér filho et al. (2012), in addition to the results obtained in the present study, agree with the results for all morphological variables evaluated. a quadratic positive response was obtained proportionally to the increase of the crf doses up to the mted (of approx­ imately 6.2 g l­1). it proves that the plant will not have higher responses if a dose higher than the mted is applied. the δsd, evaluated every 15 days after the begin­ ning the experiment, is presented in figure 5. for the plants cultivated in a greenhouse, the use of the doses of 4 and 8 g l­1 of osmocote®, incorporated into the substrate, were proven efficient for the pro­ duction of rootstocks suitable for grafting after 90 days because they presented a final sd mean of 5.1 mm and 5.5 mm, respectively. considering the aforementioned results and the relevance of the sd variable in the production of peach rootstocks, we suggest the incorporation of at least 4 g l­1 of osmocote® into the commercial sub­ strate and the cultivation of the plants in a green­ house. those conditions can increase the efficiency of the fertilizer use to obtain plants that can be graft­ ed after 90 days. the effects of different environments on the pro­ duction of peach rootstocks cv. okinawa were reported by reis et al. (2010). they reached the ideal point for grafting after 179 days in a protected envi­ ronment. schmitz et al. (2014), evaluating the pro­ duction of peach rootstocks cvs. capdeboscq and okinawa, in three production systems, reached the grafting point after 154 days. fischer et al. (2016), researching the influence of the stratification period on wet cold in the emer­ gence and production of several peach rootstocks in the field, obtained materials suitable for grafting after 240 days. these results indicate that the use of crf and a protected environment are promising in the shortening of the productive cycle, as the grafting fig. 5 ­ increase of stem diameter, over time, of peach plants cv. capdeboscq, in relation to the dose of the crf and two cultivation envi­ ronments (a) open air and (b) greenhouse. adv. hort. sci., 2020 34(2): 157­166 164 rates and accelerates the respiratory metabolism, reducing the growth rate not only for sd, but for all morphological variables (afonso et al., 2017; bassi et al., 2018). another factor that may have contributed to reduce the plants growth in external environment was the crf formulation. the granules contain a homogeneous combination of nutrients and are covered by an organic resin that regulates the nutrients release proportionally to the substrates temperature and humidity (melo júnior et al., 2014). in addition to the high tempera­ tures, the heavy rainfall can contribute to accelerate the release of crf nutrients, resulting in leaching losses. a c c o r d i n g t o t h e d a t a p r o v i d e d b y t h e agroclimatology station of pelotas (eapel, 2017), between october 2016 and january 2017, the cumu­ lative rainfall reached 549 mm, with a monthly aver­ age of approximately 137 mm, concentrated in three to four days of each month. the intense and concen­ trated precipitation in a short period of time may have caused greater leaching of the nutrients present in the soil solution that had the plants in external environment, reducing the efficiency of the fertilizer use and resulting in lower values for the evaluated variables in comparison with the plants grown in a greenhouse, which did not suffer the influence of the precipitation variable. considering the results obtained in this study, it was suggested that the control of the environment for plant cultivation provides greater efficiency in the use of productive resources (nutrients, water, tem­ perature, light and others). in addition to these fac­ tors, the use of crf incorporated into the substrate contributes to cause precocity in the production of peach rootstocks (reduction of the period to reach the grafting point) with a high­quality standard. 4. conclusions considering the results obtained in this study, the cultivation of plants in a greenhouse is proposed, since it provides the best conditions for the use of crf by the plants, and the concomitant use of the minimum dose of 4.0 g l­1 because it reduces the period to reach the grafting point to 90 days. acknowledgements t h i s s t u d y w a s p a r ti a l l y fi n a n c e d b y t h e c o o r d i n a ti o n f o r t h e i m p r o v e m e n t o f h i g h e r point can be reached after 90 days. this outcome is probably due to the ready availability of nutrients and the maintenance of the plants under favorable environmental conditions, although these results must be validated for other cultivars of [prunus persica l. batsch] rootstocks with potential use in brazil, such as cvs. flordaguard, okinawa and the tsukuba series (menegatti et al., 2019 a). the greatest δsd occurred between 30 and 60 days after transplantation (fig. 5), regardless of the dose employed, which may be due to the slow releasing of the nutrients, a main characteristic of the fertilizer used, which resulted in the greater amount of nutrients available to the plants during the experi­ ment (huett and gogel, 2000). this result also sug­ gests that it is necessary to replace the mineral ele­ ments around 60 days after the first application in order to maintain the plants optimal growth rate. therefore, studies to elucidate the best crf replace­ ment period are necessary in order to ensure contin­ ued growth through the plants different develop­ ment stages. the crf used in the production of peach root­ stocks proved to be a promising alternative in com­ parison with conventional fertilizers. the crf slowly and continuously releases nutrients to the plant, avoiding leach losses and volatilization. furthermore, it ensures a better use of nutrients and reduces the environmental and economic impact (especially by the nitrogen economy, which is an expensive and easily leachable element that has a great potential to pollute the environment). the negative aspect of using crf is the higher cost in comparison with conventional fertilizers. however, the application of the mted, with the purpose of maximizing the input use in the production of root­ stocks, has been proving to be an economically viable alternative if we consider the price increase of basic inputs. other characteristics to be considered are the conventional fertilizers high susceptibility of leaching due to the frequency of irrigation in the nursery and the need for parcelled applications, which are 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407 adv. hort. sci., 2018 32(3): 407-420 doi: 10.13128/ahs-22856 quality and nutraceutical properties of mango fruit: influence of cultivar and biological age assessed by time‐resolved reflectance spectroscopy m. vanoli 1, m. grassi 1, l. spinelli 2, a. torricelli 2, 3, a. rizzolo 1 (*) 1 consiglio per la ricerca in agricoltura e l’analisi dell’economia agraria, centro di ricerca ingegneria e trasformazioni agroalimentari (creait), sede di milano, via g. venezian, 26, 20133 milano, italy. 2 istituto di fotonica e nanotecnologie, consiglio nazionale delle ricerche (ifn-cnr), piazza l. da vinci, 32, 20133 milano, italy. 3 politecnico di milano, dipartimento di fisica, piazza l. da vinci, 32, 20133 milano, italy. key words: absorption coefficient, ascorbic acid, carotenoid composition, mangifera indica l., total antioxidant capacity, total phenols. abstract: the content and composition of the main antioxidants in the pulp of mangoes depend also on cultivar and maturity degree, the latter being non‐ destructively evaluated by the absorption coefficient measured by time‐ resolved reflectance spectroscopy (trs) at 540 nm (µa540). aiming at evaluat‐ ing the levels of antioxidants [carotenoids (car), phenols (tpc), ascorbic acid (aa)] and antioxidant capacity (tac) in relation to µa540 maturity class, select‐ ed ‘haden’ and ‘palmer’ mangoes were measured for µa540 by trs, classified based on µa540 value as less (lem), medium (mem) and more (mom) mature and analyzed for pulp firmness, pulp color (a*, h°, yellowness index), car (total and composition by hplc‐dad), tpc, aa and tac. ‘palmer’ fruit had higher tpc, aa and tac than ‘haden’ mangoes. on average mom fruit showed higher tpc, total car, total all‐trans‐violaxanthin esters and all‐trans‐β‐carotene than mem and lem fruit. lem fruit did not have compounds belonging to the 9‐cis‐violaxanthin group, while cis‐β‐cryptoxanthin was approx. 19% of total carotenoids. in mom mangoes the main carotenoid was all‐trans‐β‐carotene (53%), followed by total all‐trans‐violaxanthin esters (30%), 9‐cis‐violaxanthin group (8%) and cis‐β‐cryptoxanthin (6%). the µa540 significantly correlated (r=0.78‐0.94) with total car, all‐trans‐β‐carotene, all‐trans‐violaxanthin no.3 (both cultivars), tpc, all‐trans‐violaxanthin no.1, no.2, no.6 (‘haden’), and 9‐cis‐violaxanthin no.2, no.3 (‘palmer’). our results indicate that trs is suitable to non‐destructively measure the pulp color of mangoes and to sort fruit with different ripening degree and nutraceutical properties. 1. introduction mango (mangifera indica l.) is a climacteric fruit belonging to the family of anacardiaceae grown particularly in tropical and subtropical coun(*) corresponding author: anna.rizzolo@crea.gov.it citation: vanoli m., grassi m., spinelli l., torricelli a., rizzolo a., 2018 quality and nutraceutical properties of mango fruit: influence of cultivar and biological age assessed by time-resolved reflectance spectroscopy. adv. hort. sci., 32(3): 407-420 copyright: © 2018 vanoli m., grassi m., spinelli l., torricelli a., rizzolo a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 8 march 2018 accepted for publication 13 september 2018 ahs advances in horticultural science adv. hort. sci., 2018 32(3): 407-420 408 tries, with an estimated world production of 42 million tons per year (fao, 2015). brazil is one of the first ten largest mango producers, and more than 25% of its production is exported to europe (mitra, 2016). appreciated for its excellent eating quality due to attractive flesh color, juicy texture and sweet flavor, mango provides high contents of bioactive compounds including carotenoids, phenolic compounds, ascorbic acid and reducing sugars (rocha ribeiro et al., 2007; manthey and perkins-veazie, 2009). carotenoids are responsible for the yellow-orange color of mango pulp and their content and composition depend mainly on cultivar and maturity degree, along with edaphic and climatic factors and postharvest handling, processing and storage conditions ( o r n e l a s p a z e t a l . , 2 0 0 7 , 2 0 0 8 ; m a n t h e y a n d perkins-veazie, 2009; hewavitharana et al., 2013; liu et al., 2013; vásquez-caicedo et al., 2006; vanoli et al., 2016). during mango fruit ripening, biosynthesis of carotenoids occurs, due to chloroplasts differentiation into chromoplasts by disintegration of the thylacoid membranes and by the development of new pigment-bearing structures (vásquez-caicedo et al., 2006). this process leads to carotenoids accumulation, which is usually accompanied by color changes of the pulp turning from white to yellow-orange (ornelas-paz et al., 2008; vásquez-caicedo et al., 2006). the accumulation of carotenoids in the mesocarp shows an exponential behavior during fruit ripening and cultivar-specific relationships between total or individual carotenoid (all-trans-β-carotene, all-trans-violaxanthin and 9-cis-violaxanthin) contents and mesocarp color (a*, h°) were established in different mango cultivars (vásquez-caceido et al., 2 0 0 6 ; o r n e l a s p a z e t a l . , 2 0 0 8 ) . s i m i l a r l y t o carotenes, ascorbic acid content and total phenolic content vary according to the cultivars, maturity stage and cultural practices (rocha ribeiro et al., 2007; valente et al., 2011; liu et al., 2013; oliveira et al., 2016; septembre-malaterre et al., 2016). in order to withstand shipping to distant markets and at the same time to have the optimum eating quality when ripe, mango fruit are harvested at the hard green stage, after having reached the physiological maturity, but before the onset of the climacteric rise. if mango fruit are immature at harvest, they do not reach an eating quality when ripe and, hence, the discrimination between mature and immature fruit at harvest is very important from the marketing point of view in order to minimize qualitative losses during the supply chain. fruit shape (“shoulders” should be full), pulp color and firmness are the most used maturity indices for mangoes, but differences among cultivars and growing conditions have precluded universal maturity indices. on the other hand, the current industry measurements of firmness and pulp color have the disadvantage of being destructive of fruit; hence, the development of a non-destructive technique could help the growers to pick fruit at the proper maturity degree for the different market destinations. among the non-destructive techniques able to assess the maturity degree of fruit, time-resolved reflectance spectroscopy (trs) is gaining increasing interest (nicolai et al., 2014). trs is a non-destructive optical technique which can separate the effect of light absorption, due to chemical compounds such as pigments and water, and light scattering, due to microscopic changes in refractive index caused by membranes, vacuoles, starch granules, organelles, and air. by measuring photon time-of-flight distribution with picoseconds temporal resolution, the absorption (µa) and reduced scattering (µs) coefficients in the vis-nir wavelength range are quantified, by probing pulp at a depth of 1-2 cm with no or limited influence from the skin (cubeddu et al., 2001, torricelli et al., 2008). trs has been used to study the internal fruit attributes related to maturity in apples, peaches, nectarines and pears (rizzolo and vanoli, 2016). in nectarines, the µa measured at harvest at 670 nm (µa670), near the chlorophyll-a peak, can be considered an effective maturity index as it is linked to the biological age of the fruit (tijskens et al., 2007) and has been successfully used to predict the softening rate during shelf life in nectarines and to select fruit for different market destinations (eccher zerbini et al., 2009). previous studies carried out on mango fruit have demonstrated the potential of trs for the nondestructive determination of pulp color (vanoli et al., 2013; rizzolo et al., 2016; vanoli et al., 2016), as well as the possibility of using trs absorption in both the carotenoids (540 nm) and chlorophyll-a (670 nm) spectral regions to classify mango fruit according to maturity and to predict the ripening of individual fruit (eccher zerbini et al., 2015). the present work aimed at evaluating the levels of antioxidants and antioxidant capacity in the pulp of two cultivars of brazilian mangoes in relation to fruit maturity class assigned according to the µa540 value, along with selected quality parameters. the relationships between µa540 maturity index and total carotenoid content, total phenolic compounds, ascorbic acid and the fourteen carotenoid comvanoli et al. quality and nutraceutical properties of mangoes according to trs maturity class 409 pounds identified were also studied. 2. materials and methods mango fruit on november 2011, ‘haden’ and ‘palmer’ mang o e s w e r e p i c k e d i n e x p e r i m e n t a l o r c h a r d s o f epamig (empresa de pesquisa agropecuária de minas gerais) in minas gerais state (brazil) and transported to milan (italy) by plane soon after harvest. on arrival at crea-it lab (about 5-7 days from harvest), 60 fruits of ‘haden’ and 90 fruits of ‘palmer’ without defects were selected and measured by trs at 650 nm (‘haden’) or at 690 nm (‘palmer’) on two opposite sides in the fruit equatorial region and ranked by decreasing µa averaged over the two sides, that is from less (high µa) to more mature fruit (low µa). ‘haden’ fruit were put at 20°c for 2 days, while ‘palmer’ mangoes were randomized into 3 batches of 30 fruits, corresponding to 0, 4 and 11 days of shelf life at 20°c, in order to have the whole range of µa690 in each batch. sub-samples of 10 fruits, covering the whole range of µa, were selected for ‘haden’ and for ‘palmer’ batch held for 4 days at 20°c. each selected fruit was measured by trs at 540 nm on two opposite sides in the equatorial region; in the same positions of trs measurements, skin was removed by a slicer, and, after measuring pulp color and firmness, the whole fruit were immediately deep frozen and kept at -30°c until carotenoid, ascorbic acid and total phenolic extractions. time-resolved reflectance spectroscopy (trs) a portable compact setup working at discrete wavelengths developed at politecnico di milano (spinelli et al., 2012) was used. the light source is a supercontinuum fiber laser (sc450-6w, fianium, uk) providing white-light picoseconds pulses, with duration of few tens of picoseconds. a custom-made filter wheel loaded with 14 band-pass interference filters (nt-65 series; edmund optics) is used for spectral selection in the range 540-940 nm. light is delivered to the sample by means of a multimode graded-index fiber and diffuse remitted light is collected by 1 mm fiber placed at 1.5 cm distance from the illumination point. a second filter wheel identical to the first one is used for cutting off the fluorescence signal originated when illuminating the fruit in the visible spectral region. the light then is detected with a photomultiplier (hpm-100-50, becker & hickl, germany) and the photon time-of-flight distribution was measured by a time-correlated single-photon counting board (spc-130, becker & hickl, germany). the instrumental response function has a full width at half maximum of about 260 ps and the typical acquisition time is 1 s per wavelength. a model for photon diffusion in turbid media was used to analyze trs data to assess the bulk optical properties of the samples (martelli et al., 2009) to obtain the estimates of µa and µs at each wavelength. firmness and pulp color flesh firmness was measured using a instron utm model 4301 penetrometer (crosshead speed 200 mm min-1, 8 mm diameter plunger). data were averaged per fruit. pulp color was measured with a spectrophotometer (cm-2600d, minolta co., japan), using the primary illuminant d65 and 2° observer in the l*, a*, b* color space. hue (h°) was computed from a* and b* values according to: h°= arctangent (b*/a*) x 360/(23 x 14) yellowness index (i y ) was computed as: i y =[81.2746x−1.0574z)/y]×100 after converting l*a*b* parameters into the xyz color space (jha et al., 2006). data were averaged per fruit. carotenoids, ascorbic acid, total phenols and total antioxidant capacity analysis carotenoids, ascorbic acid, total phenols and total antioxidant capacity analysis were carried out on frozen fruit after 30 min thawing at ambient temperature, by slicing pulp portions without peel near the positions of trs measurements and pooling the slices coming from the two fruit sides. carotenoids were extracted following the procedure described by picchi et al. (2012) with slight modifications. briefly, on individual fruit, 2 g of pulp (two replicates) was extracted with 10 ml of a solution of hexane:acetone:ethyl acetate (2:1:1 v/v/v) containi n g 1 0 0 µ l o f 1 % b u t y l h y d r o x y t o l u o l ( b h t ) i n methanol, to prevent carotenoid oxidative degradation, and centrifuged at 4°c at 15,000 rpm for 20 min. the extracts were stored at -80°c until spectrophotometric and high-performance liquid chromatographic (hplc) analyses. total carotenoid content (car) was determined measuring absorbance at 450 nm using a spectrophotometer (uv-uvidec 320, jasco, japan). the hexane:acetone:ethyl acetate solution was used as the blank. total carotenoid content was estimated from a standard curve of all-trans-β-carotene and data w e r e e x p r e s s e d a s m i l l i g r a m s o f β c a r o t e n e adv. hort. sci., 2018 32(3): 407-420 410 equivalent (β-car) per kilogram of fresh weight (mg βcar kg fw-1). carotenoid composition was determined on e x t r a c t s a c c o r d i n g t o a z e v e d o m e l e i r o a n d rodriguez-amaya (2004) with some modifications. a jasco (tokio, japan) hplc system consisting of a pu1580 liquid chromatographic pump coupled to lg 1580-04 quaternary gradient unit, a model as 2055plus autosampler and an md 2010-plus multi-wavelength detector was used. separations were performed on an inertsil ods-3 column (4.6 mm i.d × 250 mm length, particle diameter 5 µm, gl science) at the temperature of 40°c which was maintained using a jasco co-1560 intelligent column thermostat. the sample injection volume was 80 µl. the column was eluted with 20% methanol and 80% of a gradient mixture of acetonitrile (a) and ethyl acetate (b) at the flow rate of 0.6 ml min-1, with 10% b at 0-25 min, 10-20% b at 25-35 min, 20-50% b at 35-40 min, 50% b at 40-45 min, 50-10% b at 45-50 min. spectra of all peaks were recorded in the 200-600 nm wavelength range, and peak areas were monitored at 450 nm. carotenoids (table 1) were identified by comparing their retention times and spectral characteristics with those of standards (all-trans-β-carotene and violaxanthin, obtained by pansy petals) and with those reported in the literature (ornelas-paz et al., 2007, 2008), considering the three maximum absorbance wavelengths (λmax) and the spectral fine structure (% iii/ii), which is the percentage of the peak height of the longest wavelength absorption band (λmax iii) to that of the middle absorption band (λmax ii), taking the maximum of the valley between peak ii and peak iii as the baseline (sajilata et al., 2008). carotenoids were quantified referring to the total carotenoid content estimated spectrophotometrically on the same extract in conjunction with the chromatogram perc e n t c o m p o s i t i o n a n d d a t a w e r e e x p r e s s e d a s milligrams of β-carotene equivalent (β-car) per kilogram of fresh weight (mg β-car kg fw-1). all the measurements were carried out in triplicate. the vitamin a value, expressed as retinol equivalent (re) was estimated from all-trans-β-carotene and cis-β-cryptoxanthin amounts using as conversion figures 6 µg for car and 12 µg for crypt (capra, 2006). ascorbic acid was extracted following the procedure described by robles-sánchez et al. (2009 a) with slight modifications. briefly, on individual fruit, 2 g of pulp (two replicates) was homogenized with 10 ml of 6% (w/v) aqueous solution of metaphosphoric acid (mpa), vortexed for 30 s, and centrifuged at 4°c at 15,000 rpm for 20 min and the extracts were kept at -20°c till hplc analysis. ascorbic acid was determined on just thawed extracts according to the conditions reported by rizzolo et al. (2002), using a jasco (tokio, japan) hplc system consisting of a pu-980 liquid c h r o m a t o g r a p h i c p u m p , a m o d e l a s 1 0 5 5 1 0 autosampler and an uv-vis 15770 detector set at 254 nm, coupled to an inertsil ods-3 column (4.6 mm i.d. × 250 mm length, particle diameter 5 µm, gl science) at the temperature of 30°c, which was eluted with 0.02 m orthophosphoric acid at a flow rate of 0.7 ml min-1. ascorbic acid was estimated from a standard curve of l-ascorbic acid in 6% mpa and data were table 1 retention time (rt, min), spectra characteristics [λmax (nm) in the mobile phase, obtained by dad, spectral fine structure (% iii/ii)] and name abbreviation of tentatively identified compounds according to ornelas-paz et al. (2007, 2008) peak no. rt λmax i λmax ii λmax iii % iii/ii tentative identification abbreviation 1 5.24-5.89 419 439 471 82 unknown unk 2 5.92-5.97 415 439 471 82 all-trans-violaxanthin various esters viol no. 1 3 6.03-6.09 415 439 471 100 all-trans-violaxanthin various esters viol no. 2 4 6.11-6.19 415 443 471 90 cis-β-cryptoxanthin crypt 5 6.23-7.29 415 439 471 93 all-trans-violaxanthin various esters viol no. 3 6 7.40-7.83 411 435 463 75 9-cis-violaxanthin 9-viol no. 1 7 8.03-8.77 415 435 467 83 9-cis-violaxanthin 9-viol no. 2 8 9.40-10.31 415 439 467 84 all-trans-violaxanthin various esters viol no. 4 9 10.32-11.15 411 435 463 80 9-cis-violaxanthin 9-viol no. 3 10 33.48-36.19 419 439 471 n.c. all-trans-violaxanthin various esters viol no. 5 11 37.97-40.61 451 479 23 all-trans-β-carotene car 12 40.90-43.60 419 439 471 100 all-trans-violaxanthin various esters viol no. 6 13 43.71-43.99 415 435 467 100 9-cis-violaxanthin 9-viol no. 4 14 44.00-44.60 415 439 467 100 all-trans-violaxanthin various esters viol no. 7 vanoli et al. quality and nutraceutical properties of mangoes according to trs maturity class 411 3. results trs optical properties in ‘palmer’ fruit, µa690 at harvest ranged from 0.074 cm-1 for the less mature fruit to 0.021 cm-1 for the more mature ones and decreased to 0.061 cm-1 and 0.019 cm-1, respectively, after 4 days of shelf life at 20°c; concomitantly, after shelf life, µa540 ranged from 0.117 cm-1 for the least mature fruit to about 0.33 cm-1 for the most mature ones. in ‘haden’ mangoes, µa650 ranged at harvest from 0.231 cm-1 to 0.030 cm-1, with the majority of the fruit in the 0.0300.065 cm-1 range; after 2 days of shelf life at 20°c, µa650 decreased only in less mature fruit, whereas in all the other mango fruit it increased to values ranging from 0.036 cm-1 to 0.053 cm-1, while the µa540 values after shelf life ranged from 0.157 cm-1 for the least mature fruit to 0.835 cm-1 for the most mature ones. the µa540 maturity index, related to carotenoids content, was then used to classify the selected fruit within each cultivar in three trs maturity classes: less mature (lem) with low µa540, more mature (mom) with high µa540 and medium mature (mem) with intermediate values of µa540. cultivar and trs maturity class influenced the value of µa540 maturity index (table 2); on average µa540 was higher in cv. h a d e n ( ‘ h a d e n ’ : 0 . 4 0 0 ± 0 . 0 2 5 c m 1; ‘ p a l m e r ’ : 0.248±0.025 cm-1) and in the mom class in both cultivars, with mom ‘haden’ fruit being characterized by the highest µa540 value. quality parameters trs maturity class and cultivar greatly affected a* and h° pulp color parameters and had only a slight influence on firmness, probably due to the high standard error values, whereas iy depended only on maturity class (table 2). on average, ‘palmer’ fruit had lower firmness and a* value, and higher h° than ‘haden’ fruit. in ‘palmer’ mangoes firmness did not vary with maturity class, while in ‘haden’ firmness showed the highest values in lem fruit and the lowest in mem and in mom ones. mom ‘palmer’ fruit had higher a* and iy and lower h° than lem and mem maturity classes, whereas lem ‘haden’ fruit had lower iy than mom fruit, and a* increased and h° decreased from lem to mem and mom maturity classes. ascorbic acid, total phenolic content and total antioxidant capacity aa content and tac were significantly influenced only by cultivar, while tpc depended by both cultivar expressed as milligram per kilogram of fresh weight (mg kg fw-1). all the measurements were carried out in triplicate. total phenol content (tpc) and total antioxidant capacity (tac) were determined on the same extract (two replicates/fruit) obtained by homogenizing 2 g of pulp with 10 ml of acidic ethanol (ethanol:0.04 m hcl, 1:1 v/v), vortexed for 30 s and centrifuged at 4°c at 15,000 rpm for 20 min. extracts were kept at -20°c till total phenol content and antioxidant capacity determinations. tpc was determined using the folinciocalteau method (singleton et al., 1999) based on the reduction of a phosphowolframate phosphomolibdate complex by phenolics to blue reaction products, and measuring absorbance at 730 nm using a spectrophotometer (uv-uvidec 320, jasco, japan). the tpc was estimated from a standard curve of gallic acid and data were expressed as milligrams of gallic acid equivalents (gae) per kilogram of fresh weight (mg gae kg fw-1). all the measurements were performed in triplicate. tac was evaluated using the free radical 1,1,-dyphenyl-2-picrylhydrazil (dpph•) according to brand-williams et al. (1995) with modifications. fifty microlitres of sample extract or trolox standard solution (0.02-0.8 mm) were added to 2 ml of ethanol and 550 μl of dpph• solution (0.05 mm in ethanol) and, during 5 min of incubation, the absorbance at 517 nm was measured with a jasco 7800 uv/vis spectrophotometer (jasco europe s.r.l., cremella, lc, italy). the dpph scavenging capacity of the samples was calculated using a standard curve of trolox, and expressed as micromoles of trolox equivalents (te) per kilogram of fresh weight (µmol te kg fw-1). all the measurements were performed in triplicate. statistical analysis the statgraphics v. 5.2 (manugistic inc., rockville, md, usa) software package was used. data were s u b m i t t e d t o m u l t i f a c t o r a n a l y s i s o f v a r i a n c e (anova) considering cultivar, trs maturation class and their interaction as source of variation. in addition, one-way anova was used to study the main factors (cultivar, trs maturity class), and the trs maturity class within each cultivar. percentage data of carotenoids were analyzed after arcsine transform a t i o n . m e a n s w e r e c o m p a r e d b y 9 5 p e r c e n t bonferroni’s test. relationships between µa540 and pulp color parameters and between µa540, a*, h°, iy and ascorbic acid, tpc, tac and carotenoids were studied using regression analysis. for each parameter, the model with the higher performance was considered. 412 adv. hort. sci., 2018 32(3): 407-420 and maturity class (table 3). on average, ‘palmer’ mangoes had higher aa, tpc and tac than ‘haden’ fruit, and lem fruit had lower tpc than mom mangoes (fig. 1). the average aa values were approx. 190 mg kg fw-1 for ‘haden’ and 390 mg kg fw-1 for ‘palmer’ and aa content did not change with maturity class in ‘palmer’ mangoes while in ‘haden’ showed the highest values in mem fruit (fig. 1). tpc was higher in ‘palmer’ than in ‘haden’ cv., being on average approx. 316 and 264 mg kg fw-1 in ‘palmer’ and ‘haden’ fruit, respectively, as well as it was higher in mom fruit from both cultivars. ‘palmer’ fruit were characterized by higher tac showing on average 2.59 times greater than that from ‘haden’. tac had significant positive correlations with tpc (r= 0.66, p= 0.002) and aa (r= 0.89, p<0.0001). carotenoids total carotenoids (car) depended only by maturity class (table 3), with lem fruit having less car than mom ones (fig. 1). the chromatographic carotenoid patterns of lem and mom maturity classes in both cultivars showed 9 common carotenoids (fig. 2) out of 14 peaks tentatively identified by comparing spectral characteristics with those previously reported u s i n g a s i m i l a r m o b i l e p h a s e ( t a b l e 1 ) . t h e carotenoid pattern includes seven all-trans-violaxanthin (viol) and four 9-cis-violaxanthin (9-viol) containing compounds, cis-β-cryptoxanthin and all-transβ-carotene. the most abundant carotenoid in both cultivars was all-trans-β-carotene (tables 4 and 5), representing 49-56% of the total carotenoid content, followed fig. 1 (a) ascorbic acid (aa), (b) total carotenoids (car), total phenol content (tpc) and total antioxidant capacity (tac) in ‘palmer’ and ‘haden’ mangoes in function of µa540 maturity class (lem, less mature; mem, medium mature; mom, more mature). bars refer to se. within each cultivar anova results are indicated as follows: *, **, ***: significant at p≤0.05, 0.01, 0.001, respectively; ns, not significant. mean±se. within each cultivar, means followed by different letters are statistically different (bonferroni's test, p≤0.05). p-value of f-ratio: ns=not significant; *p<0.05; **p<0.01; ***p<0.001. table 2 absorption coefficient at 540 nm (µa540, cm-1), flesh firmness (n) and pulp colour parameters (a*; hue, h°; yellowness index, iy) of 'palmer' and 'haden' mangoes of less mature (lem), medium mature (mem) and more mature (mom) trs maturity classes and results of multifactor anova (f-ratio value and p-value) table 3 multifactor analysis of variance (f-ratio and p-value) for ascorbic acid (aa), total carotenoids (car), total plenolic content (tpc) and total antioxidant capacity (tac) *p<0.05; **p<0.01; ***p<0.001; ns=not significant. cultivar maturity class µa540 firmness a* h° iy palmer lem 0.196±0.027 b 8.33 ±0.55 a 0.86±0.79 b 89.13±0.75 a 130.7±1.7 b mem 0.241±0.006 ab 7.70±0.45 a 3.64±1.03 b 86.85±0.89 a 153.3±1.6 ab mom 0.310±0.017 a 6.12±0.63 a 9.84±1.75 a 81.72±1.30 b 165.7±9.5 a haden lem 0.191±0.011 b 37.29±9.89 a 2.98±1.62 c 87.18±1.55 a 130.0±6.0 b mem 0.336±0.033 b 10.47±3.62 a 10.60±1.44 b 80.32±1.15 b 159.7±6.6 a mom 0.677±0.158 a 5.85±0.65 b 20.46±1.12 a 72.37±0.84 c 191.2±6.1 a anova a: cultivar 18.88 *** 4.81* 28.85 *** 32.20 *** 3.94 ns b: maturity class 23.52 *** 4.85 * 36.79 *** 32.25 *** 27.18 *** a × b 9.21 ** 3.98 * 3.94 * 4.16 * 2.01 ns factors aa car tpc tac main factors cultivar (a) 38.79 *** 0.48 ns 5.12 * 32.75 *** maturity class (b) 0.75 ns 7.28 ** 4.20 * 1.85 ns interaction a × b 0.68 ns 0.98 ns 0.81 ns 0.53 ns 413 vanoli et al. quality and nutraceutical properties of mangoes according to trs maturity class by cis-β-cryptoxanthin (6-18%) and viol no.3 (1116%). the content of all-trans-violaxanthins was higher than that of 9-cis-violaxanthins in both cultivars. the content of viol no.3, no.4 and no.6, 9-viol no.4 and all-trans-β-carotene, as well as the sums of a l l t r a n s v i o l a x a n t h i n s ( ∑ v i o l ) a n d o f 9 c i s violaxanthins (∑9-viol) depended only on maturity class, that of 9-viol no.1 only on cultivar, whereas those of 9-viol no.2 and no.3 on both cultivar and maturity class (table 4). in fact ‘haden’ mangoes had higher amounts of 9-viol no.3 (table 4) and had lower proportion of 9-viol no.4 than ‘palmer’ fruit (table 5). viol no. 1 and 9-viol no.1 were present only in ‘haden’ and 9-viol no. 2 only in ‘palmer’ fruit. fig. 2 typical chromatographic patterns at 450 nm of carotenoid extracts of (a) lem ‘palmer’, (b) mom ‘palmer’, (c) lem ‘haden’ and (d) mom ‘haden’ mangoes. for peak assignment see table 1. table 4 carotenoid compounds of 'palmer' and 'haden' mangoes (mg β-care kg fw-1) and vitamin a value (re 100 g fw-1) influence of cultivar and of trs maturity class and results of multifactor anova (f-ratio value and p-value). for identification data of each carotenoid see table 1 mean±se; 0=not detected. within cultivar and within trs maturity class means followed by different letters are statistically different (bonferroni's test, (*) p<0.10; * p<0.05; **p<0.01; ns =not significant). ∑viol= total all-trans-violaxanthin esters; ∑9-viol= total 9-cis-violaxanthin. cultivar maturity class anova palmer haden less mature medium mature more mature cultivar (a) maturity class (b) a × b viol no. 1 0±0 a 0.49±0.49 a 0 ±0 a 0±0 a 0.99±0.99 a 3.25 (*) 2.89 (*) 2.89 (*) viol no. 2 1.14±0.46 a 0.87±0.68 a 0.68 ±0.40 a 0.49±0.38 a 2.15±1.33 a 0.08 ns 2.11 ns 2.48 ns crypt 2.58±0.55 a 1.82±0.38 a 1.66 ±0.37 a 2.69±0.47 a 2.18 ±0.97 a 1.53 ns 0.89 ns 0.39 ns viol no. 3 2.43±0.31 a 2.49±0.55 a 1.53±0.28 b 2.82±0.67 ab 3.26±0.30 a 0.07 ns 3.08 (*) 2.37 ns 9-viol no. 1 0±0 b 0.81±0.42 a 0±0 a 0.67±0.42 a 0.68±0.68 a 4. 91 * 1.36 ns 1.36 ns 9-viol no. 2 0.33±0.16 a 0±0 b 0±0 b 0.06±0.06 ab 0.52±0.26 a 10.91 ** 6.23 * 6.23 * viol no. 4 0.84±0.16 a 0.91±0.40 a 0.27±0.13 b 0.88±0.40 ab 1.72±0.37 a 0.65 ns 4.90 * 0.89 ns 9-viol no. 3 0.09±0.06 b 0.35±0.15 a 0±0 b 0.21±0.11 ab 0.56±0.25 a 7.62 * 7.57 ** 2.35 ns car 9.36±1.14 a 9.63±2.72 a 5.05±0.67 b 8.85±1.81 ab 16.65±3.47 a 0.81 ns 8.37 ** 1.03 ns viol no. 6 0.64±0.14 a 0.56±0.23 a 0.08±0.08 b 0.74±0.16 ab 1.12±0.25 a 0.12 ns 10.19 ** 1.66 ns 9-viol no. 4 0.55±0.19 a 0.22±0.15 a 0±0 b 0.55± 0.21 a 0.67±0.28 a 1.58 ns 4.15 * 0.99 ns viol no. 7 0.15±0.10 a 0.19±0.10 a 0.07±0.07 a 0.17±0.11 a 0.30±0.18 a 0.19 ns 0.72 ns 0.97 ns ∑viol 5.20±0.76 a 5.51±1.95 a 2.64±0.74 b 5.11±1.22 ab 9.54±2.82 a 0.88 ns 5.69 * 2.26 ns ∑9-viol 0.98±0.35 a 1.38±0.67 a 0±0 b 1.49±0.48 ab 2.43±1.06 a 1.06 ns 4.39 ** 0.32 ns vitamin a value 258.2±22.3 a 162.0±45.3 a 85.6±11.4 b 149.7±30.5 ab 279.3±57.2 a 0.40 ns 8.38 ** 1.01 ns 414 adv. hort. sci., 2018 32(3): 407-420 lem mangoes had no 9-cis-violaxanthins and were characterized by lower contents of viol no.3, viol no.4, viol no.6 and all-trans-β-carotene than mom fruit, but higher proportion of all-trans-β-carotene than mem ones (tables 4 and 5). these carotenoids increased with advancing maturity degree, showing the highest contents in mom mangoes. on average all-trans-β-carotene corresponded to 53% of total carotenoids; the all-trans-β-carotene proportion was not significantly affected by cultivar, whereas on average was lower in mem fruit than in lem ones, while mom mangoes were not statistically different from fruit of the other two maturity classes (table 5). the vitamin a value did not differ between cultivar, but significantly increased with maturity class from 86 of lem fruit to 279 re 100 g-1 of mom mangoes (table 4). regression analysis the results of regression analysis between µa540 and pulp color parameters and between µa540, a*, h°, iy and ascorbic acid, tpc, tac and carotenoids differed for the two cultivars and data are summarized in tables 6 and 7, reporting the type of the model having the best performance. the μa540 was positively related to a* and iy and negatively to h° (table 6) with r ranging from 0.83 to 0.87 for ‘palmer’ fruit and approx. 0.98 for ‘haden’ cultivar. in ‘palmer’ mangoes (table 7) µa540, a*, h° and iy were related to total carotenoids, viol no.3 and no.4, 9-viol no.2 and no.3, all-trans-β-carotene, ∑9-viol and vitamin a value with lower r values (0.620.84) for µa540 respect to those found for pulp color parameters (0.74-0.96). only iy was related to viol no.6, 9-viol no.4 and ∑viol with r≥0.7, and only a* was related to tpc, but with r<0.6. in contrast, in ‘haden’ fruit µ a 540, a*, h° and i y were related to total table 5 carotenoid composition (percent to total carotenoids) of 'palmer' and 'haden' mangoes: influence of cultivar and of trs maturity class and results of anova (f-ratio value and p-value). for identification data of each carotenoid see table 1 table 6 results of regression analysis between absorption coefficient at 540 nm (μa540) and pulp color parameters for each regression, the following data are given: r = correlation coefficient, p = significance of the model (***, p<0.001; **, p< 0.01) and mt= model type (dr= doble reciprocal, e= exponential, l= linear, ln= logarithmic-x, m= multiplicative, rx= reciprocal-x, sc = s-curve). cultivar maturity class anova palmer haden less mature medium mature more mature cultivar (a) maturity class (b) a × b viol no. 1 0±0 a 0.87±0.87 a 0±0 a 0±0 a 0.36±0.36 a 3.25 (*) 2.89 (*) 2.89 (*) viol no. 2 6.74±2.84 a 3.13±1.49 a 2.51±0.64 a 1.04±0.43 a 2.89±0.58 a 0.21 ns 0.36 ns 1.65 ns crypt 14.87±3.01 a 15.40±2.77 a 18.18±0.53 a 15.31±0.07 a 5.88±1.04 a 0.01 ns 1.80 ns 1.40 ns viol no. 3 14.32±2.05 a 16.30±2.07 a 16.33±0.18 a 15.96±0.09 a 11.31±0.07 a 0.04 ns 1.51 ns 2.63 ns 9-viol no. 1 0±0 b 2.44±1.30 a 0±0 a 1.122±0.26 a 0.25±0.25 a 7.84 * 2.48 ns 2. 48 ns 9-viol no. 2 1.36±0.60 a 0±0 b 0±0 b 0.06±0.06 ab 1.08±0.20 a 14.55 ** 5.95 * 5.95 * viol no. 4 4.23±0.58 a 3.17±0.95 a 1.27±0.17 a 3.07±0.12 a 5.51±0.01 a 0.54 ns 2.49 ns 0.40 ns 9-viol no. 3 0.33±0.22 b 1.24±0.46 a 0±0 b 0.46±0.11 ab 1.21±0.08 a 7.51 * 8.04 ** 2.49 ns car 50.62±1.03 a 54.25±2.31 a 55.65±0.08 a 48.86±0.02 b 53.26±0.02 ab 3.22 (*) 3.28 (*) 3.38 (*) viol no. 6 3.20±0.71 a 1.98±0.68 a 0.07±0.07 b 3.27±0.10 a 3.57±0.005 a 1.66 ns 11.66 ** 0.62 ns 9-viol no. 4 2.63±0.91 a 0.71±0.52 b 0±0 b 1.73±0.23 a 1.14±0.20 ab 4.52 * 6.20 ** 2.26 ns viol no. 7 0.77±0.51 a 0.95±0.51 a 0.06±0.06 a 0.25±0.11 a 0.56±0.21 a 0.19 ns 0.46 ns 1.24 ns ∑viol 29.25±3.30 a 26.41±3.04 a 25.030±0.42 a 28.13±0.09 a 28.76±0.07 a 0.22 ns 0.17 ns 4.27 * ∑9-viol 4.31±1.33 a 4.39±1.71 a 0±0 b 6.07±0.19 a 5.79±0.21 a 0.01 ns 13.50 ** 0.00 ns mean±se; 0=not detected. within cultivar and within trs maturity class means followed by different letters are statistically different (bonferroni's test, (*) p<0.10; * p<0.05; **p<0.01; ns =not significant). ∑viol= total all-trans-violaxanthin esters; ∑9-viol= total 9-cis-violaxanthin. µa540 ‘palmer’ ‘haden’ r p mt r p mt a* 0.831 ** l 0.975 *** e h° 0.826 ** rx 0.976 *** sc i y 0.872 ** ln 0.977 *** dr vanoli et al. quality and nutraceutical properties of mangoes according to trs maturity class 415 carotenoids, viol no.2, no.3, no.4 and no.6, 9-viol no.1 and no.3, all-trans-β-carotene, ∑viol, ∑9-viol, vitamin a value and tpc, with higher r values (0.720.95) for µa540 than for pulp color parameters. in addition, only µa540 was related to 9-viol no. 4. no significant relationships were found between µa540, a*, h° and iy and ascorbic acid and tac, whatever the cultivar, suggesting that μa540 was able to reveal the carotenoids content in the pulp, as this wavelength corresponds to the tail of carotenoid absorption. independently from cultivar. rizzolo et al. (2016) also s h o w e d t h a t µ a5 4 0 m a t u r i t y i n d e x , r e l a t e d t o carotenoids content, successfully classified ‘tommy atkins’ mangoes at harvest. as for quality parameters, ‘palmer’ mangoes had firmness values typical of fully ripe fruit (beaulieu and lea, 2003), independently from maturity class, whereas in ‘haden’ fruit lem class showed firmness values typical of firm-ripe stage and mem and mom classes values typical of ready-to-eat or ripe fruit (eccher zerbini et al., 2015). pulp color parameters table 7 results of regression analysis between absorption coefficient at 540 nm (μa540), pulp color parameters and total carotenoid (car), total phenolic compounds (tpc), carotenoid compounds (for identification data see table 1) and vitamin a value µa540 a* h° iy r p mt r p mt r p mt r p mt 'palmer' car 0.78 * l 0.909 *** l 0.902 *** rx 0.897 *** l viol no.3 0.814 ** l 0.789 * l 0.783 * rx 0.735 * l 9-viol no.2 0.839 ** l 0.959 *** l 0.958 *** rx 0.823 ** l viol no.4 0.625 (*) l 0.908 *** l -0.912 *** l 0.784 * l 9-viol no.3 0.765 * l 0.871 ** l 0.871 ** rx 0.778 * l car 0.793 ** l 0.913 *** sy -0.909 *** sy 0.888 ** sy viol no.6 − − − -0.745 * rx 9-viol no.4 − − − -0.847 ** rx ∑viol 0.576 (*) log − − 0.693 * log ∑9-viol 0.812 ** l 0.873 ** l 0.86 ** rx 0.954 *** l vitamin a value 0.792 ** l 0.915 *** sy -0.912 *** sy 0.887 ** l tpc − 0.575 (*) sy − − 'haden' car 0.912 *** l 0.855 ** e 0.854 ** sc 0.876 *** e viol no.2 0.853 ** l 0.618 (*) l 0.634 * rx 0.601 (*) l viol no.3 -0.824 ** sc 0.723 * e -0.742 * e -0.805 ** sc 9-viol no.1 0.756 * l 0.666 * l 0.661 * rx 0.718 * l viol no.4 0.815 ** sx 0.789 ** l 0.79 ** rx 0.823 ** l 9-viol no.3 0.95 *** l 0.83 ** l 0.834 ** rx 0.848 ** l car 0.924 *** l 0.823 ** sy 0.817 ** sc 0.837 ** sy viol no.6 0.944 *** l 0.862 ** l 0.862 ** rx 0.873 *** l 9-viol no.4 0.700 * l − − − ∑viol 0.937 *** l 0.885 *** sy -0.886 *** e 0.905 *** sy ∑9-viol 0.848 ** l 0.723 * l 0.721 * rx 0.756 * l vitamin a value 0.923 *** l 0.823 ** sy 0.817 ** sc 0.838 ** sy tpc 0.867 ** l 0.758 * l 0.761 ** rx 0.766 ** l for each significant regression, the following data are given: r= correlation coefficient, p= significance of the model (***, p< 0.001; **, p <0.01; *, p< 0.05; (*), p<0.10) and mt= model type (dr= doble reciprocal, e= exponential, l= linear, ln= logarithmic-x, log= logistic, rx= reciprocal-x, sc= s-curve, sx= square-root-x, sy= square-root-y). 4. discussion and conclusions the absorption coefficient measured at 540 nm (µa540) showed different value ranges between ‘haden’ and ‘palmer’ mangoes and it was able to distinguish more mature fruit from less mature ones differed among maturity classes, confirming previous results obtained for ‘tommy atkins’ cultivar. in fact rizzolo et al. (2011) and vanoli et al. (2011) found that lem ‘tommy atkins’ mangoes were characterized by higher h° and lower a* and i y than mom fruit. moreover, vanoli et al. (2011) found that with fruit adv. hort. sci., 2018 32(3): 407-420 416 ripening at 20°c h° decreased and iy increased, confirming that the trend of pulp color observed in our work with the trs maturity classes was actually due to a different ripening degree. the average aa values found for ‘palmer’ and ‘haden’ fruit are comparable with the data by rocharibeiro et al. (2007) for the same cultivar, and with aa content reported for other cultivars by liu et al. (2013) and elsheshetawy et al. (2016). however, within the same variety, aa content may vary according to climatic conditions, cultural practices, maturity stage and postharvest factors. for ‘keitt’ cultivar ibarra-garza et al. (2015) found that aa content varied from about 1300 mg kg fw-1 in fruit soon after harvest, to about 2500 mg kg fw-1 till 8 days of ripeni n g a t r o o m t e m p e r a t u r e , f o l l o w e d b y a 5 4 % decrease in fully-ripe fruit. similarly, robles-sánchez et al. (2009 b) reported for ‘ataulfo’ fruit stored for 15 days at 12°c a 50% decrease of aa content at the end of shelf-life. the tpc contents found in this work are in agreement with rocha-ribeiro et al. (2007), even if these authors reported lower aa contents than those found in our work for the same cultivars. no data on tpc content in mangoes in relation to trs maturity classes, having same harvest time and same postharvest management, are available in the literature. however, the tpc increasing trend from less to more mature fruit class found in this work is in agreement with the results for the final period of shelf life/storage reported by ibarra-garza et al. (2015) and robles-sánchez et al. (2009 b) when fruit are becoming softer and with a yellower pulp color. ibarragarza et al. (2015) found higher tpc in ‘keitt’ fruit at the beginning of a 10 d shelf life period at room temperature, with a sharp decrease from 2 to 4 days of shelf life, followed by a slight but significant tpc increase till the end of shelf life; a similar trend was also found by robles-sánchez et al. (2009 b) for whole and fresh-cut ‘ataulfo’ mangoes stored at 5°c for 15 days. in agreement with data obtained for tpc and aa, ‘palmer’ fruit showed higher tac than ‘haden’ ones. the positive correlations of tac with tpc and aa found in this work are in agreement with literature data. in fact silva and sirasa (2018) reported significant correlations between ascorbic acid and tpc and frap and dpph scavenging activity measured for several fruit species, and palafox-carlos et al. (2012) b e t w e e n d p p h s c a v e n g i n g a c t i v i t y a n d t p c i n ‘ataulfo’ mangoes; on the other hand, liu et al. (2013) and ibarra-garza et al. (2015) found in mangoes correlation between phenolic concentration and antioxidant activity measured with other methods (frap, orac), but not between antioxidant activity and ascorbic acid content; in contrast rocharibeiro et al. (2007) reported that dpph radical scavenging activity of mango extracts was strongly correlated with ascorbic acid content, but not with phenolic content. liu et al. (2013) suggested that the difference in relationships between antioxidant activity and ascorbic acid and phenolic compound contents found among authors could be due to a masking effect of phenolics present in far higher concentration than ascorbic acid. our results suggest that in this work the antioxidant activity can be attributed more to ascorbic acid than to total phenols. no data on total carotenoids in relation to trs maturity classes are available in the literature. vanoli et al. (2016) for ‘palmer’ and ‘haden’ mangoes reported a wide fruit-to-fruit variability in car content for both cultivars and that car content had an increasing trend with µa540 value measured on fruit belonging to the same harvest date, i.e. that car content increases with advancing maturity degree. similarly, an exponential increase in the carotenoid content with fruit ripening has been reported for ‘ataulfo’ mangoes (ornelas-paz et al., 2008) and nine thai cultivars (vásquez-caicedo et al., 2005). as for the chromatographic carotenoid patterns, the tentative identification of peaks was carried out by comparing spectral characteristics with those previously reported using a similar mobile phase. seven peaks were tentatively identified as all-trans-violaxanthin (439 nm maximum absorption wavelength) and four as 9-cis-violaxanthin (435 nm maximum absorption wavelength) containing compounds. the spectral maximum for peak 4 was similar to that reported for cis-β-cryptoxanthin. a standard mixture of all-transβ-carotene was used for the identification of peak 11; the retention time and the spectroscopic characteristics of reference material were identical to those observed for peak 11 in all the samples. in general, the spectral fine structures (% iii/ii values in table 1) found in this work are in agreement with the values reported in the literature (ornelas-paz et al., 2007, 2008). in both cultivars the proportions of all-transβ c a r o t e n e a n d c i s β c r y p t o x a n t h i n t o t o t a l carotenoid content, as well as the higher content of all-trans-violaxanthins than that of 9-cis-violaxanthins are very similar to findings reported in ‘ataulfo’, ‘ k e i t t ’ , ‘ t o m m y a t k i n s ’ a n d ‘ k e n t ’ m a n g o e s (mercadante and rodriguez-amaya, 1998; pott et al., 2003; ornelas-paz et al., 2008). the differences in vanoli et al. quality and nutraceutical properties of mangoes according to trs maturity class 417 carotenoid composition among the maturity classes found in this research are consistent with literature data. previous researches on carotenoid composition of various mango cultivars carried out by godoy and r o d r i g u e z a m a y a ( 1 9 8 9 ) , m e r c a d a n t e a n d rodriguez-amaya (1998), yahia et al. (2006), and ornelas-paz et al. (2007) have shown that generally the most important carotenoid in mango is all-transβ-carotene and its proportion to total carotenoids depends on cultivar and fruit maturity stage. ibarragarza et al. (2015) reported for ‘keitt’ fruit that alltrans-β-carotene corresponded to 33% of total carotenoids in unripe fruit at harvest, ranged from 37 to 44% during the first 6 day-period of ripening at room temperature and reached 61% in fully ripe fruit. the contents of all-trans-β-carotene in ‘haden’ and ‘palmer’ mangoes were similar to those reported by rocha-ribeiro et al. (2007) for the same cultivars and by mercadante and rodriguez-amaya (1998) for ‘keitt’ and ‘tommy atkins’ fruit, but lower than those found for ‘haden’ and other cultivars by ornelas-paz et al. (2007). also the amounts of all-transand 9-cisviolaxanthins in both cultivars were lower than those found by ornelas-paz et al. (2007) for ‘haden’ and other cultivars, but similar to those reported by low et al. (2015) for ‘kensington pride’ mangoes. the amounts of cis-β-cryptoxanthin in both cultivars were far higher than the maximum of 0.1 µg g-1 reported by mercadante and rodriduez-amaya (1998) for ‘keitt’ and ‘tommy atkins’ mangoes from maturegreen to ripe stages, indicating that in the fruit of this experiment, not only β-carotene, but also cis-β-cryptoxanthin was a contributor to the vitamin a value for these fruit. the differences in the single carotenoid concentrations respect to literature data for the same cultivar could be due to the different maturity degree of mango fruit. in fact, referring to pulp color parameters, the fruit used in our experiment had pulp a* values similar to those reported by rocha-ribeiro et al. (2007) for the same cultivars and in this case the carotenoid amounts were similar, whereas pulp h° values for ‘haden’ fruit were higher than those reported by ornelas-paz et al. (2007), indicating a less advanced ripening degree consistent with the lower carotenoid content of our results. the results of regression analysis showed that µa540 was positively related to a* and iy and negatively to h° pulp color parameters, confirming the results obtained by spinelli et al. (2012, 2013) and vanoli et al. (2016) for the same cultivars and by vanoli et al. (2013) for ‘tommy atkins’ mangoes. color changes in the pulp of mango fruit are usually accompanied by carotenoid accumulation. in this work significant correlations between µa540, pulp color parameters a*, h°, iy and carotenoids were found for both cultivars. vanoli et al. (2016) found an increasing trend of total carotenoids content with µa540 in ‘palmer’ and ‘haden’ mangoes; they also found high positive correlations between total carotenoids and a* and iy and a higher negative correlation with h° following a logarithmic-law function with higher correlation in ‘palmer’ than in ‘haden’ cv., confirming the better relationships of pulp color in ‘palmer’ than in ‘haden’ fruits. high correlations between pulp color and all-trans-β-carotene, alltrans-violaxanthin and 9-cis-violaxanthin were also observed in ‘ataulfo’ and in ‘manila’ mangoes (ornelas-paz et al., 2008) with the highest correlation coefficients for a* and h° parameters; in ‘manila’ mangoes the best results were associated with the concentrations of all-trans-violaxanthin and 9-cis-violaxhantin, while in ‘ataulfo’ with all-trans-β-carotene, confirming that there is a cultivar specific relationship between pulp color and carotenoids content. similar correlations were also found by vasquezcaceido et al. (2005) in 9 thai mango cultivars (power law functions) and by bicanic et al. (2010) in 21 mango homogenates (second order polynomial dependence). differently from carotenoids, ascorbic acid content and total antioxidant capacity for both cultivars and tpc for ‘palmer’ were not related to pulp color, measured both by a*, iy and h° color parameters and µa540; this is not surprising as literature reported that aa and tpc contents with shelf life does not follow a well-defined increasing or decreasing trend (roblessánchez et al., 2009 b; ibarra-garza et al., 2015). in conclusion our results confirmed that the absorption coefficient at 540 nm (µa540) can be used as a non-destructive maturity index for mangoes. in fact it was able to classify intact fruit of two mango cultivars according to pulp color, the destructive maturity index commonly used for mangoes, as well as according to the contents of total carotenoids and of individual carotenoid compounds and vitamin a value. the good correlations between µa540, pulp color parameters and carotenoids indicate that trs is a suitable tool to sort fruit with different ripening degree, having specific carotenoid pattern. adv. hort. sci., 2018 32(3): 407-420 418 acknowledgements this work was funded by lombardia region (italy) a n d m i n a s g e r a i s r e g i o n ( b r a z i l ) ( p r o g e t t o d i cooperazione scientifica e tecnologica “approccio multidisciplinare per l’innovazione della filiera di frutti tropicali tropico” id 17077, rif. n° agro-16). thanks to r.m.a. pimentel, epamig (minas gerais, brazil) for ‘palmer’ and ‘haden’ mangoes supply from experimental orchards. references azevedo-meleiro c.h., rodriguez-amaya d.b., 2004 confirmation of the identity of the carotenoids of tropical fruits by hplc-dad and hplc-ms. j. food comp. anal., 17: 385-396. beaulieu j.c., lea j.m., 2003 volatile and quality changes in fresh-cut mangos prepared from firm-ripe and soft-ripe fruit, stored in clamshell containers and passive map. postharvest biol. technol., 30: 15-28. bicanic d., dimitrovski d., luterotti s., twisk c., buijnsters j.p., dóka o., 2010 estimating rapidly and precisely the concentration of beta carotene in mango homogenates by measuring the amplitude of optothermal signals, chromaticity indices and the intensities of raman peaks. food chem., 121: 832-838. brand-williams w., cuvelier m.e., berset c., 1995 use of free radical method to evaluate antioxidant activity. lwt, 28: 25-30. capra s., 2006 vitamin a, pp. 55-65. in: baghurst k. 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food chem., 53: 4827-4835. y a h i a e . m . , o r n e l a s p a z j . j . , g a r d e a a . , 2 0 0 6 extraction, separation and partial identification of ‘ataulfo’ mango fruit carotenoids. acta horticulturae, 712: 333-338. impaginato 113 adv. hort. sci., 2019 33(1): 113-122 doi: 10.13128/ahs-24787 a new device to improve the mechanical winter pruning in olive trees hedgerows a. ottanelli 1, e. marone 2 (*), p. fiorino 1 1 dipartimento di scienze e tecnologie agrarie, alimentari, ambientali e forestali, dagri, università degli studi di firenze, viale delle idee, 30, 50019 sesto fiorentino (fi), italy. 2 facoltà di bioscienze e tecnologie agro-alimentari e ambientali, università degli studi di teramo, campus coste s. agostino, via r. balzarini, 1, 64100 teramo, italy. key words: branches, fruit bearing, leaves, olea europaea, shoots, superintensive plantations. abstract: the economic success of superintensive olive plantations is mainly due to the full mechanization of the harvesting and pruning. while the advantage of straddling machines is undoubted, winter mechanical pruning determines falls in productions. this is due to the indiscriminate suppression of both fertile leafy shoots destined to fruiting and growth, and exhausted parts of the plant. to reduce this damage, an innovative device has been developed, applied to a pruning machine, able to selectively cut the “aged” parts of vegetation. the selection is achieved by an air’s fluid dynamic action obtained throughout defined and directional air jets able to push the young and flexible shoots upwards and downwards; in this way they are saved by the cut, regenerating at least one year in advance the new fruiting hedge. tests were carried out on the cultivar arbequina, tosca and sikitita, in three superintensive olive groves located in the province of grosseto, rome and latina, assessing the amount of leaves, shoots and branches, as well as fruits present at harvest, preserved from the pruning thanks to the action of the air flow, respectively for the east and west side of the rows. to get a profile of the biomass distribution along the cross section of the tree canopy, in the grosseto farm a trial was also carried out to better assess leaves, shoots and branches distribution in the canopy. the statistical data analyses immediately evidenced two different populations due to the selective pruning. the work highlighted the remarkable effectiveness of the air jet in safeguarding the flexible and leafy vegetation and allowed to quadruple leaf surface and production. 1. introduction the improved knowledge and agronomic techniques during the last decades have allowed the development of olive cultivation beyond the original distribution area (mediterranean basin), in new zones where olive growing represents one of the most promising crops (marone and (*) corresponding author: emarone@unite.it citation: ottanelli a., marone e., fiorino p., 2019 a new device to improve the mechanical winter pruning in olive trees hedgerows. adv. hort. sci., 33(1): 113-122 copyright: © 2019 ottanelli a., marone e., fiorino p. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 30 november 2018 accepted for publication 9 march 2019 ahs advances in horticultural science adv. hort. sci., 2019 33(1): 113-122 114 fiorino, 2012). since the beginning of the 1990s, the introduction o f a c a n o p y m a n a g e m e n t a n d t r a i n i n g s y s t e m defined as “superintensive” contributed to the spread of olive cultivation (tous et al., 1997; tous et al., 2010; rallo et al., 2013), as the high number of trees per hectare (over 1600), with particularly short distances between the plants on the row; the close canopies form, such as in the vine, a single hedge; the system allows the complete mechanization of the harvesting, carried out with straddling machines that detach, intercept and storage the fruits at once, working continuously; the system also allows the mechanical pruning (vivaldi et al., 2015), performed by circular saws fitted on adjustable bars, by eliminating the vegetation along horizontal (topping) or vertical (hedging) planes. the continuous harvesting yards, based on the use of straddle machines, allow strong scale economies and guarantee the greatest efficiency with respect to other systems like shakers and vibrating combs, which can work in traditional or intensive farming systems (fiorino et al., 2010). the mechanical pruning using circular saws fitted on bars, borrowed from viticulture, can be applied to both intensive (individual canopies) and superintensive (hedge) plantations. the comparison between manual and mechanical pruning highlight a greater o p e r a t i v e s p e e d o f t h e s e c o n d ( g i a m e t t a a n d zimbalatti, 1997), partially compromised by the decreasing of productivity (ferguson et al., 2002; peça et al., 2002). in fact, the mechanical pruning s i m u l t a n e o u s l y e l i m i n a t e s , t o g e t h e r w i t h t h e exhausted, also the young vegetation, influencing the canopy vegetative-productive ratios, as well as the distribution of the natural resources and of the light (cherbiy-hoffmann et al., 2012). in intensive plantations, it needs several years to evaluate the influence of regular winter pruning on plant productivity (dias et al., 2012) and, according to albarracín et al. (2017), depending on the intensity of the intervention, it can take up to two growth seasons to reconstitute a fruiting canopy and three to bring the plants back to full production. while in traditional and intensive plantations the mechanical pruning can be considered an economical and convenient alternative to the manual one, it is the only technique available to manage the canopy of adult plants grown in superintensive plantations. in this case its role changes, from tool to preserve the vegetative-productive plants equilibrium, to tool for constraining the plant dimensions within defined limits of height and width, required to perform the harvesting by straddling machines (vivaldi et al., 2015); as a consequence it is largely applied in these plantations, despite the negative consequences on the productive vegetation, due to the particular growth model of the olive tree. in fact, in the olive tree, the fruiting takes place on the vegetation of the previous year, simultaneously with the elongation of the shoots apex that will take fruits the following year; over the time, these shoot elongations progressively move away from the central leader of the tree and, as the competition between the different sinks (apical shoot growth and fruits energy demand), progressively become weak and less productive. at the same time, from the branch that originated them, some latent buds can sprout, fated to reiterate the cycle, by substituting the fertile vegetation that has already produced fruits (fiorino and marone, 2010). these vegetative structures coexist on the surface o f t h e p l a n t s , o r a l o n g a “ w a l l ” i n t h e c a s e o f hedgerows, where the renewal vegetation is dragged downwards by the weight of the sprouts, being the wood of olive branches flexible, and due to the weight of olives, which also curve the young branches. the “aged” and exhausted parts are periodically e l i m i n a t e d b y t h e w i n t e r p r u n i n g ( f i o r i n o a n d marone, 2010). an innovative device has been designed and built, inserted on a bar equipped with counter-rotating circular saws, able to delivering defined and directional air jets that push the shoots upwards and downwards, bending them. in this way the young flexible shoots inserted on the two or three year-old branches escape the action of the pruning machine blades, recovering their natural position after the course of the tool, while the short shoots inserted on the rigid vegetation that produced fruits, depleted and to remove, are cut. the present study would to test the efficiency of a new selective blower device, with the goals: a) to save leaves on the green wall, improving its efficiency in light interception, and b) to save a greater number of fertile buds since the first year after cutting and at least a part of the fruits resulting from the subsequent flowering. moreover, the study would determine the quantity of the biomass produced in olive hedgerows as byproduct of the mechanical pruning, characterized by a remarkable presence of leaves, to verify the possibility of alternative uses, in addition to those already tested for manual pruning residues that, mixed with olive pomace, contribute to the production of pellets (barbanera et al., 2016) and, as already demonstratottanelli et al. improving mechanical winter pruning 115 ed for pomace, to identify a possible use as supplements/components of livestock feed (gonzález et al., 2012; castellani et al., 2017; taticchi et al., 2017) or a s a n a t u r a l s o u r c e o f a n t i o x i d a n t c o m p o u n d s (talhaoui et al., 2015). 2. materials and methods plant material three different pruning trials were carried out in three different localities in high density plantations, on rows of olive trees trained with a central leader, allowing their natural lateral branching. 1) year 2014. the first trial was performed in the “ c a s t e l l o d i t o r r i m p i e t r a ” f a r m , l o c a t e d i n torrimpietra (rome) (41° 53 ‘43’’ north latitude, 12° 14’ 8’’ east longitude, 44 m asl), on trees of the cv. arbequina and tosca, 8 years old, with planting density of 3.8 x 1.6 m (2630 m of rows per hectare). the plants were branched about 60 cm from the soil; the thickness of the canopy walls at the time of the pruning was about 120 cm, the height of 2.20-2.40 cm. the useful height of the fruiting wall was 180 cm. the soil is sandy (over 60% sand), ph = 7.6. 2) year 2015. the second trial was performed in the “casale san giorgio” farm, located near latina (41° 37’ 7’’ north latitude, 12° 34’ 50’’ est longitude, 64 m asl), on trees of the cv. arbequina and sikitita, 5 years old, with planting density of 3.8 x 1.6 m (2630 m of rows per hectare). the plants were branched about 60 cm from the soil; the thickness of the canopy walls at the time of the pruning was 120 cm, the height of 2.00-2.20 cm. the useful height of the fruiting wall was 160 cm. the soil is clayey (36% clay), with a high percentage of sand (40%), ph = 6.7. 3) year 2018. the third trial was performed in the “tombolo” farm located in grosseto (42° 44’ 3’’ north latitude, 10° 59’ 10’’ est longitude, 8 m asl), on trees of the cv. arbequina, 10 years old, with planting density of 4.0 x 1.6 m (2500 m of rows per hectare). the plants were branched about 60 cm from the soil; the thickness of the canopy walls at the time of the pruning was 140-160 cm, the height of 2.00-2.20 cm. the useful height of the fruiting wall was 160 cm. the soil is sandy-silty (over 60% sand), ph = 7.2. the rows in the three farms are oriented northsouth. all the plants were subjected to fertigation, soil management by grassing between rows and weed control on the rows, and fight against spilocaea oleagina and bactrocera (dacus) oleae. in all trials the mechanical pruning was set to leave a maximum thickness of the canopy of the hedge of 80 cm (40 cm on the east side and 40 cm on the west side). to verify the efficiency of the air-jet apparatus, samplings were carried out to quantify leaves, shoots (up to 1 year old) and branches (over 1 year old) saved when cut as pushed by the air of the blower device towards the inside of the canopy and then come back to its original position. during the pruning, the machine proceeded along each row keeping constant the driving speed and the cutting depth of the blades, let the blower device operating for a defined number of spans (15-20 m each, depending on the plantation), alternating with spans where it remained off, and for each row on both sides (east and west orientation of the plants, respectively). the surveys were made at the center of the spans. to perform the surveys on the vegetation, a square frame (1.0 x 1.0 m) was used, placed in correspondence to the central axis of a plant, at a height of 1.0 m from the soil, and positioned at the theoretical cutting blades surface (fig. 1); all the plant material (leaves, shoots, branches) found outside this frame after the cutting was removed. table 1 shows the list of the number of plants chosen for each locality, from which the different samples of vegetal material were taken, used to carry out the experimental surveys, which concerned, for the east side and the west side, respectively: leaves number and surface, shoots length, branches length. in the third test, surveys were also made on the number of fruits present at harvest on pruned plants with and without the presence of the air flow, respectively, to evaluate the difference in the production ability of the plants determined by the two types of techniques, for the two sides of the rows (east and west). fig. 1 sampling units after the pruning in the casale san giorgio farm (2015): a) air flow off; b) air flow on. adv. hort. sci., 2019 33(1): 113-122 116 in the tombolo farm, the day before the winter p r u n i n g ( m a r c h 2 0 1 8 ) , o n 6 p l a n t s o f t h e c v . arbequina, the amount of leaves, shoots and branches on unpruned trees was determined, separately for the east and the west side, to obtain a profile of the canopy biomass distribution along the transversal section of the hedgerow. at this aim, the vegetal material present in the selected sampling units (1 m2) was collected, keeping separated the outer 15 cm of the canopy (outer layer), corresponding to a “light” pruning, the next approximately 25 cm (intermediate layer), corresponding to a “severe” pruning, and the remaining 40 cm up to the permanent structures (inner layer) of the hedge. for all the samples the leaves number and the leaf surface, the leaves dry weight, the shoots total length, the branches total length have been determined. the leaf surface was measured by collecting 50100 leaves per sampling unit; for the trials before the pruning of 2018 in the tombolo farm the leaf area was instead determined through a sampling of at least 100 leaves completely developed per sampling unit (total 600 leaves), chosen in the different sections (outer, intermediate and inner) in which the canopy has been subdivided. the leaf area was determined by scanning and analyzing the image (uthscsa image tool it version 2.03), and the dry weight was determined for the same samples. all trials and surveys were carried out in the second half of march, after the winter cold and shortly before the vegetative restart. the selective pruning device the pruning machine (bmv-fl480s), designed for use in intensive olive groves, is equipped with a removable device able to perform selective cutting, unlike commonly adopted solutions, that cut all the shoots and branches which are in a predetermined position. this is achieved by the air’s fluid dynamic action. characteristics: the pruning machine used for the tests consists of a 240 cm cutting bar fitted on a frame that allows the adjustment in lateral height and inclination, composed of six blade disks with a diameter of 500 mm, a fluid dynamic flow generator and four air flow conveyors arranged in front of the cutting bar (fig. 2). in all the trials a vehicle of more than 80 hp was used. statistical analyses the experimental design was chosen to guarantee the uniformity of sampling units, deriving from the same starting population for each locality and cultivar. average and standard deviations of raw data related to the four treatment (east air on, east air off, west air on, and west air off) for each locality and cultivar were compared; since in this experiment authors were interested in the evaluation of the effectiveness in the performances of a pruning machine (i.e. kg of production or cm of elongation tree-1, or ha-1), only raw data were submitted to the parametric test, as logarithmic transformation is most suitable to express magnitude discrepancy; for each data set, tests were carried out to evaluate the normality of the distributions, and levene’s test were performed to evaluate homoscedasticity at 95% confidence level. since in the most of cases the data distribution indicates some significant nonnormality (the standardized skewness and/or kurtosis is outside the range of -2 to +2), and since significant difference amongst the standard deviations at the 95.0% confidence level were evidenced, neither analyses of variance nor non-parametric tests to compare the medians instead of the means could be performed; as a consequence, average values and s.d. were only reported; box and whiskers plot were built up to table 1 list of the sampling units related to different cultivar and locality fig. 2 a) cutting bar equipped with the selective pruning device; b) the pruning machine working along a row. codex samples provenience east air on east air off west air on west air off total 1 cv. arbequina, torrimpietra, roma (2014) 6 6 6 6 24 2 cv. tosca, torrimpietra, roma (2014) 9 9 9 9 36 3 cv. arbequina, casale san giorgio, latina (2015) 12 12 12 12 48 4 cv. sikitita, casale san giorgio, latina (2015) 4 4 4 4 16 5 cv. arbequina, grosseto, (2018) 12 12 12 12 48 total 43 43 43 43 172 ottanelli et al. improving mechanical winter pruning 117 compare the medians, as the effect of the applied air flow to the pruning machine is so high to generate two populations different at all. this aspect is very important to consider, as the standard deviation can play a meaningful role also when it results to high to allow the application of the common descriptive stat i s t i c a l t e s t s ( m a r o n e e t a l . , 2 0 1 7 ) . t h e s a m e approach was applied to evaluate the differences in the fruit bearing parameters measured in olive trees of cv. arbequina (tombolo farm, 2018), and the same conclusions were drawn. one-way analyses of variance (anova) were performed to compare vegetative growth parameters in olive trees of cv arbequina (tombolo farm, 2018) measured in the sampling units (1 unit = 1 m2) before carrying out the pruning trials, evaluating the side effect (est/west); separation of means was performed by the fisher’s lsd test (p = 0.05). levene’s test to check the variance, and mood’s median χsquare test to evaluate the medians samples differences, if necessary, were also applied. all the computations were performed on the raw data by statgraphics centurion xv v. 15.0.04. a multivariate partial least squares-discriminant analysis (pls-da, supervised method), based on venetian blinds cross validation procedures, was applied on the d a t a o b t a i n e d f r o m a l l t h e s a m p l e s o f t h e c v . arbequina, after applying the two alternative pruning methods, coming from the sampling units collected in the three different zones, to highlight the possibility of differentiating the sampling submitted to the pruning with the air flow on, based on the chosen canopy vegetative parameters. for a description of the method, see colzi et al. (2017). data were preprocessed by a logarithmic transformation (log+1) to express magnitude discrepancy. the analysis was performed by pls-toolbox v. 8.0.2 (eigenvector research inc., west eaglerock drive, wenatchee, wa) for matlab® r2015b (mathworks inc., natick, ma, usa). a factor analysis (fa) was performed on (log+1) d a t a o b t a i n e d f r o m t h e s a m p l i n g u n i t s o f c v . arbequina (farm of tombolo, 2018), considering as factors both vegetative and productive parameters (leaves number and areas, shoots and branches length, fruits number). computations were performed by xlstat 2014.5.03. 3. results vegetative growth and fruiting parameters table 2 shows the data related to the canopy parameters measured on the (residual) vegetation outside the cutting surface: leaves number (n.) and surface (cm2), shoots total length (cm) and branches total length (cm). for each locality and cultivar, the average and standard deviation values of the sampling units (1 m2), obtained with and without air flow, are separately indicated for the two sides of the row (east/west). all the cultivars under study belong to the class of vigor suitable for their use in superintensive plantations. the data shows the great difference between the values of all the “on” tests parameters compared to those of the “off” tests. in particular, the number of leaves (and axillary buds) results in many combinations more than quadrupled in the “on” tests (table 2, fig. 3), and the same proportion is found in the leaf surface present after the cut which, in the west on test of the cv. tosca overcome the coefficient of 1.12 m2 of leaf area per m2 of cutting area. it can be also seen that, in general, the west side of the row is richer in leaves, compared to the east side, except the cv arbequina in casale san giorgio (2015) and tombolo farms (2018), which show a behavior that tends to be opposite. vegetation up to 1 year old shows the same table 2 differences in vegetative growth parameters in olive trees measured as residual vegetation in the survey units (1 m2) due to action of the selective device and the side (east/west) effect (average ± s.d.) samples 1, 3 and 5 refer to the same cv. arbequina. sample 2 to the cv. tosca, sample 4 to the cv. sikitita. treatment leaves (number) leaves (cm2) shoots (cm) branches (cm) 1 east off 99.0±65.2 471.6±350.4 273.8±201.2 21.0±35.3 east on 406.5±75.3 2060.7±359.6 1040.5±361.4 125.0±75.7 west off 179.4±100.8 795.4±434.2 368.3±226.5 27.8±20.9 west on 714.2±322.4 3322.3±1480.7 1134.0±428.5 118.2±56.8 2 east off 660.6±211.6 2542.5±926.3 782.2±220.1 66.3±40.2 east on 2358.1±564.7 8823.2±2098.2 2926.4±840.2 133.8±48.9 west off 855.7±255.9 3287.1±1124.6 855.4±233.6 78.2±33.4 west on 2980.3±665.3 11160.5±2527.3 3479.2±917.9 142.5±47.7 3 east off 115.7±65.1 542.7±305.3 270.2±83.3 5.2±9.0 east on 928.9±379.8 4356.5±1781.5 1453.9±552.6 57.08±35.9 west off 57.1±22.4 268.0±105.0 196.9±67.0 10.2±8.9 west on 596.0±232.4 2795.2±1090.2 1124.1±418.3 69.3±42.6 4 east off 15.0±10.0 46.8±31.0 200.0±92.5 9.2±14.8 east on 79.5±36.1 247.2±112.4 911.7±282.4 65.5±72.8 west off 22.3±10.9 69.5±33.9 256.5± 12.1 8.7±17.5 west on 103.3±21.4 321.3±66.4 1342.0±434.9 80.7±34.6 5 east off 35.6±15.2 167.2±71.3 99.0±34.3 2.4±4.5 east on 285.7±68.6 1335.6±321.9 572.9±223.9 48.3±37.5 west off 35.0±14.0 164.1±65.5 84.0±19.5 0.0±0.0 west on 572.7±223.9 2681.4±1050.1 943.9±295.2 29.2±12.8 118 adv. hort. sci., 2019 33(1): 113-122 trend. the amount of branches is generally negligible, except for the cv. tosca, which gives length values from 66 (east off) to 142 cm (west on). figure 4 shows the score plot and the summary statistics of the pls-da model, that compare the data related to all the samples of the cv. arbequina, coming from the sampling units collected in the 3 localities under test, after applying the two alternative pruning methods, based on the chosen vegetative parameters of the trees canopy. the model statistic indicators and the score plot clearly confirm the presence after pruning performed with and without air flow of two completely different populations. as the model is representative of three distinct agronomic situations (castello di torrimpietra, casale san giorgio and tombolo farms) and of the two sides (east/west) of the rows, this output confirms the validity of the air jet system in safeguarding a significant part of the most important vegetation useful for the fruit production and the subsequent growth of the fruiting shoots, subtracting it from the indiscriminate mechanical cutting inevitable using the current pruning machines without blower device. in the tombolo farm, the number of fruits per m2 of fruiting area (sampling unit) present on the plants of the cv. arbequina was also determined. the numerical differences between air on and air off units related to the two orientations (east and west), respectively, highlight the enormous advantage obtained using the selective blower device, exceeding 100 fruits (with an increase of 107 fruits for the east and 116 for the west) (see table in fig. 5). calculating an average increase of only 100 fruits per m2, multiplied by an useful area of about 8000 m2 ha-1, there would be a productive advantage already on the first yield after the pruning of 800000 fruits; multiplying this value by the average weight of 1 fruit of cv. arbequina in the area (about 2.0 g), gives over 1600 kg of increased product in average per hectare, compared to the production of 284 kg that would have been obtained without the support of the device, with an average increase of 412% in olive production, and a recovery of over 1.7 t of product already in the pruning year. in the figure 6 a biplot from factor analysis is shown, simultaneously representing the relationships amongst the vegetative and productive parameters measured on the cv. arbequina (tombolo farm, 2018). the first axis explains the 97.83% of the total variability in the data. the four populations deriving from the treatments and the orientation are well separated in the four plots of the diagram. the two clusters that represent the trials with the application of the blower device (on the right in the figure) are significantly related to the vegetative-productive parameters of the leaves. it is interesting to note how the parameters related to leaves, shoots and fruits are also influenced by orientation to the west, as shown by the direction and length of the arrows in the graph. fig. 3 box-and-whisker plots related to the leaves number and a r e a a s i n f l u e n c e d b y t h e f l o w ( o n / o f f ) a n d s i d e (est/west). 1-2: ‘arbequina’, 2014; 3-4: ‘tosca’, 2014; 56 : ‘ a r b e q u i n a ’ , 2 0 1 5 ; 7 8 : ‘ s i k i t i t a ’ , 2 0 1 5 , 9 1 0 : ‘arbequina’, 2018. median notches, and mean (+) ± s.d. notches added to the plot show the estimation error associated with each median; if they do not overlap, highlight which medians are significantly different from which others at 95% confidence level. mean markers (+) indicate the location of samples mean. outliers are indicated (�) if present. ottanelli et al. improving mechanical winter pruning 119 canopy distribution of biomasses in hedgerows trained olive trees table 3 compares the vegetative parameters (leaves number and area, shoots and branches length) per m2 of the three sections in which the canopy has been transversely divided. an initial evaluation seems to indicate that, for the particular area of tombolo farm, the east/west orientation plays an important role in determining the vegetative parameters taken into consideration; in fact, in the west section, regardless of the depth of the evaluation, the leaves number and surface (cm2), and the shoots length (cm) is greater. branches cannot be taken into consideration, since they are absent from the outer part, and almost absent from the intermediate section. this advantage determined by the orientation, whose causes are to better investigate, seems to be present also in the plot of castello di torrimpietra farm for both the cultivar (arbequina and tosca) and in the plot of casale san giorgio farm limited to the cv. sikitita. table 3 partition of components (leaves number and surface, shoots and branches lenght) of the three canopy transversal sections in olive trees of cv arbequina (tombolo farm, 2018) measured in the sampling units (1 unit = 1 m2), side (est/west) (average ± s.d.). average leaves dry weight for the three sections. taking into account the east/west averaged values, it results that a “light” pruning (taking away the fig. 4 score plot of pls-da model and summary statistics for the cv. arbequina samples in the three different zones after the pruning, based on the measured vegetative growth parameters: 0) air off (red), 1) air on (blue). fig. 5 differences in fruit number in olive trees of cv. arbequina (tombolo farm, 2018). in the table: average ± s.d.; in the box and whisker plot: median notches, and average (+) ± s.d. fig. 6 biplot from factor analysis. relationships among leaves number (ln), leaves area (la), shoots length (sl), branches length (bl), fruits number (fn), and the four different treatment: east air on (e/on), east air off (e/off), west air on (w/on), west air off (w/off); cv. arbequina, tombolo farm, 2018. adv. hort. sci., 2019 33(1): 113-122 120 first 15 cm of the vegetation), removes about 1/3 of the leaves thus reducing 1/3 the leaf surface of the plant, and a “severe” pruning (at a depth of about 40 cm), would remove more than ¾ of the present leaves, leaving the final permanent structure with a highly reduced leaf apparatus. even considering the shoots length (cm) it is possible to evaluate the meaningful effect of the intervention: a “light” pruning would remove about 1/5 of the present shoots, while a “severe” pruning would leave only 35% of the shoots, thus eliminating most of the vegetation potentially productive. figure 7 shows an example of partition between the different components of the canopy for each of the three considered sections. it is also interesting to consider the amount of leaves biomass that, by the pruning, can be made available for other uses: a “light” pruning, limited to a 15 cm layer, could produce, in this typology of rows, 541 kg of leaves (dry weight), and further 692 kg (dry weight) would also be removed from the intermediate section, for a total of 1.2 t of dry weight leaves to be considered an additional resource and not longer as a residual. 4. discussion and conclusions mechanical winter pruning on adult hedges in superintensive plantations, unlike manual pruning, is a useful tool to control the size of the vegetative structure, thus decreasing its traditional role of baltable 3 partition of components (leaves number and surface, shoots and branches length) of the three canopy transversal sections in olive trees of cv. arbequina (tombolo farm, 2018) measured in the sampling units (1 unit = 1 m2), side (est/west) (average ± s.d.) average leaves dry weight for the three sections *there is more than a 3 to 1 difference between the smallest standard deviation and the largest. since the levene’s test p-value is less than 0.05 (0.0004) there is a statistically significant difference amongst the standard deviations at the 95.0% confidence level. since the mood’s median χ-square test is greater than or equal to 0.05 (0.102467), the medians of the samples are not significantly different at the 95.0% confidence level. section parameter east west average leaves dry weight (g) outer section (15 cm) leaves (no.) 689.1 ± 66.2 a 815.2 ± 86.9 b 67.69 (33.4%) leaves (cm2) 3232.0 ± 310.4 a 3823.3 ± 407.5 b shoots (cm) 1234.7 ± 30.6 a 1121.5 ± 111.3 b branches (cm) 0.0 ± 0.0 0.0 ± 0.0 intermediate section (about 25 cm) leaves (no.) 680.4 ± 39.7 a 1243.4 ± 88.2 b 86.57 (42.7%) leaves (cm2) 3191.3 ± 186.2 a 5831.5 ± 413.5 b shoots (cm) 2132.7 ± 510.7 a 2979.7 ± 232.3 b branches (cm) 214.0 ± 64.1 a 240.3 ± 82.0 a inner section (40 cm from the main tree axis) leaves (n.) 367.8 ± 145.1 a 708.4 ± 206.6 b 48.43 (23.9%) leaves (cm2) 1725.1 ± 680.6 a 3322.6 ± 968.9 b shoots (cm) 2016.2 ± 423.8 a 1817.4 ± 904.2 a branches (cm) 580.2 ± 234.3* 709.2 ± 71.3* fig. 7 example of partition of components (leaves, shoots, and branches) of the canopy transversal sections in a olive tree of cv. arbequina (tombolo farm, 2018) measured in a sampling units (1 unit = 1 m2), side west. a) outer section (15 cm); b) intermediate section (25 cm); c) inner section (40 cm from the main tree axis). ottanelli et al. improving mechanical winter pruning 121 ancer between vegetative growth and production (ferguson et al., 2002). in fact, by manual winter pruning, the aged, shaded and exhausted parts of the plant are selectively eliminated, to leave light and space for the parts destined to growth and to produce the following years (peça et al., 2002). mechanical winter pruning, on the other hand, proceeds by vertical sections, indiscriminately eliminating the part exceeding the size limits imposed by the use of straddling machines; this leads, as immediate result, as pointed out by several authors (vivaldi et al., 2015; albarracín et al., 2017), a sharp decline in productivity, a long unproductive period, due to the need to regenerate a fruiting canopy and, therefore, an economic damage that decreases the benefits offered by the mechanization of the harvesting (peça et al., 2002; albarracín et al., 2017). in this work the authors tried for the first time to overcome the problem by verifying the effectiveness of a tool that combines the action of circular saws with a jet of air which push the shoots away from the cutting area, allowing to reduce the removal of plants young leafy parts. the main effect of the air flow is evidenced by the presence of two population (air on and air off), totally different and distinct. the new air-jet system, compared to traditional pruning machines, preserves over 400% of the leaf surface and relative shoots length in the canopy zones where it has been applied, determining a proportional increase in production, already economically interesting the first year after the pruning. the difference in olive produced resulted higher than 1 t ha-1 using the air jet, demonstrating the true economic advantage of the new device, able to prevent an almost total fall in production in the winter pruning year. this improves the use of agronomic practices, keeping in mind that in superintensive olive groves, fertigation, soil and pests management are always to be applied, without exceptions (vivaldi et al., 2015). at the same time, the preservation of a larger leaf surface on the vegetation of the year, also guarantees a more suitable use of the solar radiation (cherbiy-hoffmann et al., 2012), which allows both the activation and development of new growth points, and the growth of new potentially productive vegetative structures on the shoots elongations, able to guarantee an adequate continuity of the production (fiorino and marone, 2010). further research will be needed to improve the efficiency of this tool, and better understand the mechanisms and the evolution of the different buds present in the different parts of the canopy, which regulate the distribution of resources, also verifying the possible causes determining the growth differences due to the orientation east/west of the row canopy. it is also important to determine the quality of the removed biomass which, in the case of “light” pruning, is exclusively composed of leaves and shoots, and could constitute a considerable amount of vegetal material to be used as a supplement/component of the livestock feed ration, or become an important source for the growing demand for phenolic antioxidants of nutraceutical interest (talhaoui et al., 2015; castellani et al., 2017). acknowledgements the authors are grateful to: dr. ing. stefano borio, bmv di borio valerio & c. s.n.c., alba (cn), italy, for his invaluable technical support and to having provided the pruning machine equipped with the new selective device; the farms: azienda casale san giorgio, latina, azienda castello di torrimpietra, roma, azienda tombolo, grosseto, to have allowed and hosted the trials, and the olive consultant alessandro mersi, for its helpful collaboration in the organization of the tests. references albarracín v., hall a.j., searles p.s., rousseaux m.c., 2017 responses of vegetative growth and fruit yield to winter and summer mechanical pruning in olive trees. sci. hortic., 225: 185-194. barbanera m., lascaro e., stanzione v., esposito a., altieri r., bufacchi m., 2016 characterization of pellets from mixing olive pomace and olive tree pruning. renew. energ., 88: 185-191. castellani f., vitali a., bernardi n., marone e., palazzo f., grotta l., martino g., 2017 dietary supplementation with dried olive pomace in dairy cows modifies the composition of fatty acids and the aromatic profile in milk and related cheese. j. dairy sci., 100(11): 8658-8669. c h e r b i y h o f f m a n n s . u . , s e a r l e s p . s . , h a l l a . j . , rousseaux m.c., 2012 influence of light environment on yield determinants and components in large olive hedgerows following mechanical pruning in the subtropics of the southern hemisphere. sci. hortic., 137: 36-42. colzi i., taiti c., marone e., magnelli s., gonnelli c., mancuso s., 2017 covering the different steps of the adv. hort. sci., 2019 33(1): 113-122 122 coffee processing: can headspace voc emissions be exploited to successfully distinguish between arabica and robusta?. food chem., 237: 257-263. dias a.b., peça j.o., pinheiro a.c., 2012 long-term evaluation of the influence of mechanical pruning on olive growing. agron. j., 104(1): 22-25. ferguson l., krueger w.h., reyes h., metheney p., 2002 effect of mechanical pruning on california black ripe (olea europea l.) cv. ‘manzanillo’ table olive yield. acta horticulturae, 586: 281-284. fiorino p., marone e., 2010 the fate of lateral buds in t h e o l i v e ( o l e a e u r o p a e a l . s s p . e u r o p a e a v a r . europaea). a first report. adv. hort. sci., 24(1): 29-34. fiorino p., marone e., ottanelli a., 2010 mechanical harvesting, productivity and superintensive planting systems in olive groves. adv. hort. sci., 24(1): 91-94. giametta g., zimbalatti g., 1997 mechanical pruning in new olive-groves. j. agric. engen. res., 68: 15-20. gonzález e., hernández-matamoros a., tejeda j.f., 2012 two by-products of the olive oil extraction industry as oleic acid supplement source for iberian pigs: effect on the meat’s chemical composition and induced lipoperoxidation. j. sci. food agric., 92(12): 2543-2551. marone e., fiorino p., 2012 oleiculture in progress. adv. hort. sci., 26(3-4): 163-175. marone e., masi e., taiti c., pandolfi c., bazihizina n., azzarello e., fiorino p., mancuso s., 2017 sensory, spectrometric (ptr-tof-ms) and chemometric analyses to distinguish extra virgin from virgin olive oils. j. food sci. technol., 54(6): 1368-1376. peça j.o., dias a.b., pinheiro a.c., santos l., morais n., pereira a.g., reynolds de souza d., 2002 mechanical pruning of olive trees as an alternative to manual pruning. acta horticulturae, 586: 295-299. rallo l., barranco d., castro-garcía s., connor d.j., gómez del campo m., rallo p., 2013 high-density olive plantations. hortic. rev., 41: 303-384. talhaoui n., taamalli a., gómez-caravaca a.m., fernández-gutiérrez a., segura-carretero a., 2015 phenolic compounds in olive leaves: analytical determination, biotic and abiotic influence, and health benefits. food res. int., 77: 92-108. taticchi a., bartocci s., servili m., di giovanni s., pauselli m., mourvaki e., meo zilio d., terramoccia s., 2017 effect on quanti-quality milk and mozzarella cheese characteristics with further increasing the level of dried stoned olive pomace in diet for lactating buffalo. asian-australas. j. anim. sci., 30(11): 1605-1611. tous j., romero a., hermoso j.f., 2010 new trends in olive orchards design for continuous mechanical harvesting. adv. hort. sci., 24(1): 43-52. tous j., romero a., plana j., baiges j., 1997. planting density trial with ‘arbequina’ olive cultivar in catalonia (spain). acta horticulturae, 474(1): 177-180. vivaldi g.a., strippoli g., pascuzzi s., stellacci a.m., camposeo s., 2015 olive genotypes cultivated in an adult high-density orchard respond differently to canopy restraining by mechanical and manual pruning. sci. hortic., 192: 391-399. impaginato 133 adv. hort. sci., 2019 33(1): 133-138 doi: 10.13128/ahs-22530 impact of partial root-zone drying on growth, yield and quality of tomatoes produced in green house condition a. hakim 1 (*), z. qinyan 2, m. khatoon 3, s. gullo 4, 1 8624 festival drive, elk grove, ca, usa. 2 1613 holly lane, davis, ca, usa. 3 sacramento, california, usa. 4 oakwood university, cooper complex 7000 adventist blvd. nw, huntsville, al, usa. key words: conventional irrigation, fruit size, growth, tomato, yield. abstract: water resources are limited for irrigation worldwide especially for the arid and semi-arid regions; therefore, there is an urgent need to reassess an alternative technique for conventional irrigation. partial root-zone drying (prd) is considered a new water-saving irrigation technique which has been tested for some crop species. the prd technique simply requires wetting half of the rooting zone and leaving the other half dry, thereby utilizing reduced amount of irrigation water. the wetted and dry sides are interchanged in the subsequent irrigations. the focus of this article is to evaluate the effect of prd on growth, yield and quality of tomatoes as compared to conventional irrigation. to evaluate the effect of prd, a greenhouse experiment was conducted where two irrigation treatments were tested during a 160-day growing period: (1) control treatment where drip irrigation was applied to both sides of the plants; (2) prd treatment in which half of the irrigation water in drip irrigation was given alternately only to one side of the root system with each irrigation. prd treatment had 15% and 7% decreases in shoot fresh weight and leaf area of plant, respectively; however, prd had 20% higher fruit per cluster and 18% increase in fruit production in comparison to the control treatment. no significant difference was detected on fruit size between prd treated plants and control plants. but, fruits from prd treated plant exhibited better appearance, higher lycopene content, firmness, total soluble solid (tss), and tss/titratable acidity (ta) ratio than control ones. fruit from control treatment contained higher chlorophyll content than fruit from the prd treatment. postharvest storage results indicated that higher percentage of rot and chilling injury were observed in control fruits than prd treated fruits. the results of this study indicated that prd is a promising water saving irrigation technique which is able to produce higher yield and better quality tomatoes than conventional drip irrigation. 1. introduction worldwide, agriculture accounts for 70% of all water consumption, as compared to 20% for industry and 10% for domestic use. the world’s population is growing by roughly 80 million people each year. more than (*) corresponding author: hakim61@hotmail.com citation: hakim a., gullo s., khatoon m., qinyan z., 2019 impact of partial root-zone drying on growth, yield and quality of tomatoes produced in greenhouse condition. adv. hort. sci., 33(1): 133-138 copyright: © 2019 hakim a., gullo s., khatoon m., qinyan z. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 16 january 2018 accepted for publication 5 october 2018 ahs advances in horticultural science short note http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(1): 133-138 134 99% of the world’s food supply comes from the land. as the world population continues to grow geometrically, great pressure is being placed on irrigation w a t e r t o p r o v i d e a n a d e q u a t e f o o d s u p p l y . t h e r e f o r e , t h e r e i s a b i g c h a l l e n g e o n h o w t o i n c r e a s e f o o d p r o d u c t i o n w i t h l i m i t e d w a t e r r e s o u r c e s . t h i s i s e s p e c i a l l y t r u e f o r t o m a t o (lycopersicon esculentum mill), which is the second most important vegetable crop with a total world production of 130 million tons in 2016 (euro-fresh, 2016). the challenge becomes even more severe in arid and semi-arid regions where water availability is decreasing and competition for water is increasing between agriculture and industry. therefore, water resources should be used with a higher efficiency as well as a higher productivity. partial root-zone drying irrigation (prd) is one of the new efficient and productive water-saving irrigation methods that can save irrigation water up to 50% in processing tomatoes (casa and rouphael, 2015). this technique has the potential to significantly reduce crop water use (el-sadek, 2014), reduce canopy vigor, but able to maintain crop yields and quality of crops (sun et al., 2014) as compared to conventional irrigation methods. although processing tomatoes accounts for the majority of tomato tonnage, while the comparatively higher prices of fresh market tomatoes make them higher ranked in terms of value. to evaluate the effect of prd on fresh market tomatoes, an experiment was conducted in greenhouse with the objective to compare the effect of conventional drip irrigation (cdi) to prd drip irrigation on the growth, yield and quality of the fresh market tomatoes. 2. materials and methods experimental conditions the experiment was conducted in a greenhouse (approximately 24°c in day time and 15°c at night time, relative humidity (rh) 65%, 15 hour’s photoperiod and ambient light condition) at the chateau fresno nursery, 13505 south fresno, california 93609, usa from april to september 2015. seeds of the fresh market tomato (lycopersicon esculentum mill cv. vibelco) were sown on march 1st, 2015. thirty days after seeding, uniform plants were transplanted into 12 wooden boxes (2.53 m length × 0.65 m width × 0.20 m height each). each box had 4 compartments (0.50 m length × 0.50 m width × 0.20 m height) with one experimental plant per compartment. to avoid lateral water movement, a small piece of plastic (0.50 m length × 0.025 m width × 0.04 m height) was placed centrally on the base of each compartment. plants were grown in a vermicast and coconut fiber mixture (70:30 v/v). plants were fertilized with 15-15-15 (npk) at 120 kg/ha, purchased from lowes, elk grove, california, usa. bees were used for pollination. irrigation treatments two weeks after transplanting two irrigation treatments were tested. treatments were: control (conventional drip irrigation, cdi) to both sides of the root system, and half of irrigation water in drip irrigation given alternately only to one side of the root system with each irrigation (prd) (fig. 1). irrigation treatment was given 0.10 m away from the main stem and on both sides of the row. irrigation covered a total area and soil volume of 0.24 m2 and 0.048 m3, respectively. but half of it at 10:00 h and the other half at 16:00 h by manual drip irrigation system. two irrigation lines were set up and operated separately for the prd treatment. two emitters per plant (one on each line) each emitting 4 l/h were placed 0.15 m away from the main stem of each plant. irrigation in cdi treatment covered a total area and soil volume of 0.018 m2 and 0.004 m3, but half of irrigated area and soil volume was wetted in prd treatment at each irrigation. there was some drainage in all treatments, but this was not measured. however, water losses by drainage were minimized by adjusting the amount of water as the crop developed. so, values of the irrigation use efficiency fig. 1 schematic diagram of partial root-zone drying (prd) irrigation in tomato plants. hakim et al. partial root-zone drying effects on tomatoes 135 presented here might have been under-estimated considering the water losses by drainage. growth, yield, yield component, fruit firmness, fruit water content and blossom-end rot measurements growth, yield, yield components and blossomend rot were measured from twelve (12) randomly selected plants/fruits from each treatment. the plant heights were measured with a tape from the base of the plant to the tip of the plant. plant growth and development data were taken on the sampled tagged plants monthly for three months. leaf area was measured using a destructive method. total leaf area (cm2) was measured by leaf area meter (model, delta-t, cambridge, uk). the numbers of clusters were counted per plant from the first to the last cluster during the growing period. the numbers of fruits were counted when the plants started fruiting. the fruit weights were determined after harvesting the tomato using a weighing balance. after 150 days, one plant per treatment per replication was destroyed and the total vegetative f r e s h w e i g h t w a s a s s e s s e d a n d e x p r e s s e d a s kg/plant. mature green tomato fruit firmness was m e a s u r e d u s i n g a n i n s t r o n u n i v e r s a l t e s t i n g machine with a 0.5 cm2 plunger, measurement was taken at the mid-section of the fruit. water content of fruit was expressed on a dry weight basis. fruit blossom-end rot incidence was recorded and calculated in percentage of fruit affected per plant. fruit quality at harvest or postharvest for postharvest quality evaluation, six (6) replicates of five (5) mature green (cascio, 2017) fruits from each treatment were randomly chosen approximately 130 days after transplanting and were stored in a dark refrigerated room at 3°c with 95% rh. after a storage period of 2 weeks, all fruits were moved to a ventilated room without supplemental light at 24°c with 65% rh and held for 7 days. the following attributes were checked for quality measurements: weight loss, chilling injury, decay, appearance/color change, total soluble solids (tss), ph, titratable acidity (ta), chlorophyll and lycopene content. fruit weight loss was determined prior to and after storage. it was calculated as the percentage of i n i t i a l f r e s h w e i g h t . c o l o r d e v e l o p m e n t w a s observed visually using a subjective scale with mature green (mg) = 1, breaker (b) = 2, pink (p) = 3, light red (lr) = 5 and red (r) = 6 (usda, 2005). chilling injury (surface pitting) was rated visually by estimating the percentage of the injured fruits. decay (unidentified) was rated visually and calculated as a percentage of fruit affected. chlorophyll and lycopene content were determined from three randomly selected fruits from each treatment by grinding pericarp tissue (about 5 g) in 15 ml acetone. the extract was taken for centrifugation at 35,000 rpm for 10 minutes. before centrifugation, the tubes were covered with aluminum foil to prevent light-induced lycopene oxidation. after centrifugation, the supernatant was decanted and adjusted to 20 ml with acetone. absorbance of the extracts at 664 nm for chlorophyll and 503 nm for lycopene was measured with a spectrometer (model 160 a). total chlorophyll content in milligram per 100 gram of tissue was calculated according to the formula developed by holden (1976). lycopene content was calculated using the molecular extension coefficient of 3240 (davis, 1976) and expressed as micrograms per gram of fresh weight. total soluble solid (tss), ph and titratable acidity (ta) were measured on juice extracted from fruit. tss content was determined with a digital refractometer (atago, model 1, tokyo, japan). ta was determined by a metler auto titrator (model v 20) and ph was measured with an autocal ph meter (model phm 83). experimental design and data analysis a completely randomized design was used with the two treatments replicated six times with four plants per replication for each treatment. data were analyzed by a complete randomized model using the glm procedure of sas software version 8.2 (sas institute, cary, nc, usa). student’s t-tests were used to determine significant effects between two treatment means. 3. results and discussions mean plant height over the 3 months are illust r a t e d i n f i g u r e 2 . t h e m a x i m u m h e i g h t w a s obtained from tomato plant irrigated with cdi/full water regime. prd treated plants resulted in the lower height. similar results were reported by pal et al. (2016) who grown tomato plant under deficit irrigated conditions plus paclobutrazol application. prd treatment had 15% and 7% decreases in shoot fresh weight and leaf area of plant, respectively. however, prd had 20% higher fruit per cluster and 18% increase in fruit production in comparison to the cdi treatment (table 1). the difference between cdi and prd treated plant’s shoot fresh weight, leaf area, and fruit production were significantly different at adv. hort. sci., 2019 33(1): 133-138 136 0.001, 0.01 and 0.05% level respectively. the cdi treated plant appeared to have excess moisture in root zone, which causes root inactivity contributing to lower yield and delayed maturity of the crop as compared with the prd treated plant. mean cluster per plant, fruit per cluster, total fresh weight of fruit per plant and fruit weight (individual) were lower in cdi treated plants as compared to prd treated plants. the difference was statistically significant only in case of cluster per plant and total fresh weight per plant (table 1). the lower cluster number, less fruit per cluster and less weight of fruit per plant in cdi treated plant might be due to the excessive vegetative growth as a result of luxurious amount of water application. prd treated plant’s fruit exhibited significantly higher percentage of blossom-end rot as compared to the cdi treated ones. the blossom-end rot is a physiological disorder of tomato fruit caused by calcium deficiency or excessive soil moisture fluctuation which reduce uptake and movement of calcium into the plant (mathew and salvadore, 2007). the higher percentage of blossom-end rot in prd treated fruit might be due to the reduced movement of calcium to the prd treated plants. however, no calcium was analyzed either from leaf or from fruits in this study. fruit size, water content and fruit weight were influenced by prd treatment. there were some differences in fruit size, weight and water content in prd and cdi treated plant’s fruit; however, the difference were not statistically significant (table 1). reduced weight loss in the prd treated tomatoes during the storage is a positive quality attribute in tomato fruit especially for distant market (table 2). yadav and singh (2014) indicated that the weight loss of fruits in storage condition is mainly from water loss and from solid constituents. the lower water loss in prd treated fruit might be due to lesser incident of micro-cracks in the skin. however, no skin micro-cracks were examined in this study. in this study, when compared to fruits from cdi treated plants, fruits from prd treated plants exhibited significantly lower chlorophyll and higher lycopene content on the 7th day at 24°c followed by a 2-week storage period at 3°c (table 2). klunklin and savage (2017) also detected significantly higher lycopene content in prd treated tomato fruit. according to gindi et al. (2016), 63 percent of consumers’ purchase interest depends on color of fruits. table 2 demonstrated that fruits from prd treated plants exhibited less visible chilling injury (fruit surface pitting) and decay, but similar color change as fruits from cdi treated ones. in the regions of the world where irrigation water is expensive, such as arid and same-arid regions, prd treatment would be beneficial in terms of economic return, where water supply is crucial. soluble solids are an important tomato quality fig. 2 effect of cdi (conventional dripping irrigation) and prd (partial root-zone irrigation) on plant height over 3 months. physiological parameters treatment difference (prd-cdi) cdi group prd group fruit size, diameter (mm) 68.4 67.8 0.6 ns total fresh weight of fruit (kg/plant) 5.1 7.03 1.93 *** fruit water content (%) 95.2 94.8 0.4 ns fruit weight (g) 95.2 95.0 0.2 ns fruit firmness after harvest (kg/cm2) 9.1 9.0 0.1 ns cluster/plant 8.1 10.2 2.1 *** fruit/cluster 6.1 6.3 0.2 ns shoot fresh weight (kg) 10.6 9.1 1.5 *** leaf area (cm2) 451.54 418.26 33.28 * blossom end rot (%) 5.5 6.0 0.5 *** table 1 effect of prd on tomato fruit size, total fresh weight, water content, fruit firmness, cluster/plant, fruit/cluster, shoot fresh weight, leaf area, and blossom-end rot ns, *, **, *** non-significant or significant at t≤0.05, 0.01 or 0.001 respectively. hakim et al. partial root-zone drying effects on tomatoes 137 parameter. tomato flavor is generally determined by the content of soluble solids and acid (titratable acid). according to aoun et al. (2013), tomato flavor impact is co-related to total sugar and acid. in this study, total soluble solid (tss), tss/ta and ph increased, while ta decreased in prd treated fruits (table 2). in an earlier study, sun et al. (2014) also detected higher tss in tomato fruit produced under prd condition. the higher tss and lower ta in fruit from prd treated plants were probably due to the less retained water in fruit from prd treated plant than fruit from cdi treated plant. the higher ph in fruit from prd treated plant was compatible with the lower ta in fruits from prd treated plant than in fruits from cdi treated plant. 4. conclusion t h e r e s u l t o f t h i s s t u d y d e m o n s t r a t e d t h a t “vibelco” tomato plant treated with prd slowed its vegetative growth, but produced higher yield and better quality fruit at the same time saved water by 50%. this study proved that prd is a noble water saving method which can prevent excess water use and increases economic returns due to the reduction of water use. acknowledgements the author wishes to thank dr. abu helal ibrahim for his experimental assistance. references aoun a.b., bechiheb b., benyahya l., ferchichi a., 2013 evaluation of fruit quality traits of traditional varieties of tomato (solanum lycopersicum) grown in tunisia. african j. food sci., 7(10): 350-354. casa r., rouphael y., 2015 effects of partial root-zone drying irrigation on yield, fruit quality, and water-use efficiency in processing tomato. j. hort. sci. biotech., 89(4): 389-396. c a s c i o j . , 2 0 1 7 a h a r v e s t o f g r e e n t o m a t o e s . cooperative extension service, fnh-00024, university of alaska, fairbanks, usa. davis b.h., 1976 carotenoids, pp. 38-165. in: goodwin t.w. (ed.) chemistry and bio-chemistry of plant pigments. vol. 2. academic press, new york, usa, pp. 679. el-sadek a., 2014 water use optimization based on the concept of partial root zone drying. ain. shams. eng. j., 5(1): 55-62. euro fresh, 2016 tomatoes: around the world. the international distribution magazine for fresh produce & retail. euro-fresh, news, september 7. gindi a.a., abdullah a.m., ismail m.m., nawi n.m., 2016 factors influencing consumer’s retail formats c h o i c e f o r f r e s h f r u i t s p u r c h a s e i n k l a n g v a l l e y malaysia. int. j. agri. res. sustain. food sufficiency, 3(3): 52-61. holden m., 1976 chlorophyll, pp. 461-488. in: goodwin t.w. (ed.) chemistry and bio-chemistry of plant pigments. academic press, new york, usa, pp. 595. klunklin w., savage g., 2017 effect on quality characteristics of tomatoes grown under well-watered and drought stress conditions. foods., 6(8): 56. matthew d.t., salvadore j.l., 2007 blossom-end rot: a calcium deficiency. j. plant nutrition, 27(1): 123-139. table 2 effect of prd on weight loss, chilling injury, decay, chlorophyll content, lycopene content, color change, ph, titratable acidity (ta), total soluble solid (tss), and tss/ta in mature green tomatoes on the 7th day at 24°c following a 2-week storage period at 3°c physiological parameters treatment difference (prd-cdi) cdi group prd group weight loss (%) 2.15 1.96 0.19 *** chilling injury (%) 15.00 10.00 5.0 ns decay (%) 13.00 8.00 5.0 *** chlorophyll (mg/100 g fresh weight) 3.90 3.20 0.7 ** lycopene (µg/g fresh weight) 7.10 7.70 0.6 * color change 4.50 4.50 0.0 ns ph 4.40 4.55 0.15 ns ta (% citric acid) 0.49 0.46 0.03 * tss 4.30 4.50 0.2 ns tss/ta 8.77 9.80 1.01 *** ns, *, **, *** non-significant or significant at t≤0.05, 0.01 or 0.001 respectively. 138 adv. hort. sci., 2019 33(1): 133-138 pal s., zhao j., khan a., yadav n.s., batushansky a., barak s., rewald b., fait a., lazarovitch n., rachmilevitch s., 2016 paclobutrazol induces tolerance in tomato to deficit irrigation through diversified effects on plant morphology, physiology and metabolism. sci. rep., 6: 1-13. sun y., holm p.e., liu f., 2014 alternate partial rootzone drying irrigation improves fruit quality in tomatoes hort. sci., 41(4): 185-191. usda, 2005 tomatoes, shipping point and market inspection instructions. agricultural marketing service, specialty crops program, specialty crops inspection division, usda, pp. 78. yadav a.k., singh s.v., 2014 osmotic dehydration of fruits and vegetables: a review. j. food sci. technol., 51(9): 1654-1673. 313 adv. hort. sci., 2019 33(3): 313-320 doi: 10.13128/ahs-23353 influence of two training systems on growth, yield and fruit attributes of four apple cultivars grafted onto ‘m.9’ rootstock a. dadashpour 1 (* ), a.r. talaie 1, m.a. askari-sarcheshmeh 1, a. gharaghani 2 1 department of horticulture, university college of agriculture and natural resources, university of tehran, p.o. box 31587‐77871 karaj, iran. 2 department of horticultural science, college of agriculture, shiraz university, p.o. box 65186‐71441 shiraz, iran. key words: intensive planting, v-system, y-system. abstract: this research was carried out to compare several attributes pertaining to the growth, fruit and yield of four apple cultivars, i.e. ‘golab-kohans’, ‘fuji’, ‘starking’ and ‘delbar estival’. these cultivars were grafted onto m.9 rootstocks trained into ‘guttingen v-slender-spindle (or v-system) and ‘geneva y-trellis (or y-system) systems. compared to the y-system, it was observed that the v-system caused the trees to yield more fruits, dry matter, ash and total soluble solids (tss). in contrast, the y-system caused the trees to have broader trunk cross sectional areas (tcsa), along with higher yield, fruit weight, fruit diameter, fruit length and fruit firmness, compared to trees trained with the v-system. in summary, these results showed that both systems can be employed as promising approaches, but the ‘y-system’ appears to be more productive than the ‘v-system’. in addition, among the studied cultivars, it seems that the ‘delbar estival’ and ‘fuji’ were more adaptive to these intensive training systems, especially when considering the fruit traits. 1. introduction intensive training systems are particular layouts that assist orchard managers in improving the productivity of orchards (ferree and warrington, 2003). the need to improve training and pruning methods can better fit the natural growing conditions, and this can be associated with higher fruiting performances by the fruit trees (lauri, 2009). thus, modern apple orchards are planned on the basis of higher tree density than that of traditional planting systems which use dwarfing apple rootstocks (ferree and warrington, 2003). dwarfing rootstocks are increasingly becoming prevalent among the sectors of the fruit industry. they are an important factor that improve orchard productivity due to their significant effects on agro -morphological characteristics such as the yield (*) corresponding author: dadashpour@ut.ac.ir citation: dadashpour a., talaie a.r., askari-sarcheshmeh m.a., gharaghani a., 2019 influence of two training systems on growth, yield and fruit attributes of four apple cultivars grafted onto ‘m.9’rootstocks. adv. hort. sci., 33(3): 313-320 copyright: © 2019 dadashpour a., talaie a.r., askarisarcheshmeh m.a., gharaghani a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 5 june 2018 accepted for publication 22 march 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(3): 313-320 314 (barritt et al., 1995). the guttingen-v system, the ysystem (tatura), the drilling system, and the mikado system are the most popular v-shaped canopy systems, and are suggested as promising alternatives to high density orchards (robinson, 2000). dwarfing rootstocks, such as m.9 and m.27, are generally e m p l o y e d i n v s h a p e d s y s t e m s ( f e r r e e a n d warrington, 2003). v systems allow better light penetration than other training-shaped trees (robinson, 2003). the ‘geneva y-trellis’ system is a v-shaped system which uses a y shaped trellis to support the trees. the ‘guttingen v-slender-spindle’ system includes individual conic-shaped trees allowing high tree densities within multiple rows. it has been reported that the guttingen v causes the production of higher yield per hectare and thinner trunks, compared to the drilling system (sosna and czaplicka, 2008). many investigations have shown that there are significant differences between local and foreign apple cultivars in terms of growth and productivity (dadashpour et al., 2010; dadashpour et al., 2011). such reports also indicate the same with regard to apricot (strikic et al., 2007) when trained by intensive training systems. recently, it has been reported that rootstocks and training forms have significant effects on the vegetative growth, yield and fruit traits of apple cultivars (alizadeh and pirmoradiyan, 2016). it has been reported that the efficiency of several parameters can be improved by more production or by the reduction in tree size (fioravanco et al., 2016). when apple scions are grafted onto dwarfing and semi-dwarfing rootstocks, they usually produce larger fruits and more yield, compared to when scions are grafted onto non-dwarfing rootstocks (perry and byler, 2001; gjamovski and kiprijanovski, 2011). negligible differences have been reported in the cumulative yield among ‘slender spindle’, ‘hybrid tree cone’ (‘hytec’) and ‘vertical axis’ (crassweller and smith, 2004). rutkowski et al. (2009) studied nine training systems for apple trees, and reported that the growth and yield of trees may be more dependent on genetic traits, while the shapes of trees can modify the skeletal structure of an orchard. to this end, gonkiewicz (2011) showed that trees having spindle shapes can produce the best yield and fruit weight among the studied pruning systems in sweet cherry. by studying the ‘fuji’ apple, grafted onto the m.9 rootstock under five training systems, ozkan et al. (2016) reported that there were significant differences among the studied training systems in relation to canopy volume, trunk-cross sectional area (tcsa), yield, yield efficiency and fruit size. with 2.8% of the total harvestable area (134,000 ha) and 2.2% of the total production (1.7 million tons) in the world, iran is among the largest producers of apple after china, usa, turkey, poland, india and italy (faostat, 2012). the majority of apple orchards in iran are traditional ones. they are characterized by low tree densities and are commonly grown on seedling rootstocks. however, semi-intensive and intensive apple orchards are recently becoming popular among apple growers. ‘golden delicious’ and ‘red delicious’ are two apple cultivars that are planted in about 90% of cultivated areas. meanwhile, the early ripening cultivar ‘golab-kohans’ is the most prevalent, native apple cultivar in iran. it provides the summer demand for fresh apples in the market. furthermore, ‘granny smith’, ‘fuji’, ‘gala’, ‘jonagold’, and ‘braeburn’ are i n c r e a s i n g l y b e c o m i n g p o p u l a r i n t h e c o u n t r y (gharaghani et al., 2015). as the apple industry in iran is about to shift dramatically from traditional to modern production systems, e.g. semi-intensive and intensive orchard, it is important and necessary to study the performance of popular apple cultivars on different rootstocks, especially within the context of various training systems. accordingly, the objective of this study was to evaluate two training systems, i.e. ‘guttingen v-slenderspindle’ and ‘geneva y-trellis’, and compare their effects on growth characteristics, yield and fruit quality of four apple cultivars. their scions were grafted onto m.9 rootstocks in the alborz province of iran. 2. materials and methods plant materials and experimental design this research was conducted at an experimental field belonging to a horticultural research station, karaj, iran. the duration of the entire experiment took from 2007 to 2010. the average maximum temperature of the region is 13.7°c, with an annual rainfall of 254 mm. the soil in the region is classified as clay-loam. the experiments were arranged as spiltplot (main plot: training system; split-plot: cultivar) according to a randomized complete block design (rcbd) with four replicates. four apple cultivars were used, i.e. ‘delbar estival’, ‘fuji’, ‘golab-kohans’ and ‘starking’, and their scions were grafted onto dwarfing m.9 rootstocks. all trees were planted in march 2005, and trellis systems were established in june 2006. the trees were trained into two training systems, i.e. ‘guttingen v-slender-spindle’ (v-system) (0.9×3.7 m or 3000 trees/ha) and ‘geneva y-trellis’ dadashpour et al. ‐ training systems of apple cultivars 315 (y-system) (1.6×3.7 m or 1680 trees/ha), based on t h e r e l e v a n t p r o t o c o l s d e s c r i b e d b y p r e v i o u s research on apples (robinson, 2003). drip-irrigation was scheduled to operate twice a week. the soil was fertilized once in every season and was managed according to the common practice in the region. trees received their first fertilizers in the second year after planting. they were pruned during the winters, but the amount of wood being removed by pruning was not documented. fruit thinning was performed if necessary. the fruits were harvested manually. twenty representative trees within each replicate were selected for sampling and data collection. agro‐morphological and yield traits to calculate the trunk cross sectional area (tcsa), the trunk circumference was measured (20 cm above the graft union) from both sides (north-south) with a hand caliper. this was performed at the end of the growing season in the november of 2007, 2008, 2009 and 2010. the average measurement of the two sides on the trunk were taken to make trunk diameter (r) and “area= πr2”. a formula assisted in calculating the tcsa in cm2. in addition, the cumulative yield per tree and per hectare were recorded at harvest time (kg/tree and kg/ha). the yield efficiency was defined as “yield per tree divided by tcsa (kg/cm2)”. fruit properties all attributes pertaining to fruit traits were measured using 5 randomly-sampled fruits from each test tree. then, their average was recorded. the individual fruit length, the fruit diameter and the ratio of length to diameter (l/d) were calculated by a vernier caliper. the fruits fresh weight was determined using a mettler pc 8000 scale. in addition, fruit firmness w a s m e a s u r e d u s i n g a p e n e t r o m e t e r ( i n s t r o n universal machine, model 1011) and recorded as kg.cm-2. total soluble solids (tss) were measured with a bausch and lomb abbe 3l refractometer. moreover, the dry matter content was determined after the fruits were exposed to a process of drying at 70°c for 48 h. one gram of dry matter was burnt to yield ash in a gaallankamp furnace at 550°c for 6 h. titratable acidity (ta) was determined using an aminex hpx-87h column which operated at 65°c, while 4 mm sulfuric acid was used as an eluent. data analysis the data were obtained by field measurements. laboratory observations were processed by analysis of variance (anova) using the sas software and the duncan mean separation test procedure. 3. results agro‐morphological and yield traits in general, all cultivars had developed a sufficient stem diameter (data not shown). the analysis of variance signified substantial differences among the cultivars and training systems. tree vigor was affected substantially by training systems. after four years, there were significant differences in tcsa among the four cultivars. ‘golab-kohans’ exhibited the highest value of tcsa (17.12 cm2) (table 1). the apple trees that were trained by the y-system showed significantly higher tcsa values (16.41 cm2) compared to those trained by the v-system which formed thinner trunks (9.80 cm2) (table 2). the interaction between table 1 means comparison of four apple cultivars about studied characteristics in guttingen v and geneva-y trellis systems during 2007-2010 means with same letters are not significantly different. (p>0.05) using duncan multiple range test. cultivar fruit firmness (kg/cm2) fruit weight (gr) fruit diameter (cm) fruit length (cm) l/d tss ta (%) ash (%) dry matter (%) cumulative yield (kg/tree) yield efficiency (kg/cm2) tcsa (cm) delbar estival 10.00 b 130.15 b 6.57 b 5.81 a 0.86 a 14.53 a 0.45 bc 0.40 b 20.63 bc 16.4 a 0.41 a 9.58 c fuji 14.52 a 148.40 a 6.94 a 5.78 ab 0.83 b 15.33 a 0.68 a 0.35 b 23.89 ab 14.72 ab 0.1 c 14.69 b golab-kohans 8.44 c 79.25 c 5.72 c 5.01 c 0.86 a 11.23 b 0.28 c 0.38 b 19.56 c 7.72 c 0.1 c 17.12 a starking 14.37 a 143.99 a 6.63 b 5.58 b 0.82 b 14.56 a 0.47 b 0.73 a 24.14 a 10.64 b 0.22 b 10.98 c table 2 properties in guttingen v and geneva-y trellis systems during 2007-2010 means with same letters are not significantly different. (p>0.05) using duncan multiple range test. system fruit firmness (kg/cm2) fruit weight (gr) fruit diameter (cm) fruit length (cm) l/d tss ta (%) ash (%) cumulative yield (kg/tree) cumulative yield (t/ha) yield efficiency (kg/cm2) tcsa (cm) guttingen v 10.53 b 122.45 b 6.36 b 5.39 b 0.84 a 14.17 a 0.46 a 0.5 a 7.88 b 23.640 b 0.25 a 9.80 b geneva-y trellis 12.90 a 126.69 a 6.54 a 5.69 a 0.84 a 13.55 a 0.47 a 0.43 a 16.72 a 28.089 a 0.22 b 16.41 a adv. hort. sci., 2019 33(3): 313-320 316 training systems and cultivars showed that ‘fuji’ had the largest trunk diameter and the largest tcsa (19.98 cm2) (fig. 1a). regardless of the training system, ‘delbar estival’ produced the most cumulative yield (16.4 kg/tree) (table 1). table 2 shows that the y-system results in a higher average value of cumulative yield per tree (16.72 kg/tree) and per hectare (28.08 t/ha) than that of the v-system (7.88 kg/tree and 23.64 t/ha, respectively). the v-system contributed to a higher density of trees (3000 tree/ha), compared to the y-system (1680 tree/ha). results show that ‘fuji’ and ‘delbar estival’ exhibited the most cumulative yield per tree and per hectare, under the y-system and the v-system, respectively (figs. 1b and 1c). concerning the yield efficiency, during the four years, regardless of the training system, the ‘delbar estival’ yielded the highest amount of fruit per trunk cross sectional area (table 1). in addition, the v-system showed a higher yield efficiency (0.25 kg/cm2), compared to the y-system (0.22 kg/cm2). a smaller trunk diameter and a higher tree density per hectare can be reasons for the higher yield efficiency (table 2). the interaction between training systems and cultivars functioned mostly in determining the yield efficiency (0.57 kg/cm2) in the ‘delbar estival’ through the v-system (fig. 1d). fruit properties results showed that the ‘fuji’ cultivar yielded the heaviest fruit weight (148.40 gr), whereas ‘golabkohans’ had the lightest fruit (79.25 gr) (table 1). trees trained by the v-system (as a denser system in this study) developed fruits with an average lighter weight (122.45 gr), but the apples obtained from the y-system were slightly heavier (126.69 gr) (table 2). the ‘starking’ cultivar exhibited the heaviest (159.69 gr) and longest fruit (6.1 cm) by the y-system (figs. 2 a a n d 2 b ) . i n f a c t , t h e y s y s t e m c a u s e d t h e ‘starking’ to exhibit the maximum fruit length among the four cultivars. the y-system contributed to the production of fruits that were significantly longer (5.69 cm) than those obtained by the v-system (5.39 cm) (table 2). in addition, the ‘fuji’ yielded the widest fruit (6.94 cm) among the four studied cultivars (table 1). the y-system caused a greater fruit diameter (6.54 cm) than the v-system (6.36 cm) (table 2). figure 2c shows that the maximum width of fruit (7.1 cm) was recorded in the ‘fuji’ by the ysystem. the highest l/d ratio (0.87) belonged to the ‘delbar estival’ by the y-system. in general, the greatest value of fruit firmness was observed in ‘fuji’ (14.52 kg.cm-2) and the lowest was observed in ‘golab-kohans’ (8.44 kg.cm-2) (table 1). also, trees trained by the y-system yielded fruits with the greatest value of firmness (12.90 kg/cm2), compared to the function of the v-system (10.53 kg/cm2) (table 2). ‘fuji’ yielded the firmest fruits (15.96 kg/cm2) by the y-system (fig. 2d). the highest tss (15.33%) and ta (0.68%) were produced by ‘fuji’, whereas the lowest tss and ta were recorded in the fruits of ‘golabkohans’ (table 1). the content of ta also differed because of the training systems. the y-system caused higher ta values in fruits, compared to the vsystem, but this difference was insignificant (table 2) which suggests that the training system had no remarkable influence on the acidity of fruits in this fig. 1 interaction of training systems (v-system and y-system) and four cultivars (delbar estival, fuji, golab-kohans, starking) on fruit properties. dadashpour et al. ‐ training systems of apple cultivars 317 research. the ‘fuji’ yielded fruits with the highest amounts of tss and ta by the y-system and v-system, respectively. when comparing the cultivars, ‘starking’ had the best results regarding the dry matter of fruits (24.14%) and ash (0.73%) (table 1). regardless of the cultivar, the fruits contained more dry matter when the trees were trained by the v-system, compared to training by the y-system (table 2). additionally, ‘starking’ yielded the highest amount of ash by interaction with training systems examined in this study (fig. 2e). 4. discussion and conclusions the results herein suggest that the cultivars and training systems caused differences in the measured characteristics. the occurrence of more tree growth by ‘golab-kohans’ may be due to a higher degree of shading in the canopy than in other cultivars (lo bianco et al., 2007). in addition to the influence of rootstocks, cultivar vigor can be affected by training systems. a lower tcsa was observed in trees of the v-system. this can be attributed to the competition between adjacent trees which, in turn, was a result of shorter spacing between trees (0.9 m) in comparison with the y-system (1.6 m). as reported by other researchers (musacchi et al., 2015; sosna, 2017), planting the trees closer to each other might have negatively affected the stem diameter in this study. these results are in accordance with the latest findings in the available literature (robinson, 2007; ozkan et al., 2016) in which intensive cultivations had remarkable effects on tree growth. the greater yield caused by the y-system might be due to the larger (wider) tree canopy. this result is in agreement with recent reports which suggest that the number of trees per unit area has a great influence on the yield per tree and per hectare (robinson, 2007; ozkan et al., 2016). in general, a more even distribution of fruit-bearing can be observed in apple trees with vshaped canopies, as trained by the yand v-systems, compared to other popular training systems. this has been suggested before by similar research (sosna, 2017). it is known that the yield efficiency depends on the tree’s vegetative vigor and fruit production. when the cultivar has good yield and high tcsa, a lower yield efficiency occurs compared to trees of other cultivars by the same yield and lower tcsa. a lower tree vigor, as caused by the v-system, did not result in a higher yield efficiency. this can be due to a lower yield per tree. in fact, results show that a highfig. 2 interaction of training systems (v-system and y-system) and four apple cultivars (delbar estival, fuji, golabkohans and starking) on fruit properties. 318 adv. hort. sci., 2019 33(3): 313-320 er yield efficiency can be attained by increasing the number of fruits in each tree or by controlling the tree vigor by dwarf rootstocks. significant differences in yield efficiency were also reported in a previous study (fioravanco et al., 2016). it may be assumed that trees on dwarf rootstocks exhibit a weaker vegetative vigor and result in a higher amount of yield (robinson, 2007). nonetheless, the differences among cultivars in this study is likely due to the variations in morphological traits, which is in agreement w i t h p r e v i o u s s t u d i e s ( b a r r i tt e t a l . , 1 9 9 5 ; dadashpour et al., 2010). no incremental trend was observed in the fruit weight during the four years, even by the influence of training systems. the contradictory effects of planting density on the fruit weight in this study are consistent with earlier r e p o r t s ( o z k a n e t a l . , 2 0 1 2 ; s o s n a , 2 0 1 7 ) . nonetheless, fruit quality is influenced by many factors such as the specifications of a training system (robinson et al., 1991). therefore, it is natural to expect variations in the type of influence caused by the two different training systems on the measured traits in fruits. the l/d (≥1) is a criterion used for apple marketing, but all cultivars showed l/d <1 in this study. this observation is probably due to warmer nights in the climatic conditions of the experiment, resulting in insufficient cell elongation. this c o n fi r m s t h e r e s u l t s o f p r e v i o u s r e s e a r c h (dadashpour et al., 2011). based on the current discussion, the ‘delbar estival’ probably has the highest marketable value in terms of its visual appearance among the cultivars. the denser cultivation of trees in the v-system contributed to the production of fruits with lower amounts of coloration, but this was not substantially different compared to the other training system. the good quality of apples obtained from the v-system was noticed in previous studies (rutkowski et al., 2009; dadashpour et al., 2012). it seems that the climatic temperature can affect the fruit firmness. in most of the cultivars, the softest fruits were observed in 2008 (as a cool year in this experiment). however, the relation between temperature and fruit firmness is not fully understood. the y-system caused firmer fruits, compared to the v-system (table 2), and this confirms that fruits harvested from the y-system can be transported with less physical damage. significant differences in apple firmness support recent findings (talaie et al., 2011). ‘golabkohans’ was the earliest ripening cultivar and produced the softest fruits (7.25 kg.cm-2) by the v-system (fig. 2d). the ‘fuji’ produced the firmest fruits, probably because of the small fruit size, thereby confirming the findings of previous studies (drake et al., 1988; dadashpour et al., 2010). in addition, differences in fruit firmness might have been due to genetic variations among cultivars. in addition, it has been reported that fruit firmness is the first edible criterion affecting buyer acceptance (harker et al., 2008). considering the fruit sweetness, fruits and leaves that are exposed to higher light intensities may exhibit more tss (tustin et al., 1988). also, the different tss contents among cultivars may result from variations in leaf area, as suggested by previous research (hudina and stamper, 2002) or by a presumably higher canopy shading of cultivars which produce fruits of lower tss (garriz et al., 1996, 1998). although the tss was not significantly affected by the two training systems, the v-system caused slightly higher levels of tss than the y-system (table 2). among the cultivars, the ‘fuji’ produced the sourest fruits. these results show that acidity, in general, varies with cultivar, confirming previous studies (platon, 2007; dadashpour et al., 2010). the highest amount of ta was observed in fruits of the ‘fuji’ cultivar. this may have resulted from less shading in the tree canopy or because of good nutritional conditions. in general, the ‘starking’ cultivar produced the highest amount of dry matter, thereby confirming previous claims regarding the differences among cultivars in this regard (lata, 2007; palmer et al., 2010). in addition, the dry matter content varies among cultivars, and different training systems cause variations in the dry matter. the dry matter can vary from fruit to fruit and from training system to training system, in agreement with a previous study (palmer et al., 2010). in conclusion, the ‘delbar estival’ exhibited better results under intensive training systems, whereas ‘golab-kohans’ and ‘fuji’ showed the best growth characteristics. in general, the y-system was better than the v-system when considering the majority of characteristics. the two cultivars ‘fuji’ and ‘delbar estival’ were more adaptable to intensive training systems in karaj’s climatic conditions. references alizadeh a., pirmoradyian m., 2016 comparison test of training systems and its influence on yield and fruit quality of g. delicious on clonal rootstocks. sci. agri., 14: 289-292. b a r r i t t b . h . , k o n i s h i a . s . , d i l l e y m . a . , 1 9 9 5 performance of three apple cultivars with 23 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471473. robinson t.l. 2007 effects of tree density and tree s h a p e o n a p p l e o r c h a r d p e r f o r m a n c e . a c t a horticulturae, 732: 405-414. robinson t.l., 2000 v‐shaped apple planting systems. acta horticulturae, 513: 337-347. robinson t.l., 2003 apple‐orchard planting systems, pp. 345-407. in: ferree d.c., i.j. warrington (eds.) apples: botany, production and uses. cabi publishing, wallingford, uk, pp. 660. robinson t.l., lakso a.l., carpenter s.g., 1991 canopy development, yield, and fruit quality of ‘empire’ and ‘delicious’ apple trees grown in four orchard pro‐ duction systems for ten years. j. am. soc. hort. sci., 116: 179-187. rutkowski k., kantorowicz-bak m., pacholak e., 2009 effect of different tree training systems on growth and yielding of two apple cultivars. j. fruit ornam. plant res., 17: 49-59. sosna i. 2017 v‐shaped canopies in an apple orchard from the perspective of over a dozen years of research. j. agr. sci. tech., 19: 415-424. sosna i., czaplicka m., 2008 the influence of two train‐ ing systems on growth and cropping of three pear culti‐ vars. j. fruit ornam. plant res., 16: 75-81. strikic f., radunic m., rosin j., 2007 apricot growth a n d p r o d u c ti v i t y i n h i g h d e n s i t y o r c h a r d . a c t a horticulturae, 732: 495-500. talaie a.r., shojaie-saadee m., asgari-sarcheshmeh m.a., dadashpour a., 2011 fruit quality in five apple cultivars trees trained to intensive training sys‐ adv. hort. sci., 2019 33(3): 313-320 320 tem: geneva y‐trellis. genetika-belgrade, 43: 153-161. t u s t i n d . s . , h i r s t p . m . , w a r r i n g t o n i . j . , 1 9 8 8 influence of orientation and position of fruiting laterals on canopy light penetration, yield, and fruit quality of ‘granny smith’ apple. j. am. soc. hort. sci. 113: 693699. impaginato 25 adv. hort. sci., 2020 34(1): 25­33 doi: 10.13128/ahsc­8253 the effect of different colored netting on quantitative and qualitative traits of two foliage plant species (codiaeum variega‐ tum and aglaonema commutatum) s.k. abbasnia zare 1, s. sedaghathoor 1 (*), m.n. padasht dahkaei 2, d. hashemabadi 1 1 rasht branch, islamic azad university, rasht, iran. 2 agricultural and natural resources research, center of guilan, agricultural research, education, and extension organization, rasht, iran. key words: anthocyanin, brix°, croton, photoselective, yellow netting. abstract: photoselective netting is a new group of colored netting developing in the past few decades. the effect of colored (red, green, and yellow) netting was studied on physiological traits of codiaeum and aglaonema in a trial in flowers and ornamental plants research station of lahijan, north of iran. the trial was based on a split­plot experiment with two factors. the first factor was devoted to colored netting at four levels (no netting, green, yellow, and red) and the second factor was devoted to plant species at two levels of codiaeum and aglaonema based on a randomized complete block design with three replica­ tions. the results showed the positive effect of yellow netting on improving the vegetative capability of the plants, so that the highest plant height, shoot and root fresh and dry weight, and leaf area were observed in the plants grown under the yellow netting. also, the highest anthocyanin, carotenoid contents, and catalase activity were obtained from the red netting and the highest brix° and total chlorophyll from the red and yellow netting. according to the results, the highest vegetative growth rate was related to codiaeum. the application of the colored nets provided the plants with more optimal growth conditions. 1. introduction the average number of sunny days in iran annually amounts to a sig­ nificant value of 280 days, making it unavoidable to use shading to control incident radiation during sunlight exposure (forghani and kiani abri, 2005). the concept of photo­selective shade netting was first devised in israel and was tested on ornamental plants, vegetables, and fruit trees. then, the idea gradually spread to the whole world to be applied to vari­ ous plants, climatic regions, and agronomic practices (shahak, 2008). so far, black netting has mostly been used for shading and transparent net­ ting has been employed for protection against environmental or pest damages. a whole new group of protective nettings has been developed (*) corresponding author: sedaghathoor@yahoo.com citation: abbasnia zare s.k., sedaghathoor s., pada­ sht dahkaei m.n., hashemabadi d., 2020 ­ the effect of different colored netting on quantitative and qualitative traits of two foliage plant species (codiaeum variegatum and aglaonema commu‐ tatum) ­ adv. hort. sci., 34(1): 25­33. copyright: © 2020 abbasnia zare s.k., sedaghathoor s., padasht dahkaei m.n., hashemabadi d. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 27 june 2019 accepted for publication 31 october 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(1): 25­33 26 to manipulate both the quality and quantity of radia­ tion intercepted by plants and simultaneously pro­ tect them optimally (shahak et al., 2004 a, b). the technology of colored nets that selectively fil­ ters sunlight and physically protects crops is based on specific nets in which color elements are used during fabrication. this technology seeks to absorb ultravio­ let, blue, green, yellow, red, far­red, or near­infrared spectra. the direct light is intended to change into scattered light too. colored nets can only alter the components of sunlight that penetrates through their plastic strings whereas solar rays that go across the holes are not manipulated (shahak et al., 2016). it has been documented that yellow and red, as well as grey, netting can significantly increase the productivi­ ty of capsicum annuum versus black netting. these results were attributed to the increased number of fruits in a season in plants subjected to the netting (shahak et al., 2008). in another study by shahak et al. (2016), it was revealed that yellow netting outper­ formed red netting in stimulating the vegetative growth of pittosporum and among the studied nets, yellow netting exhibited much stronger strengthen­ ing effects than red netting. other studies have also shown that blue netting reduced vegetative growth and induced dwarfness in leafy ornamental plants and cut flowers whereas red and yellow nets that reduced the intensity of blue light induced vegetative growth in plants (shahak et al., 2016). photoselective nets can, according to studies, alter shade quality by scattering light and changing its spectral composition (shahak et al., 2008). wang and folta (2013) report­ ed that the colored nets had a significant effect on increasing vegetative growth rate in foliage plants as compared to the black nets with a similar coefficient of shading (oren­shamir et al., 2001). the results showed that the red and white nets increased the yield of cordyline with respect to plant height, leaf number, biomass, leaf area, photosynthesis rate, and harvest index as compared to control. also, plants under the colored nets exhibited a longer vase life than those in the open air, but there was no signifi­ cant difference between nets with different colors. according to the results, white and red nets were better for the growth of cordyline (kumar gaurav et al., 2016). since few studies have addressed the effect of colored netting on vegetative and physiolog­ i c a l t r a i t s o f f o l i a g e p l a n t s , t h e p r e s e n t p a p e r explores the impact of color netting on vegetative traits of ornamental codiaeum variegatum and a g l a o n e m a c o m m u t a t u m p l a n t s i n f l o w e r a n d ornamental plant research station of lahijan, iran. 2. materials and methods the effect of colored (red, green, and yellow) net­ ting was studied on vegetative traits of c. variegatum a n d a g l a o n e m a c o m m u t a t u m i n f l o w e r s a n d ornamental plants research station of lahijan, north of iran in the spring and of 2018. the trial was based on a split­plot experiment with two factors. the first factor was devoted to colored netting at four levels (no netting, green, yellow, and red) and the second factor was devoted to plant species at two levels of c. variegatum and a. commutatum based on a rcbd with three replications. each experimental plot was composed of four plants. the light intensity in cloudy days was in the range of 4000­5000 lux, no shade net of 6000 lux and on sunny days in the range of 20,000­ 28,000 lux and no shade net of 30,000­35000 lux. the recorded vegetative parameters included plant growth rate, plant height, shoot and root fresh a n d d r y w e i g h t a n d l e a f a r e a . d r y w e i g h t w a s obtained oven­dried at 105°c for 24 h. to determine leaf area, the leaves length (l) and widest width (w) were measured, and the leaf area (a) was calculated by the following equation (moll and kamparth, 1997): a= l x w x 0.75 the physiological traits included °brix, chlorophyll a, b and total, carotenoid, anthocyanin, flavonoid, antioxidant capacity, catalase and peroxidase. the brix° of the leaves was measured with an n­1α hand­ held refractometer (atago co., japan). to measure chlorophyll contents of the treatments, 0.5 g of sam­ ple was weighed and ground in a mortar containing 50 ml 80% acetone. then, the extract was infiltrated, adjusted to 50 ml, and poured into cuvettes. to determine chlorophyll content, it was read at 643 and 660 nm with a spectrophotometer. chlorophyll a and b and total chlorophyll contents were estimated b y t h e f o l l o w i n g e q u a ti o n s ( m a z u m d a r a n d majumder, 2003): total chlorophyll (mg/ml) = 7.12(a660) + 16.8 (a643) chlorophyll a (mg/ml) = 993(a660) ­ 0.777(a643) chlorophyll b (mg/ml) = 17.6(a643) ­ 2.81(a660) to measure the carotenoid level, the treatments were sampled. then, 0.5 g was weighed from the sample and was ground in a mortar containing 50 ml 80% acetone. then, it was infiltrated, adjusted to 50 ml , and poured into cuvettes. the extracts were read at 645, 663, and 660 nm and were placed in the fol­ l o w i n g e q u a ti o n , d e n o t e d b y a , t o d e t e r m i n e abbasnia zare et al. ‐ colored netting effects on foliage of codiaeum variegatum and aglaonema commutatum 27 carotenoid levels of the treatments (mazumdar and majumder, 2003): carotenoid level = 4.69(a660) ­ 0.268(a643) + 8.02(a663) to measure anthocyanin content, 0.5 of the sam­ ple was taken and ground in a chinese mortar con­ taining 50 ml of hydrochloric­ethanol acid (85% ethanol 95% + 15% hydrochloric acid). then, it was infiltrated, adjusted to 50 ml , and poured into cuvettes. they were placed in a refrigerator at 4°c for 24 hours followed by 2 hours in darkness. the extract was read at 535 nm with a spectrophotometer and was placed in the following equation to determine anthocyanin content (mazumdar and majumder, 2003): total absorption = e x b x c d x a where a = sample weight (0.5 g), b = the volume taken for the measurement (5 ml ), c = total volume (50 ml ), d = the fraction taken for the sample 0.1, and e = absorption read at 535 nm. total anthocyanin content of sample = total absorption of sample 98.2 to measure flavonoid content, 0.1 g of the sample was taken and ground in a mortar containing 85% methanol and was centrifuged at 8000 rpm for 5 minutes. then, it was infiltrated and the supernatant was placed in a hot bath at 80°c for 10 minutes. then, it was cooled down and its absor was read at 270, 300, and 330 nm with a spectrophotometer (humadi and istudor, 2008). a=εbc where: a= absorption rate, ε= extinction coefficient 33000 m.cm­1, b= width of the cuvette (1 cm) and c= total flavonoid content. in order to estimate antioxidant capacity, 1 g of the plant was wrapped in foil and was placed in liquid nitrogen for 2­3 minutes. then, it was ground with 10 ml methanol 85% and the samples were placed in room temperature for one hour. next, their extract was infiltrated and centrifuged for five minutes. then, 150 ml was taken from it and was added with 850 μl dpph. the solution was stirred fast and was kept in room temperature at the dark for 20 minutes. after placing the blank and resetting the instrument, first only dpph was poured into the cuvette and it was read. then, the sample was read at 517 nm by a spectrophotometer. the antioxidant capacity of the extracts was calculated by the following equation in terms of % inhibition in dpph (ramandeep and savage, 2005): %dpph = acont ­ asamp x 100 acont where %dpph = percent inhibition, acont = absorption rate of dpph, and asamp = absorption rate (sample + dpph). the activity of catalase (cat) was measured through the following stages (dazy et al., 2008): 1 g of plant tissue that had been ground in 4 ml ethanol was added with (i) 0.01 mol phosphate buffer (ph = 7), (ii) 0.5 ml h2o2 0.2 mol, and (iii) 2 ml acid reagent (dichromate/acetic acid mixture). then, its absorp­ tion was read at 610 nm with a spectrophotometer. to measure the enzymatic activity of peroxidase (pod), the extract was prepared as described above. then, the variations of od at 430 nm were read with a spectrophotometer once thirty seconds for two minutes (chance and maehly, 1955). data were sta­ tistically analyzed with mstatc software package, and the means were compared with the lsd test. 3. results according to the results of analysis of variance (anova; tables 1, 2), the simple effect of netting type and plant species and their interactions were significant (p<0.01) on plant height. the tallest plants were obtained from the yellow netting and the short­ est from no net exposure. also, means comparison for the interaction of ‘netting type × plant species’ revealed that the highest plant height was related to ‘yellow net × a. commutatum’ and the lowest to ‘no net × a. commutatum’. according to anova (table 1), plant growth rate was significantly (p<0.01) affected by shade netting, plant species, and ‘shade netting × plant species’. means comparison revealed that the highest growth rate was obtained from the yellow netting (table 3) and the lowest from no­netting treatment. means comparison for the interactive effect of ‘shade net­ ting × plant species’ on plant growth rate showed that ‘yellow netting × c. variegatum’ had the utmost plant growth rate and ‘no netting × a. commutatum’ had the lowest one. according to anova, leaf num­ ber was significantly influenced by netting type (p<0.05) and plant species (p<0.01), but the interac­ tion of these two parameters was insignificant for this trait (table 1). means comparison revealed that the highest number of leaves was observed in the plants treated with the yellow net and the lowest in adv. hort. sci., 2020 34(1): 25­33 28 the plants not treated with the nets (table 3). the effect of netting type and plant species was significant (p<0.01) on leaf size (leaf length and width), but the interaction of ‘net type × plant species’ was insignificant (table 1). the yellow net was related to the highest leaf length and width and the control to the lowest leaf size (table 3). as well, means comparison for the effect of plant species on leaf size indicated that c. variegatum produced the largest leaves (table 4). anova shows the significant (p<0.01) influence of netting type and plant species on leaf area, but the insignificant effect of ‘netting type × plant species’ on this trait (table 1). the high­ est leaf area of 138.4 cm2 was associated with the application of the yellow net, but control treatment (in which no net was applied) showed the lowest leaf area of 72.19 cm2 (table 3). means comparison for the effect of plant species on leaf area revealed that c. variegatum had the highest leaf area (table 4). anova revealed the significant (p<0.01) effect of shade netting type and plant species on shoot fresh and dry weight, but their interaction was significant table 1 ­ analysis of variance for the effect of experimental factors on the studied morphological traits ns= insignificant difference; **= significant difference at the p<0.01 level; *= significant difference at the p<0.05 level. source of variables df means of squares final height growth initial leaf no. final leaf no. leaf no. increase leaf length leaf width leaf area shoot fresh weight shoot dry weight root fresh weight root dry weight replication 2 4.76 ns 6.74 * 0.58 ns 21.15 ns 14.86 ns 0.97 ns 0.51 ns 264.38 ns 25.99 ns 0.13 ns 1.26 ns 0.006 ns netting (a) 3 202.24 ** 160.65 ** 4.20 ns 189.84 * 153.79 ** 41.01 ** 7.09 ** 4813.22 ** 3492.35 ** 17.12 ** 35.24 ** 0.173 ** error 6 5.54 0.97 5.57 37.95 15.81 0.76 0.58 226.36 85.45 0.42 1.32 0.006 plant (b) 1 1536.00 ** 997.82 ** 348.84 ** 2095.34 ** 734.27 ** 173.35 ** 19.26 ** 16949.00 ** 28295.47 ** 138.67 ** 0.39 ns 0.002 ns ab 3 42.36 ** 50.11 ** 3.75 ns 22.95 ns 11.98 ns 13.57 ns 1.89 ns 1448.57 ns 529.42 * 2.59 * 6.80 * 0.032 * error 8 1.43 2.28 4.95 25.25 10.24 5.60 0.69 517.91 82.94 0.41 1.01 0.005 c.v. (%) 3.62 14.13 26.23 25.40 28.32 12.93 11.71 22.42 18.07 18.06 21.51 21.79 table 2 ­ analysis of variance for the effect of experimental factors on the studied morpho­chemical traits ns= insignificant difference; **= significant difference at the p<0.01 level; *= significant difference at the p<0.05 level. source of variables df means of squares brix catalase peroxidase anthocyanin chlorophyll a chlorophyll b total chlorophyll carotenoid flavanoid antioxidant capacity replication 2 0.29 ns 0.001 ns 0.015 ns 1170.53 ns 1.81 ns 0.31 ns 1.62 ns 0.45 ns 0.001 ns 0.003 ns netting (a) 3 15.49 * 0.005 ** 0.014 ns 16243.31 ** 2.63 ns 0.95 ns 6.56 * 5.68 ** 0.000 ns 0.008 ** error 6 1.74 0.000 0.005 721.97 1.10 0.39 1.04 0.41 0.000 0.001 plant (b) 1 532.04 ** 0.000 ns 0.032 ns 6699.06 ** 3.06 ns 0.06 ns 1.66 ns 0.36 ns 0.000 ns 0.003 * ab 3 0.49 ns 0.000 ns 0.037 ** 48512.77 ** 2.80 ns 0.28 ns 2.13 ns 1.09 ns 0.001 ** 0.007 ** error 8 0.37 0.001 0.003 5179.48 1.19 0.27 1.92 0.47 0.000 0.000 c.v. (%) 6.97 48.63 13.11 28.83 75.90 56.85 57.60 31.23 10.53 36.02 table 3 ­ means comparison for the effect of netting type on the studied traits similar letter(s) in each column show insignificant differences according to the lsd test. 1 peroxidase enzyme unit in μm /h2o2 consumed/min/mg. netting final height (cm) growth (cm) leaf no. shoot fresh weight (g) shoot dry weight (g) root fresh weight (g) root dry weight (g) leaf length (cm) leaf width (cm2) leaf area sugar content (%) catalase (unit)1 anthocyanin (mg/100 g) total chlorophyll (mg/g) carotenoid (mg/l) antioxidant capacity (dpph%) no netting 25.25 c 3.92 c 13.50 b 23.52 c 1.65 c 2.11 c 0.14 c 14.92 c 6.04 b 72.19 c 6.66 b 0.04 b 1.37 b 0.85 b 0.77 b 9 a yellow 39.33 a 16.50 a 27.17 a 82.05 a 5.75 a 7.89 a 0.55 a 21.17 a 8.50 a 138.4 a 9.68 a 0.04 b 2.62 b 2.90 a 2.60 a 2 b red 33.92 b 11.50 b 19.63 ab 50.25 b 3.52 b 4.82 b 0.34 b 19.17 b 7.33 ab 106.5 b 10.33 a 0.10 a 4.77 a 3.09 a 2.94 a 5 ab green 33.33 b 10.88 b 18.83 ab 45.74 b 3.20 b 3.84 bc 0.27 bc 18.00 b 6.46 b 88.92 bc 8.50 ab 0.03 b 1.20 b 2.77 ab 2.50 a 9 a abbasnia zare et al. ‐ colored netting effects on foliage of codiaeum variegatum and aglaonema commutatum 29 icantly influenced by net type at the p<0.05 level and by plant species at the p<0.01 level, but the interac­ tion of ‘net type × plant species’ was insignificant for this trait (table 2). the highest degree brix was relat­ ed to the red net (10.33%) and yellow net (9.68%) and the lowest to control (6.66%) (table 3). means comparison for the effect of plant species indicated that c. variegatum had higher brix of 13.50% (table 4). the effect of net type was significant (p<0.01) on catalase enzyme, but this enzyme content was not influenced by plant species and ‘net type × plant species’ (table 2). the highest catalase enzyme con­ tent was obtained from the red netting showing insignificant differences from other treatments (table 4). anova showed that the interactive effect of ‘net type × plant species’ was significant (p<0.01) on peroxidase enzyme, but the effect of shading net and plant species was not significant (table 1). the highest peroxidase enzyme content was obtained from ‘no net × a. commutatum’ and the lowest from ‘red net × c. variegatum’, ‘green net × a. commuta‐ tum’, ‘green net × c. variegatum’, ‘yellow net × a. commutatum’, and ‘no net × c. variegatum’ (table 5). anthocyanin content was significantly (p<0.01) influenced by net type, plant species, and ‘net type × plant species’ (table 2). means comparison indicated for this trait (p<0.05) (table 1). the highest shoot fresh and dry weight was obtained from the yellow netting (table 3) as means comparison for the effect of shade net type indicated. likewise, means com­ parison for the interaction of netting type and plant species showed that ‘yellow netting × c. variegatum’ produced the highest shoot fresh and dry weight without any significant differences from that of ‘red netting × c. variegatum’ and ‘green netting × c. varie­ gatum’. the lowest shoot fresh and dry weight was obtained from ‘no netting × a. commutatum’ and ‘green netting × a. commutatum’ (table 5). the effect of net type was significant on root fresh and dry weight at the p<0.01 level and the interaction of ‘net type × plant species’ was significant for these traits at the p<0.05 level (table 1). means comparison indicat­ ed that the highest root fresh and dry weights were obtained from the yellow netting and the lowest were obtained when the plants were barely exposed to radiation (table 3). according to means compari­ son for the interactive effect of the treatments, ‘yel­ low net × a. commutatum’ produced the highest root fresh and dry weight whilst the lowest root fresh and dry weights were obtained from ‘no net × c. variega‐ tum’, ‘no net × a. commutatum’, and ‘green net × a. commutatum’ (table 5). anova indicated that degree brix (°bx) was signif­ table 4 ­ means comparison for the effect of plant species on the studied traits similar letter(s) in each column show insignificant differences according to the lsd test. plant species final height (cm) growth (cm) leaf no. shoot fresh weight (g) shoot dry weight (g) leaf length (cm) leaf width (cm) leaf area (cm2) sugar content (%) antho­ cyanin (mg/100 g) antioxidant capacity (dpph%) codaeum variegatum 40.96 a 17.15 a 29.13 a 84.72 a 5.93 a 21 a 8.98 a 128.08 a 13.50 a 3.02 a 5 b aglaonema commutatum 24.96 b 4.25 b 10.44 b 16.05 b 1.12 b 15.63 b 6.19 b 74.93 b 4.08 b 1.97 b 7 a table 5 ­ means comparison for the interactive effect of ‘netting type × plant species’ on the studied traits similar letter(s) in each column show insignificant differences according to the lsd test. 1 peroxidase enzyme unit in μm /h2o2 consumed/min/mg. plant species final height (cm) growth (cm) shoot fresh weight (g) shoot dry weight (g) root fresh weight (g) root dry weight (g) peroxidase (unit)1 ‘no netting × codaeum variegatum’ 29.33 c 6.33 cd 44.32 bc 3.10 b 2.09 c 0.15 c 0.34 b ‘no netting × aglaonema commutatum’ 21.17 d 1.50 d 2.72 d 0.19 c 2.14 c 0.15 c 0.61 a ‘yellow netting × c. variegatum’ 49.33 a 25.33 a 118.2 a 8.27 a 6.68 ab 0.47 ab 0.43 ab ‘yellow netting × a. commutatum’ 29.33 c 7.67 c 45.93 b 3.22 b 9.10 a 0.64 a 0.40 b ‘red netting × c. variegatum’ 42.83 b 17.83 b 88.31 a 6.18 a 5.20 bc 0.36 bc 0.33 b ‘red netting × a. commutatum’ 25.00 d 5.17 cd 12.19 cd 0.85 c 4.43 bc 0.31 bc 0.45 ab ‘green netting × c. variegatum’ 42.33 b 19.08 b 88.10 a 6.17 a 5.21 bc 0.36 bc 0.39 b ‘green netting × a. commutatum’ 24.33 d 2.67 cd 3.38 d 0.24 c 2.49 c 0.18 c 0.33 b 30 adv. hort. sci., 2020 34(1): 25­33 that the highest anthocyanin content was obtained from the red net (table 3), but the other treatments did not differ significantly to one another. according to means comparison for the effect of plant species on anthocyanin content, the highest content was observed in c. variegatum (table 4). also, means comparison for the interactive effect of ‘net type × plant species’ on anthocyanin content indicated that ‘red net × a. commutatum’ had the highest antho­ cyanin content and ‘green net × a. commutatum’ and ‘no net × a. commutatum’ exhibited the lowest one (fig. 1). the results of anova showed that the effect of net type was significant (p<0.05) on total chlorophyll content, but the effect of plant species, net type, and ‘net type × plant species’ was insignificant on chloro­ phyll a and b and so was the effect of plant species and ‘net type × plant species’ on total chlorophyll (table 2). means comparison for the effect of net type on total chlorophyll (table 3) indicated that the highest total chlorophyll was related to the red and yellow nets and the lowest to no­net treatment. shading net type influenced carotenoid content of plants significantly (p<0.01), but the trait was not sig­ nificantly affected by plant species and ‘net type × plant species’ (table 1). means comparison showed that the plants grown under the red nets had a high­ er carotenoid content but without any significant dif­ ferences from those grown under the yellow and green nets. the lowest carotenoid content was relat­ ed to control (table 3). the results of anova showed that the interaction of ‘net type × plant species’ was significant (p<0.01) for flavonoid content, but the effect of net type and plant species was insignificant on this trait (table 2). the highest flavonoid content was obtained from ‘green net × c. variegatum’ and the lowest from ‘red net × c. variegatum’ (fig. 2). antioxidant capacity was significantly influenced by netting type and ‘netting type × plant species’ at the p<0.01 level and by plant species at the p<0.05 level (table 2). a. commutatum plants grown under the yellow net and control plants had the highest antioxidant capacity, while the lowest capacity was related to the yellow net (table 3). also, it was found that a. commutatum had a higher antioxidant capaci­ ty than c. variegatum (table 4). means comparison for ‘net type × plant species’ revealed that the high­ est antioxidant capacity was related to ‘no net × a. commutatum’ and the lowest to ‘yellow net × c. var‐ iegatum’ and ‘yellow net × a. commutatum’ (fig. 3). 4. discussion and conclusions based on our results the highest plant height was related to ‘yellow net × a. commutatum’. in a study on the effect of colored nets on cordyline, kumar fig. 1 ­ the effect of ‘netting type × plant species’ on anthocya­ nin content. nn= no netting, yn= yellow netting, rn= red netting, gn= green netting, cv= codaeum variegatum, ac= aglaonema commutatum. fig. 2 ­ the effect of ‘netting type × plant species’ on flavonoid content. nn= no netting, yn= yellow netting, rn= red netting, gn= green netting, cv= codaeum variegatum, ac= aglaonema commutatum. fig. 3 ­ the effect of ‘netting type × plant species’ on antioxidant capacity. nn= no netting, yn= yellow netting, rn= red netting, gn= green netting, cv= codaeum variegatum, ac= aglaonema commutatum. abbasnia zare et al. ‐ colored netting effects on foliage of codiaeum variegatum and aglaonema commutatum 31 gaurav et al. (2016) stated that plants grown under colored shade nets exhibited variable growth due to the spectral effect on plant growth. they reported that the cordyline plants grown under white and red shade nets were taller than the control plants (not exposure to shade nets). it has been reported that both red and yellow shade nets enhanced the vege­ tative traits of aralia including stem length and thick­ ness, petals, and leaf dimensions and generally increased the yield of commercial cut flowers (oren­ shamir et al., 2001; shahak, 2008). shahak et al. (2016), also, found that the yellow shading net induced vegetative growth of pittosporum. the high­ est growth rate was observed in the plants exposed to the yellow nets. it has been argued that yellow nets outperform red nets in inducing vegetative growth, probably because of the inductive effect of supplementary artificial green light (oren­shamir et al., 2003; kim et al., 2004). in a four­year research study on cut flowers in besor research station (2000­ 2003), an increase was reported in stimulatory capac­ ity and in growth under yellow and red nets (shahak, 2008; ovadia et al., 2009). there is a report that let­ tuce produced the highest number of leaves under colored nets (pearl and red nets) and the lowest number in control (no net application) (ilić et al., 2017). it can be concluded that different plant species differ in their growth and development responses to the spectra generated by different col­ ored nets. leaf area is a crucial parameter for growth. it is defined as a plant’s capacity to synthe­ size dry matter in terms of radiation use rate and photosynthesis rate. kumar gaurav et al. (2016) showed that leaf area of cordyline was higher under colored shade netting than control (no shade net application). colored nets influenced both the length of growth period and morphological traits of lettuce so that they enhanced leaf area index and shortened the length of growth period significantly (ilić et al., 2017). in the present study, the yellow net resulted in higher fresh weight than the other nets and control. in addition, the highest plant growth was related to the yellow net, implying the impact of yellow nets on quantitative and qualitative traits of plants. this leads us to the conclusion that colored shade nets, espe­ cially yellow nets, can enhance plant biomass. in kumar gaurav et al.’s study (2016), the highest leaf fresh weight (85.88 percent higher in the red net t h a n i n t h e c o n t r o l ) a n d l e a f d r y w e i g h t w e r e obtained from the red net and overall, the colored nets had the strongest impact on this trait when compared to control (no net application). brix is a measure of sugar content of the solution and depends on radiation diffraction. it represents the percentage of solid material weight of a solution to the total weight of the solution. our results revealed that flowers grown under the red and yel­ low shade nets had higher degree brix and this may be related to the stimulatory effect of artificial green l i g h t u n d e r t h e y e l l o w n e t s ( k i m e t a l . , 2 0 0 4 ) . hydrogen peroxide is the most stable form of reac­ tive oxygen species. it has been suggested that hydrogen peroxide is toxic to cells. catalase and per­ oxidase play a crucial role in inhibiting the accumula­ tion of hydrogen peroxide. these enzymes are abun­ dant in aerobic microbes, but anaerobic microbes lack them (singh, 2003). it has been documented that peroxidases are involved in many cell processes such as auxin metabolism, wood formation, traverse link­ ages in plant cell walls, response to environmental stresses, and so on (yamasaki et al., 1997). thus, the higher catalase and peroxidase enzyme content in plant species plays a considerable role in inhibiting the accumulation of hydrogen peroxide in plants. a laboratory trial has shown that three properties of light color, intensity and duration affect plant growth so that red/infra­red ratio is dictated by the duration and photo flux of radiation treatments and influences anthocyanin development and synthesis significantly (mancinelli, 1990). lefsrud et al. (2008) reported that anthocyanin content of lettuce was increased in plants exposed to red led light. in the present study, plants grown under the red shade nets exhibited the highest anthocyanin content. at intense radiation, chlorophyll degradation rate in plant leaf exceeds its synthesis rate, resulting in the loss of chlorophyll content due to the inhibition of chloroplast formation (gonçalves et al., 2001; fu et al., 2012). it has been documented that colored nets increase chlorophyll content in plants. for exam­ ple, alkalai­tuvia et al. (2014) studied the effect of colored nets on peppers and reported that the chlorophyll content of the peppers grown under the pearl nets was significant higher than that of the pep­ pers grown under the black nets. the plants grown under the red nets had a higher carotenoid. it has been reported by tinyane et al. (2013) and selahle et al. (2014) that carotenoid con­ tent of tomatoes was increased under red and pearl shade nets. kong et al. (2012) reported that the yel­ low net resulted in morphological changes and leaf carotenoid increase in peppers versus the red net, which may relate to the increase in green light con­ adv. hort. sci., 2020 34(1): 25­33 32 tent under the yellow net. leaf carotenoid content was higher in lettuces grown under colored shade nets than control (no net) in ilić et al.’s study (2017), which is consistent with our findings. phytochemical biosynthesis mostly depends on light quantity and quality as was observed in lettuce plants grown under black nets. in a study, plants grown under pearl nets had significantly higher total phenol content, flavonoids, and antioxidant proper­ ties than those grown under other nets (ilić et al., 2017). in another study, an increase was observed in post­harvest flavonoid content in oregano, marjo­ ram, and coriander under pearl nets (buthelezi et al., 2016). although phytochemical content decreases slightly after harvest, a high level of post­harvest phy­ tochemical accumulation enables plants to maintain p h y t o c h e m i c a l q u a l i t y i n p o s t ­ h a r v e s t p e r i o d (buthelezi et al., 2016). the accumulation of antioxi­ dant compounds in green plants depends on many parameters such as temperature, light quantity and quality, cultivar, growing season, and metabolic fac­ tors (miller et al., 2010). the control of radiation quality by the red and pearl photo­selective netting resulted in keeping post­flowering antioxidant activi­ ty in vegetables (kong et al., 2013). kong et al. (2013) reported that peppers exhibited an elevated level of antioxidants under yellow nets implying that yellow and pearl nets were likely to enhance plant resis­ tance to biotic stresses. it seems that plants respond d i ff e r e n t l y t o d i ff e r e n t l i g h t s p e c t r a a n d p l a n t response to the elevated level of antioxidant capacity depends on the type of colored nets. the results show that among the studied colored shade nets, the yellow net outperformed the other nets in improving the vegetative capacity of the stud­ ied plants. also, the highest anthocyanin, carotenoid, and catalase contents were obtained from the red net and the highest degree brix and total chlorophyll from the red and yellow nets. overall, the application of the colored nets was more desirable for the plants than their non­application. it has been documented that the absorption rate of yellow­colored glass is higher in 360­200 nm (blue and violet) range that is lowly important radiation for photosynthesis than in 560­760 nm range that is photosynthetically impor­ tant (haghshenas and ghiabaklou, 2009) because the latter range is severely intercepted by chlorophyll and increases photosynthesis. this can be a reason for the higher efficiency of the yellow netting. the absorption rate of violet to yellow range of visible light (360­600 nm) by red glass is very similar to yel­ low glass (haghshenas and ghiabaklou, 2009). this explains the change in plants’ behavior to the radia­ tion passing through the red net because it seems that the penetration of photosynthetically active radiation through red nets (i.e. red and orange por­ tions of visible light) contributes to important plant functions such as photosynthesis and increases bio­ mass. references alkalai­tuvia s., goren a., perzelan y., weinberg t., fallik e., 2014 ­ the influence of colored shade nets on pepper quality after harvest ‐ a possible mode‐of‐ action. ­ agric. forest., 60: 7­18. buthelezi m.n.d., soundy p., jifon j., sivakumar d., 2016 ­ spectral quality of photo‐selective nets improves phytochemicals and aroma volatiles in coriander leaves (coriandrum sativum l.) after postharvest storage. ­ j. photochem. photobiol. b, biol., 161: 328­334. chance b., maehly a.c., 1955 ­ assay of catalase and peroxidase, pp. 764­775. ­ in: horecker b.l. 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pot marigold (calendula officinalis l.) under salinity conditions n. adamipour 1 (*), m. khosh-khui 1, h. salehi 1, h. rho 2 1 department of horticulture science, school of agriculture, university of shiraz, iran. 2 plant stress laboratory texas, a&m agrilife research at amarillo, 6500 amarillo blvd., west amarillo, tx 79106‐1796, usa. key words: antioxidative enzymes, chlorophyll content, fertilizer, nutrient elements. abstract: a greenhouse study was conducted in order to evaluate the interactions of vermicompost and salinity effects on morphology and physiology of pot marigold. the experiment was conducted with vermicompost treatments at five levels (0%, 5%, 10%, 15% and 20%) and salinity treatments at five levels (0, 50, 100, 150 and 200 mm nacl) in a completely randomized factorial design arrangement with four replications. results showed that increasing levels of salinity led to decline in leaf area, fresh and dry weights of flower, shoot, and root, n, p, k, fe, mg and zn concentrations, chlorophyll and carotenoid contents, while proline content increased in the plants. apx, sod, pod and cta enzyme activities significantly increased with increasing salinity from 0 to 150 mm nacl, then declined in 200 mm treatment in the plants. application of vermicompost increased the morpho-physiological indices and mineral nutrient uptake in the plants and could increase the plant yield by alleviating the harmful effects of salinity. 1. introduction calendula officinalis, known as “pot marigold”, is a plant in calendula genus of asteraceae family. it is perhaps native and widely naturalized further northern to southern europe and elsewhere in warm temperate regions of the world, and it may possibly be planted widely in gardens and landscapes (gharineh et al., 2013). among ornamental bedding plants, pot marigold is known to grow well under saline conditions. in fact, some pot marigold cultivars that are used as cut flowers or as bedding plants in landscaping can be grown by maintaining the quality of plants under saline conditions with an ecw of <8 ds m -1 (koksal et al., 2016). plants are exposed to ever-changing and often unfavorable environmental conditions, which cause both biotic and abiotic stresses such as extreme temperatures, flood, drought, and salinity. overexploitation of (*) corresponding author: nader.adamipour@shirazu.ac.ir citation: adamipour n., khosh-khui m., salehi h., rho h., 2019 effect of vermicompost on morphologi‐ cal and physiological performances of pot mari‐ gold (calendula officinalis l.) under salinity condi‐ tions. adv. hort. sci., 33(3): 345-358 copyright: © 2019 adamipour n., khosh-khui m., salehi h., rho h. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 18 july 2018 accepted for publication 23 april 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(3): 345-358 346 available water resources as well as environmental factors such as low precipitations, high temperatures and contamination from parental rocks are leading to increases in soil salinization (aroca et al., 2013). soil salinization is one of the most important agricultural and eco-environmental problems nowadays, which is increasing steadily in many parts of the world. saline soils have been estimated to occupy more than 7% of the earth’s land surface and it is expected to be increased by up to 50% by the middle of the twentyfirst century (ruiz-lozano et al., 2012). salinity stress is one of the major abiotic threats to plant life and agriculture worldwide and significantly reduces crop yield in the affected areas. excessive salt above what plants need limits plant growth and productivity and can lead to plant death. about 20% of all irrigated land is affected by soil salinity, decreasing crop yields. plants are affected by salinity stress in two main ways: osmotic stress and ionic toxicity. these stresses affect all major plant processes, including photosynthesis, cellular metabolism, and plant nutrition (aslamsup et al., 2011). amelioration of salt-affected soils can be accomplished through many effective methods, such as water leaching, chemical remediation, and phytoremediation (qadir et al., 2007). the amelioration of salt-affected soils using chemical agents, including gypsum, calcite, calcium chloride and organic matter, is a successful approach that has been implemented worldwide (sharma and minhas, 2005; tejada et al., 2006). according to a study, the application of organic matter conditioners has become a common practice in salt-affected areas in the last several decades and constitutes an important method of soil regeneration and fertility enhancement (melero et al., 2007). organic matter is very important for maintaining structural stability in soils as well as improving the physical, chemical and biological properties of soils. salt-affected soils generally exhibit poor structural stability due to low organic matter. the addition of organic materials (e.g. green, farmyard and poultry manures, compost, food processing wastes, etc.) has been suggested for improving structural stability of soils by many researchers (tejada et al., 2006). barzegar et al. (1997) found that addition of plant residues improved the water-stable aggregate in soils because of increased organic matter content and decreased soil salinity. the application of organic matter for soil remediation is considered essential for sustainable land use and crop productivity. given the importance of pot marigold in green space, and their being placed in saline soils in most cultivable regions, so far, enough research has not been conducted to understand morpho-physiological properties of pot marigold under salinity stress conditions and determine the status of vermicompost in reducing the devastating effects of salinity stress in pot marigold. for this purpose, an experiment was conducted to determine the effects of vermicompost on some morphological and physiological characteristics of pot marigold under salinity stress conditions. 2. materials and methods to investigate the effects of vermicompost on some physiological characteristics of pot marigold (calendula officinalis l. cv. candyman orange) under salinity stress, a greenhouse experiment was conducted in a completely randomized factorial design including vermicompost at five levels (0%, 5%, 10%, 15% and 20%) and salinity stress at five levels (including 0, 50, 100, 150 and 200 mm nacl) with four replicates. this study was conducted at the research greenhouse of the department of horticultural science, college of agriculture, shiraz university, shiraz, iran (52°32´e and 29°36´n). after disinfection of pots (20 and 30 cm in diameter and length, respectively), 5%, 10%, 15% and 20% (v/v) vermicompost from kian pars shiraz company, were dried in the shade and were mixed with four kilograms of a native soil. the main physical and chemical characteristics of the vermicompost and soil mixture are shown in table 1. then, four marigold seeds were planted in each pot. the pots containing seeds were kept in a greenhouse with 27/18°c (day/night) temperature, 16 h light conditions, and 35% relative humidity. after germination of seeds one plant was selected in each pot and the three other plants were removed. when the plants reached the four-leaf stage, the plants were treated with four levels of salinity stress. table 1 some physico-chemical properties of vermicompost and soil sample vermicompost soil organic matter % 33 0.41 ph 8.1 7.05 ec (d/sm) 1.7 0.6 p (%) 1.8 pwp (%) 5.1 k (%) 1.2 fc (%) 14 c/n 13 total n (%) 1.5 soil texture sandy-loam adamipour et al. ‐ effect of vermicompost on performances of pot marigold 347 salinity stress treatment was applied by adding net quantities of sodium chloride (nacl) to the irrigation water so that pots were irrigated with 50, 100, 150, and 200 mm nacl containing water based on field capacity of the soil, and the amount of the decreased water obtaining by the salinity treatment resulted in the average electrical conductivity (ec) of 3.62, 6.27, 9.36 and 12.71 ds/m in each level of the treatment, respectively. the control treatment was applied using distilled water. after 35 days of the treatments, the plants were harvested in order to measure morphological and biochemical traits. growth parameters growth parameters including, flower diameter (mm), leaf area (cm2) and fresh and dry weights of flower, shoot, and root (g) were measured. for dry weight determination, the shoots, roots, and flowers were dried in an oven at 70°c for 48 h and weighed. proline content the leaf proline content was determined using the method of bates et al. (1973). proline was extracted from leaf samples of 100 mg weight fresh with 2 ml of 40% methanol. 1 ml of the extract was mixed with 1 ml of a mixture of glacial acetic acid and orthophosphoric acid (6 m) (3:2, v/v) and 25 mg ninhydrin. after 1 h of incubation at 100°c, the tubes were cooled, and 5 ml toluene was added. the absorbance of the upper phase was spectrophotometrically determined at 528 nm. the proline concentration was determined using a standard curve. chlorophyll and carotenoid contents chlorophyll and carotenoid contents were estimated by the method of hiscox and israelstam (1979). fresh leaf and petal material (1 mg) and 10 ml dmso were taken in vials and kept in an oven at 65°c for 4 h. absorbance was read at wavelengths of 665 and 649 for leaves, and 480 nm for petals using a spectrophotometer (beckman du 640 b, fullerton, usa). the following equations were used to calculate each compound. chl a= (12.47×a665)-(3.62×a649) chl b= (25.06×a649)-(6.5×a665) total chlorophyll (mg g-1 f.w.) = chla + chlb carotenoids (mg g-1 f.w.) = where a stands for the absorbance reading of a sample at 665, 649 and 480 nm of wavelength. antioxidant analysis fresh leaf samples were homogenized in extraction buffer (0.1 m phosphate buffer ph 6.8) with a mortar and pestle on ice. the homogenate was then centrifuged at 12,000 g for 15 min at 4°c and the supernatant was used as the crude extract for the superoxide dismutase, guaiacol peroxidase, ascorbate peroxidase and catalase. the superoxide dismutase (sod), guaiacol peroxidase (pod), ascorbate peroxidase (apx) and catalase (cat) enzymes were estimated using the methods previously described by b e a u c h a m p a n d f r i d o v i c h ( 1 9 7 1 ) , c h a n c e a n d maehly (1955), nakano and asada (1981), and dhindsa et al. (1981), respectively. plant nutrient element analysis leaf material was ground to pass 0.5 mm sieve in a cyclone laboratory mill, weighed into ceramic crucibles, ashed overnight at 550°c in a muffle furnace, and the ash was suspended in 2m hcl for determination of mineral nutrients. then, total nitrogen (n), phosphorus (p) and potassium (k) were determined in the leaf using the kjeldahal, colorimetrically and ammonium acetate methods respectively., zinc (zn), magnesium (mg) and iron (fe) were determined by atomic absorption spectroscopy (baumard et al., 1998). statistical analyses the data were analyzed using one-way analysis of variance at p < 0.05 significance with sas version 9.4 software (sas institute inc., cary, nc). fisher’s lsd test was conducted to determine the statistical differences among different treatments. 3. results and discussion leaf area and fresh and dry weight of shoots the growth (leaf area and fresh and dry weight of shoots) was significantly affected by the treatments (table 2). the treatment of plants with nacl significantly reduced the growth parameters and the decrease was proportional to the concentration of nacl. the highest concentration (200 mm nacl) was the most deleterious and decreased the leaf area by 53.32%, fresh weight by 72.76%, and dry weight by 48.74% as compared to those of the control plants. however, all the above parameters were significantly enhanced by the vermicompost treatments. the highest and the lowest leaf area and fresh and dry weight of shoots were obtained in the 20% vermicompost and the control treatment, respectively (table 2). leaf area and fresh and dry weight of shoots increased (12.03%, 39.52% and 38.96%, respectively) at the 20% vermicompost treatment compared to the control plants. researches showed 1000 480 1 29 53 78 220      a chla chlb. . adv. hort. sci., 2019 33(3): 345-358 348 that soil salinity reduces the growth of plant shoots. this water potential reduction in the soil or osmotic effect is due to the presence of salt in the soil which limits the root water absorption (nguyen et al., 2015). in the salt stress, the plant’s hormonal system which synthesizes and transmits a number of hormones such as cytokinins is impaired and their transport from roots to the upper parts of the plant is limited. the termination of the hormone transfer from roots to branches and the consequent reduced water absorption capacity can lead to a decline in plant growth. reduction of leaf area can be explained by decreased cell growth or a reduced cell division rate due to decreased cellular turgor. decrease in leaf area also reduces the rate of photosynthesis, resulting in decreased fresh and dry weight of plants (zarei et al., 2016). in addition, alshammary et al. (2004) showed that decrease in growth was due to reduction of flexibility, development of cells, and reduction of auxins. vermicompost, due to its high microbial activity resulting from the presence of fungi, bacteria, yeasts, actinomycetes, and algae, can produce different growth regulators such as auxins, gibberellins, and cytokinins all of which may have positive effects on plant growth and development (xu et al., 2016; ullah et al., 2018). thus, the cause of rises in height and leaf area of the plants treated with vermicompost is probably from the stimulation of production of growth regulators including auxins and gibberellins. furthermore, vermicompost contains humic substances that increase the availability of plant n, p, k, and in particular zn for the synthesis of tryptophan, a precursor to auxins that are used for rooting and plant growth (sharifianpour et al., 2015; scaglia et al., 2016). fresh and dry weight of roots the fresh and dry weight of roots of the pot marigold plants was lower with the salinity stress treatments as compared to that of the plants under non-saline conditions (table 3). fresh and dry weight of roots decreased (51.91% and 77.98%, respectively) table 2 effect of salinity and vermicompost and their interaction on leaf area and shoot fresh and dry weight * in each variable, data followed by the same letters (small letters for interactions and capital letters for means) are not significantly different using lsd at 5% level. data represent the mean value ± s.d. the mean of four replicates. vermicompost (%) salinity (mm) mean vermicompost 0 50 100 150 200 leaf area 0 36.10±1.33 gh 36.10±0.62 gh 35.12±0.62 i 27.15±0.62 m 15.91±0.62 q 30.05±8.10 e 5 37.05±0.62 f 36.71±0.62 fg 35.01±0.62 gh 27.85±0.62 m 16.61±0.62 q 30.03±8.08 d 10 38.39±0.62 de 38.05±0.62 e 37.16±0.62 f 29.19±0.62 l 17.95±0.62 p 32.14±8.08 c 15 39.28±0.62 bc 38.94±0.62 cd 38.05±0.62 e 30.08±0.62 k 18.84±0.62 o 33.03±8.08 b 20 40.40±1.18 a 40.06±1.18 ab 39.17±1.18 cd 31.20±1.18 j 19.96±1.18 n 34.16±8.08 a mean salinity 38.24±1.73 a 37.95±1.65 a 37.06±1.65 b 29.09±1.65 c 17.85±1.65 d shoot fresh weight (g) 0 59.92±1.18 h 57.79±1.18 i 54.37±1.18 k 27.19±1.18 n 9.79±1.18 p 44.05±1.92 e 5 60.62±0.62 h 58.49±0.62 i 55.07±0.62 kj 39.11±0.62 m 10.49±0.62 p 44.75±1.92 d 10 70.35±0.82 e 68.22±0.82 f 64.22±0.82 f 48.84±0.82 l 20.22±0.82 o 54.49±1.92 c 15 76.27±0.91 ab 74.14±0.91 c 74.14±0.91 c 54.76±0.91 kj 26.14±0.91 n 60.41±1.92 b 20 77.32±0.75 a 75.19±0.75 bc 75.19±0.75 bc 55.8±0.751 j 16.22±0.75 n 61.46±1.92 a mean salinity 68.89±7.67 a 66.76±7.67 b 63.34±7.67 c 47.38±7.67 d 18.76±7.67 e shoot dry weight (g) 0 13.18±1.18 gh 12.84±1.18 hi 11.38±1.1 8 jk 8.92±1.18 mn 5.8±1.18 1p 10.42±3.01 e 5 13.88±0.62 fg 13.54±0.62 fgh 12.08±0.62 ij 9.62±0.62 lm 6.5±0.621 p 11.12±2.87 d 10 15.22±0.62 cd 14.88±0.62 de 13.42±0.62 gh 10.96±0.62 k 7.85±0.62 o 12.46±2.87 c 15 16.11±0.62 b 15.77±0.62 bc 14.31±0.62 ef 11.85±0.62 j 8.74±0.62 n 13.35±2.87 b 20 17.23±0.62 a 16.89±0.62 a 15.43±0.62 bcd 12.97±0.62 h 9.62±0.62 lm 14.48±2.87 a mean salinity 15.12±1.65 a 14.78±1.65 a 13.32±1.65 b 10.86±1.65 c 7.75±1.6 5 d adamipour et al. ‐ effect of vermicompost on performances of pot marigold 349 vated the amount of biomass; the total fresh and dry weightwere 31.37% and 79.04% higher than those of the control plants, respectively. the vermicompost treatments also improved the amount of biomass in the plants that were subjected to the salinity stress treatments (table 3). edwards and burrows (1988) reported that increase in fresh and dry weight of roots depends on increase in the activity of hormonal substances such as auxins, cytokinins, and gibberellins as well as vitamin b12. flower diameter and fresh and dry weight of flower flower diameter and fresh and dry weight of flower of the pot marigold plants were found to significantly decrease as the salt concentration was raised. these parameters decreased (33.58%, 67.94% and 27.48%, respectively) at the salinity level with 200 mm nacl compared to the control treatment (table 3 and 4). several reports have shown that salinity decreases the flower diameter and fresh and dry weight of flower in (zinnia elegans) (carter and grieve, 2010), madagascar periwinkle (cathasanthus r o s e u s ) ( j a l e e l e t a l . , 2 0 0 8 ) a n d g a r d e n m u m (chrysanthemum× morifolium) (lee and van iersel, 2008). the vermicompost applications significantly at salinity level of 200 mm nacl compared to the control treatment. ionic toxicity, nutritional imbalance, and osmotic osmolality adjustment are the negative effects of salinity stress on plant metabolism. roots are the organ responsible for absorbing water and minerals, and salinity stress affects plant shoots more than the root system; however, the root system is the first organ that is exposed to salinity stress (demiral and türkan, 2005). one of the most effective indices in salinity tolerance is the maintenance of cellular turgor and osmotic regulation due to salt absorption and organic matter production. plants, for production of organic materials such as glycine betaine, sorbitol, mannitol, and proline, spend a large amount of energy to regulate osmotic resistance in response to salinity stress. as a consequence, plants under salinity stress conditions reduce root efficacy in supplying nutrients and water to other organs. this reduces growth of shoots and dry matter production, and eventually reduces the transfer of nutrients from roots to shoots, and thus leads to a reduction in dry weight of roots and stems of plants (claussen, 2005; butt et al., 2016; sattar et al., 2016). the vermicompost treatments of the plants grown under stress-free conditions significantly eletable 3 effect of salinity and vermicompost and their interaction on root fresh and dry weight and flower diameter * in each variable, data followed by the same letters (small letters for interactions and capital letters for means) are not significantly different using lsd at 5%level. data represent the mean value ± s.d. the mean of four replicates. vermicompost (%) salinity (mm) mean vermicompost 0 50 100 150 200 root fresh weight (g) 0 13.33±0.54 bcd 13.25±0.54 bcd 13.18±0.54 cde 9.28±0.54 fg 6.26±0.54 i 11.06±2.96 d 5 13.33±1.32 bcd 13.46±1.32 bcd 12.08±1.32 ij 9.49±1.32 fg 6.30±1.32 i 11.24±3.18 d 10 14.46±0.73 b 14.38±0.73 bc 14.31±0.73 bc 10.41±0.73 f 6.47±0.73 i 12.00±3.34 c 15 16.01±1.24 a 15.93±1.24 a 15.86±1.24 a 11.96±1.24 e 7.85±1.24 h 13.52±3.48 b 20 17.02±0.66 a 16.94±0.66 a 16.87±0.66 a 12.97±0.66 de 8.86±0.66 gh 14.53±3.36 a mean salinity 14.87±1.69 a 14.79±1.69 a 14.72±1.69 a 10.82±1.69 b 7.15±1.36 c root dry weight (g) 0 6.50±0.54 d-g 6.38±0.54 efg 6.07±0.54 fg 2.17±0.54 jk 0.83±0.54 l 4.39±2.51 d 5 6.71±1.32 e-g 6.59±1.32 e-g 6.28±1.32 fg 2.38±1.32 ijk 0.89±1.32 l 4.57±2.73 d 10 7.63±0.73 cd 7.51±0.73 de 7.20±0.73 def 3.30±0.73 ij 1.30±0.73 ik 5.38±2.76 c 15 9.18±1.24 ab 9.06±1.24 ab 8.75±1.24 bc 4.85±1.24 h 3.43±1.24 i 6.85±3.02 b 20 10.19±0.66 a 10.07±0.66 a 9.76±0.66 ab 5.86±0.66 gh 7.85±0.66 h 7.86±2.87 a mean salinity 8.04±1.69 a 7.92±1.69 a 7.61±1.69 a 3.71±1.69 b 1.77±1.28 c flower diameter (mm) 0 65.50±1.91 cde 61.50±1.91 def 59.50±1.91 fg 52.50±1.91 i 41.50±1.91 k 55.50±8.20 c 5 62.50±2.08 cde 61.50±2.08 def 59.50±2.08 fg 52.50±2.08 i 41.50±2.08 k 55.50±8.23 c 10 63.25±2.98 bcd 62.25±2.98 c-f 60.25±2.98 ef 53.25±2.98 i 42.25±2.98 k 56.25±8.45 c 15 65.00±3.55 abc 64.00±3.55 bcd 62.00±3.55 def 55.00±3.55 hi 44.00±3.55 kj 58.00±8.62 b 20 67.00±1.41 a 66.00±1.41 ab 64.00±1.41 bcd 57.00±1.41 gh 46.75±1.41 j 60.15±7.85 a mean salinity 65.05±2.85 a 63.05±2.85 a 61.05±2.85 b 54.05±2.85 c 43.20±3.03 d 350 adv. hort. sci., 2019 33(3): 345-358 increased these parameters compared to the control under both absence and presence of the salinity stress treatments. the 20% vermicompost treatment under salinity stress conditions gave the higher values for these parameters than those with the other t r e a t m e n t s ( t a b l e 3 a n d 4 ) . t h e e x t e n t o f t h e increase in the values mentioned above was by 8.37%, 56.53% and 6.19 in the 20% vermicompost treatment as compared with those of the control, respectively. hidalgo et al. (2006) stated that using vermicompost fertilizer increased flower diameter and fresh and dry weight of flower in marigold which is consistent with the findings of this study. proline content as shown in table 4, proline content of the pot marigold plants was markedly increased by the salinity stress treatments. the maximum and the minimum proline content were observed in the 200 mm nacl and the control treatments, respectively. proline content increased 447.87% at 200 mm nacl compared to the control treatment (table 4). proline content increase is one of the protective mechanisms of this plant against salinity stress. it has been reported when plants are exposed to salinity stress, the breakdown of proteins and thus the increase of amino acids and amides accelerates, one of which is proline (iqbal et al., 2015). protein degradation, decrease in proline oxidase enzyme activity, and exacerbation of p5cs gene expression are the most important factors affecting proline concentration under stress conditions. increase in p5cs gene expression is one of the most important factors affecting proline concentration under stress conditions (kubala et al., 2015). the proline content was clearly affected by the vermicompost treatments. as compared to the control, the increase was 1.28% at 20% vermicompost. interaction between levels of salinity and vermicompost resulted in the highest and the lowest proline content in the plants with 200 mm nacl and 20% vermicompost treatment and with 0 mm nacl and 0% vermicompost treatment, respectively (table 4). in this study, the application of vermicompost increased proline content and reduced the damaging effects of salinity stress. vermicompost increases the amount of n available in plants due to the presence of n in the proline structure, which leads to increased proline synthesis under salinity conditions and increased plant resistance to salinity stress (rafiee et al., 2017). table 4 effect of salinity and vermicompost and their interaction on flower fresh and dry weight and proline content * in each variable, data followed by the same letters (small letters for interactions and capital letters for means) are not significantly different using lsd at 5%level. data represent the mean value ± s.d. the mean of four replicates. vermicompost (%) salinity (mm) mean vermicompost 0 50 100 150 200 flower fresh weight (g) 0 4.35±0.57 ef 4.28±0.57 ef 3.88±0.57 fg 2.89±0.57 ij 1.04±0.57 m 3.29±1.37 d 5 4.71±0.65 de 4.64±0.65 de 4.24±0.65 ef 3.25±0.65 ih 1.56±0.65 lm 3.68±1.33 c 10 5.07±0.65 d 5.00±0.65 d 4.60±0.65 de 2.37±0.65 jk 1.92±0.65 kl 3.79±1.51 c 15 6.25±0.47 bc 6.18±0.47 bc 5.78±0.47 c 3.55±0.47 gh 2.03±0.47 kl 4.76±1.77 b 20 6.93±0.82 a 6.86±0.82 a 6.46±0.82 ab 3.27±0.82 ih 2.22±0.82 kl 5.15±2.16 a mean salinity 5.46±1.14 a 5.39±1.14 a 4.99±1.14 b 3.07±0.74 c 1.75±0.65 d flower dry weight (g) 0 1.26±0.07 e-h 1.25±0.07 e-h 1.20±0.07 hi 1.11±0.07 jkl 0.84±0.07 p 1.13±0.07 c 5 1.27±0.06 e-g 1.26±0.06 e-g 1.21±0.06 gh 1.12±0.06 jk 1.05±0.06 lm 1.16±0.07 b 10 1.30±0.05 cde 1. 30±0.05 cde 1.23f±0.05 gh 1.14±0.05 ji 0.88±0.05 op 1.18±0.07 ab 15 1.30±0.01 cde 1.30±0.01 cde 1.28±0.01 e-g 1.07±0.01 klm 1.04±0.01 m 1.20±0.08 a 20 1.40±0.05 a 1.38±0.05 ab 1.36±0.05 bcd 0.96±0.05 n 0.92±0.05 no 1.20±0.09 a mean salinity 1.31±0.22 a 1.29±0.13 a 1.25±0.16 b 1.08±0.10 c 0.95±0.16 d proline content (µmol g‐1 f.w.) 0 4.19±0.12 k 4.31h±0.12 ij 4.76±0.12 f 10.32±0.12 d 23.18±0.12 b 9.35±7.47 d 5 4.20±0.11 k 4.32±0.11 hi 4.77±0.11 f 10.33±0.11 d 23.19±0.11 b 9.36±7.47 d 10 4.22±0.10 k 4.35±0.10 h 4.80±0.10 f 10.35±0.10 d 23.22±0.10 b 9.39±7.47 c 15 4.28±0.08 j 2.40±0.08 g 4.85±0.08 e 10.41±0.08 c 23.27±0.08 a 9.44±7.47 b 20 4.30±0.07 ij 4.42±0.07 g 4.87±0.07 e 10.43±0.07 c 23.30±0.07 a 9.47±7.47 a mean salinity 4.24±0.10 e 4.36±0.10 d 4.81±0.10 c 10.37±0.10 b 23.23±0.10 a adamipour et al. ‐ effect of vermicompost on performances of pot marigold 351 chlorophyll content the plants treated with nacl exhibited a significant decrease in chlorophyll content and the greatest damage was caused by 200 mm nacl treatment. in comparison with the control, chlorophyll content decreased by 28.12% with 200 mm nacl treatment (table 5). whereas, the vermicompost treatments reversed the adverse effects of nacl and caused a significant increase in chlorophyll content in the salttreated plants (table 5). the highest value of chlorophyll content in leaves (18.71%) was recorded in the vermicompost-treated plants over the control plants. usually, decreasing chlorophyll when plants face stress conditions may be due to an alternation in n metabolisms in relation to the production of compositional compounds such as proline, which are used in osmosis regulation, because an increase in proline production causes glutamate to less involve in the chlorophyll biosynthesis pathways (jaleel et al., 2008; håkanson and eklund, 2010). in addition, increased oxidative stress that is caused by reactive oxygen species damage to the chloroplast structure reduces the concentration of chlorophyll. reduction of chlorophyll content has been reported in plants under salinity stress conditions due to the activity of chlorophyllase enzyme. furthermore, some growth regulating agents such as abscisic acids and ethylene stimulate the activity of this enzyme (ali et al., 2004; zhao et al., 2007). the application of vermicompost significantly increased chlorophyll content in the leaves in the present study. vermicompost increases the synthesis of chlorophyll under salinity stress conditions by providing nutritious elements such as fe, zn, mg, and n directly and indirectly (nadi et al., 2011; narkhede et al., 2011). total carotenoid content the content of carotenoid decreased in the plants that received 50, 100, 150 and 200 mm nacl concentrations. the content of carotenoid was the lowest (37.16% over control) for the plants that received the highest level of the salinity treatment (200 mm nacl) (table 5). the vermicompost treatments not only improved the production of total carotenoid under saline-free conditions, but also successfully ameliorated the adverse effects caused by the salinity stress treatments on the plants (table 5). the content of carotenoid increased in the vermicompost-treated plants grown with the 20% level by 1.30% compared to that in the control plants. carotenoids act as * in each variable, data followed by the same letters (small letters for interactions and capital letters for means) are not significantly different using lsd at 5%level. data represent the mean value ± s.d. the mean of four replicates. table 5 effect of salinity and vermicompost and their interaction on chlorophyll content, total carotenoid content and superoxide dismutase vermicompost (%) salinity (mm) mean vermicompost 0 50 100 150 200 chlorophyll content (mg g‐1 f.w.) 0 2.09±0.11 k 2.06±0.11 l 1.97±0.11 m 1.77±0.11 r 1.46±0.11 v 1.87±0.26 e 5 2.17±0.11 g 2.14±0.11 i 2.05±0.11 l 1.85±0.11 p 1.54±0.11 u 1.95±0.26 d 10 2.24±0.11 e 2.21±0.11 f 2.12±0.11 j 1.92±0.11 o 1.60±0.11 t 2.02±0.26 c 15 2.27±0.11 d 2.24±0.11 e 2.15±0.11 h 1.95±0.11 n 1.64±0.11 s 2.05±0.26 b 20 2.44±0.11 a 2.41±0.11 b 2.32±0.11 c 2.12±0.11 j 1.81±0.11 q 2.22±0.26 a mean salinity 2.24±0.15 a 2.21±0.15 b 2.13±0.15 c 1.92±0.15 d 1.61±0.15 e total carotenoid content (μg∙g‐1 f.w.) 0 12.48±0.92 k 12.46±0.92 l 12.43±0.92 o 12.35±0.92 r 7.82±0.92 w 11.51±2.06 e 5 12.50±0.92 i 12.49±0.92 j 12.45±0.92 m 12.37±0.92 q 7.84±0.92 v 11.53±2.06 d 10 12.52±0.92 f 12.51±0.92 h 12.48±0.92 k 12.40±0.9 2p 7.87±0.92 u 11.55±2.06 c 15 12.57±0.92 d 12.55±0.92 e 12.52±0.92 g 12.44±0.92 n 7.91±0.92 t 11.60±2.06 b 20 12.63±0.92 a 12.61±0.92 b 12.58±0.92 c 12.50±0.92 i 7.97±0.92 s 11.66±2.06 a mean salinity 12.54±0.82a 12.52±0.82 b 12.49±0.82 c 12.41±0.82 d 7.88±0.82 e superoxide dismutase (ug‐1 f.w.) 0 146.00±1.92 m 152.00l±1.92 m 156.00±1.92 kl 440.00±1.92 d 104.00±1.92 q 199.60±1.25 c 5 155.50±0.80 kl 161.50±0.80 ijk 165.50±0.80 ghi 449.50±0.80 c 113.50±0.80 p 209.10±1.24 b 10 15.50±0.80 jkl 163.50±0.80 hij 167.50±0.80 f-i 451.50±0.80 bc 115.50±0.80 o 211.10±1.24 b 15 163.50±0.80 hij 169.50±0.80 e-h 173.50±0.80 ef 457.50±0.80 ab 121.50±0.80 n 217.10±1.24 a 20 165.50±0.80 ghi 171.50±0.80 efg 175.50±0.80 e 459.50±0.80 a 123.50±0.80 n 219.10±1.24 a mean salinity 157.60±1.22 d 163.60±1.22 c 167.60±1.22 b 451.60±1.22 a 115.60±1.22 e adv. hort. sci., 2019 33(3): 345-358 352 helper pigments in chloroplasts, but their most important role is antioxidant properties. because of the oxidative stress caused by salinity stress in plant tissues, carotenoid activity in both antioxidant enzymatic and non-enzymatic systems decrease (pant et al., 2009). in similar studies in summer savory (satureja hortensis l.) (najafi and khavari-nejad, 2010), wheat (triticum vulgare l.) (reddy and vora, 2005), and marjoram (origanum majorana) (baatour et al., 2010), reduction of carotenoids under salinity conditions has been reported. in a study consistent with the findings of the present study, ayyobi et al. ( 2 0 1 4 ) s t a t e d t h a t v e r m i c o m p o s t i n c r e a s e d carotenoid content in peppermint (mentha piperita l.) leaves. enzyme activities the activities of apx, pod, cat and sod in pot marigold plants were significantly affected by the salinity stress and vermicompost treatments. with the increasing extent of salinity stress, the activities of apx, pod, cat and sod increased markedly and then significantly decreased (table 5 and 6). apx, pod, cat and sod enzyme activities increased (12.08%, 13.67%, 36.08% and 186.54%, respectively) in the plants at salinity level of 150 mm nacl compared to those in the control plants. usually one of the biochemical changes that occur in plants under stress conditions is the accumulation of reactive oxygen species such as superoxide, hydrogen peroxide, and radical hydroxyl, all of which are highly toxic and reactive, and disrupt the normal metabolism of the cells. these radicals create secondary oxidative stress, through peroxidation of lipids, resulting in membrane degradation, protein degradation, deactivation of enzymes, elimination of pigments, and disruption of dna, leading to serious damage to the structure of cells and eventually to the whole plant. one strategy of plants to counteract this stress is the accumulation of antioxidant enzymes (kang et al., 2014). similar results have been reported in pot marigold (calendula officinalis l.) (hemmati et al., 2018) and chickpea (cicer arietinum l.) (sadak et al., 2017). the vermicompost treatments significantly enhanced the activities of apx, pod, cat and sod under salinity stress conditions, and the increases in the activities of apx, pod, cat and sod were 2.90%, 21.08%, 2.41% and 9.76% in the plants under the 20% vermicompost treatment compared with those in the control plants (table 5 and 6). similar results table 6 effect of salinity and vermicompost and their interaction on catalase, peroxidase and ascorbate peroxidase * in each variable, data followed by the same letters (small letters for interactions and capital letters for means) are not significantly different using lsd at 5%level. data represent the mean value ± s.d. the mean of four replicates. salinity (mm) sanility mean vermicompost 0 50 100 150 200 catalase (ug‐1 f.w.) 0 31.65±1.58 r 31.85±1.58 p 32.16±1.58 m 43.18±1.58 e 20.78±1.58 w 31.92±7.4 e 5 31.80±1.58 q 32.00±1.58 o 32.31±1.58 k 43.33±1.58 d 20.93±1.58 v 32.07±7.4 d 10 31.85±1.58 p 32.05±1.58 n 32.36±1.58 j 43.38±1.5 8 c 20.98±1.58 u 32.13±7.4 c 15 32.05±1.58 n 32.26±1.58 l 32.56±1.58 h 43.58±1.58 b 21.19±1.58 t 32.33±7.4 b 20 32.41±1.58 i 32.61±1.58 g 32.92±1.58 f 43.94±1.58 a 21.54±1.58 s 32.69±7.4 a mean salinity 31.95±1.43 d 32.16±1.43 c 32.46±1.43 b 43.48±1.43 a 21.08±1.43 e peroxidase (ug‐1 f.w.) 0 68.40±0.93 o 69.07±0.93 no 70.80±0.93 mn 78.62±0.93 gh 41.48±0.93 t 65.68±12.99 e 5 70.75±3.98 nm 71.42±3.98 lm 73.15±3.98 kl 80.97±3.98 ef 43.83±3.98 s 68.03±13.43 d 10 75.03±3.98 jk 75.71±3.98 ij 77.44±3.98 hi 85.26±3.98 c 48.12±3.98 r 72.31±13.43 c 15 77.59±3.98 hi 78.27±3.98 gh 80.00±3.98 fg 87.82±3.98 b 50.67±3.98 q 74.87±13.43 b 20 82.25±3.98 ef 82.93±3.98 de 84.66±3.98 cd 92.48±3.98 a 55.33±3.98 p 79.53±13.43 a mean salinity 74.80±5.96 c 75.48±5.96 c 77.21±5.96 b 85.03±5.96 a 47.89±5.96 d ascorbate peroxidase (ug‐1 f.w.) 0 869.60±1.51 s 875.40±1.51 q 880.40±1.51 n 976.10±1.51 e 459.60±1.51 x 812.20±1.85 e 5 873.20±1.51 r 878.90±1.51 p 883.90±1.51 m 979.60±1.51 d 463.20±1.51 w 815.80±1.85 d 10 879.60±1.51 o 885.40±1.51 l 890.40±1.51 k 986.10±1.51 c 469.60±1.51 v 822.20±1.85 c 15 885.40±1.51 l 891.10±1.51 j 896.10±1.51 h 991.80±1.51 b 475.40±1.51 u 827.90±1.85 b 20 893.20±1.51 i 898.90±1.51 g 903.90±1.51 f 999.60±1.51 a 483.20±1.51 t 835.80±1.85 a mean salinity 880.20±1.6 d 885.90±1.6 c 890.90±1.6 b 986.60±1.6 a 470.20±1.6 e adamipour et al. ‐ effect of vermicompost on performances of pot marigold 353 have been reported in cowpea(vigna unguiculata), rice (oryza sativa l.), and tall fescue (festuca arundi‐ nacea schreb.) (cavalcanti et al., 2004; garcía et al., 2014; adamipour et al., 2016). nutrients in leaf tissue n, p, k, fe, mg and zn concentrations significantly declined by the increasing salinity stress level from 0 to 200 mm nacl (table 7 and 8). their concentrations table 7 effect of salinity and vermicompost and their interaction on nitrogen, phosphorus and potassium table 8 effect of salinity and vermicompost and their interaction on zinc, magnesium and iron * in each variable, data followed by the same letters (small letters for interactions and capital letters for means) are not significantly different using lsd at 5%level. data represent the mean value ± s.d. the mean of four replicates. vermicompost (%) salinity mean vermicompost 0 50 100 150 200 nitrogen (%) 0 2.01±0.04 ghi 1.99±0.01 hi 1.90±0.01 j 1.01±0.01 op 0.97±0.01 q 1.58±0.49 e 5 2.03±0.02 g 2.02±0.009 gh 1.93±0.009 j 1.04±0.009 o 1.00±0.009 pq 1.60±0.49 d 10 2.15±0.04 e 2.10±0.01 f 2.01±0.01 ghi 1.12±0.01 m 1.08±0.01 n 1.69±0.49 c 15 2.40±0.08 c 2.38±0.02 c 2.29±0.02 d 1.40±0.02 k 1.36±0.02 l 1.97±0.49 b 20 3.01±0.02 a 2.99±0.01 a 2.90±0.01 b 2.01±0.01 gh 1.97±0.01 i 2.58±0.49 a mean salinity 2.32±0.38 a 2.30±0.38 b 2.21±0.38 c 1.32±0.38 d 1.28±0.38 e phosphorus (%) 0 0.15±0.005 ijk 0.14±0.005 lm 0.12±0.005 no 0.11±0.005 p 0.08±0.005 r 0.12±0.02 e 5 0.15±0.006 hi 0.15±0.006 jkl 0.13±0.006 mn 0.11±0.006 op 0.09±0.006 qr 0.13±0.02 d 10 0.16±0.009 gh 0.15±0.009 ij 0.14±0.009 klm 0.12±0.009 no 0.10±0.009 q 0.13±0.02 c 15 0.19±0.006 e 0.18±0.006 f 0.16±0.006 g 0.15±0.006 ij 0.12±0.006 no 0.16±0.02 b 20 0.24±0.005 a 0.23±0.005 b 0.22±0.005 c 0.20±0.005 d 0.18±0.005 f 0.22±0.02 a mean salinity 0.18±0.03 a 0.17±0.03 b 0.15±0.03 c 0.14±0.03 d 0.11±0.03 e potassium (%) 0 3.93±0.14 fgh 3.89±0.14 ghi 3.84±0.14 hi 2.93±0.14 mn 2.11±0.14 p 3.34±0.74 d 5 4.11±0.18 d-h 4.07±0.18 d-h 4.02±0.18 e-h 3.11±0.18 lm 2.29±0.18 op 3.52±0.75 c 10 4.22±0.12 de 4.18±0.12 def 4.13±0.12 d-g 3.22±0.12 kl 2.40±0.12 o 3.63±0.74 c 15 4.63±0.22 b 4.59±0.22 bc 4.54±0.22 bc 3.63±0.22 ij 2.81±0.22 n 4.04±0.76 b 20 5.32±0.31 a 5.28±0.31 a 5.23±0.31 a 4.32±0.31 cd 3.50±0.31 jk 4.73±0.78 a mean salinity 4.44±0.54 a 4.40±0.54 a 4.35±0.54 a 3.44±0.54 b 2.62±0.54 c * in each variable, data followed by the same letters (small letters for interactions and capital letters for means) are not significantly different using lsd at 5%level. data represent the mean value ± s.d. the mean of four replicates. vermicompost (%) salinity mean vermicompost 0 50 100 150 200 zn (mg/kg) 0 91.47±0.47 efg 91.42±0.47 fg 91.34±0.47 fg 90.35±0.47 i 88.88±0.47 k 90.69±1. 10e 5 91.78±0.51 def 91.73±0.51 def 91.65±0.51 def 90.66±0.51 hi 89.19±0.51 jk 91.00±1. 10d 10 92.14±0.32 d 92.09±0.32 d 92.01±0.32 de 91.02±0.32 gh 89.55±0.32 j 91.36±1. 06c 15 93.25±0.86 c 93.20±0.86 c 93.12±0.86 c 92.13±0.86 d 90.66±0.86 hi 92.47±1. 28b 20 95.44±0.51 a 95.39±0.51 a 95.31±0.51 a 94.32±0.51 b 92.85±0.51 c 94.66±1. 11a mean salinity 92.82±1. 56 a 92.77±1. 56 a 92.69±1. 56 a 91.70±1. 56 b 90.23±1. 56 c mg (mg/kg) 0 1.69±0.09 efg 1.64±0.09 e-h 1.60±0.09 e-h 1.46±0.09 ghi 1.23±0.09 i 1.52±0.18 d 5 1.72±0.06 efg 1.69±0.06 efg 1.65±0.06 e-h 1.51±0.06 f-i 1.28±0.06 i 1.57±0.17 cd 10 1.82±0.12 cde 1.79±0.12 c-f 1.75±0.12 d-g 1.61±0.12 e-h 1.38±0.12 i 1.67±0.20 c 15 2.10±0.20 c 2.07±0.20 c 2.03±0.20 cd 1.89±0.20 cde 1.66±0.20 e-h 1.95±0.24 b 20 3.38±0.58 a 3.35±0.58 a 3.31±0.58 a 3.17±0.58 ab 2.94±0.58 b 3.23±0.54 a mean salinity 2.14±0.70 a 2.11±0.70 a 2.07±0.70 a 1.93±0.70 b 1.70±0.70 c fe (mg/kg) 0 122.05±1.68 ij 121.92±1.68 ij 120.69±1.68 j 115.50±1.68 k 100.27±1.68 m 116.09±8.60 e 5 124.19±1.96 hi 124.06±1.96 i 122.83±1.96 ij 117.64±1.96 k 102.41±1.96 m 118.23±8.65 d 10 128.42±2.33 g 128.29±2.33 g 127.06±2.33 gh 121.87±2.33 ij 106.64±2.33 l 122.45±8.72 c 15 154.19±2.69 d 154.06±2.69 d 152.83±2.69 d 147.64±2.69 e 132.41±2.69 f 148.23±8.80 b 20 196.53±4.15 a 196.40±4.15 a 195.17±4.15 a 189.98±4.15 b 174.75±4.15 c 190.56±9.24 a mean salinity 145.07±29.02 a 144.94a±29.02b 143.71±29.02 b 138.52±29.02 c 123.29±29.02 d adv. hort. sci., 2019 33(3): 345-358 354 decreased (44.82%, 36.06%, 40.99%, 15.01%, 20.56% and 2.79%, respectively) in the plants at 200 mm nacl compared to those in the control plants. n, p, k, fe, mg and zn concentrations in the leaf tissues of pot marigold increased significantly with increasing vermicompost composition in the media compared to those in the control plants. the highest and the l o w e s t c o n c e n t r a ti o n s o f t h e e l e m e n t s w e r e observed in the 20% and the 0% vermicompost treatments, respectively (table 7 and 8). furthermore, n, p , k , f e , m g a n d z n c o n c e n t r a ti o n s i n l e a v e s increased 46.20%, 80.32%, 4.61%, 64.14%, 112.50%, and 4.37% in the plants with the 20% vermicompost treatment compared to those in plants with the control treatment, respectively. interaction between the levels of salinity and vermicompost resulted in the highest and the lowest nutrient concentrations in the plants with the 0 mm nacl and 20% vermicompost and with the200 mm nacl and 0% vermicompost treatments, respectively (table 7 and 8). plant growth is reduced in salt-affected soil because of the excess uptake of potentially toxic ions. soil salinity is characterized by high amounts of na, mg, ca, cl, hco3 and b ions in soil which have negative effects on plant growth. eventually, high salt concentrations in soil reduce the absorption of nutrients of plants which negatively affects the fertility of the soil (zhao and ren, 2007). in this study, the increase of vermicompost levels increased the mineral nutrition. vermicompost contains humic substances that have multiple effects in the soil. it may improve soil properties such as micronutrient transport and availability (aşık et al., 2009). chen and aviad (1990) summarized the effects of humic substances on plant growth and mineral nutrition, pointing out the positive effects on the uptake of macro(such as n, p, and k) and micro-elements. vermicompost may enhance the uptake of nutrients and reduce the uptake of some toxic elements of plants under salinity stress conditions. chamani et al. (2008) reported that addition of vermicompost to soils resulted in increased mineral contents in the substrate and higher concentrations p, ca, mg, cu, mn and zn in shoot tissues of red clover and cucumber. correlation analysis the results of the pearson correlation analysis between vermicompost abundance and the measured morphological traits and elements showed a positive and significant correlation. therefore, a positive relation between the increases of the amount of vermicompost and improvement in the morphological traits and measured elements can be inferred. according to the results, shoot fresh weight compared to shoot dry weight (r=0.89**) and flower diameter (r=0.57**) showed the highest and the lowest correlations among the morphological traits, respectively (table 9). in the measured elements under the vermicompost treatments, the highest correlation was observed between shoot fresh weight and k content (r=0.81**) while the lowest correlation was seen in mg content (r=0.67**) (table 9). this improvement can be attributed to the presence of macro-micro elements, the release of elements in the soil and soil amendment of physical and biological conditions which have been previously reported in other plants table 9 correlation coefficients for evaluated traits on pot marigold in vermicompost treatment traits shoot fw shoot dw leaf area root fw root dw flower diameter fruit fw fruit dw zn fe mg ca k p n shoot fw 1 shoot dw 0.891** 1 leaf area 0.886** 0.992** 1 root fw 0.798** 0.799** 0.777** 1 root dw 0.798** 0.799** 0.777** 1.00** 1 flower diameter 0.574** 0.603** 0.586** 0.519* 0.519* 1 fruit fw 0.811** 0.795** 0.801** 0.618** 0.618** 0.334 ns 1 fruit dw 0.618** 0.728** 0.695** 0.734** 0.734** 0.536* 0.417 ns 1 zn 0.789** 0.883** 0.875** 0.864** 0.864** 0.623** 0.778** 0.722** 1 fe 0.809** 0.836** 0.822** 0.825** 0.825** 0.624** 0.818** 0.686** 0.951** 1 mg 0.679** 0.797** 0.781** 0.764** 0.764** 0.538* 0.733** 0.749** 0.927** 0.907** 1 ca 0.789** 0.883** 0.875** 0.864** 0.864** 0.623** 0.778** 0.722** 1.00** 0.951** 0.927** 1 k 0.818** 0.851** 0.842** 0.799** 0.799** 0.561** 0.778** 0.765** 0.911** 0.929** 0.884** 0.911** 1 p 0.815** 0.847** 0.835** 0.815** 0.815** 0.554* 0.831** 0.698** 0.937** 0.981** 0.921** 0.937** 0.938** 1 n 0.805** 0.834** 0.814** 0.831** 0.831** 0.604** 0.810** 0.704** 0.956** 0.989** 0.933** 0.956** 0.916** 0.981** 1 fw = fresh weight; dw= dry weight. ns= not significant, * and **: significant at 5% and 1% respectively. adamipour et al. ‐ effect of vermicompost on performances of pot marigold 355 (bachman and metzger, 2008). in this correlation analysis, peroxidase activity (r=0.79**) and proline content (r=0.44**) had the highest and the lowest correlations with shoot fresh weight. further, insignificant correlations were observed in carotenoid content and catalase enzyme activity (table 10). it seemed the positive correlations in activities of antioxidant enzymes, chlorophyll and proline content in the plant was due to increased concentrations of the elements which could be provided by vermicompost. because in the structure of the mentioned traits, there are a variety of macroand microelements that vermicompost provides directly and indirectly to plants (hidalgo et al., 2006). the results of a simple correlation analysis between the morphological traits and the studied elements under salt stress conditions showed that shoot fresh weight had a positive and significant correlation with all the morphological indices and concentration of elements. in this analysis, the highest and the lowest correlations were seen in leaf area (r=0.99**) and fresh flower weight (r=0.91**), respectively (table 11). also, the highest correlation of shoot fresh weight was observed in fe content (r=0.98**) and the lowest correlation was found in n content (r=0.88**) (table 11). there are many reports of positive correlations between plant yield and concentration of elements under salinity stress conditions. increase in soil ph, reduction in the amount of absorbent elements in the soil, increase in toxic elements of soil such as cl and na, and osmotic stress in the soil that prevents water absorption are the most c r i ti c a l f a c t o r s f o r t h i s p h e n o m e n o n i n p l a n t s (edwards et al., 2010). analysis of correlations of shoot fresh weight with chlorophyll and proline contable 11 correlation coefficients for evaluated traits on pot marigold in salinity stress conditions traits shoot fw shoot dw leaf area root fw root dw flower diameter fruit fw fruit dw zn fe mg ca k p n shoot fw 1 shoot dw 0.939** 1 leaf area 0.995** 0,956** 1 root fw 0.966** 0.886** 0.961** 1 root dw 0.923** 0.868** 0.921** 0.986** 1 flower diameter 0.986** 0.977** 0.991* 0.952** 0.919** 1 fruit fw 0.918** 0.830** 0.906** 0.844** 0.798** 0.865** 1 fruit dw 0.942** 0.947** 0.948** 0.901** 0.855** 0.972** 0.779** 1 zn 0.938** 0.947** 0.956** 0.850** 0.802** 0.941** 0.923** 0.892** 1 fe 0.985** 0.940** 0.987** 0.914** 0.851** 0.977** 0.909** 0.948** 0.963** 1 mg 0.895** 0.910** 0.803** 0.762** 0.897** 0.919** 0.856** 0.969** 0.912** 0.857** 1 ca 0.938** 0.947** 0.956** 0.850* 0.802** 0.941** 0.923** 0.892** 1.00** 0.963** 0.969** 1 k 0.981** 0.961** 0.991* 0.954** 0.928** 0.982** 0.915** 0.927** 0.966** 0.967** 0.932** 0.966** 1 p 0.936** 0.877** 0.911** 0.933** 0.926** 0.919** 0.843** 0.858** 0.804** 0.879** 0.756** 0.804** 0.894** 1 n 0.885** 0.870** 0.892** 0.941** 0.976** 0.886** 0.809** 0.796** 0.831** 0.811** 0.781** 0.923** 0.817** 0.891** 1 fw = fresh weight; dw= dry weight. ns= not significant, * and **: significant at 5% and 1% respectively. table 10 correlation coefficients for evaluated traits on pot marigold under salinity stress conditions (above diameter) and vermicompost treatment (below diameter) ns= not significant, * and **: significant at 5% and 1% respectively. traits shoot fresh weight chlorophyll proline carotenoid ascorbate perodidase peroxidase catalase superoxide dismutase shoot fresh weith 1 0.913 ** -0.904 ** -0.874 ** 0.872 ** 0.990 ** 0.905 ** 0.861** chlorophyll 0.664 ** 1 -0.992 ** 0.857 ** -0.856 ** 0.888 ** 0.888 ** 0.626** proline 0.444 ** 0.176 ns 1 0.854 ** 0.680 ** -0.816 ** 0.714 ** 0.571** carotenoid 0.059 ns 0.488 * 0.454 * 1 0.801 ** 0.651 ** -0.544 * -0.346 ns ascorbate peroxidase 0.490* 0.064 ns 0.038 ns -0.435 ns 1 0.760 ** 0.407 ns -0.085 ns peroxidase 0.794 ** 0.480 * 0.540 * 0.093 ns 0.746 ** 1 0.458* -0.020 ns catalase 0.131 ns 0.444 * -0.341 ns 0.252 ns 0.430 ns 0.317 ns 1 -0.006 ns superoxide dismutase 0.514 * 0.793 ** -0.210 ns 0.131 ns -0.002 ns 0.241 ns 0.355 ns 1 adv. hort. sci., 2019 33(3): 345-358 356 tent, and antioxidant enzyme activity showed significant positive and negative correlations. from the viewpoint of biochemistry, most of significant correl a ti o n s w e r e f o u n d i n c o n t e n t o f c h l o r o p h y l l (r=0.91**), proline (r= -0.90**), carotenoid (r= -0.87**), in activity of ascorbate peroxidase (r=0.87**), peroxidase (r=0.99**), catalase (r=0.90**), and superoxide dismutase (r=0.86**) compared to the shoot fresh weight (table 10). there were also few negative correlations in which proline and carotenoid content can be attributed to their function in dealing with osmotic stress in the plants due to increased salinity in the soil. in similar studies, positive correlation of antioxidant enzyme activity under salinity stress conditions in german chamomile (matricaria recutita l.), sunflowers (helianthus annuus l.) and basil (ocimuum basilicum l.) have been reported (baghalian et al., 2008; heidari et al., 2011; heidari, 2012). 4. conclusions salinization of soil is a serious land degradation problem and is increasing steadily in many parts of the world, particularly in arid and semiarid areas. soil salinity affects the establishment, growth, and development of plants leading to huge losses in productivity. vermicompost is one of the major organic fertilizer which can improve growth and salinity tolerance with containing plenty nutrition elements, hormones, and organic materials. in this study, increase in salinity stress significantly led to decline in morphological and physiological indices of pot marigold. application of vermicompost under salinity conditions increased m o r p h o p h y s i o l o g i c a l i n d i c e s i n t h i s p l a n t . vermicompost increased the activity of the antioxidant system, the content of proline and chlorophyll in the plant by increasing the nutrients in the soil environment that could increase the plant yield and alleviating the harmful effects of salinity. references adamipour n., heiderianpour m., zarei m., 2016 application of vermicompost for reducing the destructi‐ ve effects of salinity stress on tall fescue turfgrass (festuca arundinacea schreb. 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ind. crop. prod., 94: 574585. zarei m., azizi m., rahemi m., tehranifar a., 2016 evaluation of nacl salinity tolerance of four fig genoty‐ pes based on vegetative growth and ion content in lea‐ ves, shoots, and roots. hortscience, 51: 1427-1434. zhao g., ma b., ren c., 2007 growth, gas exchange, chlorophyll fluorescence, and ion content of naked oat in response to salinity. crop sci., 47: 123-131. impaginato 171 adv. hort. sci., 2019 33(2): 171-177 doi: 10.13128/ahs-23326 relationship between chlorosis, photosynthesis and the nutrient content of plane trees in the presence of chemical and organic fertilizers h. aalipour (*), a. nikbakht, n. etemadi department of horticulture, college of agriculture, isfahan university of technology, 8415683111 isfahan, iran. key words: mycorrhizal fungi, nutrient acquisition, organic matter, symbiosis, urban trees. abstract: chlorosis disorder is a major problem affecting the growth and physiological processes of many trees including plane trees (platanus orientalis l.). this experiment was conducted to study the relationship between leaf chlorosis disorder and the nutritional status and some important physiological characteristics of plane trees. the experiment was arranged in a randomized complete block design with six replications and four treatments including control, manure (m), manure + fertilizer (20-5-10) (mf), and manure + fertilizer + mycorrhizal fungi (mfa) (glomus intraradices + g. mosseae). the results showed that although all treatments significantly improved the nutrients content, soluble carbohydrates content, photosynthesis rate and chlorophyll content in the leaves, they mostly reached their peak in the mycorrhizal inoculated plants. nitrogen (n), phosphorus (p) and zinc (zn) were increased in the amf amended trees compared to the control plants. the photosynthesis rate was enhanced by all the mixtures at least by 60% compared to the control. the most chlorosis (17.5%) to the leaves recorded on the control plants, while leaf damage dropped to less than 2.9% at mycorrhizal treatment leading to the improved nutritional balance in the plane trees. the results proved the effectiveness of including mycorrhizal inoculation to the common fertilization practices to prevent leaf chlorosis in the plane trees. 1. introduction plane tree (platanus orientalis l.) is among the most common ornamental and street trees planted in the urban landscape in iran and some mediterranean countries (anselmi et al., 1994; khorsandi et al., 2016). they are known for their longevity and wide distribution in the temperate zones. however, the chlorosis as an important physiological disorder in the plane trees has affected a majority of them in iran in recent years (khorsandi et al., 2016). the problem is a common physiological disorder affecting many plants around the globe. it is especially a major problem in (*) corresponding author: h.ali@ag.iut.ac.ir citation: aalipour h., nikbakht a., etemadi n., 2019 relationship between chlorosis, photosynthesis and the nutrient content of plane trees in the presence of chemical and organic fertilizers. adv. hort. sci., 33(2): 171-177 copyright: © 2019 aalipour h., nikbakht a., etemadi n. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 31 may 2018 accepted for publication 10 january 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(2): 171-177 172 the calcareous soils and soils with high ph (wallace, 1982). the chemical properties of the soil and the adequate supply of nutrients are major factors affecting natural plant growth and extension (cekstere and osvalde, 2013), therefore important factor in nutrient uptake is the availability of the nutrients in the soil. most trees cultivated in the alkaline and calcareous soils are exposed to the incidence of chlorosis which is reported to be basically due to fe deficiency (mortvedt, 1986). several factors can contribute to leaf chlorosis including nutritional disorders and a disorder in the chlorophyll biosynthesis. indeed, the lack of some nutrient elements such as nitrogen (n), zinc (zn) and expecially iron (fe), lead to the chlorosis in plants (godde and dannehl, 1994). moreoever, following the lack of sufficient chlorophyll, the affected plant will not be able to operate photosynthesis process, resulting in stunted growth (miller et al., 1984). arbuscular mycorrhizal fungi (amf) are obligate biotrophs that colonize the roots of the most land plants and increase host nutrient acquisition (desiro et al., 2014) and it is claimed that virtually all trees acquire nutrients through symbiotic mycorrhizal fungi (brundrett, 2009). mycorrhizal inoculation is documented as a method to the improve nutrient uptake in many plants (lehmann et al., 2014; varga, 2015; young et al., 2015). amf are effective symbionts for plants, and their symbiotic relationship can i n c r e a s e p l a n t g r o w t h ( v a f a d a r e t a l . , 2 0 1 4 ) . moreover, there is a lack of information on symbiosis relationship between the plane tree and amf fungi. in the present study, we added amf to the common fertilization program of the plane trees in the urban landscape to study the following items. firstly study the effect of mycorrhizal association on the trees response and then, observe how nutrient content and different physiological processes are associated with the leaf chlorosis disorder. to the best of knowledge, this is the first report attempting to discover the correlation between different plane tree physiological processes and leaf chlorosis disorder under amf inoculation. 2. materials and methods experimental site and treatments the experiment was conducted during 2013-2014, o n t h e c a m p u s o f t h e i s f a h a n u n i v e r s i t y o f technology in isfahan (32°39’ n, 51°40’ e; 1600 m), iran. the site is characterized as having an arid climate with cold winters, 122.8 mm average annual rainfall and 23.4°c average annual temperature. twenty-four uniform 15-year-old plane trees (p. orientalis l.) were selected. the experiment was a randomized complete block design (rcbd) with four treatments. treatments included control, manure (m), manure + fertilizer (water soluble 20-5-10 n-p-k compound fertilizer with 12.8% sulfur, 1.3% magnesium oxide, novatec solub, compo, germany) (mf), and manure + fertilizer + mycorrhizal fungi (mfa). six replications were prepared for each treatment. the plants were inoculated with two amf inoculations including glomus intraradices and g. mosseae (both of them have been transferred to new genera, so index fungorum considers them now as rhizophagus intraradices (n.c. schenck & g.s. sm.) and funneliformis mosseae (nicolson & gerd.) (schüßler and walker, 2010). the am fungi were provided by the institute of soil and water research, tehran, iran. inoculum was comprised of a mixture of spores (80 spores g-¹ for g. intraradices and 80 spores g-¹ for g. mosseae). the mixtures of filling materials were placed into 0.5 × 0.5 m holes, depending on the treatment in early spring. this technique provides a nutrients in a zone in and around each hole. with the first wetting, the nutrients are released from the fertilizer into the soil and the manure slowly lower the ph of the soil surrounding the hole. over a period of time, a zone of soil around each hole is modified to be lower in ph and rich in micronutrients in approximately the correct proportions. two identical holes were drilled around each tree about one meter away from the tree trunk and filled up with the corresponding mixture. during the process, we avoided drilling into large buttress roots. in m treatment, trees received 5 kg of manure per hole mixed with the soil of the drilled hole. in mf treatment, 100 g of fertilizer per hole was added to the manure. trees of mfa treatment received the amf inoculums by adding 250 g of mycorrhizal inoculums into each hole mixed with manure and fertilizer (500 grams of inoculum per each tree in total). the control group did not receive any treatment (two identical holes were drilled). the trees were irrigated once a week. some chemical and physical properties of the soil and cow manure are presented in table 1. measurements various morphological and physiological parameters were measured 5 months after treatment. the aalipour et al. platanus orientalis disorders and nutritional status 173 mineral contents of the plant leaves were determined in the second year of the experiment. plant samples were oven-dried at 65°c for 48 h and then were ground to determine their mineral composition. the determination of the total n in the leaf samples was based on the kjeldahl method (baker and thompson, 1992). the extraction of p, k, fe, and zn from the plant tissue material was performed by using 2 m hydrochloric acid (hcl) after dry ashing at 550°c for 5.5 h. the concentrations of fe and zn were determined by atomic absorption spectrophotometer (670 shimadzu, kyoto, japan) (aoac, 2006). p concentration was determined by vanado molybdate phosphoric acid method with a spectrophot o m e t e r ( u v 1 6 0 a u v v i s i b l e r e c o r d i n g s p e c t r o p h o t o m e t e r , s h i m a d z u , t o k y o , j a p a n ) (cottenie, 1980). lci portable photosynthesis and transpiration rate analyzer (li 6400; licor, lincoln, ne, usa) was used to measure the net photosynthesis rate (a) between 09.30-11.30 h on 10 fully expanded current-season leaves situated at the midcanopy height. the soluble sugars were measured according to the phenol-sulfuric acid method (dubois et al., 1956). the extraction of the leaf chlorophyll pigments was carried out using 100% acetone according to lichtenthaler (1987). for evaluation of the leaf chlorosis extent, 100 leaves from each tree were selected randomly and both leaf surfaces were scanned by a scanner (canon i-sensys mf4010, canon inc., korea). the leaf chlorosis was determined by digital image processing u s i n g m a t l a b s o f t w a r e . a r a n g e o f c o l o r w a s defined for the leaf chlorosis in the program and total leaf area was examined pixel by pixel by the software and the percentage of pixels which was defined as chlorotic areas were calculated by the software (rathod et al., 2013). data analysis data were assessed for normality and log-transformed used to make data conform to normality when necessary prior to analysis. non-homogeneity data were observed in leaf chlorosis, being the data transformed with the formula arcsin ö(leaf chlorosis/100) to obtain homogeneity. the experimental data were statistically analyzed by the analysis of variance ( a n o v a ) . t h e s i g n i f i c a n c e o f t h e d i f f e r e n c e s between treatments was estimated using the least significant difference (lsd) test at p≤0.05, and graphs were drawn using excel 2010. statistical correlation was calculated by pearson’s correlation coefficient (r). this test was used to measure the strength of a linear association between the leaf chlorosis and other variables including nutrients content, photosynthesis rate and chlorophyll and soluble sugar contents. the value r = 1 means a perfect positive correlation and the value r = -1 means a perfect negative correlation. the experimental data were statistically analyzed with statistical analysis systems (sas) software, version 9.1 and statistics, version 8.0. 3. results amf inoculation increased all nutrients content including p, n, fe and zn in the leaves of the treated trees (table 2). n, p and zn reached their peak value only when the fertilizer mix amended by mycorrhizal inoculums. p and zn increased by 424% and 425% factors texture ph ec (ds m-1) organic matter (%) n (%) p-available (mg kg-1) k-exchangeable (mg kg-1) fe (mg kg-1) zn (mg kg-1) soil clay 7.9 1.53 1.15 0.15 140 235 1400 21 manure 8.02 15.23 20.4 3.07 791 2030 12300 194 table 1 some chemical and physical properties of the soil and cow manure used in research treatment nutrient n (g kg-1) p (g kg-1) fe (mg kg-1) zn (mg kg-1) control 17.47 d 1.85 c 54.93 b 4.82 d manure 19.14 c 5.25 b 148.99 a 15.09 c manure + fertilizer 19.65 b 7.31 b 146.71 a 17.32 b manure + fertilizer + amf 20.39 a 9.71 a 170.14 a 25.31 a table 2 influence of arbuscular mycorrhizal (am) fungi and other treatments on nutrient uptake of plane tree (platanus orientalis l.) means in the same column followed by the same letters are not statistically different at p≤0.05 by the least significant difference test (lsd). adv. hort. sci., 2019 33(2): 171-177 174 respectively, compared to the control plants. all treatments (regardless of the composition of the mixture) successfully enhanced fe and n contents in the leaves compared to the control plants (table 2). all treatments significantly improved the soluble carbohydrates content in the leaves; however, it reached the peak in the mycorrhizal inoculated plants. the soluble carbohydrates content increased by 35.44% and 13.82% compared to the control of non-fertilized treatment and non-inoculated plants, respectively (fig. 1). all treatments increased the photosynthesis rate at least by 60% compared to the control, although no significant difference was observed between the treatments (fig. 2). the same trend was observed in the case of chlorophyll content (fig. 3), where it increased at least by 32% compared to the control. leaf chlorosis was influenced dramatically by the treatments (fig. 4). the most leaf chlorosis was recorded in control non-fertilized treatments. leaf chlorosis reduced to less than 2.9% on inoculated trees by amf, while 17.5% of leaves tissue were affected by chlorosis in the control plants. the amf inoculated plants showed an increase of 13.44% compared to the plants that received manure + fertilizer. a strong relationship was found between leaf chlorosis and fe, n and zn contents (fig. 5). a significant and linear relationship was also found between chlorosis and the chlorophyll content and net photosynthesis in the plane tree leaves (fig. 6). leaf chlorosis in the plane trees resulted in a dramatic and linear decline in the soluble carbohydrate in the leaves (fig. 6). 4. discussion and conclusions two explanations can be presented for increasing fe content under any treatments (except for the config. 1 influence of arbuscular mycorrhizal (am) fungi and other treatments on soluble carbohydrate content in plane tree (platanus orientalis l.). means are separated by lsd test at p≤0.05. fig. 2 influence of arbuscular mycorrhizal (am) fungi and other treatments on photosynthesis rates in plane tree (platanus orientalis l.). means are separated by lsd test at p≤0.05. fig. 3 influence of arbuscular mycorrhizal (am) fungi and other treatments on chlorophyll content in plane tree (platanus orientalis l.). means are separated by lsd test at p≤0.05. fig. 4 influence of arbuscular mycorrhizal (am) fungi and other treatments on chlorosis damage in plane tree (platanus orientalis l.). means are separated by lsd test at p≤0.05. aalipour et al. platanus orientalis disorders and nutritional status 175 trol) in this experiment (fig. 1). firstly, as the soil of the site was a calcareous and compacted soil, any treatment increasing the air flow in the soil could improve fe content in the plant (lucena, 2003). secondly, using the manure itself can increase fe content in the plants (mortvedt, 1986). it is documented that the manure provides micronutrients including fe and improves the structure of the soil (lucena, 2003). our results at least partly confirm the findings that combining fe fertilizers with the organic matter is more favorable in terms of fe uptake than fe sources applied alone (mortvedt, 1986). it has been reported that the application of feso4 (4-8 kg tree-¹) mixed with manure, cotton seed cake, or other organic substances in 8 to 10 holes in the soil around the crowns of apple trees (malus sylvestris mill.) r e s u l t e d i n m a r k e d c o r r e c t i o n o f f e c h l o r o s i s (zheng-qing and cang-zhen, 1982). it is also well established that as a result of the decomposition of organic matters in the soil, compounds such as humic acid (ha) and fulvic acid (fa) are produced in the soil (nardi et al., 2002). these acids are well known as naturally-occurring chelating agents (mortvedt, 1986; nardi et al., 2002). there are many reports showing enhanced micronutrients uptake by the plants receiving ha, fa (nikbakht et al., 2008; 2014) or organic matter (atiyeh et al., 2002). it is shown that inoculation turfgrass (lolium prenne l.) with amf receiving ha not only improved plant growth but also showed more elevated nutrients content in the leaves than in non-inoculated (control) plants or plants receiving only ha (nikbakht et al., 2014). researchers believed that the role of amf in no-3 t r a n s p o r t t o t h e r o o t s u r f a c e i s s i g n i fi c a n t (subramanian and charest, 1999; javaid, 2009). they especially insist that the role of amf is of value and importance in nitrate uptake in mediterranean and (semi-) desert ecosystems which are characterized by calcareous soils. n, p and zn uptake reached their peak value when the fertilizer mixture amended by amf (table 2). these results confirm the well-documented effect of amf inoculation on nutrients uptake (brundrett, 2009; varga, 2015; young et al., 2015). a strong relationship between leaf chlorosis and fe, n and zn contents implies that the chlorosis is not only because of fe deficiency in the plant, but also other nutrients including n and zn play they own role (fig. 5). it indicates that leaf chlorosis in the plane trees was not simply due to fe deficiency. it is well documented that fe is an essential element for many vital processes in a plant including photosynthesis, respiration, n fig. 5 relationship between chlorosis with fe (a), zn (b) and n concentration (c) in plane tree (platanus orientalis l.). fig. 6 relationship between chlorosis with photosynthesis rate (a), chlorophyll content (b) and soluble carbohydrate (c) in plane tree (platanus orientalis l.). 176 adv. hort. sci., 2019 33(2): 171-177 fixation, chlorophyll and hormone synthesis; fe is also a constituent of heme proteins (cytochromes, catalase, and peroxidase) (briat and lobreaux, 1997). as a result, affected plants by fe deficiency suffer severe metabolic and structural disorders (javaid, 2009). there are also some reports indicating that the major cause of fe deficiency is the very low solubility of fe oxides in the soil (mortvedt, 1986). it shows the importance of the fact that the role and priority of each element in the plane tree chlorosis remain to be investigated further. fe deficiency depresses the synthesis of chlorophyll, which results in the decrease of photosynthetic products, which in turn affect plant growth (wang et al., 2008). as a result of carbohydrates synthesis reduction in chlorotic leaves, which slows the movement of k+ from the leaf to the phloem vessels, a decline in the production of biomass is reported (maldonado-torres et al., 2006). these explain why we found a relationship between chlorosis and chlorophyll content, net photosynthesis and soluble c a r b o h y d r a t e c o n t e n t o f t h e l e a v e s ( f i g . 6 ) . moreover, increased photosynthetic capacity by amf is in agreement with the results of the previous study by birhane et al. (2012). it seems this process has improved nutrition, leading to higher photosynthetic rates (vafadar et al., 2014). to the best of our knowledge, no similar information has yet been provided for interaction effect of fertilizers and amf inoculation on plane trees and its relationship with the leaf chlorosis disorder. this study demonstrated that amf inoculation added to the common fertilizer program served successfully as a biological and environmental-friendly method to overcome chlorosis disorder of the plane trees. in addition, the findings of this study suggest that in calcareous soils drill hole nutrition should be considered as a standard method to prevent nutritional disorders in the urban landscape. the results also revealed that fe is not the only nutrient participating in the leaf chlorosis of plane trees. it suggests further investigations to study the weight and importance of each nutritional element in chlorosis disorder of the plane trees. in this study we mainly focused on the effect of improved media around the plane trees, rather than specific effect of amf. indeed, this study was one of the primary trail in a series of experiments we want done later. in the later, we specifically will studied the amf effect on the plane trees. our experiment creates a paradigm for future studies of relationship between plane trees and microorganisms. references anselmi n., cardin l., nicolott g., 1994 plane decline in european and mediterranean countries: associated pests and their interactions. eppo bull., 24: 159-171. aoac, 2006 official methods of analysis. association of official analytical chemists, 18th edition, gaithersburgs, md, usa. atiyeh r.m., lee s., edwards c.a., arancon n.q., metzger j.d., 2002 the influence of humic acids derived from earthworm-processed organic wastes on plant growth. bioresour. technol., 84: 7-14. baker w.h., thompson t.l., 1992 determination of total nitrogen in plant samples by kjeldahl, pp. 13-16. in: plank c.o. 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zheng-qing z., cang-zhen l., 1982 studies on the application of ferrous sulphate for controlling chlorosis of apple tree on calcareous soils. j. plant nutr., 5: 883896. impaginato 87 adv. hort. sci., 2019 33(1): 87-95 doi: 10.13128/ahs-23794 comparison of salinity effects on grafted and non-grafted eggplants in terms of ion accumulation, mda content and antioxidative enzyme activities m. talhouni 1 (*), k. sönmez 2, s. kiran 3, r. beyaz 4, m. yildiz 5, ş. kuşvuran 6, ş.ş. ellialtıoğlu 7 1 national agricultural research center (narc), horticulture directorate, amman, jordan. 2 eskişehir osmangazi university, faculty of agriculture, department of horticulture, eskişehir, turkey. 3 soil, fertilizer and water resources central research institute, ankara, turkey. 4 ahi evran university, faculty of agriculture, department soil science and plant nutrition, kırşehir, turkey. 5 ankara university, faculty of agriculture, department of agronomy, ankara, turkey. 6 cankiri karatekin university, kizilirmak vocational high school, cankiri, turkey. 7 ankara university, faculty of agriculture, department of horticulture, ankara, turkey. key words: apx, cat, eggplant, lipid peroxidation, nacl, na+, cl-, k+, ca++, scion/rootstock combination, sod. abstract: grafting onto resistant/tolerant rootstocks is known to alleviate the negative effects of abiotic stress factors like salinity by enhancing their enzymatic antioxidant defense system and having more efficient nutrient uptake. t h i s s t u d y w a s c a r r i e d o u t u n d e r g r e e n h o u s e c o n d i t i o n s , d i f f e r e n t rootstock/scion eggplant combinations were grown under two salinity treatments 1.8-2 ds/m (control) and 6-7 ds/m (stress) with seven eggplant genotypes as rootstocks (commercial and turkish genotypes). two genotypes were used as the scion. leaf mda and ions (na+, cl-, k+ and ca++) content, antioxidant enzymes activity were evaluated as indicators for plant tolerance level. it was found that the rootstock-grafted plants were more efficient in preventing na+ ions to be transferred to the plants upper parts and had higher sod, cat, and apx activity levels compared to the selfand non-grafted plants which resulted in better tolerance and growth in these plants. 1. introduction eggplant (solanum melongena l.) is an important vegetable crop (*) corresponding author: manar.alhouni@gmail.com citation: talhouni m., sönmez k., kiran s., beyaz r., yildiz m., kuşvuran ş., ellialtioğlu ş.ş., 2019 comparison of salinity effects on grafted and non-grafted effplants in terms of ion accumulation, mda content and antioxidative enzyme activities. adv. hort. sci., 33(1): 87-95 copyright: © 2019 talhouni m., sönmez k., kıran s., beyaz r., yıldız m., kuşvuran ş., ellialtıoğlu ş.ş. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 9 april 2018 accepted for publication 12 december 2018 ahs advances in horticultural science adv. hort. sci., 2019 33(1): 87-95 88 worldwide, its production reaches about 48.5 million ton while in turkey eggplant production reaches 800900 thousand tons (tuik, 2015). one of the most stress factors that affect eggplant production is salinity. salinity is a major environmental factor limiting plant growth and productivity, especially in the arid a n d s e m i a r i d r e g i o n s ( p a r i d a a n d d a s , 2 0 0 5 ) . conflicting literature exists on eggplant tolerance to soil salinity and this difference could be related to the varieties or cultivars used and to the different environmental conditions in those studies (ünlükara et al., 2010). overcoming salt stress problems would have a positive impact on agriculture production. attempts have been made to improve salt tolerance of crops by traditional breeding programs but with limited success due to the complexity of the trait (flowers, 2004). even the use of genetic transformation of plants to raise their tolerance, despite its success in some cases (rus et al., 2001); the complexity of the trait and lack of public acceptance, limiting its wide spread and use (munns, 2002). one way of avoiding or reducing losses in production caused by salinity would be to use the tolerant rootstocks. in relation to salt tolerance, many studies have been conducted to determine the response of grafted plants to salinity. according to these studies, the improvement of salt tolerance by grafting is related to the capability of rootstocks to reduce toxicity of na+ and/or clthrough exclusion and/or reduction of absorption of clby the roots, and the replacement or substitution of total k+ by total na+ in the foliage (estañ et al., 2005; martinez-rodriguez et al., 2008). it is supposed that useful rootstocks should be able to reduce the uptake and transport of saline ions to the shoot, which will slow or prevent the accumulation of the toxic salt ions in the leaves (usanmaz and abak, 2018). salt stress causes a range of adverse effects in plants, mainly ionic disorders, osmotic stress and nutritional imbalance. a common feature of these effects is the overproduction of reactive oxygen species (ros) such as singlet oxygen (1o2), superoxide anion (o2−), hydrogen peroxide (h2o2), and hydroxyl radical (oh) which are highly reactive and toxic and cause damage to proteins, lipids, carbohydrates and dna which ultimately results in oxidative stress (ashraf and foolad, 2007). salt stress causes stomatal closure, which reduces the co2/o2 ratio inside leaf tissues and inhibits co2 fixation (hernández et al., 2000). plants antioxidant enzymes such (superoxide dismutase, sod; catalase, cat; ascorbate peroxidase, apx; glutathione reductase, gr; monodehydroascorbate reductase, mdhar; dehydroascorbate reductase, dhar; glutathione peroxidase, gpx; guaicol peroxidase, gopx and glutathione-stransferase, gst) work in concert to control the cascades of uncontrolled oxidation and protect plant cells from oxidative damage by scavenging of ros (scandalios, 1997; dixit et al., 2001; shalata et al., 2001). superoxide dismutase (sod) reacts with the superoxide radical at almost diffusion-limited rates to produce h2o2 (scandalios, 1993). h2o2 is scavenged by peroxidases, especially ascorbate peroxidase (apx), and catalase (cat). in the present study, we exposed non-grafted, self-grafted and rootstock-grafted eggplants to conditions of salt stresses to investigate whether grafted plants could improve tolerance to salinity by alleviating the expression of antioxidant enzymes. using local genotypes in breeding programs is of vital importance to find new rootstocks with the ability to alleviate the effects of salinity and reduce its effect on plant growth and productivity. in the present study, two eggplant genotypes were grafted onto seven rootstocks to compare the ability of the different rootstock genotypes in increasing eggplant tolerance as it is related to the ability of the rootstock to 1) control the transport of na+ and cl-, 2) to maintain better k+ and ca++ uptake, 3) to increase the enzymatic defense mechanism scavenging the ros induced by oxidative stress resulting in less leaf malodialdehyde (mda) content. another goal was to evaluate the potential of the turkish genotypes burdur and mardin as rootstocks under salinity conditions in comparison to the commercial genotypes. 2. materials and methods the field part of the experiment was carried out between august-november 2014, in a 300 m2 plastic house belongs to the private sector (genta general agricultural products marketing co.) in antalyaturkey while laboratory works and analysis were carried out in ankara university faculty of agriculture, departments of horticulture and agronomy laboratories. plant material two eggplant (solanum melongena l.) genotypes, naomi f1 cv., a commercial cultivar, and artvin, a salt sensitive breeding line, were used as scions. and for rootstocks five commercial genotypes were used, talhouni et al. salinity effects on grafted and non-grated effplants 89 agr703 (solanum aethiopicum), köksal f1, yula f1 and vista (s. incanum x s. melongena hybrids), and hawk (s. torvum), these genotypes are the most common used rootstocks in grafting eggplants due to their tolerance. in addition two local salt-tolerant genotypes burdur and mardin (s. melongena l.) were used as rootstocks, their tolerance to salinity were confirmed in previous studies screening for turkish tolerant genotypes (yaşar, 2003). grafting and salt treatment eggplant seeds were sown in germination trays filled with 2:1 peat:perlite. after sowing trays were kept under controlled conditions of temperature (25°c) and humidity (80%). when the seedlings reached 2-4 true leaves stage, grafting was carried out. the tube-grafting was used in this study because i t i s t h e m o s t w i d e l y u s e d g r a f t i n g m e t h o d i n solanaceae family (rivard et al., 2009). then grafted seedlings were kept under controlled conditions of humidity (90%) for four days, then seedlings were transferred to the greenhouse under shading before planting for acclimatization. ten days after grafting seedlings were placed in the plastic house and were ready for transplantation in 8 l plastic pots filled with 3:1 perlite:vermiculite. in one of our earlier studies we examined these genotypes tolerance under salinity conditions at seedling stage in a hydroponic experiment (talhouni, 2016), in this study we wanted to assess these same genotypes at flowering and fruit set stage. when plants reached the flowering and fruit set stage salinity treatment began. 6-7 dsm-1 water ec was used as the stress treatment by solving nacl into the nutrition solution; according to the volume of barrels used in fertigation about 8 kg of iodine-free sodium chloride were required , while for the control the ec level was kept at 1.8-2 dsm-1 (no nacl was added). leaf ion concentrations after 60 days from salinity stress application, samples were taken from the control and the salinity treated plants for the different analysis. for the leafna+, cl-, k+ and ca++ concentration measurements, leaves were dried at 65°c for 48 hours, grounded, dissolved in 1% (v/v) hcl. for the analysis of na+, k+ and ca++ contents, atomic absorption spectrophot o m e t e r ( v a r i a n s p e c t r a a a 2 2 0 f s ) w a s u s e d (kuşvuran, 2012). while for cl-, titration procedure was followed as described by taleisnik et al. (1997) using buchler cotlove chloridometer. enzyme extractions and assays fresh leaf samples were submersed for 5 min in liquid nitrogen. the frozen leaves were kept at -80°c for further analyses. enzymes were extracted from 0.5 g leaf tissue using a mortar and pestle with 5 ml extraction buffer containing 50 mm potassium phosphate buffer, ph 7.6 and 0.1 mm na-edta. the homogenate was centrifuged at 15,000 g for 15 min and the supernatant fraction was used to assay for the various enzymes. all steps in the preparation of enzyme extracts were performed at 4°c. apx activity was determined by measuring the consumption of ascorbate by following absorbance at 290 nm. one unit of apx activity was defined as the amount of enzyme required to consume 1 μmole ascorbate min-1 (cakmak and marschner, 1992). s o d w a s a s s a y e d a c c o r d i n g t o c a k m a k a n d marschner (1992), by monitoring the superoxide radical-induced nitro blue tetrazolium (nbt) reduction at 560 nm. one unit of sod activity was defined as the amount of enzyme which causes 50% inhibition of the photochemical reduction of nbt. catalase (cat) activity was measured as the decline in absorbance at 240 nm due to the decomposition decline of extinction of h2o2. the reaction was started by adding h2o2. lipid peroxide content lipid peroxidation was measured as the amount of malondialdehyde (mda) determined by the thiobarbituric acid (tba) reaction. frozen samples were homogenized in a pre-chilled mortar with two volumes of ice-cold 0.1% (w/v) tricloroacetic acid (tca) and centrifuged for 15 min at 15000 x g. assay mixture containing 1 ml aliquot of the supernatant and 2 ml of 0.5% (w/v) thiobarbituric acid in 20% (w/v) tricloroacetic acid (tca) was heated to 95°c for 30 min and then rapidly cooled in an ice-bath. after centrifugation (10000 x g for 10 min at 4°c), the supernatant absorbance (532 nm) was read and values corresponding to non-specific absorption (600 nm) were subtracted. the mda content was calculated according to the molar extinction coefficient of mda (155 mm-1 cm-1). statistical analysis randomized complete block design with three replicates was used. each replicate included 108 pots (18 rootstock/scion combination * 2 salinity level * 3 plants of each combination) with one plant/pot. data were subjected to duncan’s multiple range tests adv. hort. sci., 2019 33(1): 87-95 90 using the sas program (p≤0.01)(version 6.12, sas institute inc., cary, usa). 3. results na+ concentrations in general, the concentrations of na+ in the leaves increased significantly due to increased nacl concentration (table 1) with significant differences between grafting combinations, and a significant ‘salinity x scion/rootstock combination’ interaction at p≤0.01. after 60 days of salinity stress application, the combinations that showed the least concentrations o f n a + i n t h e i r l e a v e s w e r e ; ( r o o t s t o c k / s c i o n ) k ö k s a l / a r t v i n , v i s t a / n a o m i , k ö k s a l / n a o m i , agr703/naomi and agr703/artvin (9.75, 9.65, 9.92 1 0 . 0 2 a n d 1 0 . 4 6 µ g / m g f w r e s p e c t i v e l y ) w i t h increase rates of 3511, 1035, 4623, 1721, 3506% respectively (fig. 1) which indicated that these rootstock genotypes could limit na+ to the leaves more successfully. no significant effects of grafting per se were noticed, no differences were observed between non-grafted and self-grafted combinations. clconcentration as in leaf na+ concentration, leaf clconcentration also increased significantly under salinity treatment in all combinations, with a significant differences and a significant ‘salinity x scion/rootstock combination’ interaction at p≤0.01 (fig. 1). the highest clconcent r a t i o n w a s o b s e r v e d i n a r t v i n , n a o m i , (rootstock/scion) artvin/artvin, naomi/naomi, mardin/artvin, mardin/naomi (11.83, 10.81, 9.54, 9.06, 8.83 8.12 µg/mg fw) combinations, while the lowest was observed köksal/artvin, agr703/artvin, agr703/naomi and burdur/artvin (5.06, 5.06, 5.27, 5.49 µg/mg fw respectively) and there were no significant differences between nonand self-grafted combinations (table 1). k+ concentrations the amounts of k ion measured in leaf samples taken from plants treated with ec 6-7 ds / m nacl gave lower values in some combinations than control plants (table 1), the highest decrease in leaf k+ content was observed in non-grafted artvin (-9.82%).followed by (rootstock/scion) artvin/artvin with decrease rate of (-6.25%) (fig. 1). the highest values were obtained from (rootstock/scion); köksal/artvin, köksal/naomi, mardin/naomi, agr703/artvin, yula/naomi (4.86, 4.64, 4.36, 4.30, 4.27 µg/mg fw, respectively) (table 1). among these combinations, köksal/artvin, agr703/artvin and yula/naomi had the highest k+ ions increase rate (47.27%, 22.16%, 20.96%) (fig. 1). combinations that gave the lowest k+ ion amount measurements were (rootstock/scion) hawk/artvin, naomi, burdur/naomi, naomi/naomi, table 1 leaf ions concentration (µg/mg fw); in the different grafting combinations under control and salinity treatments grafting combination na+ k+ ca++ clcontrol salinity control salinity control salinity control salinity köksal/artvin 0.27±0.00 a 9.75±0.25 a 3.30±0.15 ab 4.86±0.33 b 0.50±0.03 f 0.48±0.03 e 0.06±0.02 a 5.06±0.64 a agr703/artvin 0.29±0.03 a 10.46±0.53 a-c 3.52±0.34 a-c 4.30±0.26 ab 0.49±0.02 f 0.41±0.04 de 0.06±0.02 a 5.06±0.90 a vista/artvin 0.51±0.03 ab 12.14±0.86 b-e 3.89±0.11 a-e 3.93±0.53 a 0.46±0.02 b-f 0.41±0.03 de 0.12±0.04 a-d 6.30±1.10 a-c yula/artvin 0.24±0.02 a 11.08±0.58 a-e 3.74±0.18 a-e 3.89±0.12 a 0.43±0.01 a-c 0.35±0.03 b-d 0.19±0.02 c-f 6.63±0.75 a-d burdur/artvin 0.49±0.01 b 10.91±0.53 a-d 3.57±0.12 a-c 3.97±0.24 a 0.49±0.02 ef 0.39±0.05 c-e 0.09±0.03 ab 5.49±0.13 ab mardin/artvin 0.61±0.09 b-d 12.36±0.29 c-e 3.70±0.23 a-d 4.22±0.09 ab 0.44±0.01 a-d 0.35±0.06 b-d 0.14±0.03 a-e 8.83±1.08 c-f hawk/artvin 0.26±0.05 a 10.89±0.18 a-d 4.05±0.28 c-e 3.84±0.31 a 0.46±0.02 c-f 0.41±0.03 de 0.11±0.02 a-c 6.45±1.50 a-d artvin/artvin 0.83±0.08 e-g 12.36±0.70 c-e 4.32±0.05 de 4.05±0.15 ab 0.41±0.01 a 0.35±0.02 b-d 0.19±0.02 c-f 9.54±1.21 e-g artvin 0.88±0.04 fg 12.77±0.46 de 4.38±0.28 e 3.95±0.13 a 0.40±0.01 a 0.29±0.02 ab 0.22±0.02 ef 11.83±0.62 g köksal/naomi 0.21±0.02 a 9.92±0.37 ab 4.11±0.22 c-e 4.64±0.23 ab 0.49±0.02 d-f 0.31±0.01 a-c 0.09±0.04 ab 6.06±0.38 ab agr703/naomi 0.55±0.12 ab 10.02±1.26 ab 3.87±0.06 a-e 3.96±0.28 a 0.48±0.01 d-f 0.36±0.02 b-d 0.10±0.04 a-c 5.27±0.55 a vista/naomi 0.85±0.05 fg 9.65±0.71 a 3.67±0.15 a-d 4.15±0.08 ab 0.44±0.02 a-e 0.36±0.02 b-d 0.15±0.03 b-f 6.13±0.19 ab yula/naomi 0.73±0.04 d-f 10.93±0.97 a-d 3.53±0.10 a-c 4.27±0.29 ab 0.42±0.01 a-c 0.25±0.02 a 0.18±0.05 b-f 7.51±0.54 a-e burdur/naomi 0.49±0.05 b 11.14±1.16 a-e 3.50±0.36 a-c 3.88±0.23 a 0.48±0.01 d-f 0.40±0.01 c-e 0.13±0.01 a-d 6.37±1.01 a-c mardin/naomi 0.68±0.04 c-e 12.07±0.13 b-e 3.95±0.32 b-e 4.36±0.24 ab 0.43±0.01 a-c 0.34±0.03 a-d 0.14±0.03 a-e 8.12±0.63 b-e hawk/naomi 0.80±0.10 e-g 11.14±1.20 a-e 3.80±0.11 a-e 4.09±0.27 ab 0.41±0.01 ab 0.35±0.01 b-d 0.20±0.02 d-f 7.29±0.24 a-e naomi/naomi 0.94±0.04 g 12.34±0.84 c-e 3.33±0.20 ab 3.96±0.36 a 0.40±0.01 a 0.34±0.01 a-d 0.22±0.02 ef 9.06±1.06 d-f naomi 0.52±0.06 ab 13.30±0.73 e 3.27±0.29 a 3.85±0.30 a 0.40±0.02 a 0.28±0.03 ab 0.23±0.03 f 10.81±1.41 fg cv (%) 43.07 9.78 8.81 6.83 8.01 15.22 38.38 27.17 treatment ** ** ** ** combination ** ** ** ** combination x treatment ** ** ** ** talhouni et al. salinity effects on grafted and non-grated effplants 91 agr703/naomi, artvin (3.84, 3.85, 3.88, 3.96, 3.96, 3.96 85 µg/mg fw). no significant differences were obtained for self-grafted combinations. ca++ concentrations ec 6-7 dsm-1 nacl treatment led to a decrease in leaf ca++ concentration in all combinations (fig. 1) with significant differences between treatments and combination (p≤0.01) and with a significant ‘salinity x scion/rootstock combination’ interaction. the highest leaf ca++ concentrations were obtained in (roots t o c k / s c i o n ) a g r 7 0 3 / a r t v i n , k ö k s a l / a r t v i n , burdur/naomi, hawk/artvin and vista/artvin (0.48, 0.42, 0.41, 0.41 µg/mg fw, respectively). no significant effects were observed in self-grafted combinations. antioxidant enzyme activities salt treatments increased superoxide dismutase (sod) activities in all of the plants (table 2). however, fig. 1 leaf ions concentration (µg/mg fw); in the different grafting combinations under control (blue) and salinity treatments (red). table 2 sod, cat, and apx enzymes activities (µmol/g fw) in the different grafting combinations under control and salinity treatments eggplant sod cat apx mda control salinity control salinity control salinity control salinity köksal/artvin 217.03±8.00 a 635.46±27.84 gh 139.90±13.74 ab 593.91±28.44 h 2042.77±12.67 h 5387.96±560.98 e 5.12±0.29 a-d 10.44±0.54 a agr703/artvin 207.06±6.28 a 645.64±38.29 h 129.82±10.00 ab 576.91±27.73 gh 2020.91±97.38 h 4820.66±545.40 de 5.02±0.13 ad 10.23±1.24 a vista/artvin 215.92±11.75 a 464.88±44.39 c-f 113.03±11.99 a 484.17±25.96 c-f 1758.36±28.48 e-h 3829.29±568.57 a-d 4.86±0.27 ab 12.83±0.26 ab yula/artvin 193.54±6.29 a 414.49±26.02 b-d 103.86±6.46 a 409.13±1.88 bc 1765.71±141.18 e-h 3307.96±550.14 a-c 5.12±0.26 a-d 10.14±0.61 a burdur/artvin 208.97±10.32 a 508.88±55.72 d-f 131.44±4.26 ab 548.67±19.02 e-h 1882.51±52.64 f-h 3839.81±57.28 a-d 5.19±0.10 a-d 11.16±0.44 a mardin/artvin 195.91±10.91 a 470.99±16.48 c-f 138.76±14.25 ab 434.69±6.55 b-d 1395.16±235.41 b-d 3105.27±93.76 ab 5.49±0.25 a-e 11.50±0.21 a hawk/artvin 203.44±10.29 a 436.05±50.00 b-e 118.68±15.42 a 464.12±25.38 c-e 1926.37±199.83 gh 4303.30±32.77 b-e 4.80±0.31 a 11.95±0.98 ab artvin/artvin 186.29±10.04 a 341.33±58.60 ab 123.87±6.38 ab 350.16±52.43 ab 1134.59±102.54 ab 2731.28±37.49 a 6.32±0.13 fg 12.54±0.15 ab artvin 189.95±12.89 a 295.15±16.97 a 113.49±7.29 a 374.84±27.07 b 903.43±37.94 a 2614.61±37.06 a 6.34±0.33 g 14.81±1.23 b köksal/naomi 190.92±25.52 a 553.92±32.30 f-h 116.33±14.37 a 553.17±30.84 f-h 1968.59±112.34 g-h 4427.68±48.03 c-e 4.95±0.18 a-c 10.93±1.20 a agr703/naomi 213.81±4.16 a 491.39±39.05 d-f 165.69±17.37 b 503.21±14.17 d-g 1924.53±144.55 gh 4326.69±614.68 b-e 4.87±0.23 ab 10.54±1.04 a vista/naomi 194.09±17.87 a 457.84±29.92 c-f 124.91±15.83 ab 405.95±29.79 bc 1547.82±154.87 c-f 3072.32±48.96 ab 5.59±0.25 b-f 11.18±0.16 a yula/naomi 200.41±10.91 a 427.66±11.33 b-e 99.34±6.30 a 401.18±19.92 bc 1499.31±168.98 c-e 3663.61±635.13 a-d 5.26±0.28 a-d 11.06±0.93 a burdur/naomi 196.48±16.49 a 534.86±35.10 e-g 117.08±28.01 a 503.48±27.99 d-g 1813.06±32.67 e-h 3789.02±90.92 a-d 5.09±0.39 a-d 10.36±0.85 a mardin/naomi 191.44±20.27 a 378.05±10.08 a-c 104.70±15.90 a 367.67±14.99 ab 1608.48±112.47 d-g 3448.81±529.02 a-c 5.71±0.19 d-g 10.86±0.74 a hawk/naomi 193.35±9.53 a 421.65±43.89 b-d 140.49±24.51 ab 360.07±7.01 ab 1695.49±80.45 d-h 4471.40±613.60 c-e 4.80±0.12 a 10.89±0.92 a naomi/naomi 201.11±19.08 a 272.74±22.75 a 125.68±6.97 ab 366.67±43.39 ab 1205.04±58.49 a-c 2693.56±51.33 a 6.05±0.16 e-g 14.66±2.05 b naomi 199.68±10.99 a 271.57±36.99 a 123.98±8.26 ab 289.02±39.88 a 1033.83±54.71 ab 2782.04±593.00 a 5.66±0.18 c-g 14.91±1.65 b cv (%) 4.65 24.53 12.87 19.94 21.9 21.7 9.37 13.58 treatment (t) ** ** ** ** combination (c) ** ** ** ** c x t ** ** ** ** 92 adv. hort. sci., 2019 33(1): 87-95 in the rootstock-grafted plants, sod activity increased faster and with higher rates than in the nonand selfgrafted plants. köksal/artvin and agr703/artvin had the highest sod activity level (645.64 and 635.46 u m o l / m i n / m g f w r e s p e c t i v e l y ) . f o l l o w e d b y köksal/naomi, burdur/naomi, and burdur/artvin c o m b i n a t i o n s ( 5 5 3 . 9 2 , 5 3 4 . 8 , a n d 5 0 8 . 8 8 umol/min/mg fw respectively). the highest increase rate in sod activity was obtained for agr703/artvin combination (211.8%) while naomi/naomi and naomi combinations had the lowest increase with rates of 35.62 and 36% respectively (fig. 2). naomi and self-grafted plants had the lowest values of the enzyme activity with no significant differences between nonand self-grafted combinations. catalase (cat) activity increased under salt treatment in all combinations compared to the control plants (table 2), (rootstock/scion) köksal/artvin and agr703/artvin had the highest cat activity (593.91 and 576.91 μmol/min/mg fw respectively). followed by köksal/naomi, burdur/artvin, agr703/naomi and burdur/naomi. on the other hand artvin and naomi (non-grafted) and the self-grafted plants had the lowest cat activity. burdur/naomi had the highest increase rate in cat activity (330%) while non-grafted naomi had the lowest rate (133%) (fig. 2). no significant differences were observed between nonand self-grafted combinations. under nacl-salinity conditions, ascorbate peroxidase (apx) activity was increased in all plants (fig. 2). however, agr703/artvin and köksal/artvin had the highest apx activity levels indicating their better tolerance level (4820.66 and 5387.96 μmol/min/mg fw respectively) as shown in table 2, while the lowest apx enzyme activity was found in non-grafted plants naomi and artvin indicating their poor tolerance. no significant differences were obtained between nonand self-grafted combinations. malondialdehyde (mda) salinity resulted in a significant increase on the mda content compared to the controls due to oxidative stress induced peroxidation (fig. 2). with regard to the mda, significant differences were found among grafting combinations and a significant ‘salinity x rootstock/scion combination’ interaction at p≤0.01 (table 2). according to the results naomi, naomi/naomi and artvin were found to be more sensitive with mda content increase rate of 163.4, 142.3, 133.6% respectively (fig. 2). no significant differences were observed between nonand self-grafted combinations. 4. discussion and conclusions as a result of high salinity level, na+ and clions can be accumulated in toxic levels in plant tissues depends on the plant species. even though these two ions are suitable for osmotic adjustment, excess concentrations will be toxic enough to prevent plant growth. in the study, combinations vista/artvin, köksal/artvin, vista/naomi, and köksal/naomi had less na+ accumulation in their tissues which indicates that these combinations were able to keep na+ ions a w a y f r o m t h e i r l e a v e s . c o n c e r n i n g l e a f c l -, vista/artvin combination had the least concentration. grafted plants tend to hold na+ and clions in their root tissues preventing them from being translocated to the shoots and leaves in high concenfig. 2 sod, cat and apx enzymes activities (µmol/g fw) in the different grafting combinations under control (blue) and salinity treatments (red). talhouni et al. salinity effects on grafted and non-grated effplants 93 trations (levitt, 1980; estañ et al., 2005). most vegetables like, cucumbers, melons, tomatoes and eggplant are injured by excess na+ ions (tester and davanport, 2003). in giuffrida et al. (2009), increased nacl level led to na+ concentration increase in tomato leaves and fruits. kuşvuran et al. (2007), under salinity conditions na+ and clwere accumulated in higher rates in the salinity-sensitive melon plants compared to the salinity-tolerant ones. k+ decrease rate was different between the different combinations. akinci and lösel (2012), different eggplant genotypes showed different tolerant level to salinity. pala cv. showed better tolerance to salinity compared to kemer and aydın siyahı cultivars with better k+/na+ ratio. yaşar et al. (2006), in tissue culture study on eggplant, there was an increase in na+ and cltissues concentrations with decrease in k+ and ca++ due to salinity. however there were significant differences between different genotypes, the salinitytolerant mk and bb showed higher k+ and ca++ concentrations compared to the salinity sensitive gr and ah genotypes. consequently mk and bb had higher k+ and ca++ uptake decreased under stress treatment (savvas and lenz, 2000). in a similar study on pepper, the same results were obtained (aktaş et al., 2002). combinations with the highest leaf ca++ concent r a t i o n s u n d e r s a l i n i t y w e r e v i s t a / n a o m i , agr703/naomi and burdur/artvin. and maybe for this reason burdur genotype can be considered a potential rootstock for increasing eggplant tolerance against salinity. the decrease in ca++ uptake due to nacl salinity was observed by many authors, and in contrary to k+, the decrease was not due to the competition between na+ and k+ at the absorption site on the root surface, it was always found because of the decline in the transpiration rate under stress conditions (maggio et al., 2007). in gao et al. (2005), under stress conditions of low temperatures (5°c) grafted eggplants maintained higher leaf ca++ concentrations compared to the non-grafted plants which gave the grafted plants higher tolerance under such stress conditions. plant adaptation to salinity may depend on different mechanisms, including the capacity to maintain high levels of antioxidants and/or through the induction of antioxidant enzymes (sod, cat, gr, and apx, etc.) (sevengör, 2010). in the present study, rootstock-grafted plants had higher activity of antioxidant enzymes under salinity conditions, which was translated to lower mda content in their leaves which means these combinations, were less affected by the ros-induced lipid peroxidation and they were more tolerant to salinity than the nonand self-grafted plants. mda content always found higher in salinity-sensitive plants compared to salinity-tolerant ones (yaşar, 2003; kuşvuran et al., 2015) and a significant relation between mda content and antioxidant enzymes activity is first proven by shalata and tal (1998). meloni et al. (2001) in cotton, yaşar (2003) in eggplant, doğan (2004) in tomato, and sevengör (2010) in pumpkin, all found that mda content was low in plants with high antioxidant enzymes activity under salinity stress conditions. in this study, antioxidant enzymes activity showed a higher increase in rootstock-grafted plants compared to the nonand self-grafted plants, this increase was significantly different between grafting combinations. in another study where cucumber was grafted onto salinity-tolerant rootstock, h2o2 level w a s f o u n d t o b e l o w , w h e r e a s s o d , c a t , p o d enzymes activity level were found higher. öztekin and tuzel (2011), cat activity level differed according to the rootstock genotypes, but always was higher in the grafted plants compared to the non-grafted plants. all results indicated that grafting per se had no significant role in alleviating negative effects of salinity as there were no significant differences between nonand self-grafted combinations in all parameters measured in this study. in general, local genotypes (landraces) are adapted to prevailing environmental conditions like salinity. in this work, the local turkish genotype burdur showed a good potential to compete commercial genotypes. on the other hand, mardin was way behind and did not show enough potential in this study. acknowledgements this research was supported by the coordination u n i t o f t h e s c i e n t i f i c r e s e a r c h p r o j e c t s o f t h e university of ankara (project no: 15h0447001). references akinci ş., lösel d.m., 2012 plant water-stress response mechanisms, pp. 15-42. in: rahman i.m.m. 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stress phd dissertation, yüzüncü yıl university, institute of natural and applied sciences, van, turkey, pp. 138. yaşar f., ellialtioğlu s., kuşvuran s., 2006 ion and lipid peroxide content in sensitive and tolerant eggplant callus cultured under salt stress. europ. j. hort. sci., 71(4): 169-172. impaginato 61 adv. hort. sci., 2020 34(1): 61­69 doi: 10.13128/ahsc­8402 production of seed­propagated com­ pact potted corylopsis plant in one year j.h. kim 1, j.k. suh 1, s.t. yoon 2, m.s. roh 3 (*) 1 department of environmental horticulture, dankook university, cheonan, chungnam, 31116, korea. 2 department of crop science and biotechnology, dankook university, cheonan, chungnam, 31116, korea. 3 the institute of natural resources development, mokpo national university, cheonggye‐myeon, muan‐gun, jeonnam, 58554, korea. key words: corylopsis coreana, corylopsis sinensis var. calvescens, new orna­ mental plant, pinching, plant growth regulator, slow release fertiliz­ er. abstract: the feasibility to produce compact corylopsis sinensis var. calvescens and c. coreana plant in a 10 cm pot in one year from transplanting seedlings with maximized number of short shoots and inflorescences was investigated. corylopsis sinensis var. calvescens was selected as a suitable species to produce compact plant with inflorescences. slow release fertilizer (srf) at a rate of 0, 0.125, 0.25, and 0.5 g per pot was applied to the surface of the growing medi­ um (expt. 1). shoots were pinched 2 (feb. 28), 4, 6, and 8 weeks (may 16) (expt. 2) after transplanting, and ancymidol, paclobutrazol, chlormequat, and daminozide plant growth retardants were treated (expt. 3). application of a srf at 0.5 g per pot and pinching four times at 2­week intervals before may 16 effectively increased the flowering percentages and the number of stems with inflorescences, to accelerate flowering, and also produced a compact plants. paclobutrazol at 10­20 mg/l applied as soil drench was effective in inhibiting stem elongation in the first year; however, higher concentrations should be avoided to prevent excessive reduction in the growth of shoots and production of malformed inflorescences. 1. introduction the genus corylopsis siebold & zucc., commonly known as winter hazel that flowers early in the spring in china and korea, is a shrub or small tree. most of the corylopsis species grows tall reaching a height of 2­4 meters (bean and anisko, 2014). flowers are bisexual and seeds are produced. among 29 species, 19 species are endemic in china (zhang et al., 2003). chinensis sinensis hems. var. calvescens rehder & e.h. wilson is growing in the mountains in guangxi, sichuan, and jiangxi, among other provinces in china (zhang et al., 2003) and c. coreana uyeki (son et al., 2016) in a rather restricted area in korea. all species are deciduous shrubs producing light yellow pendant racemes (inflorescence) measuring (*) corresponding author: marksroh@gmail.com citation: kim j.h., suh j.k., yoon s.t., roh m.s., 2020 ­ production of seed‐propagated compact potted corylopsis plant in one year. ­ adv. hort. sci., 34(1): 61­69. copyright: © 2020 kim j.h., suh j.k., yoon s.t., roh m.s. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 19 june 2019 accepted for publication 25 october 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(1): 61­69 62 about 5 cm in length, followed by appearance of leaves (fig. 1). corylopsis, one of many germplasms native and indigenous to china that includes an endangered acer pentaphyllum diels (roh et al., 2008 b), are not well known to horticulturist, growers, and landscape industry, but has a great potential to develop as a new ornamental plant. although corylopsis may be available from rooted cuttings or tissue­cultured propagules for a mass propagation (moon et al., 2002; koh and lim, 2006), the success of the rooting of cuttings depends on the season when cuttings were collected and may not provide a large number of plants (kwon et al., 2011) and acclimatization of tissue culture propagules is not easy (moon et al., 2002; koh and lim, 2006). therefore, seeds are a viable alternative source for mass propagation and for forcing seedlings to flower. suitable species should first be identified and then excessive stem elongation must be controlled using plant growth retardants (currey and lopez, 2016), and pinching combined with plant growth retardant treatment (jeong, 2000). stem elongation can be inhibited by practices such as pinching shoots in many floral and ornamen­ tal plants, resulting in short plant height (lee et al., 2006; latimer and whipker, 2013). growth and flow­ ering is also affected by treatment with plant growth regulators. among the many growth retardants, ancymidol (ɑ­cyclopropyl­ɑ­(4­methoxyphenyl)­5­ pyrimidinemethanol), daminozide (butanedioic acid mono(2,2­dimethylhydrazide), chlormequat (2­ chloro­n,n,n­trimethylethanaminium chloride), and paclobutrazol [(2rs, 3rs)­1­(4­chlorophenyl)­4,4­ dimethyl­2­(1h­1,2,4­triazol­1­yl) penta­3­ol] have been used in many floral and ornamental plants to inhibit stem elongation (currey and lopez, 2016). generally, ancymidol and paclobutrazol is effective when applied as a soil drench, and daminozide and chlormequat when applied as a foliar spray. germination of corylopsis seeds as affected by warm and cold stratification and the x­ray imaging to separate full seeds from empty seeds is well docu­ mented (kim et al., 2015, 2017, 2018). to produce compact and flowering corylopsis plants in small pots in one year after transplanting seedlings, selection of proper species and the most suitable cultural prac­ tices should be identified. however, there is no report on the growth and flowering of corylopsis starting from small propagules regardless of propaga­ tion methods: seed propagation, rooting of cuttings, and in vitro propagation. shortening the total pro­ duction time from 2­3 years to one year while ensur­ ing the qualities of plants at flowering from seedlings in lilium longiflorum thunb. bulbils in l. ×elegans thunb. and tissue­cultured propagules in interspecif­ ic hybrids between l. longiflorum and l. ×elegans was reviewed (roh, 1992, 1996). production of corylopsis in a small pot with inflo­ rescence will attract and enable consumers to pur­ chase at the nursery or the garden center in the early spring, and then plant them in the garden to enjoy the beauty of flowers for many years. the objectives of this research were (1) to select the species to grow starting from seeds in small pots, and to study the effect of (2) slow release fertilizer (srf) treatments, (3) the pinching frequencies, and (4) plant growth retardant treatments to produce compact seed­prop­ agated corylopsis plants in 10 cm pots in one year from transplanting seedlings. 2. materials and methods preliminary field evaluation to select a suitable species for final evaluation seeds of 45 accessions (data not presented) including c. glabrescens (na50804, longwood 1997­ 0068b, longwood chimes), c. spicata (na37208, na40102, arnold 7950a), c. pauciflora (na37205, l o n g w o o d 1 9 4 4 ­ 0 2 1 3 * h ) , a n d c . v i e t c h i a n a (na37208, na65619) were sown between oct. 2 and nov. 2 and planted into 10 cm pot filled with promix bm (premier horticulture inc., quakertown, pa, usa) between mar. 1 and apr. 2, and grown in the field. the final evaluation based on the number of plants that flowered and the growth habits, c. coreana and c. sinensis var. calvescens (na 57391) (roh et al., fig. 1 ­ appearance of c. sinensis var. calvescens at anthesis pro­ duced in a 10 cm pot from seeds. clusters of flower buds (inflorescences) are well developed on long shoots. kim et al. ‐ compact corylopsis plant production 63 2008 a) were selected (table 1) for evaluation in the next three experiments. effect of slow release fertilizer treatment on growth and flowering (expt. 1) a b o u t 2 0 0 s e e d s e a c h o f c . s i n e n s i s v a r . calvescens and c. coreana were sown on oct. 20, 2009 in a 15 cm pot and received temperature treat­ ments [20oc (oct. 21 ­ dec. 1) and 5oc (dec. 2, 2009 ­ feb. 16, 2010)]. one seedling was transplanted per 10 cm pot filled with promix bm on mar. 4, 2010. on mar. 18, 2010 when seedlings formed 4 nodes, the main shoot was pinched leaving two pair of leaves. slow release fertilizer (srf; osmocote, 14 n ­ 6.2 p ­ 11.6k; scotts co., marysville, oh, usa) was applied to the surface of the growing medium at transplant­ ing seedlings at a rate of 0, 0.125, 0.25, and 0.5 g per 10 cm pot (table 2). during the culture, plants were fertilized with 1.33 g/l of 15n ­ 7p ­ 12.8k water solu­ ble fertilizer once a month. greenhouse day temperature was maintained at 21­22oc on oct. 1, 15­17oc on nov. 1, 13­14oc on nov. 16, 10­12oc on dec. 1, 7­8oc on dec. 16, 2010 and at 4­5oc on jan. 1, 21­24oc on apr. 16, 2011, and was raised by 2.5oc every 2 weeks until sept. 1, 2011. night temperature was maintained 2oc lower than the day temperature. the number of weeks to flower counted from the date of transplanting seedlings to pots, and the number and length of nodes with inflo­ rescences from the three longest shoots, and the number of nodes with 2 inflorescences was recorded from 15 plants per treatment. flowering date was recorded when two florets each from two inflores­ cences reached anthesis, and data were subjected to t h e r e g r e s s i o n a n a l y s i s f o r e a c h s p e c i e s u s i n g statistical analysis system program (sas, 2002). effect of pinching frequencies on growth and flower‐ ing (expt. 2) after sowing about 200 seeds as described in expt. 1, seedlings were transplanted. on mar. 18, 2010, 0.25 g of slow release fertilizer was applied to the surface of growing medium and the effect of pinching frequencies on c. sinensis var. calvescens was evaluated. shoots were either not­pinched or pinched 2 (feb. 28), 4 (apr. 18), 6 (may 2), and 8 weeks (may 16) after transplanting as outlined (table 3). to the surface of the growing medium at trans­ planting seedlings 0.8 g of slow release fertilizer per pot was applied, and plants were fertilized with 1.33 g/l of 15n ­ 7p ­ 12.8k water soluble fertilizer once a month. the number of weeks to flower, and the total number of shoots with flowers and flower buds, and the length and number of flowers from the first and second longest shoots was recorded from 15 plants per treatment. the number of days to flower was counted from the date of transplanting. data were subjected to the analysis of variance (anova) and means were compared with tukey’s honestly signifi­ cant difference (hsd) test. effect of growth retardants treatment on growth and flowering (expt. 3) corylopsis sinensis var. calvescens seeds were sown and transplanted, and pinched as described in expt. 1, and pinched again on may 26, 2010. plants w e r e g r o w n i n g r e e n h o u s e m a i n t a i n e d a t 1 8 ­ 21oc/16­19oc (day/night) and then in greenhouse maintained at 22­25oc/20­23oc until july 6. to the surface of the growing medium at transplanting seedlings 0.8 g of slow release fertilizer per pot was applied, and plants were fertilized with 1.33 g/l of 15n ­ 7p ­ 12.8k water soluble fertilizer once a month. growth retardants were applied on jul. 7, when new shoots were about 5­8 cm long. each pot was treated with 25 ml of ancymidol [0.026% active ingre­ dient (a.i.)] and paclobutrazol (0.4% a.i.) at 0, 10, 20, 40, and 80 mg/l was applied as a soil drench. daminozide (85% a.i.) and chlormequat (11.8% a.i.) at 0, 2,500, table 1 ­ evaluation of flowering and growth habit in the field c. coreana and c. sinensis var. calvescens z range and mean of flowering. y plants that produced inflorescence and the total number of plants evaluated (parenthesis). x number of plants ( parenthesis) showing upright and prostrate growth characteristics. species seed harvest 2008 2009 germi­ nation trans­ planting flowering z no. of plants y growth characteristics x c. coreana 2007 mar. 6 mar. 12 mar. 19­25 (mar. 21) 9 (21) upright (12), prostrate/upright (1) c. sinensis var. calvescens 2007 mar. 1 apr. 2 mar. 27­apr. 7 (apr. 2) 14 (16) upright (13), prostrate/upright (1) 2008 feb. 26 apr. 8 mar. 22­apr. 3 (mar. 28) 16 (16) upright (16) adv. hort. sci., 2020 34(1): 61­69 64 5,000, 7,500, and 10,000 mg/l was applied as a foliar spray, and 200 ml of solution was applied to 15 plants. on nov. 20, 2010, plants were grown in a greenhouse maintained at 4­5oc for cold treatment until mar. 1, 2011. dates of flowering, when two florets from an inflorescence reached anthesis were recorded and the lengths of two longest shoots (shoot length a) per plant and shoots longer than 3 cm were counted on jan. 16. plants were moved outdoors on mar. 27, and the new growth of two longest shoots (shoot length b) was also recorded on may 10, 2011. data collected from 15 plants per treatment were ana­ lyzed by two­way anova with plant growth retar­ dants and concentration as variables. 3. results selection of a suitable species for final evaluation following evaluation of 45 accessions including c. glabrescens,c. spicata, c. pauciflora, and c. vietchi‐ ana (data not presented), c. sinensis var. calvescens and c. coreana showing upright growth characteris­ tics of shoots and flowering response were selected­ for the final evaluation. all accessions grew taller than 1.3 m and spread over 65 cm wide in case of c. spicata, but with a few inflorescence (data not pre­ sented). the selection criteria were based on the number of plants that had flowered exhibiting upright growth characteristics. in less than one year counting from the time of transplanting, 14 from 16 c. sinensis var. calvescens plants flowered showing up­right growth (table 1). corylopsis coreana was also selected for its large foliage for its good fall foliage color, even though only nine out of 21 plants had flowered. effect of slow release fertilizer treatment on growth and flowering (expt. 1) the number of weeks to flower in 52 to 53 weeks in c. sinensis var. calvescens and c. coreana was not affected by the rate of srf treatments (table 2). the number of total shoots and of shoots with inflores­ cences increased linearly with srf treatment from 2.5 to 4.1 in c. coreana and from 2.1 to 4.1 in c. sinensis var. calvescens. the lengths of the three longest shoots also increased in both species, from 11.9 to 25.6 cm for the longest shoot, from 4.8 to 15.8 cm for the third shoots in c. coreana, and from 12.8 cm to 29.3 cm for the longest shoot in c. sinen‐ sis var. calvescens. the number of nodes with inflorescences in all the three shoots of c. coreana received 0.5 g srf treat­ ment was 0.3 or less than 0.3 and there was only one node with more than 2 inflorescences. however, the number of nodes with inflorescence produced and the number of nodes with 2 inflorescences was high­ er in c. sinensis var. calvescens than in c. coreana. the number was increased to 4.8 nodes in the first table 2 ­ the effect of slow release fertilizer treatment on the growth and flowering of corylopsis coreana and c. sinensis var. calvescens z z there was a significant difference between two species; data for each species were subjected to the linear regression analysis. y the number of weeks to flower was counted from the date of transplanting seedlings. x nodes with inflorescence that were formed on new growth by pinching. w non­significant (ns), significant at p≤0.05 (*) and p≤0.01 (**). species slow release fertilizer (g/10 pot) no. of weeks to flower y no. of total shoots length (cm) of three longest shoots (sh) no. of nodes with inflorescences x sh 1 sh2 sh 3 sh1 sh2 sh 3 c. coreana 0 52 2.5 11.9 6.7 4.8 0.1 0.1 0.0 0.125 52 3.3 14.4 9.8 7.4 0.3 0.1 0.0 0.25 53 4.6 20.3 13.6 10.1 0.1 0.0 0.0 0.5 52 4.1 25.6 22.0 15.8 0.1 0.3 0.3 regression analysis ­ linear effect w ns * ** ** ** ns ns ns c. sinensis var. calvescens 0 53 2.1 12.8 6.7 4.5 0.1 0.0 0.0 0.125 53 2.9 14.3 9.9 7.5 0.8 0.7 0.0 0.25 52 3.4 21.3 13.0 10.4 3.1 2.8 1.8 0.5 52 4.1 29.3 21.8 19.8 4.8 4.4 2.7 regression analysis ­ linear effect w ns ** ** ** ** * * * kim et al. ‐ compact corylopsis plant production 65 control. t h e l e n g t h o f t h e fi r s t a n d t h e s e c o n d n o n ­ pinched shoot was 49.3 and 33 cm, respectively, with a difference of 16.3 cm (table 3). however, when pinched 4 times, the lengths were 38.0 and 31.8 cm with a difference of 6.2 cm. the number of inflores­ cences in non­pinched and pinched shoots, which was 9.2 and 7.1 in the first shoot and 4.0 and 6.9 in the second shoot, respectively, did not vary signifi­ cantly. however, the difference in the number of inflorescences (0.2) between the first and the second shoot was significantly lower in the pinched shoot compared with the non­pinched shoot (5.2). in gen­ eral, when pinched, the difference in the inflores­ cences between the first and the second shoot was less than 1.9, which was significantly less than that of the control. effect of growth retardant treatments on growth and flowering (expt. 3) w h e n p l a n t s w e r e t r e a t e d w i t h a n c y m i d o l , chlormequat, and daminozide, flowering took 22 to 28 days regardless of treatment concentrations, which was not significantly different from that of control (table 2). however, soil drench treatments with paclobutrazol (20 mg/l or higher concentra­ tions) took longer than 36 days. flowering percent­ age was higher than 60% when plants were treated with ancymidol, chlormequat, and daminozide, regardless of treatment concentrations.treatment with 20 mg/l of paclobutrazol severely inhibited the extension of peduncle bearing inflorescence trigger­ ing the death of inflorescence immediately after shoot and to 4.4 nodes in the second shoot following treatment with 0.5 g of srf and the number of nodes with more than 2 inflorescences was also increased. c. sinensis var. calvescens treated with 0.125, 0.25, and 0.5 g srf has the potential to produce a small potted plant (fig. 2). effect of pinching frequencies on growth and flower‐ ing (expt. 2) regardless of frequencies and timing of pinching, the flowering of c.sinensis var. calvescens occurred in 53 weeks (table 3). flowering ranged between 73 and 93%, and the highest flowering rate was record­ ed when pinched for 4 times at 2, 4, 6, and 8 weeks, yielding a significantly higher number of shoots with inflorescences (5.3) and consequently the highest number of inflorescences (22.1) compared with the fig. 2 ­ appearance of the corylopsis sinensis var. calvescens plants in a 10 cm pot treated with 0.125, 0.25, and 0.5 g of slow release fertilizer per pot prior to leaf emergence and anthesis. table 3 ­ the effect of pinching frequencies on growth and flowering of corylopsis sinensis var. calvescens z not pinched (x) or pinched (o) 2 (feb. 28), 4 (apr. 18), 6 (may 2), and 8 weeks (may 16) after transplanting. y the number of weeks to flower was counted from the date of transplanting seedlings. x nodes with inflorescence induced by pinching. w non­significant (ns) or significance at p≤0.05, f­test. pinching z no. of weeks to flower y flowering % total shoots no. with inflores­ cences flowers (total no.) shoot length (cm) no. of nodes withinflorescences x 2 weeks 4 weeks 6 weeks 8 weeks first shoot second shoot difference first second shoot difference x x x x 53 87 2.9 17.2 49.3 33.0 16.3 9.2 4.0 5.2 o x x x 53 87 2.1 12.8 49.0 38.8 10.2 6.9 6.8 0.1 o o x x 54 73 2.4 13.1 47.1 35.5 11.6 6.4 4.8 1.6 o o o x 53 87 3.5 18.5 41.6 35.2 6.4 6.8 5.7 1.1 o o o o 53 93 5.3 22.1 38.0 31.8 6.2 7.1 6.9 0.2 o x o o 53 87 4.3 17.3 32.4 29.2 3.2 5.7 5.2 0.5 o o x o 54 80 3.1 10.4 37.3 26.0 11.3 4.7 3.9 0.8 o x o x 53 73 3.3 14.8 39.5 29.8 9.7 6.5 6.3 0.2 o x x o 53 87 3.3 16.3 39.0 22.2 16.8 7.6 5.7 1.9 level of significance w hsd at p<0.05 ns ­ 0.94 3.58 8.25 5.61 5.82 4.27 3.84 1.59 66 adv. hort. sci., 2020 34(1): 61­69 e m e r g e n c e f o l l o w i n g l e a f e m e r g e n c e ( f i g . 3 ) . therefore, days to flower were estimated on the date of leaf emergence. flowering percentage was significantly reduced to less than 30% when plants were treated at 20 and 40 mg/l paclobutrazol. when shoot lengths following 80 mg/l ancymidol were recorded on jan. 16 (26 weeks after growth retardant treatment), the length of the first and the second longest shoots was significantly reduced from 26.3 cm to 15.6 cm and from 16.4 cm to 12.2 cm, respectively (table 4). the length of the two longest shoots treated with daminozide and chlormequat showed similar trends as observed in plants treated with ancymidol. the length of the two longest shoots was significantly reduced to 12.9 cm and 9.8 cm following treatment with 10 mg/l paclobutrazol (fig. 3), responding to the quadratic effect of concentrations. the length of shoot b showing new growth on may 10 was signifi­ cantly inhibited to less than 5.0 cm when treated with paclobutrazol. the number of shoots longer than 3 cm varied from 4.8 to 5.7, from 3.8 to 4.8, 4.3 to 5.5, and 5.0 to 3.9 upon treatment with ancymi­ dol, chlormequat, daminozide, and paclobutrazol, respectively. the numbers were not affected by con­ centrations of these three retardants (data not pre­ sented). 4. discussion and conclusions successful acclimatization rate of in vitro propa­ gated c. coreana was low (moon et al., 2002) and limited time of the season to propagate by rooting of cuttings (kwon et al., 2011) are the limiting factors for mass propagation to secure sufficient and uni­ form propagules for experiments, and further, reports are not available on flowering of in vitro propagules and rooted cuttings. this clearly indicates that seeds can be used as a propagule to produce sufficient number of seedlings to produceflowering plants in a year from transplanting seedlingsby vari­ ous cultural practices reported in this study. the mor­ p h o l o g i c a l c h a r a c t e r i s ti c s o f c . s i n e n s i s v a r . calvescens are suitable to produce in 10 cm pots compared with c. coreana, if stem elongation can be controlled and many shoots with well­developed inflorescences can be formed (fig. 1). growth and flowering as influenced by slow release fertilizer (srf) there is a clear difference between c. coreana and c. sinensis var. calvescens responding to srf a p p l i c a ti o n p e r 1 0 c m p o t . r e s p o n d i n g t o t h e increased rates of slow release fertilizer, especially at 0.5 g srf application, and c. sinensis var. sinensis is recommended to produce as a 10 cm potted plant in one year as the number of inflorescences and of n o d e s w i t h m o r e t h a n t w o i n fl o r e s c e n c e s a r e increased. production of c. coreana may not be rec­ ommended due to fewer numbers of nodes with inflorescences and only 0.1 node produced more than 2 inflorescences. growth and flowering as influenced by pinching and growth retardant treatments stem length is one of the limitations to produce compact c. sinensis var. calvescens in small pots, which can be reduced either by pinching or growth retardant treatments. manual or mechanical pinch­ i n g i s a s s o c i a t e d w i t h i n c r e a s e d l a b o r c o s t s . treatment with growth retardant may not induce branching when compared with pinching. pinching shoots four times in 2, 4, 6, and 8 weeks prior to may 2 is an effective cultural practice to pro­ duce compact plants for small pots without affecting days to flowering and flowering percentage. the increase in the number of shoots with inflorescences, the total number of inflorescences, and the number of nodes with inflorescences may result from an increased number of shoots that are formed prior to the development of inflorescence, which may occur fig. 3 ­ corylopsis sinensis var. calvescens in a 10 cm pot treated with 25 ml of 40 mg/l paclobutrazol (a). blasted inflore­ scences (arrow) and emerging of dark green leaves of reduced size indicate excessive doses of paclobutrazol (b). photographed on mar. 27, 2011. kim et al. ‐ compact corylopsis plant production 67 after may 2. since the time of floral bud initiation has not been examined anatomically, it requires further studies. increase in the number of lateral shoots and flowers were increased as the pinching frequencies in sedum rotundifolium d.b. was increased (jeong, 2000). the length of shoots exceeding 30 cm follow­ ing pinching is considered excessive for producing corylopsis in a 10 cm pot. flowering of c. sinensis var. calvescens plants t r e a t e d w i t h a n c y m i d o l , c h l o r m e q u a t , a n d daminozide that produced higher than 60% plants with inflorescences regardless of treatment concen­ trations did not differ significantly from that of con­ trol. the longest shoot length (a) on jan. 16 respond­ ing linear effect to ancymidol, chlormequat and daminozide was the shortest, especially when treat­ ed with 80 ppm ancymidol. ancymidol is,therefore, recommended for c. sinensis var. calvescens in 10 cm pot. since the length of new shoot (b) on may 16 was not affected, the effect of these three plant growth table 4 ­ growth and flowering of corylopsis sinensis var. calvescens as influenced by growth retardant treatments plant growth regulator no. of days to flower (mar. 1) (flowering percent) shoot lenght a (cm) (jan. 16, 2011 z) shoot length b (cm) of new growth (may 10, 2011) fir y sec fir sec ancymidol (soil drench) (mg/l) 0 22 (60) 26.3 16.4 21.0 20.0 10 23 (80) 28.4 17.5 20.2 19.4 20 24 (70) 27.9 21.7 20.2 20.0 40 25 (70) 23.9 19.1 21.9 17.9 80 25 (60) 15.6 12.2 16.5 16.3 regression analysis ns l* l* ns ns paclobutrazol (soil drench) (mg/l) 0 22 (60) 24.7 20.0 24.0 20.0 10 28 w (50) 12.9 9.8 5.0 3.5 20 38 w (30) 11.2 8.1 1.7 1.2 40 36 w (10) 10.3 7.9 1.3 0.8 80 36 w (30) 11.0 8.5 1.1 0.6 regression analysis x ­ q** q** q** q** chlormequat (foliar spray) (mg/l) 0 22 (70) 28.9 20.3 23.1 20.9 2.5 22 (80) 35.1 24.9 22.9 20.1 5 25 (70) 26.5 20.0 18.3 18.1 7.5 27 (60) 23.7 14.5 17.9 17.0 10 25 (70) 24.3 18.5 20.2 19.9 regression analysis ns l* ns ns ns daminozide (foliar spray) (mg/l) 0 23 (60) 29.2 23.0 21.0 19.9 2.5 27 (80) 33.3 23.5 23.5 18.5 5 25 (70) 28.5 22.0 22.5 18.9 7.5 25 (60) 29.3 22.7 22.7 19.1 10 27 (70) 25.5 19.5 21.2 18.5 regression analysis ns l* ns ns ns level of significance v growth retardant (pgr) ns *** *** *** *** concentratio ns ** * * * pgr × concentration ns ** ** ** ** z data collected from over­wintered plants. y length of the first (fir) and second (sec) longest shoots. x analysis was not carried out due to the estimated days of flowering and low flowering percentage, and regression analysis was per­ formed for each growth retardant. linear (l) and quadratic (q) effect. w days to flower following paclobutrazol treatment were recorded upon leaf emergence following the death of inflorescence. generally, about 15 days elapsed between flowering and the appearance of leaf emergence. v non­significant (ns) or significance at p<0.05 (*), 0.01 (**) or 0.001 (***), f­test. adv. hort. sci., 2020 34(1): 61­69 68 retardants may not last long when compared with paclobutrazol. a single application of ancymidol is not effective and may require two treatments to pro­ duce quality mussaenda ‘queen sirikit’ as a short­ stemmed potted plant without reducing the number of flowers per plant and delaying the flowering (cramer and bridgen, 1998). application of ancymidol requires further testing since growth retarding effects of ancymidol do not persist as reported in l. lancifolium thunb. (roh, 1979) and shoot length is increased under a long day photoperiod during june o r j u l y a s o b s e r v e d i n l . l o n g i fl o r u m ( r o h a n d wilkins, 1977). soil drench treatment with paclobutrazol 20 mg/l or higher concentrations which took longer than 36 days to flower (table 4) compared with 22 days with the control. when treated with 20 mg/l of paclobu­ trazol, the extension of peduncle­bearing inflores­ cence was severely inhibited resulting in the death of inflorescence immediately following leaf emergence, thus lowering the flowering rate from 60% to 10% ( f i g . 3 ) . s h o o t l e n g t h w a s a r r e s t e d u n d e r a n y paclobutrazol treatment which is undesirable. although 10­20 mg/l paclobutrazol as a soil drench is considered effective to reduce shoot length, the growth of new shoots and inflorescence development is significantly arrested even a year later, which may require double treatments at low concentrations, i. e., 5 mg/l to avoid severe growth retardation and malformation of inflorescence. a sin­ gle foliar spray of 500 mg/l paclobutrazol may be used to test produce compact flowering plants as reported in rhododendron hybrids, which is a woody ornamental (wilkinson and richards, 1991). generally paclobutrazol was not effective in mussaenda at 0.125­0.25 mg a.i. per pot as a soil drench compared with ancymidol and daminozide (cramer and bridgen, 1998), which is considered effective in producing compact plants with accelerat­ ed flowering although 0.4 g a.i. per pot increased the number of flowers, producing malformed and unac­ c e p t a b l e o f r h o d o d e n d r o n ‘ s i r r o b e r t p e e l ’ (wilkinson and richards, 1991). shoot length of poin­ settia (euphorbis pucherrima wild. ex klotzch) was reduced by daminozide and chlormequat treatments without affecting the flowering (lewis et al., 2004). c o m b i n e d t r e a t m e n t w i t h c h l o r m e q u a t a n d daminozide can also be considered as reported to be effective to retard stem elongation of zonal (cutting) geraniums [pelargonium ×hortorum (l.h. bailey)] (tayama and carver, 1990). d u e t o t h e l o n g ­ l a s ti n g i n h i b i t o r y e ff e c t o f paclobutrazol on shoot elongation and reduction in leaf size when applied as a soil drench, a malforma­ tion of inflorescence and formation of inflorescences after leaf emergence result in a lower percentage of plants with inflorescences in both species in this study and in other woody ornamentals such as dissotis rotundifolia (sm.) triana and tibouchina f o r t h e r g i l l a e × p i l o s a ( h a w k i n s e t a l . , 2 0 1 5 ) . therefore, paclobutrazol is not recommended to use as a soil drench in corylopsis. the optimum dosages require further study comparing the effect of soil d r e n c h a n d f o l i a r s p r a y . a s p r a y t r e a t m e n t o f paclobutrazol may be considered as the quality of d i a n t h u s c a r y o p h y l l u s l . , c v . m o n d r i a a n w a s improved (bañón et al., 2002). this is the first report providing practical and hor­ ticultural strategies to produce flowering corylopsis plants in small pots with a great potential to utilize under­utilized native plants as ornamental and nurs­ ery plant starting from seeds. corylopsis sinensis var. calvescens indigenous to china is a suitable species as compared to c. coreana native to korea to pro­ duce from seeds with application of slow release fer­ tilizer at 0.5 g per pot and pinching for four times at 2­week interval before may 16 to reduce shoot elon­ gation, to increase flowering percentage, and to accelerate flowering with increased number of inflo­ rescences. treatments with ancymidol as a soil drench and daminozide and chlormequat as a foliar spray at all concentrations evaluated in this study were not effective to produce compact plants as compared to paclobutrazol treatment. soil drench treatment with paclobutrazol at 10­20 mg/l is considered effective in reducing shoot elongation the first year. however, the inhibitory effects of paclobutrazol last longer than a year, and appear resulting in malformation of inflorescences in the second year. therefore, investi­ gation on selecting appropriate treatment methods andconcentrations of paclobutrazol to reduce shoot elongation without triggering malformation of inflo­ rescence is needed. the time of floral initiation and development in relation to pinching treatment needs to be determined as well. references bañón s., gonzalez a., cano e.a., franco j.a., 2002 ­ growth, development and colour response of potted dianthus caryophyllus cv. mondriaan to paclobutrazol treatment. ­ scientia hort., 94: 371­377. kim et al. ‐ compact corylopsis plant production 69 bean e., anisko t., 2014 ­ captivating corylopsis. ­ https://www.amerinursery.com/growing/captivating ‐ corylopsis/. cramer c.s., bridgen m.p., 1998 ­ growth regulator effects on plant height of potted mussaenda ‘queen sirikit’. ­ hortscience, 33: 78­81. currey c.j., lopez r.g., 2016 ­ commercial greenhouse and nursery production. applying plant growth retar‐ dants for height control. ­ purdue extension. hd­248­ w. purdue university. https://www. extension.pur­ due.edu/extmedia/ho/ho­248­w.pdf. hawkins s.m., ruter j.m., robacker c.d., 2015 ­ spray and drench treatments of paclobutrazol influence of growth of dissotis and tibouchina. ­ hortscience, 50: 1514­1517. jeong j.h., 2000 ­ effects of pinching and growth regula‐ tors on growth and flowering of sedum rotundifolium. ­ j. korean soc. hort. sci., 41: 105­108 (in korean with english summary). kim j.h., kim k.o., lee a.k., roh m.s., suh j.k., 2017 ­ germination of corylopsis seeds evaluated by x‐ray imaging and cold stratification. ­ hort. sci., (prague), 44:105­111. kim j.h., lee a.k., suh j.k., 2015 ­ effect of warm and cold stratification, ethanol treatment on germination of corylopsis seeds. ­ hort. sci. (prague), 43: 84­92. kim j.h., suh j.k., yoon s­t., jourdan p., roh m.s., 2018 ­ germination of fully developed corylopsis seeds influenced by harvest date and cold stratification. ­ hortscience, 53: 1360­1363. koh g.c., lim d.k., 2006 ­ in vitro propagation of korean native winter hazel (corylopsis coreana). ­ kor. j. hort. sci. technol., 24: 272­278. kwon s.t., kim j.c., jeong j.h., 2011 ­ change of root potential as affected by rooting promoter treatment in cuttings of corylopsis coreana. ­ kor. soc. floric. sci., 19(4): 202­205 (in korean with english abstract). latimer j., whipker b., 2013 ­ selecting and using plant growth regulators on floricultural crops. ­ virginia polytechnic institute and state university, blacksburg, va, usa, publication 430­102 (hort­43p), pp. 32. lee j.h., kim k.s., song c.y., 2006 ­ effects of pinching and retardants on growth and flowering of celosia argentea native to korea. ­ kor. j. plant res., 19: 573­579 (in korean with english abstract). lewis k.p., faustand j.e., sparkman iv j.d., 2004 ­ the effect of daminozide and chlormequat on the growth and flowering of poinsettia and pansy. ­ hortscience, 39: 1315­1318. m o o n h . k . , n o h e . w . , h a y . m . , s h i m k . k . , 2 0 0 2 ­ micropropagation of juvenile and mature tree of corylopsis coreana by axillary bud culture. ­ kor. j. plant biotechnol., 29: 117­121 (in korean with english abstract). roh m.s., 1992 ­ a method for producing lilium elegans. ­ u.s. patent no. 5,138,794. roh m.s., 1996 ­ new production technology of lilium. ­ a r e v i e w o n p r o p a g a ti o n a n d f o r c i n g . ­ a c t a horticulturae, 414: 219­228. roh m.s., lee a.k., suh j.k., bordelon c.m., 2008 a ­ i n t e r s p e c i fi c v a r i a ti o n s i n s e e d g e r m i n a ti o n o f corylopsis. ­ scientia hort., 118: 347­350. roh m.s., mcnamara w.a., picton d., yin k., wang q., 2008 b ­ assessment of genetic variation in acer penta­ phyllum based on amplified fragment length polymor‐ phisms. ­ j. hort. sci.& biotech., 83: 725­731. roh s.m., 1979 ­ various factors influencing the growth and flowering of lilium lancifolium thunb. ‐ j. kor. soc. hort. sci., 20: 72­83. roh s.m., wilkins h.f., 1977 ­ the influence and interac‐ tion of ancymidol and photoperiod on growth of lilium longiflorum thunb. ­ j. amer. soc. hort. sci., 102: 255­ 257. sas, 2002 ­ sas proprietary software version 9.00. ­ statistical analysis system institute, inc., cary, nc, usa. son s.­w., kim h., kim y.­s., 2016 ­ corylopsis coreana. t h e i u c n r e d l i s t o f t h r e a t e n e d s p e c i e s 2 0 1 6 : e.t72136190a72136238.­ http://dx.doi.org/10.2305/ iucn.uk.2016­1.rlts.t72136190a72136238.en. tayama h.k., carver s.a., 1990 ­ zonal geranium growth and flowering responses to six growth regulators. ­ hortscience, 25: 82­83. wilkinson r.i., richards d., 1991 ­ influence of paclobu‐ trazol on growth and flowering of rhododendron ‘sir robert peel’. ­ hortscience, 26:282­284. zhang z., zhang h., endres p.k., 2003 ­ hamalelidaceae. ­ flora of china, 9: 18­42. 16. http://flora.huh.harvard. edu/china/mss/volume09/hamamelidaceae.pdf. http://https://www.amerinursery.com/growing/captivating-corylopsis/ http://https://www.amerinursery.com/growing/captivating-corylopsis/ http://dx.doi.org/10.2305/iucn.uk.2016-1.rlts.t72136190a72136238.en http://dx.doi.org/10.2305/iucn.uk.2016-1.rlts.t72136190a72136238.en http://dx.doi.org/10.2305/iucn.uk.2016-1.rlts.t72136190a72136238.en http://hortsci.ashspublications.org/search/?author1=r.i.+wilkinson&sortspec=date&submit=submit impaginato 53 adv. hort. sci., 2021 35(1): 53­60 doi: 10.36253/ahsc­9663 endogenous hormone causes flower and fruit drop of wax apple (syzygium samarangense cv. citra) e. wukir tini (*), t.a. dwi haryanto, sakhidin, saparso agriculture faculty, jenderal soedirman university, purwokerto, central java, indonesia. key words: acc, cytokinins, fruit drop, iaa, ga3, wax apple. abstract: the aim of this study was to obtain information about the content of endogenous hormones that causes flowers and fruit drop of wax apple. the variables observed in the six stages of flower and fruit development that drop easily and retention of indole­3­acetic acid (iaa), cytokinin (zeatin and kinetin), gibberellins (ga3), 1­amino cyclopropane­1­carboxylic acid (acc), total sugar, and starch. six stages of development of wax apple fruit: (1) bud development (initial flowering) 0­3 days before anthesis. (2) anthesis (perfect blooming flow­ ers), 0­7 days after anthesis. (3) fruit set, 7­14 days after anthesis. (4) fruit development, 14­28 days after anthesis. (5) fruit maturation, 28­35 days after anthesis. (6) fruit ripening, 35­50 days after anthesis. the results showed that the content of iaa, zeatin, ga3, and total sugar of flowers and fruit of wax apple at 6 stages that would fall smaller than those of retention and acc con­ tent and starch was higher in flower and fruits that drop easily than retention. the kinetin content in the flower development that drop easily is smaller than the retention but in the fruit development the kinetin content is not significant­ ly different between those that drop easily and retention. 1. introduction water apple originates from the southeast asian region (indonesia and malaysia), then spreads to the islands of the pacific and north and central america. generally, wax apple fruit is consumed as fresh fruit, but it can also be made for salad and preparations such as pickles, syrups, jel­ lies, and cocktails. water apple fruit is not only sweet and refreshing but has diversity in appearance. types of water apple are syzygium aqueum (water apple) and syzygium samarangense (wax apple). the varieties of syzygium samarangense include delima, lilin, camplong, cincalo, citra, kesuma, and madu, (kuswandi, 2008). corona virus disease (covid­19) is originating from wuhan, china began to spread throughout the world from january 2020 to 31 december 2020, so there were 83,264,353 cases, with 1,816,164 deaths from 218 affected countries (world health organization, 2020). no vac­ cine has been found to treat patients with the covid­19 virus. according (*) corresponding author: etik.unsoed@gmail.com citation: wukir tini e., dwi haryanto t.a., sakhidin, saparso, 2021 ­ endogenous hormone causes flower and fruit drop of wax apple (syzygium samarangense cv. citra). ­ adv. hort. sci., 35(1): 53­60 copyright: © 2021 wukir tini e., dwi haryanto t.a., sakhidin, saparso. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 3 september 2020 accepted for publication 27 january 2021 ahs advances in horticultural science https://doi.org/10.36253/ahsc-9663 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2021 35(1): 53­60 54 to the ministry of health of the republic of indonesia (2020), prevention of viruses can be done by fre­ quently washing hands, wearing masks, consuming vegetables and fruit, exercise, and adequate rest. one of the prevention efforts that can be done by consuming fruits that contain vitamin c to enhance immunity. every 100 g of wax apple fruit contains 22.3 µg of vitamin c (asia­pacific association of agricultural research institutions, 2014). therefore wax apple is a tropical fruit that is cheap, easily obtained, and contains vitamin c which can be used as an alternative to increasing body immunity as a preventative measure against the covid­19 virus. the main problem in the cultivation of wax apple plants is the high drop of flowers and fruit. the high rate drop of flowers and fruit causes a few numbers of wax apple fruit that can be produced. wax apple drop fruit rate reaches 52% (khandaker et al., 2016). fruit drop also occurs in other fruits, such as durian flowers reach 95 to 100% (suparto and sakhidin, 2013), lychee fruit is 90 to 97% (stern et al., 1995), star fruit is 25 to 30% (kurniawati and hamim, 2009), mangosteen flower is 14.1% and mangosteen fruit is 70.1% (rai et al., 2008). dropped fruit is a natural phenomenon that occurs in almost all types of fruit. drop fruit, especially at the beginning of fruit growth, is a mechanism for regulat­ ing autoregulation in each plant (davarynejad et al., 2009). physiologically the loss of flowers and fruit cor­ relates with the limited supply of photosynthate and nutrient adequacy (iglesias et al., 2007), as well as h o r m o n a l r e g u l a ti o n i n t h e a b s c i s s i o n z o n e (bangerth, 2000). flower and fruit drop are also influ­ enced by endogenous hormone content in plants due to high ethylene concentrations and low concentra­ tions of auxin and gibberellins. auxin, gibberellins, and ethylene are hormones that are directly related to the process of flowers and fruit drop (bangerth, 2000). complex hormonal interactions occur during fruit development. gibberellins and cytokines gener­ ally stimulate fruit growth and auxin as a growth stim­ ulator and also as a fruit drop agent. abscisic acid (aba) and ethylene are also involved in the process of loss (sakamoto et al., 2008). high ethylene concentra­ tions, low concentrations of auxin and gibberellins, and high aba concentrations in plants are the main causes of drop fruit (iglesias et al., 2007). the causes of flower and fruit drop of the wax apple due to imbalance of plant growth regulator as well as cultivation and environmental engineering factors, including pollination, fertilization, fruits set, lack of water and nutrition supply, pest attack, rain­ fall and wind (khandaker et al., 2016). research on the use of synthetic growth plant regulators (gpr) to reduce the fruit drop of wax apple has been conduct­ ed by khandaker et al. (2013), khandaker et al. (2016), 50 mg l­1 gibberellins (ga3), ga3 produces the lowest flower and fruit buds loss of 29% compared to controls (not given ga3 36%). ga3 20 mg l ­1 reduced f r u i t l o s s 3 2 % c o m p a r e d t o 5 2 % c o n t r o l . 2 ­ 4 ­ dichlorophenoxyacetid (2,4 d) 5 mg l­1 reduces flower and fruit bud loss by at least 30% and 18% compared to 35% and 40% controls. naphthaleneacetid acid (naa) 5 mg l­1 reduced the flower buds and fruits drop as lower as 28% and 30% compared to 30% and 52% controls. the causes of flowers and fruit drop of wax apple are because the content of endogenous hormones is not yet known, so the application of growth plant regulator given is not appropriate to reduce the flowers and fruit drop. singh et al. (2017) the results of research on the application of growth plant regulator on khasi man­ darin (citrus reticulata blanco) showed an increase in fruit retention and the number of fruit with the appli­ cation of urea 1% + 2, 4­d 15 ppm (45.4% and 244, 3 fruits/tree), application of naa 5 ppm + 2,4­d 10 ppm (44.3% and 241.6 fruit/tree) compared to con­ trols (17.3% fruit retention and 181 fruit/tree). naa treatment 15 ppm increased the proportion of cape gooseberry cv. aligarh is retention (71.40%) (kaur and kaur, 2016). the purpose of this study was to obtain informa­ tion on hormones that causes flower and fruit drop at several stages of the development of wax apple fruit and to obtain differences in the hormone con­ tent at how many stages of flower and fruit develop­ ment are easily dropped and retention. 2. materials and methods the research used an experimental method from may 2019 to april 2020. the experimental design used was a completely randomized design (crd) with the treatment being tried, namely flower and fruit retention and drop easily of wax apple at six stages fruit development: (1) bud development (from 0 to 7 days before anthesis); (2) anthesis (full bloom), from 0 to 7 days after anthesis; (3) fruit set (the for­ mation of finished fruit), from 7 to 14 days after anthesis; (4) fruit development, from 14 to 28 days after anthesis; (5) fruit maturation, from 28 to 35 wukir tini et al. ‐ hormone endogenous causes flower and fruits drop 55 days after anthesis; (6) fruit ripening (from 35 to 50 days after anthesis). experiment with 4 replications and data analysis using t­test and advanced test dmrt (duncan multiple range test). flower and fruit samples were taken from 46 wax apple plants, kajongan village, bojongsari district, purbalingga regency, central java. the variables observed were auxin content (iaa), cytokinins (zeatin and kinentin), gibberellin (ga3), acc (1­aminocyclopropane 1­ carboxylic acid), total sugar, and starch. the samples used for the study were flowers and fruits from 6 stages of development of flowers and water guava. plants are 5 years old, plant spacing 8 m x 9 m, with a height of ± 7 m and have been fruiting for the last 4 years. temperature 25.8°c with 76% humidity, sunlight intensity 571 lux outside the canopy, and under the canopy 529 lux and soil ph 6.8. organic cultivation of wax apple uses goat manure at a dose of 100 kg per plant per year. irrigation in the dry season with springs that are channeled from a hose from a pipe made by the gar­ den. the results of soil analysis conducted at the postharvest center of the ministry of agriculture, inceptisol soil type, ph 6.14, organic c 0.769%, total n 0.082%, available n 0.025%, total k 55.960 ppm, k available 30.277 ppm, total p 45.189 ppm, and 1.321% organic matter. the samples drop easily are taken from the flower and fruit which when the branches or twigs are shak­ en fall out and after the fall there is a black abscission layer on the flower stalk or fruit. the retention sam­ ples are wax apple flower and fruit which if the branches or twigs are shaken not fall off, are still attached to the plant, and usually, the flower stalk or fruit is still present. the sample is placed in an icebox containing blue ice and then taken to the integrated research laboratory, jenderal soedirman university to be dried in a 2kxc bench top vacuum freeze dryer. wax apple samples for bud development, anthesis, and fruit set for 25 hours, and fruit development, fruit maturation, fruit ripening for 55 hours with a temperature of ­70°c, and a pressure of 13,332.2 pa the contents of auxin (iaa), ga3, zeatin, kinetin, acc, total sugar, and starch were carried out at the c h e m i c a l l a b o r a t o r y , c e n t e r f o r p o s t h a r v e s t , ministry of agriculture, bogor, west java, indonesia. procedure for measuring hormone content (auxin, c y t o k i n i n , a n d g i b b e r e l l i n s ) u s i n g h p l c ( h i g h ­ pressure liquid chromatography) at a wavelength of 214 nm, sample temperature of 10°c and column temperature of 25°c, stationary phase using c­18. the formula for hormone content by hplc is sample area divided by standard area multiplied by standard concentration (harborne, 1973). measurement of acc content according to lizada and yang (1979), using gc (gas chromatography). the formula for acc content in gc is sample area divided by standard a r e a m u l ti p l i e d b y s t a n d a r d c o n c e n t r a ti o n . m e a s u r e m e n t o f t o t a l s u g a r a c c o r d i n g t o t h e indonesian national standard (1992) using the luff schoorl method. starch calculation according to horwitz and latimer (2006). 3. results table 1 shows that the fruit drop easily having lower iaa, zeatin, and ga3 content than fruit reten­ tion in all phases of fruit development. the kinetin content at the bud development, anthesis, and fruit set stages that drop easily is lower than the reten­ tion, but the kinetin content at the fruit develop­ ment, fruit maturation and fruit ripening stages is not significantly different in the fruit that drop easily dan retention. while the acc content of fruit that drops easily is greater than that of retention. table 2 shows that the total sugar content at vari­ ous stages of flower and wax apple fruit develop­ ment in flower and fruit retention is higher than that of drop easily. the content of starch in fruit retention of various stages of development of the flower and fruit is lower than those that are of drop easily. 4. discussion and conclusions the iaa content in wax apple flower and fruit which drops easily on bud development, anthesis, fruit set, fruit development, fruit maturation, and fruit ripening is lower than the retention flower and fruit. this is according to rai et al. (2008). the iaa content of mangosteen flowers falling 3.37 ng g­1 is lower than the retention rate of 8.80 ng g­1 dry weight and iaa content in mangosteen fruit falling 0.83 ng g­1 is lower from the iaa content of 6.43 ng g­1 retention fruit. sakhidin et al. (2011), the content of iaa in mango fruit loss is lower than that of reten­ tion. gadung 21 and lali jowo cultivars that will fall age 6 and 9 days after anthesis are 5.96 and 3.86 10­1 µg g­1 sample fresh weight and 4.48 and 4.12 10­1 µg g­1 sample fresh weight. mango fruit retention of 10.32 and 8.12 (10­1 µg g­1 sample fresh weight and adv. hort. sci., 2021 35(1): 53­60 56 10.08 and 7.98 10 ­1 µg g ­1 sample fresh weight. according to kurniawati and hamim (2009), star fruit with an application of 15 ppm 2, 4­dichlorophenoxy­ acetic acid (2,4­d) contains 227 ppm iaa and 60 ppm ga3 applications have 221 ppm iaa. the formation of the absicission layer at the stem point that causes fruit drop is an imbalance of auxin, cytokinin, and gibberellins (chen et al., 2006). flower and fruit loss is also influenced by high ethylene con­ centrations and low iaa type auxin concentrations and low gibberellins. auxin and ethylene are hor­ mones that are directly related to the process of a drop of flowers and fruit (bangerth, 2000). auxin is often reported to delay or to induce fruit, it increases cell enlargement rather than cell division. this observation might indicate that auxin is related to cell enlargement, an essential factor controlling f r u i t s i z e d u r i n g t h e r a p i d f r u i t g r o w t h p h a s e . enlargement of fruits treated with auxin seems to be due to cell expansion rather than cell division (iglesias et al., 2007). table 1 shows that the content of zeatin type cytokinins and fruit kinetin that drop easily is smaller than retention. this is because cytokinins function to inhibit aging (kieber and schaller, 2018) so that the flowers and fruits of wax apple which will fall at sev­ eral stages of development have a smaller zeatin content compared to the retention seen in table 1. the cytokinin content of both zeatin and kinetin at the stage of development flowers and fruit of wax a p p l e w a s t h e h i g h e s t a t t h e f r u i t s e t s t a g e . according to chen (1983), the cytokinin content table 1 ­ the content of several hormones at the stage of development fruit of wax apple var. citra the number followed by the same letter in the same column for each fruit development is not significantly different from the t­test at p<0.05. treatment flower and fruit development bud development anthesis fruit set fruit development fruit maturation fruit ripening iaa (ppm) retention 69.50 a 33.72 a 47.40 a 57.02 a 42.86 a 42.10 a drop easily 1.42 b 0.73 b 0.68 b 1.22 b 0.78 b 0.41 b zeatin (ppm) retention 2.18 a 2.25 a 3.29 a 2.19 a 2.59 a 2.04 a drop easily 0.17 b 0.50 b 0.66 b 0.36 b 0.03 b 0.46 b kinetin (ppm) retention 4.36 a 2.46 a 11.31 a 3.17 a 3.30 a 3.93 a drop easily 2.16 b 1.38 b 2.96 b 3.05 a 2.75 a 2.39 a ga3 (ppm) retention 79.65 a 68.08 a 90.47 a 68.25 a 63.45 a 57.72 a drop easily 28.63 b 16.13 b 20.14 b 26.69 b 17.57 b 8.97 b acc (ppm) retention 19.18 b 17.26 b 14.21 b 14.13 b 13.31 b 12.14 b drop easily 48.39 a 38.09 a 37.99 a 35.75 a 34.14 a 30.92 a table 2 ­ total sugar content and starch content on stages of wax apple flower and fruit development the number followed by the same letter in the same column each fruit development is not significantly different in the t­test at the level of p<0.05. treatment flower and fruit development stage bud development anthesis fruit set fruit development fruit maturation fruit ripening total sugar content (%) retention 6.87 a 8.72 a 11.38 a 23.35 a 28.93 a 32.59 a drop easily 5.80 b 7.60 b 8.60 b 20.61 b 26.36 b 28.66 b starch content (%) retention 17.23 b 16.29 a 14.52 b 13.11 b 11.02 b 8.77 b drop easily 17.90 a 16.40 a 15.80 a 13.89 a 12.62 a 11.21 a wukir tini et al. ‐ hormone endogenous causes flower and fruits drop 57 increased in mango flowers maximum at 10 days after anthesis and decreased gradually up to 50 days after anthesis. this is consistent with trueman ( 2 0 1 1 ) , t h a t h i g h c y t o k i n i n c o n c e n t r a ti o n s i n macadamia fruit are retention. cytokinins are pro­ duced in roots and young fruit (pratima and chawla, 2019). table 1 shows that the ga3 content in several stages of flower and fruit development will fall lower than the retention of flowers and fruit. the ga3 con­ tent in fallen fruit was lower than that in retention (bains et al., 1997). the role of ga3 is able to stimu­ late plant growth and flowering, increase flowering and reduce drop flower (budiarto and wuryaningsih, 2007). according to kurniawati and hamim (2009), gibberellin and auxin can support fruit retention by inducing the enzyme α­amylase to hydrolyze starch to sugar that is needed for fruit growth and develop­ ment (subiyanto, 1991). this can be seen in table 1 that the increased auxin content also increases the gibberellin content in retention flowers and fruits compared to drop easily and in table 2 flowers and fruits retention have a higher total sugar content than drop easily. the acc content of wax apple flowers and fruits that drop easily is higher than the retention of flow­ ers and fruits are seen in table 1. this is because the acc which is an ethylene precursor makes the drop of flowers or fruits become higher (wang et al., 2002). the mango acc content that drops easily is higher than that of retention (sakidin et al., 2011). the acc content of gadung 21 and lali jiwo cultivars which drop easily higher at the age of 3, 9, 12 days after anthesis are 26.81, 35.78, 43.41 (10­1 mg g­1) sample fresh weights and 29.13, 29.35, 35.79 (10­1 mg g­1) sample fresh weight. the acc content of gadung 21 and lali jiwo cultivars with age retention of 3, 9, 12 days after anthesis is 26.81; 35.78; 43.41 (10­1 mg g­1) fresh sample weights and 10.13; 13.66; 11.94 (10­1 mg g­1) sample fresh weights (sakhidin et al., 2006). f l o w e r a n d o v a r y a b s c i s s i o n o c c u r i n a z ­ a (abscission zone) located between branches and flower stalks. absence in the az­a zone starts from the fruit set period and this negative effect is regulat­ ed by the content of gibberellins in the ovaries. fruitlet abscission during fruit drop in june at the end of the fruitset period on az­c which is located in the petals, between the flower disks and the ovarian wall, and highly dependent on carbohydrate avail­ ability. sugar in mature leaves is transported for fruit­ let growth and activates az­c (iglesias et al., 2007). according to iglesias et al. (2007), the lack of car­ bon in fruitlets induces an increase in abscisic acid (aba) and acc (1­aminocyclopropane 1­carboxylic acid) which are ethylene precursors. these precur­ sors are then oxidized to ethylene (c2h4) and release of gases that cause fruit abscission. in contrast to the development of fruitlets, in the process of cooking the fruit accumulation of sugar in ripe fruit has a role to induce the activation of abscission before harvest. the balance between c2h4 as a process accelerator, and auxin (aux) as an inhibitor, is one of the main factors in the regulation of ripe fruit abscission. synthesis of auxin in young leaves and transported to adult fruit as an inhibition inhibitor that protects az from high c2h4 content. the role of the regulation of jasmonic acid (ja) in fruit reduction is thought to be mediated through the stimulation of c2h4 biosynthe­ sis. the balance between aux and aba in mature fruits is also important in determining the sensitivity of az­c for abscission stimulus because acc is an ethylene precursor (wang et al., 2002). fruit abscission that occur during fruit develop­ ment due to the active abscission zone. the process is induced by several environmental factors, competi­ tion in the use of assimilates, and internal hormone content. the abscission zone on the mango is located on the fruit stalk with a distance of several mm from the fruit concave (where the fruit is attached to the fruit stalk). from the biochemical and molecular aspects, abscission occurs due to the active enzyme ß­1,4­endoglucanase (eg) and polygalacturonase (pg). the two hydrolase enzymes are involved in damage to plant cell walls that are responsible for the drop of flowers and fruits. the specificity of the abscission zone in responding to organ drop depends on the sensitivity of the layer to ethylene (bonghi et al., 2000). control of fruit growth and abscission in oranges is due to three regulatory factors: genetic, metabolic, and environmental. all three of these affect hormon­ al signals in citrus plants (iglesias et al., 2007). overall, these studies show that a complex set of hormonal interactions occur during fruit develop­ ment. thus, gibberellins (gas) and cytokinins are generally considered positive fruit growth regulators while auxin has been reported to act as a growth stimulator and also as an abscess agent. abscisic acid (aba) and ethylene have been involved in several ways in an abscess. concentrations of iaa and ga3 in fruit and fruit stalks that drop easily are lower than 58 adv. hort. sci., 2021 35(1): 53­60 those in fruit retention and fruit stalks, fruits that will fall out have high abscisic acid content. drop fruit is also caused by an increase in ethylene production. table 2 shows that the flower and fruit drop had a lower total sugar content but a higher starch content than the retention. this is following stopar et al. (2001), apples that drop easily have higher starch content and lower sugar content compared to reten­ tion. mangoes that pre­abscission have a lower total sugar content and a higher starch than retention (sakhidin et al., 2011). total sugar content in flower and fruit develop­ ment of wax apple var. citra of bud development, anthesis, fruit set, fruit development, fruit matura­ tion, and fruit ripening stages 5.8, 7.6, 8.6, 20.16, 26.36, 28.66% lower in fruits that drop easily with fruit retention of 6.87, 8.72, 11.38, 23.35, 28.93, 32.59%. this is in accordance with rai et al., (2008), carbohydrate content (total sugar) in leaves in flower branches and mangosteen fruit which is lower (40.3 and 52.3 mg g­1 leaf dry weight) compared to leaves in branches that are flower and fruit retention (41.2 and 59.1 mg g­1 leaf dry weight). the retention of glu­ cose, fructose, and inverted sugar of wax apple fruit was as follows: 8.9%, 8.9%, 8.7% var. giant green, 9.83%, 9.9%, 9.6% var. masam manis pink, and 9.61%, 9.6%, 9.3% var madu red (khandaker et al., 2011). this shows that the drop of flowers and fruit is associated with a low supply of carbohydrates. limited carbohydrate sources affect the formation and development of fruit (pawar and rana, 2019). increasing the rate of photosynthesis is very important to produce carbohydrates during the development of fruit sets (iglesias et al., 2002). the limited supply of photosynthate and nutritional sta­ tus of plants can limit the number of flowers that develop into fruit that can be harvested (rai et al., 2008). in oranges, carbohydrate deficiencies produce young fruit abscess by triggering an increase in acc levels (gómez­cadenas et al., 2000). auxin applied to trees that have yellow leaves due to low carbohy­ drate cannot prevent pre­harvest loss (sakhidin et al., 2006). table 2 shows that the starch content in flower and fruit development stages of bud development, anthesis, fruit set, fruit development, fruit matura­ tion, and fruit ripening 16.29, 14.52, 13.11, 11.02, 8.77% in fruit retention. the starch content in wax a p p l e i s g e tti n g s m a l l e r a s t h e f r u i t d e v e l o p s . according to mureşan et al. (2015), the starch con­ tent was reduced in the early stages of fruit develop­ ment in the 3 apple varieties studied, then the starch concentration increased significantly from 35 to 65 days after anthesis. starch concentration gradually decreases as the fruit ripens. this can be seen in table 2 that the starch content during the develop­ ment of the wax apple fruit is seen to decrease from flower buds to fruit maturity, and in fruits that drop easily the starch content is higher than that of reten­ tion. it was concluded the content of iaa, zeatin, kinetin, ga3, and total sugars at several stages of the flower and fruit wax apple which drop easily lower than that of retention and the acc content and starch are higher in fruit which drops easily than retention. the endogenous hormone content of wax apple flower and fruit that drop easily are as follows iaa 0.41 to 1.42 ppm, zeatin 0.03 to 0.66 ppm, kinetin 1.38 to 2.96 ppm, ga3 8.97 to 28.63 ppm, and acc 30.92 to 48.39 ppm. the wax apple fruit that retention iaa content of 30.92 to 48.39 ppm, zeatin 2.04 to 3.29 ppm, kinetin 2.46 to 11.31 ppm, ga3 57.72 to 79.65 ppm, and acc 30.92 to 48.39 ppm. the total sugar content of wax apple flower and fruit which drop easily from 5.8 to 28.66% is lower than the retention rate of 6.87 to 32.59%. the content of fruit starch that drops easily is 11.21 to 17.9% higher than the retention of 8.77 to 17.23%. actions that can be taken to prevent the drop of wax apple fruit by giving natural endogenous hormones contained in other plant materials. this can be done after knowing the content of auxin, zeatin, kinetin, gibberellins, and acc in wax apple fruit that drop easily or retention. acknowledgements the research was supported by research funds from the directorate of research and community service, ministry of education and culture doctoral dissertation research scheme for the 2020 budget year through a decree of the chairperson of the research and community service institute, 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chrysanthemum to syn­ thesized silver nanoparticles (agnps) n. rashidiani 1, f. nazari 1 (*), t. javadi 1, s. samadi 2 1 department of horticultural science, college of agriculture, university of kurdistan, sanandaj, iran. 2 department of chemistry, faculty of science, univesity of kurdistan, sanandaj, iran. key words: cut flowers, ethylene, microbial growth, silver nanoparticles, vase life, water relations. a b s t r a c t : c a r n a ti o n ( d i a n t h u s c a r y o p h y l l u s l . ) a n d c h r y s a n t h e m u m [dendranthema grandiflorum (ramat.) kitam.] cut flowers are among the most important commodities that dominate flower markets throughout the world. two major problems in the transportation and marketing of these flowers are their relatively short vase life and the rapid decline of their aesthetic value. in this respect, the current study investigates the effects of silver nanoparticles (agnps) on ethylene­sensitive (carnation) and ethylene­insensitive (chrysanthe­ mum) cut flowers. specifically, this research examines their morpho­physico­ chemical characteristics, antioxidant enzyme activities and vase life. here, the agnps were synthesized by chemical methods and then applied on both flow­ ers by a pulsing method. the treatments involved two concentrations of agnps (0.04 and 0.08 g l­1) along with the control (deionized water), and the duration of exposure lasted for 24 h. then, the flower stems were placed in an aqueous sucrose solution (4%) until the end of the experiment. all traits, except the vase life, were evaluated after 0, 3, 6 and 9 d following the treatments during the vase period. during this time, the control groups of both flowers showed con­ siderable amounts of decrease in the relative fresh weight (rfw), vase solution uptake (vsu), flower diameter, membrane stability index (msi) and total solu­ ble carbohydrate (tsc). meanwhile, there were increases in hydrogen peroxide content (h2o2) and peroxidase (pod) activity. the bacterial population of the stem end and total soluble protein (tsp) increased in carnation petals, but decreased in chrysanthemum petals. the activity of superoxide dismutase (sod) dropped in carnation petals, whereas it rose in chrysanthemum petals. using agnps at concentrations of 0.08 and 0.04 g l­1 can optimally extend the vase life of carnation and chrysanthemum, respectively. 1. introduction nowadays, the floriculture industry is one of the most profitable sec­ tors in horticulture. its financial turnover in all respects amounts to over 300 billion dollars, and one­third of which is related to cut flowers (chandler and sanchez, 2012).consumer demand for cut flowers and (*) corresponding author: f.nazari@uok.ac.ir citation: rashidiani n., nazari f., javadi t., samadi s., 2020 ­ comparative postharvest responses of car‐ nation and chrysanthemum to synthesized silver nanoparticles (agnps). ­ adv. hort. sci., 34(2): 133­145 copyright: © 2020 rashidiani n., nazari f., javadi t., samadi s. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 25 november 2019 accepted for publication 1 april 2020 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(2): 133­145 134 ornamental plants are increasing, and the value of production is inevitably becoming higher. in this regard, flower quality is one of the most important indices in the sale and marketing of cut flowers. this makes flower quality an important factor in attracting customers over and over again (scariot et al., 2014). therefore, the advancement of post­harvest pro­ grams is a definite requirement within the cut flower market. even after their separation from mother plants, cut flowers continue to be metabolically active and proceed with all of their vital processes by consuming the available food in their tissues. carbohydrates, proteins and fat can be used by cut flowers to meet their metabolic demands. the increase in vase life and the delay in flower senescence can be achieved by upholding the normal rate of water uptake, pre­ venting the depletion of carbohydrate storage and limiting the exposure of flowers to ethylene (halevy and mayak, 1981). water balance is an essential factor in determin­ ing the quality and vase life of flowers. it should be maintained between water uptake and transpiration (lu et al., 2010). water uptake can be reduced due to the occlusion phenomenon generated by bacterial accumulation, physiological factors or air embolism (damunupola and joyce, 2008). post­harvest senes­ cence occurs within a few days, and is a major limita­ tion in the marketing of cut carnation and chrysan­ themum flowers. stem end blockage is one of the important factors in early wilting of leaves and inflo­ rescences in some cut flowers. van doorn and vaslier (2002) reported stem blockage is a major factor caus­ ing severe leaf wilting of chrysanthemum. the in­rolling of petal margin and wilting of whole petals in senescence process in some cut flowers (e.g. carnation), is associated with the self­regulation of ethylene production (yang and hoffman, 1984), as a hormone, implying that the flowers are sensitive to ethylene. on the other hand, there are flowers such as chrysanthemum, pericallis and narcissus that initi­ ate senescence without being influenced by ethyl­ ene, and thus are considered insensitive to ethylene (dole and wilkins, 2005; jones 2013). therefore, the short vase life and early wilting of inflorescences of carnation and chrysanthemum may be due to sensi­ tivity of ethylene and stem end blockage, respective­ ly. in fact, in ethylene­sensitive flowers, ethylene is a necessary requirement for the initiation and suste­ nance of senescence­based processes. the mecha­ nisms by which ethylene can stimulate senescence involve changing the cell structure and increasing the concentration of reactive oxygen species (ross) such as superoxide radicals, hydroxyl radicals and hydro­ gen peroxide (abeles et al., 1992). s t u d i e s h a v e a l s o s h o w n t h a t p h y s i o l o g i c a l changes occur during senescence. these include chlorophyll decomposition, decreased activity of antioxidant enzymes, increased productions of ethyl­ ene and ross, along with membrane damage to the cells of cut flowers (prochazkova and wilhelmova, 2007). advancements in nanotechnology, especially the development of silver nanoparticles (agnps), have led to a wide range of nanocomposites with antimi­ crobial properties. the high surface­to­volume ratio of these particles makes them biologically more active and increases their contact with fungi and bac­ teria (chau et al., 2007). agnps are becoming popular in the flower industry for their ability to inhibit the synthesis of ethylene and delay the senescence in cli­ macteric flowers (kim et al., 2005). in addition, agnps tend to regulate the stomatal aperture, main­ tain the chlorophyll content, preserve the relative fresh weight (rfw) and membrane stability index (msi), reduce the transpiration rate, weight loss, hydrogen peroxide(h2o2), malondialdehyde (mda) and ross, and increase the activity of antioxidant enzymes along with their effects on the vase life of different cut flowers such as gerbera (liu et al., 2009; solgi et al., 2009; nazari and koushesh saba, 2017), rose (lu et al., 2010; nazemi rafi and ramezanian, 2013; hassan et al., 2014), chrysanthemum (carrillo­ lópez et al., 2016), carnation (naing et al., 2017; lin et al., 2019 b), gladiolus (li et al., 2017), peony (zhao et al., 2018) and cut gardenia foliage (lin et al., 2019 a). the application of agnps in vase solution of carna­ tion alleviated vascular occlusion by inhibiting bacter­ ial colonisation and biofilm formation on stem­end cut surfaces and in the xylem vessels (lin et al., 2019 b). maity et al. (2019) also showed that the piper betle silver nanoparticles (pbsnps) in vase solution of gladiolus have played an important role for scaveng­ ing ross by enhancing antioxidant enzyme activities that led to decrease in mda and increased the msi. nanosponges are new nano­sized colloidal carri­ ers synthesized from ß­cyclodextrins that have been prepared for delivering preservative and anti­ethyl­ ene compounds (devecchi et al., 2009). devecchi et al. (2009) evaluated the effect of nanosponges including anti­ethylene molecules, such as 1­methyl­ cyclopropene (1­mcp), 1­methylcyclopentene (1­ mcpt), 2,5­norbornadiene and agno3 on vase life of carnation flowers. they concluded that, 1­mcp­ rashidiani et al. ‐ silver nanoparticles effects on ethylene sensitive and no sensitive cut flowers 135 nanosponge complex outperformed the other treat­ ments in extending the vase life. in addition, seglie et al. (2011) reported that 1­mcp in cyclodextrin­based nanosponges improved the vase life of carnation cut flowers. nonetheless, no report has so far described com­ parisons between the responses of ethylene­insensi­ ti v e a n d e t h y l e n e ­ s e n s i ti v e fl o w e r s t o a g n p s . therefore, the current research is aimed an evaluat­ ing how the cut flowers of carnation (highly sensitive to ethylene) and chrysanthemum (insensitive or slightly sensitive) respond to the application of agnps by the pulsing method. comparisons are made between the two types of cut flowers by measuring their morphological, physiological and biochemical properties, as well as their enzymatic activities. 2. materials and methods plant materials and application of treatments the cut flowers of carnation (dianthus caryophyl‐ l u s c v . y e l l o w v i a n a ) a n d c h r y s a n t h e m u m (dendranthema grandiflorum cv. boris becker sunny) were harvested at their commercial harvesting stage from a soilless­cultured greenhouse. they were taken to the laboratory and the basal ends of the stems were immediately cut to reduce the stem length to 40 cm. apart from 3­4 leaves on the top of each stem, all other leaves were removed. then, agnps were applied at two concentrations (0.04 and 0.08 g l­1) by the pulsing method for 24 h. the flowering stems were individually put in a bottle vase contain­ ing 300 ml deionized water. this also contained 4% (w/v) sucrose until the end of the experiment. to prevent the evaporation and contamination of the vase solution, the vase opening was covered with aluminum foil. all traits, except for vase life, were evaluated after 0, 3, 6 and 9 d following the applica­ tion of treatments. the ph of vase solution at the first day (d 0) was 7.82 and 8.76 for carnation and chrysanthemum, respectively. on d 9 of the vase period the ph of vase solution was 3.95 for carnation and 4.06 for chrysanthemum. the pulsing treatments and vase life were evaluated at 23±2°c, 50%±10% rh and 12 h of 15­20µmol m­2s­1irradiance from cool­ white fluorescence lamps. synthesis of agnps to a solution of 0.265 mm (0.045 g) of silver nitrate in 100 ml distilled water, 10 ml of a trisodium citrate aqueous solution (1%) was added slowly at room temperature. after 10 minutes, 0.2 ml of ascorbic acid (0.005 m) was added to the mixture of reaction and stirred for 1 h until a yellow­green agnps colloid was formed (tavallali and pouresmaeil, 2012). the surface morphology of agnps was indicat­ ed by scanning electron microscopy (sem) (tscan, czech republic) and showed in figure 1, it is exhibited that agnps have a size of about 8­80 nm. measurement of flower and stem diameter the flower diameter was measured in two direc­ tions, and the stem diameter was measured in three parts (i.e. under peduncle, middle, and end of the stem). these measurements were repeated every three days and the average of values were reported. measurement of relative fresh weight the relative fresh weight (rfw) of flowering stems in both types of cut flower was calculated every three days by the following formula: rfw (%) = (wt/wt0)×100 fig. 1 ­ the sem image of agnps. adv. hort. sci., 2020 34(2): 133­145 136 where wt is the weight of the stem (g) at t = d 0, 3, 6, and 9,while the wt0is the weight of the same stem (g) at t = d 0 (he et al., 2006). measurement of vase life the vase life of both types of cut flower was mea­ sured by counting the number of days from the beginning of the experiment until 50% of the flower had wilted. in carnations, the occurrence of in­rolling and browning petals by more than one­third was considered as the end of the vase life (naing et al., 2017). in chrysanthemum, however, the vase life ended when 50% of petals had wilted and the leaves had yellowed (carrillo­lópez et al., 2016). measurement of vase solution uptake the weight of the vase with and without the flower shoots was recorded daily, and the following formula was used for calculating the vase solution uptake (vsu): vsu (mg g­1 stem f. w.) = (st−1­st); where st is the weight of vase solution (g) at t = d 0, 3, 6 and 9, st−1 is the weight of vase solution (g) on the previous day (he et al., 2006; lu et al., 2010). measurement of membrane stability index to measure the membrane stability index (msi) of petals, this method involved the preparation of petal discs (measuring 1 cm in diameter) which were placed in falcons containing 20 ml distilled water. after this, a series of falcons were placed in a warm bath (40°c) for 30 min and their electrical conductivi­ ty (ec) was read by a conductivity meter (c1) after the falcons had cooled down to 25°c. then, the sec­ ond series of falcons was placed in a warm bath of 100°c for 20 min and their ec was read after having cooled down to 25°c (c2). in the end, the msi was calculated using the following equation (sairam et al., 2002): msi= [1­(c1/c2)] 100 c1 = ec after exposure to 40°c and c2= ec after expo­ sure to 100°c. measurement of bacterial population of stem end to measure the bacterial population on the stem end, one gram of the stem end was homogenized and diluted with peptone water until a concentration of 10­3was reached. subsequently, 1 ml of this solu­ tion was transferred to petri dishes and then a vol­ ume of 10 ml sterilized plate count agar medium was added to each petri dish. these were slowly mixed for 5 to 10 s. the cultured petri dishes were then kept in an incubator at 32°c for 2 d and, after count­ ing the bacterial colonies, the results were reported as log cfu g­1 (balestra et al., 2005; liu et al., 2009). measurement of total soluble protein according to the bradford method (1976), 0.5 g of petal tissue was powdered with liquid nitrogen, and then 0.25 g of polyvinylpyrrolidone (pvp) was added to the solution when stirring the 1.5 ml of potassium phosphate buffer containing sodium metabisulfite (0.019 g per 100 ml buffer). the homogenized sam­ ples were centrifuged (hettch, germany) at 4°c for 20 min at 15,000 g. then, 50 μl of supernatant was mixed with 950 μl of bradford solution and, after 15 min, the light absorption was read at 595 nm by a spectrophotometer (unico 2100, usa). measurement of peroxidase activity to measure the peroxidase (pod) enzyme activity, according to a method reported by hemeda and klein (1990), first 400 μl of 50 mm potassium phos­ phate buffer (ph 7) was mixed with 40 μl of 1% gly­ col and 40 μl of 0.3% hydrogen peroxide in an ice bed. then immediately, 65 μl of protein extract was added to the mentioned composition. the changes in light absorption were read at 120 nm by a spec­ trophotometer within a wavelength of 470 nm. measurement of superoxide dismutase activity the activity of superoxide dismutase enzyme (sod) was measured by the beyer and fridovich (1987) method. according to this method, the solu­ tion used for the reaction was prepared by mixing 25 ml of 50 mm phosphate buffer (ph 7) with 0.0035 g l­methionine (9.9 mm), 0.004 g nbt (57 μm) and 7.5 μl triton x­100. one ml of the reaction mixture was blended with 10 μm riboflavin and 20 μl of protein extract. the mixture was placed on a shaker at a dis­ tance of 30 cm from a 20­watt fluorescent lamp for 10 min. then, the light absorption was measured at 560 nm by a spectrophotometer. measurement of total soluble carbohydrate to measure total soluble carbohydrates (tsc), 0.5 g of petal tissue was crushed using liquid nitrogen along with 5 ml of 95% ethanol which helped obtain uniform extracts. the supernatant extract was cen­ trifuged for 10 min at 3,500 g. then, 1 ml of this extract was combined with 3 ml of anthrone before being transferred to a warm bath of 100°c. the heat caused the appearance of a colored phase after 10 min. subsequently, the samples were removed from the warm bath and were allowed to cool down at room temperature. their light absorption was read at 625 nm. the tsc content of petals was determined by creating a standard curve using standard glucose. rashidiani et al. ‐ silver nanoparticles effects on ethylene sensitive and no sensitive cut flowe 137 the results were expressed as mg g­1 f.w. (irigoyen et al., 1992). measurement of hydrogen peroxide in order to measure the amount of hydrogen per­ oxide (h2o2), the method was similar to the one used by alexieva et al. (2001). accordingly, 0.2 g of petal ti s s u e w a s c o m p l e t e l y g r o u n d w i t h 5 m l trichloroacetic acid (tca). the extract was obtained and centrifuged at 10,000 g for 5 min. then, 250 μl of the supernatant was mixed with 250 μl of 100 mm phosphate­potassium buffer (ph7) and 500 μl of 1 m of potassium iodide (ki). the absorbance of each sample was read by a spectrophotometer within a wavelength of 390 nm. statistical analysis this research was conducted as a factorial based on a completely randomized design (crd), it had three factors that the first factor was the type of flower at 2 levels (carnation and chrysanthemum), second factor was agnps at 3 levels (0.04 and 0.08 g l­1 along with deionized water) and the third factor was the sampling time at 4 levels (0, 3, 6 and 9 d of the vase period) with 3 replications, each of which included 8cut flower stems. for measuring the vase life, two factors of the type of flower and the sam­ pling time were not considered and its design was as a crd with three treatments (agnps at 0.04 and 0.08 g l­1 along with deionized water). data were analyzed using sas software and a comparison of mean values was made by the lsd test at 5% probability level. 3. results relative fresh weight based on the comparison of mean values, the rel­ ative fresh weight (rfw) of both cut flowers gradual­ ly decreased in the control treatment during the vase period. a faster rate of this decline was observed in carnations, as compared to the chrysanthemum. from the initial days to the ninth day, the decrease of rfw in carnations was about twice as much as the decrease in chrysanthemum. the application of0.04 and 0.08 g l­1 agnps caused the percentage of rfw to remain relatively constant in both cut flowers until t h e t h i r d d a y s , b u t t h e n t h e r f w g r a d u a l l y decreased. furthermore, treating the cut flowers with agnps caused a better preservation of their rfw from d 3 to d 9 of the vase period, as compared to the control, but the difference between the two concentrations was no significant. on d 9 of the vase fig. 3 ­ effect of two concentrations of agnps (0.04 and 0.08 g l­1) along with the control (deionized water) on vsu of two cut flowers during the vase period. values represent means ± standard error (n=3). lsd at p≤0.05 was used for means comparison. period, the rfw of chrysanthemum was heavier than that of carnations, and the differences were signifi­ cant (fig. 2). vase solution uptake there was an increase in the rate of vase solution uptake (vsu) by both flowers until the third day of the vase period, by which time the vsu in carnations was almost twice as much as that in chrysanthemum. from the third day onward, the vsu in both flowers decreased. the application of agnps caused an increase in the vsu in both flowers compared to the control. on d 9 of the vase period, the vsu in carna­ tions had been significantly affected by both concen­ trations of agnps, whereas the chrysanthemum was only affected by 0.08 g l­1 agnps to a substantial degree (fig. 3). fig. 2 ­ effect of two concentrations of agnps (0.04 and 0.08 g l­1) along with the control (deionized water) on rfw of two cut flowers during the vase period. values represent means ± standard error (n=3). least significant differen­ ce (lsd) at p≤0.05 was used for means comparison. 138 adv. hort. sci., 2020 34(2): 133­145 bacterial population of stem end the bacterial population of the stem end in carna­ tions increased throughout the vase period. in chrysanthemum, however, the same trend continued until the third days before declining. both concentra­ tions of agnps reduced the bacterial population of the stem end in chrysanthemum during the vase period, making it significantly different when com­ pared to the control on d 9. on the other hand, this trait in carnations was only affected by 0.08 g l­1 agnps to make a significant difference compared to the control. in general, in both flowers, 0.08 g l­1 agnps was more effective than the 0.04 g l­1 in reducing the bacterial population of the stem end. the highest of this trait among both cut flowers was observed in the control group of chrysanthemum on d 3, whereas the lowest of this value was caused by 0.08 g l­1 agnps and measured on d 9 (fig. 4). changes in the stem and flower diameter the stem diameter was one of the traits which was not significantly affected by agnps in both flow­ ers. even a comparison between d 0 and 9 of the control groups showed no significant difference. despite the fact that 0.08 g l­1 agnps in both flowers caused the stem diameter to become thicker in com­ parison with the stems of plants treated by 0.04 g l­1 and the control, there were no significant differences between these groups (data not shown). from d 3 onward, the diameter of both flowers decreased. however, it occurred more dramatically in carnations which shrank twice as much as the chrysanthemum. the effect of agnps on changing the flower diameter was significant only in carnations, during their vase period. although agnps increased the diameter of flowers in both species, as compared with their respective control groups, the diameters had finally decreased by d 9 of the vase period. on the mentioned day, these treatments had made a significant difference in carnations only, as compared to the carnations control group. in general, the biggest flower diameter was observed in chrysanthe­ mum on d 0 after being treated with 0.04 g l­1 agnps, and the smallest of all diameters was obtained in the control group of carnations on d 9 (data not shown). membrane stability index the decline in values of the membrane stability index (msi) occurred in both flowers during the vase period, and no significant differences were observed between the two flowers in this respect. in the begin­ ning of the vase period (d 0), the msi in carnations was higher than in chrysanthemum, but the value of this trait decreased in both flowers through time. on d 9, this decrease was twice as much in carnation when compared to chrysanthemum. in both flowers, agnps caused the msi to increase during the vase period, as compared to the control, but chrysanthe­ mum responded more strongly to the treatment than the extent to which carnations did. in response to agnps, chrysanthemum showed a gradual increase in the msi value ­ so much so that it became slightly ­1 agnps can be a successful treatment for increasing the msi in carnations. in contrast, however, the con­ centration of 0.04 g l­1 worked optimally on chrysan­ themum. in general, the highest value of msi was observed in carnations on d 0 when treated with 0.08 g l­1 agnps, whereas the lowest value occurred in the control on d 9 (fig. 5). total soluble protein a comparison of the mean values showed that the fig. 4 ­ effect of two concentrations of agnps (0.04 and 0.08 g l­1) along with the control (deionized water) on bacterial population of the stem end of two cut flowers during the vase period. values represent means ± standard error (n=3). lsd at p≤0.05 was used for means comparison. fig. 5 ­ effect of two concentrations of agnps (0.04 and 0.08 g l­1) along with the control (deionized water) on msi of two cut flowers during the vase period. values represent means ± standard error (n=3). lsd at p≤0.05 was used for means comparison. rashidiani et al. ‐ silver nanoparticles effects on ethylene sensitive and no sensitive cut flowe 139 content of total soluble protein (tsp) in chrysanthe­ mum petals was about 4 times higher than that of carnation petals on d 0. in carnations, the tsp con­ tent of the petals increased during the vase period. meanwhile, chrysanthemum underwent a different pattern of change, whereby the content of tsp increased from d 0 to d 3, but then decreased sharply until d 6, and continued to decline at a gradual rate until d 9. the agnps increased the tsp in both flow­ ers during the vase period, as compared to the con­ trol. generally, the highest value of tsp was observed in chrysanthemum on d 3 in the treatment group of 0.04 g l­1 agnps, whereas the lowest value was observed in the control on d 9 (fig. 6). total soluble carbohydrate the total soluble carbohydrate (tsc) content in carnation petals was more than the content in chrysanthemum petals. in the control groups of both flowers, the content of tsc decreased. the use of agnps in both flowers did not cause a significant dif­ ference in tsc on d 0, as compared with the control, but thereafter the difference gradually became signif­ icant until d9. applying the agnp at 0.08 g l­1 on car­ nations and at 0.04 g l­1 on chrysanthemum signifi­ cantly increased the tsc in comparison with their respective control groups. in general, carnations responded more strongly to the use of agnps, and the increase in their tsc content was much greater than in the case of chrysanthemum. the highest tsc content was measured in carnations on d 3 of the vase period after being treated with 0.08 g l­1 agnps. the lowest content was measured in the control group of chrysanthemum on d 9 (fig. 7). hydrogen peroxide content the petals of both flowers initially contained simi­ lar amounts of hydrogen peroxide (h2o2) which grad­ ually increased during the vase period. the rate of this increase was higher in carnations compared to the chrysanthemum. when comparing the h2o2 con­ tent between d 0 and d 9, its increase in carnations was about three times more than the increase mea­ sured in chrysanthemum. the application of agnps on both flowers reduced the h2o2 content in their p e t a l s , a s c o m p a r e d w i t h t h e c o n t r o l , b u t t h e increase was not prevented completely. on the last day of the vase period, the application of agnps on carnations led to a significant difference in h2o2 con­ tent when compared with the control. however, this was not the case in chrysanthemum. in general, the highest amount of h2o2 was measured in the control group of carnations on d 9 of the vase period. its low­ est amount was obtained in chrysanthemum on d 6 by 0.08 g l­1 agnps (fig. 8). fig. 6 ­ effect of two concentrations of agnps (0.04 and 0.08 g l­1) along with the control (deionized water) on tsp of two cut flowers during the vase period. values represent means ± standard error (n=3). lsd at p≤0.05 was used for means comparison. fig. 7 ­ effect of two concentrations of agnps (0.04 and 0.08 g l­1) along with the control (deionized water) on tsc of two cut flowers during the vase period. values represent means ± standard error (n=3). lsd at p≤0.05 was used for means comparison. fig. 8 ­ effect of two concentrations of agnps (0.04 and 0.08 g l­1) along with the control (deionized water) on h2o2 con­ tent of two cut flowers during the vase period. values represent means ± standard error (n=3). lsd at p≤0.05 was used for means comparison. adv. hort. sci., 2020 34(2): 133­145 140 peroxidase activity based on the results, peroxidase (pod) activity in both flowers increased until d 3 of the vase period and then decreased until d 6 before increasing again thereafter. using agnps on chrysanthemum, unlike carnation, significantly increased the activity of pod in comparison with the control. when treated with 0.04 g l­1 agnps, the chrysanthemum showed a level of pod activity on d 9 that was about 2.5 times greater than the activity in carnations on the same day. in general, the highest level of pod activity was measured in chrysanthemum on d 9 after the treat­ ment with 0.04 g l­1 agnps, whereas the lowest level of activity was measured on d 6 in the control (fig. 9). superoxide dismutase activity based on the results, that the superoxide dismu­ tase (sod) activity in carnation petals was about 2.5 times higher than that of chrysanthemum petals on d 0, but on d 9 it was completely different, and the activity of this enzyme in chrysanthemum was more than 4.5 times that of carnation. generally the activi­ ty of sod dropped in carnation petals, whereas it rose in chrysanthemum petals. the agnps decreased the sod in both flowers during the vase period, as compared to the control. generally, the highest value of sod was observed in chrysanthemum on d 9 in the control treatment, whereas the lowest value was observed in carnation on this day of vase period in the treatment group of 0.08 g l­1 agnps (fig. 10). vase life clearly, agnps caused the vase life of both flowers to increase. both concentrations had a significant effect on carnations, while chrysanthemum was sig­ nificantly affected by the 0.04 g l­1 only. in carna­ tions, the vase life increased parallel to the increase in applied concentrations of agnps from 0.04 to 0.08 g l­1, the effects of which were significantly different compared to each other. using 0.08 g l­1 agnps yield­ ed more appropriate results in carnations. on the other hand, the vase life of chrysanthemum was slightly affected in a negative manner as the concen­ tration of agnps rose from 0.04 to 0.08 g l­1, but the difference between the two concentrations was insignificant. nonetheless, both caused significant differences in comparison with the control (fig. 11a and b). 4. discussion and conclusions in this study, the cut flowers of carnation and chrysanthemum showed various levels of decrease in rfw (fig. 2), vsu (fig. 3) and flower diameter throughout the vase period. similar results have been reported after assessing the rfw and vsu of gerbera cut flowers (liu et al., 2009), rose (chamani et al., 2005) and cut gardenia foliage (lin et al., 2019 a) dur­ ing the vase period. the current study showed that using the agnps reduced the bacterial population of the stem end in both flowers, while the rfw was improved as compared to the control. previous stud­ ies confirm such findings on the use of agnps in cut flowers such as gerbera (liu et al., 2009; solgi et al., 2009; nazari and koushesh saba, 2017) and rose (lu et al., 2010; nazemi rafi and ramezanian, 2013; hassan et al., 2014). these studies showed that the vase life improves when the bacterial population of the stem end decreases, besides when the vsu and rfw increase. fig. 9 ­ effect of two concentrations of agnps (0.04 and 0.08 g l­1) along with the control (deionized water) on pod acti­ vity of two cut flowers during the vase period. values represent means ± standard error (n=3). lsd at p≤0.05 was used for means comparison. fig. 10 ­ effect of two concentrations of agnps (0.04 and 0.08 g l­1) along with the control (deionized water) on sod activity of two cut flowers during the vase period. values represent means ± standard error (n=3). lsd at p≤0.05 was used for means comparison. rashidiani et al. ‐ silver nanoparticles effects on ethylene sensitive and no sensitive cut flowe 141 the decrease in the rfw of cut flowers marks the beginning of senescence in flowers, and the more the flowers become closer to the process of senescence, their ability to take up water from the vase is reduced. the imbalance between water uptake and transpiration distorts cell turgor, thereby causing the flowers to wither (reid and jiang, 2012). the survival of cut flowers depends largely on a positive water balance: an increase in water uptake and a decrease in water loss (halevy and mayak, 1981; van doorn, 1997). after being placed in the vase, the flower loses rfw in part because of vascular occlusion and the growth of microorganisms which grow in the vase solution. they reduce the water uptake by blocking the stem end and vessels, thereby causing water stress which is a main factor in the reduction of vase life (van doorn, 1997; macnish et al., 2005). adding a germicide to the vase solution may control the activi­ ty of microbes. it has been reported that adding antimicrobial compounds containing ag+ ions to the vase solution improves the vase life of cut flowers (hassan et al., 2014). agnps nanoparticles function effectively because of their high surface­to­volume ratio and their crystallographic surface structure (rai et al., 2009). furthermore, ag+ ions cause bacterial cell death by affecting membrane structure and per­ meability, inhibiting dna transcription, disrupting transport activity and changing cellular content and atp (feng et al., 2000; sondi and salopek­sondi, 2004; rai et al., 2009; dakal et al., 2016). this difference in the bacterial population of the stem end of the two cut flowers may depend on the genotype of the plant, as well as the extract and metabolites that are secreted from the stem end and are released into the vase solution, thereby affecting the growth of bacteria. phenolic compounds are sec­ ondary metabolites that are involved in plant defense against pathogens (naczk and shahidi, 2004). in tradi­ tional chinese medicine, the flowering heads of chrysanthemum indicum are used as a source of bac­ tericide, with additional antifungal and antiviral prop­ erties (shunying et al., 2005). in agreement with our results, shunying et al. (2005) showed that the chemi­ cal composition and secondary metabolites of ch. indicum possess antimicrobial activity. flower diameters in this study were significantly influenced by agnps in carnation flowers, butthe effect on chrysanthemum was not significant. dar et al. (2014) reported that the flower diameter in dianthus chinensis first increased and then finally decreased during post­harvest development and senescence, respectively. the current research showed that the flower diameter of carnations tends to increase after being treated with agnps. this accords closely witha previous report on polianthes tuberosa which was treated with 0.015 g l­1 agnps and led to a significant increase in the diameter of flowers (bahrehmand et al., 2014). it is well known that the msi gradually decreases from the time when the flowers open to the time of their senescence. such a trend occurs evidently in certain flowers such as lilium (bieleski and reid, 1992), rose (hassan et al., 2014) and iris (ahmad and tahir, 2016). the senescence process is mainly asso­ ciated with protein loss, increased lipid peroxidation, membrane leakage, cell wall component degradation a n d c e l l u l a r m e m b r a n e d i s r u p ti o n ( b u c h a n a n ­ wollaston, 1997). agnps may preserve the mem­ brane stability by curbing the peroxidation of lipids (hatami and ghorbanpour, 2013). there was a lower percentage of msi in carnation petals, compared to chrysanthemum, which may be due to the fact that carnations are highly sensitive to ethylene. the rea­ son becomes clear when knowing that ethylene fig. 11 ­ effect of two concentrations of agnps (0.04 and 0.08 g l­1) along with the control (deionized water) on vase life of carnation (a) and chrysanthemum (b). values repre­ sent means ± standard error (n=3). lsd at p≤0.05 was used for means comparison. adv. hort. sci., 2020 34(2): 133­145 142 degrades the cell membrane and increases its leak­ age (van doorn and woltering, 2008), thereby result­ ing in a lower value of msi. however, agnps acts against the production of ethylene (hassan et al., 2014). in fact, agnps induce an efficient cellular elec­ tron exchange mechanism which reduces electron leakage and, subsequently, limits the creation of ross. agnps also dys regulate lipid peroxidation and have a propensity to maintain the msi (lu et al., 2010; hassan et al., 2014). previous studies have reported a decrease in tsp content and an increase in protease activity during the vase life of cut flowers (wagstaff et al., 2005; dar et al., 2014; zhao et al., 2018). quite differently, however, we concluded that changes in the tsp con­ tent may vary depending on the type of flower, species and cultivar. still, contrary to our results, dar et al. (2014) showed that protein degradation in dianthus chinensis is a key factor in regulating the senescence process of flowers. shahri et al. (2011) also reported that tsp decreased during the senes­ c e n c e p r o c e s s o f h e l l e b o r u s o r i e n t a l i s c v . ‘olympicus’, while there was an increase in its low­ molecular­weight proteins. accordingly, realizing an increase of tsp content in carnation petals could be a result of these low­molecular­weight proteins. i t h a s b e e n s h o w n t h a t i n fl o w e r s s u c h a s helleborus orientalis and dianthus chinensis, the tsc is reduced during the senescence process (shahri et al., 2011; dar et al., 2014).there was a lower content of tsc in chrysanthemum petals, as compared with carnations, which may be due to the genetic differ­ ences between the two plants, as well as the differ­ ence in the rate of polysaccharide decomposition during the opening and development of flowers and petals. in agreement with our results, it has been found that the starch and fructan polysaccharides are degraded and reduced during the development of flowers and the expansion of petals in chrysanthe­ mum (trustyl and miller, 1991). therefore, in this study, agnps in both flowers may have benefited the vase life by maintaining the content of tsc in petals and by promoting the mechanisms through which carbohydrates stabilize the cell membrane (ashraf et al., 2010). furthermore, agnps regulate and protect the cellular osmotic potential, inhibit the formation of free radicals (parida and das, 2005) and regulate the expression of genes (rahdari et al., 2012). usually, the amount of h2o2 increases in plant cells during senescence (ezhilmathi et al., 2007; saeed et al., 2014). it can be suggested that high lev­ els of h2o2 in carnation petals occur because of the plants sensitivity to ethylene. this can be compared with chrysanthemum which is not sensitive to the hormone. our results showed that the use of agnps on both flowers reduced the production of h2o2 in petals, as compared with the control. these results accord closely with a previous report by hassan et al. (2014) where the production of h2o2 became signifi­ cantly limited in roses because agnps was used. senescence is an oxidation process in which ros and antioxidant systems are involved (buchanan­ wollaston, 1997). plant cells have developed a series of antioxidant mechanisms for defense to prevent the production of ross and to limit their destructive effects on proteins, fats and nucleic acids (arora et al., 2002). the enzymatic part of this system consists of antioxidant enzymes such as sod, pod, catalase (cat) and ascorbate peroxidase (apx) which degrade all types of ross (balakhnina and borkowska, 2013). depending on the species or type of cultivars, the pod activity and sod enzymes can exhibit different patterns of change in cut flowers during the senes­ cence process (hassan et al., 2014). as the produc­ tion of h2o2 increased in the petals of both flowers during the vase period, the pod enzyme likewise increased its activity to scavenge the higher amounts of h2o2. furthermore, hassan et al. (2014) reported similar results which indicate that petals of the rose cut flower cv. ‘first red’ ­ which is sensitive to ethyl­ ene (chamani et al., 2005) ­ showed declining levels of sod activity during the vase period. however, con­ trary to our results on the incremental trend of pod in carnations and chrysanthemum during the vase period, hassan et al. (2014) reported that the pod activity in the rose cultivar decreases. it has also been reported that sod activity in ethylene­sensitive flowers such as carnation is relatively lower com­ p a r e d t o e t h y l e n e ­ i n s e n s i ti v e fl o w e r s s u c h a s chrysanthemum. this comprises a major factor in accelerating the senescence (bartoli et al., 1995). in addition, it may be suggested that in ethylene­ insensitive flowers such as chrysanthemum, ros cause the greatest amount of damage to the cell components, thereby leading to a shorter vase life. for this reason, the sod activity has to increase so as to create a parallel level of scavenging. in certain flowers such as carnations, which are sensitive to ethylene, the level of sod activity may not be pro­ nounced as much. as previously mentioned, agnps cause the plants to acquire an efficient cellular elec­ tron exchange mechanism, whereby the electron leakage and ros production are reduced (hassan et al., 2014). perhaps, the activity of sod does not rashidiani et al. ‐ silver nanoparticles effects on ethylene sensitive and no sensitive cut flowe 143 increase in carnations even when agnps are applied. agnps increased the vase life of chrysanthemum and carnation (figs. 11a and b) by increasing the rfw (fig. 2) and reducing the bacterial population of the stem end (fig. 4). the scientific literature con­ tains several reports that mention the efficiency of agnps in increasing the vase life of gerbera (liu et al., 2009; solgi et al., 2009; nazari and koushesh saba, 2 0 1 7 ) , r o s e ( l u e t a l . , 2 0 1 0 ; n a z e m i r a fi a n d ramezanian, 2013; hassan et al., 2014), chrysanthe­ mum (carrillo­lópez et al., 2016), carnation (naing et al., 2017) and gladiolus (li et al., 2017). in conclusion, agnps improved the vase life of both cut flowers by contributing to the values of their rfw, vsu, flower diameter, msi, tsc, tsp and pod activity and by limiting their bacterial population of the stem end and h2o2 levels, as compared to the control. it is highly probable that agnps are capable of reducing the expression of genes responsible for the production of ethylene, as well as limiting the rate of transpiration and the opening of stoma. furthermore, agnps have roles in regulating the aperture of the stoma and in reducing mda produc­ tion, thereby prolonging the vase life of cut flowers. ultimately, this research revealed that the use of agnps at 0.04 g l­1and 0.08 g l­1 can extend the vase life of at least two cultivars belonging to chrysanthe­ mum and carnation, respectively. acknowledgements the authors wish to thank the university of kurdistan for providing facilities and financial support (grant no. crc97­00242­1) of this research. references abeles f., morgan p., saltveit j.r.m., 1992 ­ ethylene in plant biology. ­ academic press, san diego, ca, usa, pp. 399. ahmad s.s., tahir i., 2016 ­ increased oxidative stress, lipid peroxidation and protein degradation trigger senescence in iris 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i f e s c i e n c e s a n d e n v i n r o n m e n t a l sustainability, university of parma. parco area delle scienze, 11/a, 43124 parma, italy. key words: genetic diversity, phenotype, pomegranate, rapds, ssrs, varieties. abstract: the italian research on p. granatum l. is still limited, although the study of local germplasm is extremely important in order to preserve the exist­ ing biodiversity and to identify potential useful characters for a renewed indus­ try. the study aimed at characterizing for the first time ancient pomegranates, grown in emilia romagna (italy), through 38 quantitative morphometric descriptors related to leaf, flower, fruit and seed, 42 rapd and 12 ssr markers. morphological analyses showed large variation of traits among accessions and the descriptors related to fruit and seed had the highest power of discrimina­ tion. the considerable variation found was consistent with anova and pca results. among all rapds tested, 7 were selected for their polymorphism; whereas among selected ssrs, 8 presented differences in the genetic profiles allowing a good discrimination of the local pomegranate accessions. the genet­ ic relationships among pomegranates were studied by upgma cluster analysis and the accessions were clearly regrouped in four different genotypes. the study has highlighted significant differences and interesting pomological char­ acteristics in the local pomegranates, which confirmed the good potential of this germplasm for the pomegranate industry. 1. introduction pomegranate (punica granatum l.) is one of the world most ancient domesticated fruit crops and it is believed to have been first grown in the region between the caspian sea and the caucasus (zohary and spiegel­ roy, 1975). its diffusion occurred across the millennia due to man’s activi­ ties or gene flow in quite varied environments as concerns climatic condi­ tions, has produced a rich and diversified germplasm. p. granatum l. has (*) corresponding author: deborah.beghe@unipr.it citation: beghè d., fabbri a., petruccelli r., marieschi m., torelli a., ganino t., 2019 ­ morphological and molecular characterization of ancient pome‐ granate (punica granatum l.) accessions in northern italy. ­ adv. hort. sci., 33(4): 581­592. copyright: © 2019 beghè d. , fabbri a., petruccelli r., marieschi m., torelli a., ganino t. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 27 june 2019 accepted for publication 22 october 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(4): 581­592 582 a l a r g e g e n e ti c p o o l , r e p r e s e n t e d b y o v e r 5 0 0 described cultivars throughout the world, and by a wide amount of wild plants, and its germplasm is so far only partially explored (beghè et al., 2016). in spite of the wide genetic diversity, only 50 cultivars were widely cultivated in the main growing areas at the time of the last official survey made by interna­ tional institutions (ipgri, 2001). consequently, the risk of a drastic loss of the existing biodiversity is high. in the last decade the interest on pomegranate has grown and the world production (estimated to be around 1.5 million tonnes, of which 90% provided by the main producers: india, china, iran) has rapidly increased (da silva et al., 2013). the renewed interest in this crop is to be ascribed to socio­economical and cultural factors, which led to a change in food habits in the west, with a growing attention to the nutrition­ al quality of foods (negri, 2003). pomegranate, in this respect, is considered one the fruits with the most valuable nutritional properties (calani et al., 2013). pomegranate cultivation is also growing in italy, and in a ten­year period (since 2008) the surface cov­ ered, from a mere 7 ha (2 ha in calabria and 5 in sicily), passed to 1142 ha (mainly in southern italy, sicily (364 ha) and apulia (363 ha) and, with minor productions, also in venetia (152 ha), latium (101 ha), emilia romagna (44 ha), tuscany (21 ha) and lombardy (20 ha). productions rose from only 69 tons to over 12531 tons (agristat, 2017). one of the trends of recent years has been to import from israel pomegranate cultivars created and patented by israel breeders, to be utilised in italian orchards. although this strategy has led to an increase in production, it hinders any attempt at exploiting italian cultivars, which have been known for centuries but are not utilised in commercial culti­ vation. in italy, there are several local cultivars which are little known and scarcely diffused in the country. scarce research, mostly confined to southern italy, has been devoted to the characterization of p. grana‐ tum l. italian diversity, by means of molecular, mor­ phological and biochemical markers (adiletta et al., 2018). a research on the whole national territory is unavoidable to select autochthonous genotypes suit­ ed to the different environments, and to promote a pomegranate industry able to satisfy the internal demand and to compete with the international product. the best suited italian areas for pomegranate growing are the central and southern regions, char­ acterized by a mediterranean climate, unlike the northern regions, which have a continental climate, with cold and snowy winters. however, in some zones of emilia­romagna (44°­45° n and 11°­12° e, northern italy) microclimate conditions are such as to allow the cultivation of this species and of other mediterranean species like olive (lona et al., 1981; calani et al., 2013). ancient pomegranate trees, survived for hun­ dreds of years and adapted to local conditions have been retrieved in these areas. this local germplasm is of particular interest, being the result of a selection process occurred during many centuries in the unfa­ vorable conditions of this territory. the present research is part of a multidisciplinary project aiming at valorising ancient pomegranate cul­ tivars of northern italy. we utilized morphological a n d m o l e c u l a r m a r k e r s ( r a n d o m a m p l i fi e d polymorphic dna (rapds) and simple sequence repeat (ssrs)) to characterize ancient pomegranate accessions present in emilia­romagna region. the pomological comparisons also had the purpose of determining the peculiar features and the potential of these plants, for a possible introduction in commer­ cial plantings or for their use in breeding programs. 2. materials and methods plant materials the pomegranate germplasm subject of this study was represented by very ancient trees located in a small hill area of parma province (emilia­romagna) (44°69 n, 10°02 e), at an altitude from 150 to 250 m a.s.l. eight accessions of p. granatum l., tagged with an alpha­numerical code (id): me1, me2, me3, me4, me5, me6, me7 and me8 (table 1), were studied during two seasons, 2014 and 2015. the selected plant material was maintained at pomegranate germplasm collection field in the low hills of the emilia appennins, where each accession was repli­ cated 4 times. the pomegranate trees were planted at a spacing of 5 x 5 m and trained to form a bush. morphological characterization the accessions were characterized according to the guidelines proposed by the project ec project genres 29 “conservation, evaluation, exploitation and collection of minor fruit tree species” (bellini and giordani, 1998), integrated by the list of characters p r o p o s e d b y b e l l i n i e t a l . ( 2 0 0 7 ) a n d b y t h e i n t e r n a ti o n a l u n i o n f o r t h e p r o t e c ti o n o f n e w varieties of plants (upov, 2012). p l a n t m a t e r i a l w a s r a n d o m l y s a m p l e d f r o m beghè et al. ‐ characterization of ancient pomegranates in northern italy 583 around the canopy of four plants per each accession, by collecting 40 flowers (20 hermaphrodite also called “long­styled” and 20 male also called “short­ styled”) at full bloom, on june 1st, 40 adult leaves, from the middle part of the shoot in summer and 12 fruits, at ripening, in the first decade of october. all seeds were extracted from each fruits and 25 of them were randomly selected; arils (the seed fleshy coats, containing edible juice, that represent the seed outer integument or testa) were hand removed to analyze also the tegmen (seed lignified inner integument). the morphological characters evaluat­ ed included 38 quantitative traits (table 2). the linear dimensions were determined with a caliper, and the weight was measured using a semi­analytic electronic scale. from these values other indices have been cal­ culated as indicated in table 2. furthermore, some qualitative characters were observed; these traits are reported in table 1. dna extraction and molecular characterization total cellular dna was extracted from young leaves following the ctab (cetyl trimethylammonium bromide) as reported in ganino et al. (2008). f o r t y ­ t w o d e c a m e r o l i g o n u c l e o ti d e p r i m e r s belonging to the ai, ah, opa, opc, opx and opk series (table s1 of supplementary data) and twelve couples of ssr primers belonging to the pgaer (çalişkan et al., 2017), pgkvr (ravishankar et al., 2015), pom (hasnaoui et al., 2012), pom­agc (currò et al., 2010) and pg (ebrahimi et al., 2010) were used for polymorphism detection on the samples. rapd a m p l i fi c a ti o n s w e r e p e r f o r m e d a s r e p o r t e d i n marieschi et al. (2016). the rapd profiles obtained with each utilized primer were analyzed by comparison with gene ruler 100 pb dna ladder plus marker (m­medical, milano, it), with the kodak digital sciences 1 d images analysis software, calculating the size in base pairs (bp) of each amplicone present in the elec­ trophoretic run of each sample. ssr amplification reaction was performed as reported in ganino et al. (2008). the amplification condition, for the pgkvr, pgaer, pom­agc and pg series, were: a first step at 95°c for 5 min followed by 35 cycles of 45 s at 94°c, 45 s at 57°c, 45 s at 72°c, for denaturation, annealing, and primer extension; the last step included 8 min of incubation at 72°c. for the “pom” serie, the following thermal cycling proto­ col was used: a first step at 95°c for 3 min followed by 10 touchdown cycles of 30 s at 94°c, 40 s at 65°c (­1°c per cycle), 30 s at 72 and 25 cycles of 30 s at 94°c, 30 s at 55°c, 40 s at 72°c with final extension time of 8 min at 72°c. the amplification products were separated with a ceq 2000 genetic analysis system (beckman coulter, inc.) sequencer on acry­ lamide gel ceq separation gel lpa­1 (beckman coulter, inc.). a marker ceq dna size standard kit 400 (beckman coulter, inc.) was used to estimate the molecular weight of the amplified products. data analysis the quantitative morphological characters were evaluated: means, minimum and maximum, standard deviation. the coefficient of variation (cv) was calcu­ lated as indicator of variability. all data were subject­ ed to one way analysis of variance (anova) followed by tukey test to determine the statistically significant differences (p≤0.05). correlation analyses between descriptors to reveal possible relationships were car­ table 1 ­ punica granatum l. accessions studied, coded (id) and main qualitative characteristics of their fruit, leaf and flower id fruit shape size epicarp colour calyx type leaf shape petiol colour mucro blade colour flower petal colour shape short­ stiled shape long­ stiled me1 oblate/rounded­ spheroid large/very large reddish­yel­ low/red semi­closed/ closed elliptic yellow no yellow red/orange medium bell sinuolate jug me2 oblate/rounded­ spheroid large/very large reddish­yel­ low/red semi­closed/ closed elliptic red no yellow red/orange medium bell sinuolate jug me3 oblate/rounded­ spheroid large reddish­yel­ low/red semi­closed/ closed elliptic red no yellow red/orange medium bell sinuolate jug me4 oblate/rounded­ spheroid large/very large reddish­yel­ low/red semi­closed/ closed elliptic red no yellow red/orange medium bell sinuolate jug me5 oblate/rounded­ spheroid large/very large reddish­yel­ low/red semi­closed/ closed elliptic red no yellow red/orange medium bell sinuolate jug me6 oblate/rounded­ spheroid large reddish­yellow semi­ closed/ open elliptic red no yellow red/orange broad bell jug with base me7 oblate/rounded­ spheroid very small reddish­yel­ low/red open elliptic yellow no yellow red/orange narrow bell sinuolate jug me8 oblate/rounded­ spheroid very small reddish­yel­ low/red open elliptic yellow no yellow red/orange narrow bell sinuolate jug adv. hort. sci., 2019 33(4): 581­592 584 ried out using a bilateral pearson correlation. the same characters were also submitted to a principal component analysis (pca) to evaluate the relation­ ship between pomegranate accessions. the analysis w a s p e r f o r m e d u s i n g x l s t a t 2 0 0 9 s o ft w a r e (addinsofttm1995­2009). rapd bands were treated as binary characters (present = 1 or absent = 0), xlstat 2009 software was used to estimate genetic similarities/dissimilari­ ties using jaccard’s similarity coefficient and cluster analysis by using the unweighted pair­group method with arithmetic mean (upgma) algorithm. the size of ssr fragments was determined using a conservative binning approach (kirby, 1990) through the statistical r software. the information content of the ssr markers under study was evaluated according to number of alleles per locus (na), observed (ho) and expected (he) heterozygosity, and polymorphic infor­ mation content (pic) (botstein et al., 1980) using the cervus 3.0 software (kalinowski et al., 2007). the level table 2 ­ quantitative traits used for characterizing pomegranate accessions and their descriptive statistics analysis using the mean, minimum, maximum, standard deviation (sd) and coefficient of variation (cv) (z) aril weight were calculated by subtracting tegmen fresh weight from whole seed fresh weight. trait trait code mean minimum maximum sd cv (%) leaf leaf fresh weight (g) lfw 0.10 0.070 0.160 0.029 29.13 leaf length (cm) ll 5.44 4.450 6.920 0.828 15.21 leaf width (cm) lw 1.62 1.260 2.150 0.278 17.21 leaf shape (length/diameter) ls 3.50 2.970 3.910 0.341 9.76 flower flower diameter long­styled (cm) fdl 1.51 1.220 1.810 0.221 14.57 flower length long­styled (cm) fll 4.75 2.900 5.950 1.193 25.11 petal number long­styled (cm) pnl 6.52 5.670 7.500 0.713 10.93 pistil length long­styled (cm) pll 1.75 1.500 2.030 0.231 13.20 flower diameter short­styled (cm) fds 1.48 1.200 1.770 0.163 11.06 flower length short­styled (cm) fls 3.73 2.840 4.490 0.564 15.10 petal number short­styled pns 6.50 6.000 7.400 0.510 7.86 pistil length short­styled (cm) pls 0.43 0.310 0.600 0.105 24.43 fruit fruit weight (g) fw 274.02 87.010 388.730 120.303 43.90 fruit diameter equatorial (cm) fd 8.22 5.070 9.600 1.799 21.88 calyx diameter equatorial (cm) cd 2.14 1.430 2.840 0.545 25.50 fruit height without calyx (cm) fl1 6.64 4.400 8.120 1.380 20.80 total fruit length (cm) fl2 8.11 5.570 9.530 1.568 19.34 calyx height (cm) cl 1.56 1.170 2.220 0.323 20.67 fruit skin thickness equatorial (mm) ft 0.42 0.300 0.550 0.096 22.69 fruit skin and carpellary membranes weight (g) scw 129.94 28.980 191.810 66.526 51.20 number of carpel in equatorial section nc 6.88 5.330 8.000 0.993 14.43 fruit shape index (height/diameter) fsi 0.81 0.730 0.890 0.059 7.21 calyx shape index (height/diameter) csi 0.80 0.610 0.880 0.095 11.93 % skin and carpellary membranes sc (%) 45.10 33.300 53.400 7.499 16.63 % seeds s (%) 54.92 46.600 66.700 7.491 13.64 total seeds weight (g) stw 141.918 58.076 202.556 53.394 37.62 seed weight (g) sw 0.27 0.144 0.381 0.080 29.57 seed length (cm) sl 0.96 0.786 1.080 0.113 11.73 seed diameter (cm) sd 0.69 0.547 0.804 0.090 13.04 tegmen weight (g) tw 0.02 0.016 0.029 0.005 19.95 tegmen length (cm) tl 0.65 0.544 0.769 0.074 11.35 tegmen diameter (cm) td 0.31 0.253 0.416 0.052 16.88 woody portion index (tegment weight/aril weight ) wpi 0.09 0.071 0.121 0.021 22.12 seed shape (length/diameter ) sl/sd 1.39 1.310 1.540 0.083 5.93 tegment shape (length/diameter) tl/td 2.15 1.880 2.410 0.176 8.19 aril weight (g) z aw 0.25 0.120 0.350 0.080 32.49 aril weight/tegmen weight aw/tw 10.04 7.400 12.390 2.162 21.53 % aril a (%) 90.35 88.200 92.600 1.653 1.83 beghè et al. ‐ characterization of ancient pomegranates in northern italy 585 wpi, aw, aw/tw). these results are consistent with previous studies (zamani et al., 2007, mansour et al., 2011). the mean leaf quantitative values are reported in table s2 and the traits values presented significant differences between the accessions. moreover, leaf blade margin color and petiole color next to the shoot was red for accessions me1, me7 and me8, and yellow for the others. all accessions have an elliptic shape and absence of mucro (table 1). the flower characteristics are reported in table 1 and table s3 and the observed values are comparable with those of lebanese genotypes studied by dandachi et al. (2017). the flowers of me7 and me8 accessions showed a much smaller size than that of the flowers of the other, but presented a similar style length, a feature that favors pistil pollination. moreover, the long­styled flowers presented a “sinu­ ate jug”, except the flowers of me6 accession that pre­ sented “jug with base”. me7 and me8 presented a “narrow bell” shape in the short­styled flowers where­ as me6 presented “broad bell” shape and other acces­ sions presented flowers with “medium bell” shape. the mean values of quantitative fruit and seed traits are reported in tables 3 and s4. significant vari­ ability was observed in total fruit weight (fw), in maximum equatorial diameter (fd) and in fruit length, with calyx (fl2) and without calyx (fl1). in particular, these characters have lower values for me7 and me8. all accessions showed fruits with shape “oblate or rounded­spheroid” and “closed or semi­ closed calyx”, except me7 and me8 that have a majori­ ty of fruits with “open calyx”. the number of locules (nc) was higher in fruits of higher total weight. of similarity/dissimilarity among examined accessions was obtained through the genetic similarity matrix uti­ l i z i n g m a n h a tt a n d i s t a n c e a n d c l u s t e r a n a l y s i s (upgma) algorithm, with xlstat 2009 software. finally, to test the correlations between genetic distance matrices and between the morphological and genetic distance matrices among accessions, mantel tests were performed (mantel, 1967). each matrix dis­ tance was obtained by calculating pearson’s index. mantel tests were performed with 100,000 permuta­ tions (p = 0.05). pearson’s r­value was used to mea­ sure linear correlation between two matrices. 3. results and discussion morphological characterization the data resulting from the 2­year study were grouped and the average values were used for statis­ tical analysis. the accessions showed significant vari­ a b i l i t y i n m a n y o f t h e c h a r a c t e r s a n a l y z e d . descriptive values for each quantitative trait are recorded in table 2. the coefficient of variation (cv) was used to determine the total variability present in each trait. the cv varied from 1.83% (a%) to 51.20% (scw%), with seven traits having cv between 15 and 20% and fourteen traits with cv value higher than 20%. according to audergon (1987), the descriptors with a high cv are more discriminating than the other ones, and can be reliable markers for the char­ acterization of pomegranate accessions. the highest cv values were evident in traits involving fruits and the lowest were in flowers (except fll and pls), leaves (except lfw) and seeds (except stw, sw, table 3 ­ mean values, standard deviation and anova analysis for fruit characteristics the same letter show no statistically significant differences (p<0.05). (z) for explanation of character symbols, see table 2. id fw (z) fd cd fl1 fl2 cl ft scw nc fsi csi sc% s% me1 373.48 ±49.78 a 9.60 ±0.62 a 2.68 ±0.51 a 7.00 ±1.14 a 9.22 ±1.28 a 2.22 ±0.62 a 0.55 ±0.10 a 191.81 ±19.64 a 8.00 ±1.58 a 0.73 ±0.12 a 0.84 ±0.27 a 53.4 ±0.03 a 46.6 ±0.03 d me2 303.53 ±93.46 a 8.83 ±1.17 a 2.40 ±0.62 a 7.83 ±0.72 a 9.53 ±1.06 a 1.70 ±0.43 a 0.53 ±0.11 ab 144.42 ±51.87 a 6.67 ±0.58 ab 0.89 ±0.07 a 0.70 ±0.03 a 47.2 ±0.03 ab 52.8 ±0.03 cd me3 335.73 ±13.03 a 9.10 ±0.52 a 2.43 ±0.55 a 7.10 ±0.17 a 8.53 ±0.61 ab 1.43 ±0.49 a 0.44 ±0.10 bc 163.25 ±11.59 a 7.67 ±0.58 a 0.78 ±0.06 a 0.61 ±0.23 a 48.6 ±0.02 ab 51.4 ±0.02 cd me4 388.73 ±89.49 a 9.40 ±0.84 a 2.15 ±0.78 a 8.12 ±1.03 a 9.00 ±1.27 ab 1.61 ±0.41 a 0.48 ±0.11 abc 185.73 ±46.34 a 7.60 ±0.55 a 0.86 ±0.06 a 0.82 ±0.31 a 48.3 ±0.03 ab 51.7 ±0.02 cd me5 345.68 ±87.85 a 9.50 ±1.14 a 2.84 ±0. 94 a 7.47 ±0.67 a 9.24 ±0.81 a 1.63 ±0.55 a 0.41 ±0.09 bc 181.74 ±47.63 a 7.25 ±0.58 ab 0.80 ±0.05 a 0.85 ±0.27 a 52.5 ±0.01 a 47.5 ±0.01 d me6 266.81 ±23.78 ab 8.60 ±0.71 a 1.70 ±0.14 a 6.45 ±0.64 ab 7.95 ±0.78 ab 1.50 ±0.14 a 0.37 ±0.08 bcd 111.03 ±6.31 ab 7.00 ±0.45 ab 0.75 ±0.01 a 0.88 ±0.01 a 41.9 ±0.06 bc 58.2 ±0.06 bc me7 87.01 ±21.77 b 5.67 ±0.57 b 1.43 ±0.06 a 4.73 ±0.38 b 5.82 ±0.58 b 1.25 ±0.11 a 0.30 ±0.09 d 28.98 ±7.48 b 5.33 ±0.58 b 0.84 ±0.02 a 0.88 ±0.19 a 33.3 ±0.01 d 66.7 ±0.01 a me8 91.21 ±27.45 b 5.07 ±0.11b 1.47 ±0.06 a 4.40 ±0.10 b 5.57 ±0.23 b 1.17 ±0.15 a 0.30 ±0.79 d 32.54 ±10.48 b 5.50 ±0.55 b 0.87 ±0.03 a 0.80 ±0.13 a 35.5 ±0.02 cd 64.5 ±0.02 ab 586 adv. hort. sci., 2019 33(4): 581­592 according to the list of “pomegranate descriptors” of bellini et al. (2007), the fruits were classified as “large or very large” (me1, me2, me3, me4, me5, me6) and “very small” (me7, me8). the first group had a total weight mean values of about 335 g, comparable to those of the fruits of many italian and spanish cultivars (martinez et al., 2006 and ferrara et al., 2014). moreover, the epicarp (or “skin”) of the local fruits has presented different colors, ranging from reddish­yel­ low to red. the size of the fruit and the color of the epicarp are two important parameters considered in the international market as concerns the quality of the fresh product (mansour et al., 2011). another impor­ tant parameter for fruit quality was the descriptor “skin thickness”. me7 and me8 fruits have a thinner “skin thickness” than other accessions, and in field their fruits were more subjected to cracking at the first rainfall in october. significant variability was observed in seeds total weight (stw), skin and carpellary membranes weight (scw) and seeds percentage (s%) (tables s4 and 3). the mean s% found was of 54.91% and it was similar to that reported in another italian study (cristofori et al., 2011). furthermore, our mean values were lower than those reported on italian and iranian cultivars (ferrara et al., 2014). the seed descriptors showed, for the majority of traits, significant differences between two groups of accessions: me1, me2, me3, me4, me5, me6 and me7, me8; the former had sl, sd, sw, tl, tw greater than the latter (table s4). tegmen index (wpi), aril percentage (a%), aril weight (aw) and aril/tegmen ratio (aw/tw) are very important parameters from a qualitative point of view. the wpi is a parameter that refers to the quantity of lignified tissue contained in the seed compared to total seed weight, and consumers greatly appreciate seeds with a limited amount of lignified tissue (martinez et al., 2006). accessions me1, me2, me3, me4, me5 had a higher quantity of aril (aw) and a lower percentage of the tegmen index (wpi) than me6, me7 and me8. the wpi presented an average value of 7.7% in the first group of accessions, and an average value of 11.9% in the accessions me6, me7 and me8. these values were in agreement with those of other italian (5.4 to 10%), spanish (7.4 to 9.7%), moroccan (6.1 to 10.7%) and iranian (5.4 to 7.5%) accessions (martinez et al., 2006; 2012; sarkhosh et al., 2009). the aril is a tissue valued for the high pro­ duction of juice; aw and a%, with reference to the individual seed, were high in all accessions, and a% showed an average value of about 90%; a value com­ parable and higher than that of other italian and spanish genotypes (la malfa et al., 2009). correlation among morphometric traits the correlations found between the quantitative variables, significant at p < 0.05, are reported in fig­ ure 1. the correlation coefficient can provide infor­ fig. 1 ­ pearson's correlation matrix of the quantitative traits in pomegranate accessions, visualized as a heat map plot. for explanation of character symbols, see table 2. beghè et al. ‐ characterization of ancient pomegranates in northern italy 587 mation on the traits that are most important in assessing accessions (norman et al., 2011). skinner et al. (1999) recommended analyzing correlation coeffi­ cients close to 0.7: in these conditions, the variance of one trait is strongly dependent on the others. according to this criterion, we estimated two hun­ dred and fifteen valid correlations. most of the signif­ icant correlations among traits coincide with those from the same plant organ, in particular the variables relative to fruits and seeds. a strong correlation value was found for fruit and seed descriptors, and these were also positively correlated with each other (e.g. fw was positively correlated with other fruit descrip­ tors, fd, fl1, fl2, cl, scw, nc, sc%, s% and seed descriptors, stw, sl, sd, sw, tw, a%, aw, aw/tw). the character wpi was negatively correlated with fruit traits relative to dimensions and with sw, sl, sd. the same results were observed in several for­ mer works that analyzed accessions from different countries (martinez et al., 2006; zarei et al., 2013). moreover, the trait wpi could be an index of seed hardness, because this feature is strongly dependent on the trait wpi, as reported in martinez et al. 2012 (r= 0.63 p ≤ 0.01). in flowers, positive correlations were found among the variables relative to dimen­ sions: between fls and fds and between fll and fdl. in leaves, a positive correlation was between ld and ll, actually all accessions showed an elliptic leaf shape. finally, some correlations were observed between seed (sw, a%, aw, tl, tw) and flower char­ acteristics (fdl, fll, fds, fls, pns); we noted that accessions with small flowers (me7­me8) presented fruits and seeds of smaller dimensions. in fact, fruit growth potential is largely determined genetically through the ovary size in several species (rosati et al., 2009). these correlations between different traits could be due to genetic linkage or to a pleiotropic effect (i.e., when one gene influences two or more seemingly unrelated phenotypic traits) (iezzoni and pritts, 1991). principal component analysis the results of pca revealed the existence of large variability among accessions. the total variance explained by the first three principal components (pcs) in the model was 83.53%. a plot of the percent­ age of variance explained by seven pcs and eigenval­ ues associated with the first seven pcs for each quan­ titative trait are reported in supplementary material ( f i g . s 1 a n d t a b l e s 5 ) , r e s p e c ti v e l y . t h e p c 1 explained the 54.60 % of total variance and the traits with the greatest weight on this component were related to fruit (fw, fd, cd, fl1, fl2, cl, ft, scw, nc, sc%), seed (stw, sl, sd, sw, tw, wpi, a%, s%) and some flower traits (fdl, fll, fls). the pc2 explained 15.82% of the variability, and showed a strong negative load for td, ld and pns, whereas a strong positive load was present for sl/sd ,tl/td, fsi, and ls. finally, ld, ll relating to the leaf and pls relating to the flower showed the highest contribu­ tion to pc3 (13.11% of the variability). the compari­ son of plot scores for pc1, pc2 and pc3 in figure 2 permits to obtain a view of accession dispersion and their clustering based on morphological traits. the accessions, for the first two pcs, were grouped into two main groups highly dissimilar: the first group consists of me7 and me8, the second group consists of three sub­groups (sub­group me6; sub­group me2, me3, me4, me5 and sub­group me1). the groups plotted for the pc1 and pc3 were very similar to t h o s e o n p c 1 ­ p c 2 p l o t , t h o u g h a c c e s s i o n m e 6 showed less differences with the sub­group me2, me3, me4, me5. as already reported in the literature (martinez­nicolas et al., 2016), fruit characteristics fig. 2 ­ loading plots of the first, second and third principal component showing the position of accessions. adv. hort. sci., 2019 33(4): 581­592 588 had the highest loading values for the first compo­ nent in principal component analysis. our results confirmed that the traits related to fruit and seed had the highest power of discrimination, and were, therefore, the most useful for characterization of this local germplasm. rapds and ssrs characterization and genetic rela‐ tionships only 7 rapd oligonucleotides (ai08, ai12, ai105, ah17, opa19, opb08, opc16) out of 42 belonging to the ai, ah, opa, opc, opx and opk series, showed polymorphism in two or more accessions, by produc­ ing polymorphic and reproducible amplification pat­ terns. the 7 oligonucleotides amplified a total of 84 rapd fragments, 14 of which were polymorphic, making 16.67% polymorphism (table 4). the number of polymorphic fragments found per primer was between 1 (opb8, ai05 and ah17) and 3 (ai12, opc16, ai08), with a mean of 2 (table 4) and their size ranged from 250 to 3000 bp. the rapd markers have been applied in many investigations aimed at the study of polymorphism in pomegranate, for their simplicity and low cost (kathuria et al., 2017), but these markers have often shown low polymorphism in this species. as reported in literature it is neces­ sary an initial screening by a high number of rapd primers to detect a discrete number of discriminating markers (zamani et al., 2010). the level of polymor­ phism detected in our study is lower than that reported in other works (sarkhosh et al., 2006; zamani et al., 2007). however, a percentage of poly­ morphism similar to ours was detected by hasnaoui et al. (2010). in agreement with these authors we hypothesized that the slightly lower percentage of polymorphism detected could be due to the reduced dimension of the sample collection and to the limited v a r i a b i l i t y i n t e r m s o f g e o g r a p h i c a l o r i g i n . relationships among accessions were studied by clus­ ter analysis (upgma) based on jaccard’s coefficient, and following statistical analysis a dendrogram was produced (fig. 3a). the genetic distance among the accessions ranged from 0 to 0.8, showing genetic diversity among the pomegranate accessions. in the dendrogram, two main clusters could be identified: the first cluster included only two accessions (me7 and me8) at a 0 dissimilarity level (genetic identity). the second cluster comprised all other accessions which presented a level of dissimilarity that varied between 0 and 0.6. this last cluster presented three subgroups: me1, me6 and a subgroup that included accessions with genetic identity or with a very little (0.10) dissimilarity’s distance (me2, me3, me4, me5). the ssr molecular technique was utilized as a sec­ ond molecular method to discriminate the pome­ granates and to characterize their genetic profile. although for p. granatum there is not yet a set of the best ssr primers with validity recognized at the inter­ national level, these markers have been successfully employed by several researchers to characterize the pomegranate germplasm (beghè et al., 2016). ssrs utilized in this study have been chosen between the primers which had shown a high discriminating capacity and yielded a total number of 31 repro­ ducible fragments, which allowed a good discrimina­ fig. 3 ­ upgma clusters of 8 pomegranate accessions generated by rapd markers using the jaccard similarity coefficient (a) and by ssr markers using manhattan distance (b). table 4 ­ primer sequence of the most informative primers and level of polymorphism found by the rapd analysis rapds sequence total n° of bands n° of polymorphic bands opa19 5ʹ­d[caaacgtcgg]­3ʹ 13 2 opb08 5ʹ­d[gtccacacgg]­3ʹ 12 1 opc16 5ʹ­d[cacactccag]­3ʹ 13 3 ah17 5ʹ­d[cagtggggag]­3ʹ 13 1 ai08 5ʹ­d[aagcccccca]­3ʹ 15 3 ai12 5ʹ­d[gacgcgaacc]­3ʹ 15 3 ai105 5ʹ­d[gtcgtagcgg]­3ʹ 3 1 tot 84 14 mean 12 2 beghè et al. ‐ characterization of ancient pomegranates in northern italy 589 tion of the local accessions (table 5). among 12 selected microsatellites, 10 showed polymorphism and 8 showed differences in the pomegranates genetic profiles. the alleles obtained by amplification of ssrs loci produced four different genetic profiles from eight ancient accessions analyzed (table s6 of supplementary data). the number of alleles at each locus (na) varied between 1, for loci pgkur114 and pgkur127, and 5, for loci ssroeua­pg6 and pom021, with an average value of 2.58 and their size ranged from 155 to 319 bp. the values of expected (he) and observed (ho) heterozygosity were always above 0.500, except for the locus pgkvr027, where was not observed heterozigosity, and obviously for the two monomorphic loci (pgkvr114 and pgkvr127). it is important to underline that four primers (pg6, pom021, pom045, pgaer154) were highly polymor­ phic, showing a pic> 0.5, as defined by botstein et al. (1980) (table 5). these last markers showed genetic parameters (ho, he, pic) higher (or similar) to those reported in previous studies where they have been developed (ebrahimi et al., 2010; hasnaoui et al., 2012; caliskan et al., 2017). instead, the pgkvr­ primers presented genetic parameters lower to these reported in literature (ravishankar et al., 2015); these primers had low polymorphism; of 6 primers 2 r e s u l t e d m o n o m o r p h i c m a r k e r s a n d o n l y 3 (pgkvr027, pgkvr064 and pgkvr165) showed dif­ ferences in the studied accessions. similarly, the na, he, ho and pic values in other studies of pomegranate varieties also varied according to the primers tested and the geographic origin of population analyzed (caliskan et al., 2017). these results confirm the necessity to test different series of ssrs to obtain the best set of markers for each local germplasm. the upgma cluster based on ssr data divided the set of pomegranate accessions into two main cluster at a dissimilarity level of 45 (fig. 3b). in the ssrs den­ drogram, the first cluster included only two acces­ sions (me7 and me8) with genetic identity. the sec­ ond cluster instead presented all other accessions which a level of dissimilarity that varied between 0 and 38%. this last cluster comprised three genetic subgroups: me1, me6 and a subgroup that included accessions with genetic identity (me2, me3, me4, me5). as confirmed by mantel’s test (r = 0.399; p ≤ 0.033), the ssrs clustering was very similar to that performed by rapd markers. in fact, it showed the distinction of the same four genetic groups (fig. 3a and b). comparison between morphological and molecular based clusters and potential use of pomegranate genetic resources it is known that rapd fragments derived from any r e g i o n o f t h e g e n o m e a n d t h a t s s r f r a g m e n t s derived only from non­transcribed regions, therefore post­transcriptional modifications and non­nuclear inheritance of some characteristics can’t be detected by these markers (sarkhosh et al., 2006). for these reasons, in literature there are contrasting results about the correlation between these molecular and morphological descriptors (sarkhosh et al., 2009; zamani et al., 2010; basaki et al., 2013). however, researchers agreed that the combination of morpho­ logical and molecular techniques are essential for a p r o p e r a n d c o m p l e t e c h a r a c t e r i z a ti o n o f t h e germplasm of this species (beghè et al., 2016). in this study, the rapd and ssr analysis reflected the main morphological differences observed among the local accessions studied; the molecular cluster analyses confirmed the same two main clusters detected with pca analysis using quantitative mor­ phological traits. moreover, molecular analyses allowed to detect clearly four different genotypes. analysis of correlation between distance matrices (morphological traits and molecular markers) by mantel’s test confirmed a high statistical significance (r = 0.412; p ≤ 0.034 and r = 0.583; p ≤ 0.002 for ssrs and rapds, respectively). it is important to stress that in populations adapt­ ed to difficult ecological conditions, as the germo­ plasm in study, the polyphenolic content was high (calani et al., 2013). in this previous study the me1, me3, me5 and me8 accessions were subjected to phytochemical discrimination fingerprinting in pome­ ssrs na size he ho pic pgkvr027 2 236­242 0.429 0 0.305 pgkvr064 2 239­241 0.536 0.750 0.359 pgkvr065 2 202­204 0.571 0 0.375 pgkvr114 1 258 ­ ­ ­ pgkvr127 1 246 ­ ­ ­ pgkvr165 2 307­319 0.571 1.000 0.375 pom­agc11 2 183­185 0.536 0.250 0.359 pg4 2 198­244 0.571 1.000 0.375 pg6 5 191­199 0.786 0.750 0.653 pom021 5 203­211 0.857 1.000 0.712 pom045 3 155­163 0.750 0.750 0.581 pgaer154 4 262­300 0.786 1.000 0.630 tot 31 mean 2.58 0.533 0.542 0.394 table 5 ­ number of alleles (na), size (bp) expected (he) and observed (ho) heterozigosity, polymorphic information content (pic) at 12 loci in pomegranate accessions adv. hort. sci., 2019 33(4): 581­592 590 granate juices. the emilian pomegranates have pre­ sented interesting and peculiar phytochemical pro­ files. moreover, the juices were rich in ellagitannins and had high total phenol content and total antioxi­ dant capacity, especially me8 pomegranate. for these reasons, the local germplasm studied could be considered a source of useful traits (e.g. resistance to diseases, frost tolerance, polyphenol synthesis) for genetic improvement of this species. according to pomological descriptors and phytochemical charac­ teristics, we could appreciate peculiar features of these plants. indeed, the me7 and me8 accessions showed some characteristics (e.g. small size of fruits, high woody portion in seeds, low ph) that make the fruits unlikely to be used for direct consumption, but have a juice with high antioxidant capacity, and could be successfully employed for the preparation of nutraceutical products or for industrial blending of juices. the other pomegranates (except me6 acces­ sion that presented a high woody portion in seeds) presented good pomological characteristics for which a fresh use of the fruit could also be expected. 4. conclusions the present work is a first contribution to the genetic and morphological characterization of the pomegranate germplasm still present in emilia romagna region. the morphological traits, in particu­ lar those related to fruit and seed, seven rapds and eight ssrs have allowed to characterise the genetic diversity of ancient pomegranate accessions. the study, although preliminary and limited to a restrict­ ed area, highlighted significant differences and inter­ esting pomological traits in local pomegranates. these results, presented in association with the study of calani et al. 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10.13128/ahs-22792 impact of light quality on the physiological characteristics of capsicum chinense seeds d.c. fontana 1 (*), c.e. becker 2, m.v.m. pinheiro 2, m.m. pretto 2, j. dos santos 2, b.o. caron 2, d. schmidt 2 1 top school in agriculture luiz de queiroz, university of são paulo, piracicaba, sp, brazil. 2 federal university of santa maria, campus of frederico westphalen, departament of agronomic and environmental sciences, linha sete de setembro, br 386 km 40, 98400-000, frederico westphalen, rs, brazil. key words: germination, leds bulbs, pepper, vigor. abstract: the objective of this work was to evaluate the physiological quality of capsicum chinense seeds submitted to different light spectral qualities. it was used a completely randomized design, in a 4x5 factorial scheme, with four pepper cultivars [brs moema biquinho yellow (biq. yellow), airetama biquinho red (biq. red), boyra habanero red (boyra hab. red), brs seriema tupã bode red (tupã bode red)] and five light spectral qualities, being blue leds (b-leds); red leds (rleds); blue+red leds (br-leds); white leds (w-leds) and fluorescent lamp (fl) carried out germination and vigor analysis, with four replicates of 50 seeds. for this, the seeds were conditioned inside gerbox® boxes and kept in a growth room. the biq. yellow and boyra hab. red peppers showed the highest potential of germination and vigor, respectively, indicating high physiological quality. in general, the light spectral qualities provide differentiated responses in the initial development of the pepper cultivars, being the reduction of the percentage of dead seeds favored by the spectrum br-leds and w-leds. the root fresh mass is increased by all lights, except r-leds. the fresh mass of the aerial part presents positive results in the fl lamps. shoot length is favored by the r-leds. 1. introduction capsicum peppers are closely related to the brazilian richness culture and are a valuable part of the biodiversity heritage, being cultivated an immense variety, sizes, colors, flavors and pungences (neitzke et al., 2008). the brazilian production is around 11,071 tons (conab, 2015), being the state of são paulo considered the largest producer. among the factors that regulate plant production, the light plays an important role because it is an important regulator of growth and development of the plant, as it regulates morphological characteristics and acts as an energy source in the primary metabolism and in the photosynthetic process (simlat et al., 2016). the qualitative or quantitative characteristics of growth and morphogenesis are influenced by the quality of the supplied light, affecting the plants development, mainly, by photomorphogenic alterations (heo et al., 2002; rezende et al., 2008). (*) corresponding author: daani_fontana@hotmail.com citation: fontana d.c., becker c.e., pinheiro m.v.m., pretto m.m., dos santos j., caron b.o., schmidt d., 2019 impact of light quality on the physiological characteristics of capsicum chinense seeds. adv. hort. sci., 33(2): 235-243 copyright: © 2019 fontana d.c., becker c.e., pinheiro m.v.m., pretto m.m., dos santos j., caron b.o., schmidt d. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 1 march 2018 accepted for publication 22 february 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(2): 235-243 236 in recent years, light-emitting diodes (led) have been widely used as alternative light source potential for plants (simlat et al., 2016). among the advantages of leds systems are visible light emission and low heat production for long periods, with a specific wavelength, color and lighting flexibility, reduction of electrical consumption and toxic substances, as well as improved lifetime (carvalho, 2007; yeh and chung, 2009). this technology becomes promising for the growth of plants in a controlled environment, such as in tissue culture and also in the supplementation of growth chambers and greenhouses (yeh and chung, 2009). different wavelengths of light can trigger a variety of responses in plants (simlat et al., 2016). for example, red, blue, green and white leds lights were tested in different species, mainly forest ones, demonstrating the promotion of seed germination and subs eq u en t d ev el o p m en t ( g o n ç a l v es et a l . , 2 0 0 6 ; victório and lage, 2009). red light may promote seed germination and root development (daud et al., 2013), shoot elongation (kim et al., 2004; araújo et al., 2009), fresh mass increment (sorgato et al., 2016), and an increase in shoot length (cybularzurban et al., 2007), among others. combinations of blue and red leds may promote biomass increase (gu et al., 2012; maluta et al., 2013; da silva et al., 2016) and increase of the root system (gu et al., 2012). the blue wavelengths tend to improve stomatal conductance (hogewoning et al., 2010), affect phototropism (johkan et al., 2010), and increase the r a t e o f p h o t o s y n t h e t i c p i g m e n t s p r o d u c t i o n . however, plants exhibit a wide range of morphological and phytochemical plasticity in response to each type of wavelength of light (macedo et al., 2011). many studies report that leds can modify seed germination and plant growth and development (gonçalves et al., 2006; victório and lage, 2009; daud et al., 2013; da silva et al., 2016). although there are reports on the development of seedlings of the genus capsicum spp. in light qualities (da silva et al., 2016), the effects of the light spectral qualities on the germination and vigor of capsicum chinense have not yet been analyzed. thus, the objective of this work was to verify the impact of the light spectral qualities on the physiological quality of capsicum chinense seeds. 2. materials and methods plant material and conduction of the experiment the work was conducted at the plant tissue culture laboratory of the universidade federal de santa maria, campus frederico westphalen rs (federal university of santa maria, campus frederico westphalen rs), in november 2016. the experiment was conducted in a completely randomized design, in a 4x5 factorial scheme, with four pepper cultivars (capsicum chinense) and five light spectral qualities, totaling 20 treatments, with four replicates of 50 seeds each tratament, totalizing 4000 seeds tested. four cultivars of pepper were used [brs moema biquinho yellow (biq. yellow), airetama biquinho red (biq. red), boyra habanero red (boyra hab. red) and brs seriema tupã bode red (tupã bode red)], and five light spectral qualities [tec-lamp® blue leds (450 nm) b-leds; red leds (660 nm) r-leds; blue (450 nm) + red (660 nm) brleds in the ratio of 40% and 60%, respectively; white leds w-leds; and special daylight type fluorescent fl (osram®, brazil)]. the seeds were placed inside gerbox® boxes with l i d s ( 1 1 x 1 1 x 3 c m ) c o n t a i n i n g t w o s h e e t s o f germitest® paper (in box dimensions), moistened with 0.2% kno3 solution (dissolved in distilled water), in proportion to 2.5 times the dry paper weight, as described in the regras analis de sementes (rules for seed analysis) (mapa, 2009). the gerbox boxes were maintained in a growth room under temperature of 25±2°c and a luminous intensity of 36 µmol m-2 s-1 for 14 days. analyzed variables for the germination test, counting was performed by seven and 14 days after the test installation. at the first count (fc), the normal seedlings were counted and the values expressed as a percentage (%), at 14 days the following variables were analyzed: percentage of germination (pg), percentage of abnormal seedlings (pas), percentage of hard seed (phs) and percentage of dead seeds (pds) (mapa, 2009). according to mapa (2009), dead seeds are the seeds that do not germinate at the end of the test, are neither hard nor dormant, and are usually softened, attacked by microorganisms and show no signs of germination. already, the hard seeds are those that remain without absorbing water for a longer period than normal and are therefore at the end of the test with the appearance of seeds newly placed on the substrate. for the root length (rl) and shoot length (sl) variables, 10 seedlings of each replicate were measured for all light qualities, being measured with a digital caliper. for the fresh mass of the aerial part (fmap) and root fresh mass (rfm), the same seedlings were fontana et al. light quality impact on capsicum chinense seeds 237 used for the sl and rl measurement, with the values r e f e r r i n g t o t h e 1 0 s e e d l i n g s . a f t e r w a r d s , t h e seedlings were conditioned in paper bags and kept in a forced air oven at 60°c, until constant weight was reached, to determine the dry mass of shoot (dms) and dry mass of the root (dmr). the germination speed index (gsi) was calculated by the sum of the number of germinated seeds per day, divided by the number of days between sowing and germination, following the maguire’s methodology (1962). gsi= (g1 / n1) + (g2 / n2) + ⋯ + (gn / nn) (1) where gsi = g 1 , g 2 , ..., gn = number of seedlings computed in the first, second, third and last count; n 1 , n 2 , ..., nn = number of days of sowing to the first, second, third and last count. the obtained data were submitted to analysis of variance, and the interaction between pepper cultivars and light spectral qualities was evaluated, and when they were significant, the averages were compared by the tukey’s test, at 5% of error probability, using the statistical program assistat 7.7 beta. 3. results the analysis of variance showed significant interaction for the pepper cultivar factors x light spectral qualities only for the fresh mass of the aerial part (fmap) and shoot length (sl) variables. the variables of the first count (fc), percentage of germination (pg), normal seeds (ns), abnormal seeds (as), hard seeds (phs), germination speed index (gsi), root fresh mass (rfm) and root length (rl) were significant only for the pepper cultivars factor. root length (rl), root fresh mass and percentage of dead seeds (pds) variables were significant for the light spectral qualities factor. on the other hand, the dry mass of the aerial part (dmap) and dry mass of the root (dmr) variables were not significant (data not shown) by the f test, at 5% of error probability. the pepper cultivars differed for the percentage of normal seeds (ns) and abnormal seeds (as). the highest percentages of ns were observed for biq. yellow pepper with approximately 88%, being the same as biq. red pepper (85%), and differing from the others (p<0.05) (fig. 1a). for the variable as, the tupã bode red cultivar presented the highest values, with 12.30% of abnormality, being higher than the others, and the lowest percentages were verified for biq. yellow pepper, with 4.40% (fig. 1b). regarding the percentage of hard seeds, the boyra hab. red cultivar had the highest values, with an average of 11.8%, differing significantly from the others (fig. 1c). the biq. yellow pepper cultivar showed the highest percentages of germination in the evaluation of the first count (fc), with 94.8%, differing significantly from the boyra hab. red cultivar (fig. 1d). fig. 1 percentage of normal seeds (ns-a), abnormal seeds (asb), hard seeds (hs-c) and germinated (gs-d) of four pepper cultivars, being biq. yellow, biq. red, boyra hab. red and tupã bode red, submitted to different light spectral qualities. *different letters represent significant statistical difference (tukey’s test p<0.05; bars=sd). adv. hort. sci., 2019 33(2): 235-243 238 for the root length variable, the boyra hab. pepper presented the highest values with approximately 51 mm in length, being statistically different from the other cultivars. the lowest averages were observed in biq. yellow and biq. red peppers with 36 and 35 mm, respectively (fig. 2a). the boyra hab. red pepper again stood out for the root fresh mass variable, with 0.072 gram for 10 seedling differing significantly from the other ones evaluated (fig. 2b). for the germination speed index (gsi), the biq. yellow pepper stood out, presenting 23.70, being significantly similar to the biq. red and tupã bode red peppers, differing only from boyra hab. red pepper (fig. 2c). for the first counting of normal seeds the biq. yellow pepper showed the highest values with 84.1% of normal seedlings, differing from the other ones (fig. 2d). the light spectral qualities of br-leds and w-leds provided higher root length (rl), with 43.37 and 42.97 mm respectively, differing statistically from the fl (fig. 3a). for the root fresh mass variable, it was observed that the red spectrum promoted mass fig. 2 root length (a), root fresh mass (g for 10 seedlings) (b), germination speed index (c) and first counting of normal seeds (d) of four pepper cultivars, being biq. yellow, biq. red, boyra hab. red and tupã bode red, submitted to different light spectral qualities. *different letters represent significant statistical difference (tukey’s test p<0.05; bars=sd). fig. 3 root length (a), root fresh mass (g for 10 seedlings) (b) and percentage of dead seeds (c) of four pepper cultivars submitted to different light spectral qualities, being b l e d s , r l e d s , b r l e d s , w l e d s a n d f l l a m p s . *different letters represent significant statistical difference (tukey’s test p<0.05; bars=sd). fontana et al. light quality impact on capsicum chinense seeds 239 (table 1). r-leds spectral quality provided the highest averages for the shoot length variable, being higher to the other spectra. in this spectrum, the boyra hab. red and tupã bode red were superior to the other peppers. for biq. yellow pepper the r-leds, fl lamp and br-leds spectra conditioned the larger shoot length, statistically differing from the b-leds and w-leds. for biq. red pepper, the r-leds spectrum was statistically superior to the others (table 2). the boyra hab. red pepper presented superior performance to the others, presenting the highest average in shoot lenght (sl), being favored by the leds spectra and in disadvantage by the fl, differing significantly. shoot length of tupã bode red cultivar was favored by the r-leds light spectra qualities, differing significantly from the other spectra (table 2). 4. discussion and conclusions the biq. yellow pepper cultivar was superior to gsi, fc, ns, pg, presenting the lowest percentage of abnormal (as) and hard (hs) seeds (fig. 1). however, the boyra hab. red pepper cultivar has been highlighted for the rl (figs. 2a, 3a), sl (table 2), fmap (table 1), rfm (figs 2b, 3b) variables. the results sugreduction, with 0.013 gram for 10 seedling, differing statistically from the other light spectra, which presented higher mass (fig. 3b). correspondingly, it was observed that the red spectrum conditioned the highest percentage of dead seeds, with 6%, differing statistically from the br-leds and w-leds. the beleds and w-leds spectra conditioned low percentage of seed mortality (fig. 3c). for the biq. yellow pepper cultivar the light spectral qualities of w-leds, fl and br-leds showed the highest values of fresh mass of the aerial part (fmap), differing significantly from r-leds (table 1). for biq. red and boyra hab. red peppers, the fl light provided greater accumulation of fresh mass, presenting 0.194 and 0.283 gram for 10 seedlings, respectively, being statistically higher to the others (table 1). as for the tupã bode red pepper, the wleds light conditioned the largest fresh mass of the aerial part, differing significantly from the b-leds and r-leds (table 1). it was observed that the br-leds and fl lights provided a greater increment of fresh mass for boyra hab. red pepper, with 0.236 and 0.283 g for 10 seedling, respectively, differing significantly from the r-leds spectrum. the tested peppers presented similar responses in relation to the w-leds light spectrum, not statistically different from each other table 1 fresh mass of the aerial part (g for 10 seedlings) of four pepper cultivars, being biquinho yellow, biq. red, boyra hab. red and tupã bode red, submitted to different light spectral qualities, b-leds, r-leds, br-leds, w-leds and fl lamps cultivars fresh mass of the areal parts b-leds r-leds br-leds w-leds fl biq. yellow 0.144 bab 0.114 bb 0.182 ba 0.191 aa 0.192 ba biq. red 0.1233 bc 0.139 abbc 0.179 bab 0.169 aabc 0.194 ba boyra hab. red 0.196 abc 0.176 ac 0.236 aab 0.204 abc 0.283 aa tupã bode red 0.099 bc 0.138 abbc 0.160 bab 0.200 aa 0.183 bab cv 15% * different lowercase letters in the column or uppercase letters in the row represent significant statistical difference (tukey’s test p<0.05). table 2 shoot lenght of four pepper cultivars, being biquinho yellow, biq. red, boyra hab. red and tupã bode red, submitted to different light spectral qualities, being b-leds, r-leds, br-leds, w-leds and fl lamps * different lowercase letters in the column or uppercase letters in the row represent significant statistical difference (tukey’s test p<0.05). cultivars shoot lenght (mm) b-leds r-leds br-leds w-leds fl biq. yellow 28.66 abb 34.28 ba 30.62 abab 29.26 bcb 34.15 aa biq. red 26.56 bc 37.65 ba 30.34 bbc 30.65 abbc 33.33 ab boyra hab. red 30.74 ab 43.14 aa 34.59 ab 33.92 ab 25.52 bc tupã bode red 29.075 abc 42.10 aa 33.89 abb 26.18 cc 26.53 bc cv 6.71% 240 adv. hort. sci., 2019 33(2): 235-243 gest that biq. yellow pepper has a higher germinative potential, while boyra hab. red pepper has greater vigor. the characteristics of germination and vigor are individual for each cultivar and variables between them. for most of them, the speed, uniformity and germination rate depends on external and internal factors to the seed (plue et al., 2010; demotesmainard et al., 2016), such as temperature, humidity, light, viability of the embryo and genetic factors, characterizing in this way, the differences verified between the pepper cultivars. in general, the use of high vigor seeds results in a good performance of the crops in the field through better establishment of seedlings and survival of seedlings. in this way, germination and vigor tests are important in order to choose the best pepper to be used. specific light spectra can act positively stimulating the germination process in some species (gonçalves et al., 2006), or can be indifferent to others. in general, species whose seeds present sensitivity to the light quality, the positive photoblastics, are considered pioneers in nature, since they require light stimulus to initiate their germination process (rebouças and santos, 2008). for the capsicum chinense pepper plant, as observed in this study, only the percentage of dead seeds was influenced by the luminous spectra tested where the r-leds spectrum caused a high number of dead seeds whilst the br-leds and wleds reduced the percentage of mortality. the other germination variables evaluated did not present responses to the spectra. red light has been reported to stimulate seed germination and root development (bewley and black, 1994; abdullateef and osman, 2011; daud et al., 2013). in addition to the quality, the luminous intensity in which the seeds and plants are submitted can also promote differentiated responses in the plant, as verified for capsicum chinense habanero, which presented increase in growth with the light intensity of 28 μmol m-2 s-1 (barrales-lópez et al., 2015). the luminous spectra used in this experiment were larger, with 36 µmol m-2 s-1, which may have masked the plant response. it is known that light in excess can result in reduction of the net photosynthetic rate, causing oxidative damage to the foliar tissues; only under appropriate light plants can be fully self-regulated to obtain the best status for absorption and transformation of light energy (yao et al., 2017). it is known that excess light can also promote photovoltaic changes in plants, leading to the production of reactive oxygen species (ros), which may have promoted mortality in seeds submitted to the red spectrum. after germination, it was possible to observe changes in the morphological characteristics due to the different light spectral qualities in which they were submitted. fluorescent light (fl), for example, provided a higher increase of fmap in the evaluated peppers. this spectral quality is the most used for in vitro growth of plant species. positive results in the increase of fresh mass of the aerial part were already found for curcuma longa in fluorescent light, followed by red light (~ 625-440nm) and yellow light (~565590nm) (de souza ferrari et al., 2016). naturally plants develop themselves under varied lights composed of a mixture of quality and quantity, which promotes the activation of several photoreceptors, among them phytochromes (rockwell et al., 2006). the highest values of shoot length were verified in r-leds spectral quality for capsicum chinense peppers, corroborating with other studies, which found an increase in root formation in cultures such as jatropha curcas and protea cynaroides (daud et al., 2013; wu and lin, 2013), and stevia rebaudiana (simlat et al., 2016). kim et al. (2004), observed stretching of the aerial part of chrysanthemums cultivated in vitro, under light in the red band. when the plants were submitted to the r-leds spectrum, some authors verified elongation in cattleya loddigesii (araújo et al., 2009), increase of fresh mass in dendrobium phalaenopsis (sorgato et al., 2016) and increase of shoot length for cattleya (cybularz urban et al., 2007), corroborating with the results found in this work. red light is effective for photosynthesis as the red emission spectrum fits perfectly with the photon energy required to reach the first excited status of a and b chlorophyll (singh et al., 2015). lights in blue and red spectra too are strongly absorbed by phyt o c h r o m e t h r o u g h s p e c i f i c p h o t o r e c e p t o r s (mathews, 2010). these photoreceptors activate enzymes associated with the synthesis of auxins, growth hormone, and greater photosynthetic efficiency (sun et al., 1998), promoting an increase in growth, justifying the results found. in this way, plants that grow under these conditions have a good initial development, such as a well-formed root system, which allows for faster acclimation and better survival rates in the field (chandra et al., 2010; gruszecki et al., 2010). positive results for root fresh mass and root length were observed in br-leds spectrum. some studies indicate that combinations of leds with blue (30%) and red (70%) spectrum promoted an increase fontana et al. light quality impact on capsicum chinense seeds 241 in biomass of fragaria x ananassa and saccharum officinarum (nhut et al., 2003; maluta et al., 2013). results for anthurium andraeanum favored gains in fresh and dry matter in the combination of rb-leds, followed by white light (gu et al., 2012). when rbleds have been used in vitro propagation, a large part of the works observed a mass gain in both the root and the aerial part (gu et al., 2012; maluta et al., 2013; da silva et al., 2016). for chili culture (capsicum annuum l. cv. rubi gigante), shoot length and collar diameter were favored by treatment with br-leds light compared to white fluorescent light (fl) (da silva et al., 2016). for this species, the authors also point out that the different qualities of the light spectrum have little effect on the growth and development of seedlings. the photosynthetic pigments absorb light in the red and blue range, the red quality increas the photosynthetic rate, while blue quality improves the chloroplast development, chlorophyll biosynthesis and stomatal opening, thus, increasing the content of p h o t o s y n t h e t i c p i g m e n t s ( j o h k a n e t a l . , 2 0 1 0 ; hogewoning et al., 2010; daud et al., 2013). these luminous spectra influence the primary and secondary metabolism in plant development, however, plants exhibit a wide range of morphological plasticity, and phytochemical in response to a given type and wavelength of light (macedo et al., 2011). in this way, some cultures are favored by the supply of these wavelengths, increasing their growth. the tested peppers showed good results for the root length and root fresh mass variables when submitted to the w-leds spectrum. positive results with the use of w-leds were obtained by wilken et al. (2014), in which they found more vigorous growth of musa spp. compared to the use of fluorescent lamps. for sugarcane plants, w-leds lamps promoted a higher number of shoots, in addition to a higher content of chlorophylls and carotenoids (ferreira et al., 2017). the emitted photons in the combination of brleds, suppose that more photoreceptors of the pepper seedlings received stimuli, which may have triggered some morphogenetic mechanism in more photoreceptor cells than when exposed to only one spectrum. similar assumptions were made by shimokawa et al. (2014) and chen et al. (2017). in this way, the positive results found for rfm and rl and the low seed mortality (sm), in the br-leds combination were explained. light is a signal that is received by photoreceptors, which regulate plant differentiation and growth (li et al., 2013). the quality of the emitted light by leds has promoted significant improvements in morphogenesis and differentiation in different species grown in vitro (gupta and jatothu, 2013). however, the effects and mechanisms associated with light quality may be peculiar to each species or cultivar (li et al., 2013; da silva et al., 2016). some explanations may be generalized, however, this specificity seems to be related to the different responses that were found for the vigor of the pepper cultivars. the biq. yellow and boyra hab. red present high potential of germination and vigor, respectively, indicating high physiological quality. in general, the light spectral qualities provide differentiated responses in the initial development of the peppers, being the reduction of the percentage of dead seeds favored by the spectrum br-leds and w-leds. the root fresh mass is increased by the all lights, except r-leds. the fresh mass of the aerial part presents positive results in the fl lamps. shoot length is favored by the r-leds. acknowledgements the authors would like to thank the coordenação de aperfeiçoamento de pessoal de nível superior (capes) for the scholarship. references abdullateef r.a., osman m.b., 2011 effects of visible light wavelengths on seed germinability in stevia rebaudiana bertoni. int. j. biol., 3(4): 83. araújo a.g., pasqual m., rodrigues f.a., rodrigues j.d., castro e.m., santos a.m., 2009 crescimento in vitro de cattleya loddigesii lindl. em diferentes espectros luminosos associado a ácido giberélico. rev. ceres, 56(5): 542-546. barrales-lópez a., robledo-paz a., trejo c., espitiarangel e. rodríguez-de la o.j.l., 2015 improved in vitro rooting and acclimatization of capsicum chinense jacq. plantlets. 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n., chung j.p., 2009 high-brightness leds energy efficient lighting sources and their potential in door plant cultivation. renew. sust. energ. rev., 13(8): 2175-2180. impaginato 257 adv. hort. sci., 2019 33(2): 257-262 doi: 10.13128/ahs-23240 tumv as an efficient transient vector for expressing heterologous proteins in nicotiana tabacum and n. benthamiana m. modarresi 1, m. jalali-javaran 1 (*), m. shams-bakhsh 2, s. zeinali 3, m. mirzaee 1 1 d e p a r t m e n t o f p l a n t b r e e d i n g a n d b i o t e c h n o l o g y , f a c u l t y o f agriculture, tarbiat modares university, tehran, i.r. iran. 2 department of plant pathology, faculty of agriculture, tarbiat modares university, tehran, i.r. iran. 3 department of molecular medicine, pasteur institute of iran, tehran, i.r. iran. key words: green fluorescent protein, recombinant protein, tobacco plant, transient expression, viral vector. abstract: nowadays the production of recombinant proteins such as drugs and commercial protein compounds in plants is called molecular farming. it has some benefits such as fast and large quantity production of recombinant proteins with low cost. in this research, the green fluorescent protein (gfp) was transiently expressed in two tobacco species via turnip mosaic virus (tumv) derived vector, a virus which can infect a wide range of plant species. florescence microscopy results indicated that tumv could infect tobacco plants and accumulate gfp protein in plant leaves. in addition, rt-pcr, dot-blot and elisa assays demonstrated the recombinant gene transcription, translation and stability. this is the first report of using tumv-based viral vectors for producing recombinant proteins in tobacco. optimized tumv-based viral vectors could be used for producing recombinant proteins in tobacco. 1. introduction various expression systems, such as bacteria, yeast, plants, insects and mammalian cell cultures can produce recombinant proteins. the benefits of expressing recombinant proteins in plants include economic, agricultural scale, safe and authentic production (sijmons et al., 1990; ma et al., 2003; mardanova et al., 2015;). molecular farming (also known as molecular pharming or biopharming) uses genetically engineered plants for the production of biopharmaceutical products, vaccine subunits, industrial enzymes therapy peptides and other compounds of interest (boothe et al., 1997; wang and ma, 2011; yarbakht et al., 2015). recombinant proteins in plants may be gained by stable genetic transformation (nuclear or plastid) or through transient expression. transient expression is usually used for fast and flexible expression of genes of (*) corresponding author: m_jalali@modares@ac.ir citation: modarresi m., jalali-javaran m., shamsbakhsh m., zeinali s., mirzaee m., 2019 tumv as an efficient transient vector for expressing heterologous proteins in nicotiana tabacum and n. benthamiana. adv. hort. sci., 33(2): 257262 copyright: © 2019 modarresi m., jalali-javaran m., shamsbakhsh m., zeinali s., mirzaee m. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 23 march 2018 accepted for publication 7 august 2018 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(2): 257-262 258 interest (goi), evaluation of expression system performance and components such as promoter and enhancers (chiera et al., 2008). in plants, a number of virus-based vectors are utilized for the transient expression of foreign genes, such as tobacco yellow dwarf virus (tydv) for transient expression of chalcone synthase in petunia hybrida (atkinson et al., 1998), tobacco mosaic virus (tmv) for transient expression of gfp (jellyfish, aequorea victoria greenfluorescent protein) in tobacco (shivprasad et al., 1999), bean pod mottle virus (bpmv) for expression the gfp in the soybean (zhang et al., 2010), wheat streak mosaic virus (wsmv) for expression the gfp in cereals (tatineni et al., 2011) etc. turnip mosaic virus (tumv) belongs to potyviridae family and infects a wide range of plant species especially cruciferous (brassicaceae family). it is a positive-sense single stranded rna virus with a linear and monopartite genome and average length of 720 nm (brunt et al., 1996). previously beauchemin et al. (2005) strongly expressed gfp and gus (bacterial βglucuronidase) reporters genes in brassica perviridis plants via tumv virus. furthermore, chen et al. (2007) introduced gfp in some brassica hosts such as b. campestris and b. juncea and high levels of the recombinant protein expression were observed. therefore, in this study, to investigate the performance of recombinant protein production, the gfp reporter gene was introduced into the tobacco (nicotiana tabacum and n. benthamiana) plants by using tumv vector. 2. materials and methods plant material and growth conditions nicotiana tabacum cv. xanthi and cv. samsun and n. benthamiana seeds were grown in pots containing autoclaved soil, including 40% farm soil, 30% peat moss and 30% perlite. they were kept at 25°c in a phytotron under a 16-hour photoperiod (16:8 h l: d). plasmid and viral constructs the tumv-gfp construct (fig. 1) was kindly prov i d e d b y d r . s h y i d o n g y e h , p l a n t p a t h o l o g y d ep a rt men t , na t i o n a l c h en g h s i n g un i vers i t y, taichnug, taiwan. the plasmid contains a cauliflower mosaic virus 35s promoter (camv 35s) and gfp coding sequence between the nib (nuclear inclusion protein b) and cp (coat protein) positions. recombinant viral construct, ptumv-gfp, was transferred into bacterial (escherichia coli dh5α) competent cells (sambrook and russell, 2001). bacteria were grown in 200 ml luria-bertani medium and then, ptumvgfp was extracted (engebrecht et al., 1991). plant rub-inoculation with tumv-derived vector wild-type tumv (for control plants) and ptumvgfp was mechanically inoculated on upper surface of two top leaves (10 µg in 10 µl per leaf), using a cotton stick and carborandum powder according to hosseini et al. (2013). systemically infected (noninoculated leaves) were used for further analysis. total rna extraction and reverse transcription-polymerase chain reaction (rt-pcr) the presence of the gfp gene in inoculated leaves was determined by rt-pcr. total rna was extracted from inoculated and control plant leaves (five independent samples) by qiagene kit (south korea) twelve days after incubation according to the manufacturer’s instruction. rna was extracted from noninoculated leaves for confirming replication and movement of the virus. after treating with dnase i (thermo fisher scientific, usa), cdna was synthesized using the revertaid reverse transcriptase (thermo fisher scientific, usa) and gfp reverse primer (5’ –ttg tac tcc agc ttg tgc cc-3’) according to the producer’s instructions. rt-pcr was conducted using the cdna and the following forward and reverse primers under the following cycling conditions: forward (5’acg acg gca act aca aga cc 3’) and reverse (5’ttg tac tcc agc ttg tgc cc 3’). pcr cycling conditions were as follows: 94°c for 3 min for initial denaturation; 35 cycles of 94°c for 30 s, 51°c for 30s, and 72°c for 30 s; and 72°c for 10 min for a final extension. then pcr products were analyzed by 1% tae agarose gel. fluorescence microscopy leaves from tumv-based vector inoculated and control plants were observed under an olympus fluorescent microscope 6 (version ix71, tokyo, japan). fig. 1 schematic representation of the viral construct containing gfp under the 35s promoter that was used in this expression analysis. the foreign gene insertion site is between nib (nuclear inclusion protein b) and cp (the virus coat protein gene) provided by ncoi and nhei restriction endonuclease enzymes. modarresi et al. heterologous proteins production via viruses 259 the fluorescence photographs were taken using a mounted high-resolutionm7 olympus dp70 dp71 digital camera at 12 days post-inoculation (dpi). protein extraction and gfp analysis proteins were extracted from 0.5g tissues of control and inoculated tobacco leaves (five independent samples) with extraction buffer, including 0.2m trishcl (ph 8.0), 5mm ethylenediaminetetraacetic acid ( e d t a ) , 1 0 0 m m s u c r o s e , a n d 0 . 1 m m 2 m e r capthoethanol (abdoli-nasab et al., 2013) and the concentration was assessed by bradford’s assays (bradford, 1976). dot-blot (stott, 1989) and indirect enzyme-linked immunosorbent assay (elisa) (wang and gonsalves, 1990) were carried out to quantitative detection of the gfp protein in the inoculated tobacco plants. statistical analysis all experiments were done according to a completely randomized design at five independent samples. data analyses were performed using microsoft excel program software and spss version 22. when significant differences were found least significant difference (lsd) test at p<0.05 was applied to separate means. 3. results and discussion the main benefits of the plant made proteins (pmps) are lower costs and potential to produce a very large scale of recombinant proteins. viral vectors have the ability to express transgenes in hosts and they are suitable and rapid platform for production high-level of recombinant proteins. in this research, we utilized a tumv viral vector (fig. 1) under the control of the camv 35s promoter for transient expression of the gfp in tobacco plants. although systemic symptoms of tumv were not observed on infected plants, gfp was detected by the fluorescence microscopy (fig. 2) twelve days post-inoculation. this research has displayed for the first time that recombinant protein (gfp) can accumulate in tobacco plants via tumv based viral vector with camv 35s promoter. tumv can infect tens different plant species (chen et al., 2007) (to compare common viral vectors which can affected specific plant species), therefore, tumv based viral vector can be economical. in this study, two different tobacco species, n. benthamiana and n. tabacum (two different cultivars xanthi and samsun) were investigated. fluorescence microscopy analysis of gfp (fig. 2), rt-pcr (fig. 3), dot-blot analyses (fig. 4) and elisa assay (fig. 5) indicated that recombinant protein expression in tobacco plants leaves occrued. rt-pcr (fig. 3) showed that, as expected, 160 bp bands were found in infected plants, while not observed in the negative control (wild type). it shows that the tumv virus can infect the plant and replicate its genome. viruses (like tumv from potyviridae family) have developed proteins such as helper component proteinase (hcpro), fig. 2 fluorescence microscopy analysis of gfp expression in tobacco plant's leaves which infected by ptumv-gfp. (a) nicotiana benthamiana, (b) n. tabacum cv. xanthi, (c) n. tabacum cv. samsun (d) negative control (tobacco plant infected with wild-type tumv). green color indicated gfp expression and the red indicated chlorophyll autofluorescence. fig. 3 rt-pcr amplified a 160 bp fragment from the gfp with specific primers in 1% agarose gel. c1= negative control (water template), c2= negative control (rna template), c3= negative control (wild type (non-inoculated plant)), c+= positive control, lane 1= nicotiana benthamiana transformed plants, lane 2= n. tabacum cv. xanthi, lane 3= n. tabacum cv. samsun transformed plants, m= molecular weight marker (1 kb standard generuler). adv. hort. sci., 2019 33(2): 257-262 260 which suppress the plants silencing defense (voinnet, 2001). furthermore, hcpro has protease activity and it is necessary for virus genome replication and viral movement and transmission (klein et al., 1994; chiera et al., 2008). dot-blot assay (fig. 4) indicated that gfp protein was recognized by specific antibody and developed brown color in transformed plants and positive control. elisa assay indicated that expression levels of gfp was estimated approximately ≤0.5% of total soluble protein (tsp) of fresh weight of tobacco leaves. these results show lower accumulation of recombinant proteins compared with a number of previous studies which expressed by viral vectors such as artichoke mottled crinckle virus (lombardi et al., 2009), beet curly top virus (kim et al., 2012) etc. some strategies, such as codon-optimization (love et al., 2012) and the use of improved viral vector elements including strong viral promoters (gleba et al., 2007) can increase the expression of recombinant protein. it seems that, n. benthamiana lacks rna-dependent rna polymerases (rdrps) which required for defense against viruses, therefore n. benthamiana infected plant displays more strong symptoms and its products more than do other tobacco species (yang et al., 2004). in many previous studies (kumar and kirti, 2010; sasaki et al., 2015; vojta et al., 2015; park et al., 2 0 1 6 ) , r h i z o b i u m r a d i o b a c t e r ( f o r m e r l y agrobacterium tumefaciens) delivery systems has been used to express the transient expression of recombinant protein using a viral vector. however, in this study, a direct virus inoculation system via dusted with carborundum has been used. our study indicated that this method is useful to accelerate the production of recombinant proteins in tobacco plants. 4. conclusions in conclusion, our results showed that tumv, as a virus that could infect a wide range of plant species, could be used to produce recombinant proteins in tobacco. in this investigation, all inoculated plants expressed gfp protein. results showed that incubated n. benthamiana has more accumulated recombinant protein compared to the two n. tabacum cultivars. although the level of expression is low and should be optimized for future studies. acknowledgements the authors thank dr. sayed mohsen nassaj hosseini (environmental research institute, acecr, rasht, iran) and mrs. m. azmoodeh (laboratory of biotechnology) for their helpful advice and assistance. we thank dr. shyi-dong yeh for kindly provided tumv-gfp vector. furthermore, we thank the plant breeding and biotechnology department at tarbiat modares university for their support. references abdoli-nasab m., jalali-javaran m., cusidó r., palazón j., baghizadeh a., alizadeh h., 2013 expression of the truncated tissue plasminogen activator (k2s) gene in tobacco chloroplast. mol. biol. rep., 40: 5749-5758. atkinson r.g., gleave l.r., janssen b.j., morris b.a., 1998 post-transcriptional silencing of chalcone synthase in petunia using a geminivirus-based episomal vector. plant j., 15: 593-604. beauchemin c., bougie v., laliberté j.f., 2005 simultaneous production of two foreign proteins from a potyvirus-based vector. virus res., 112: 1-8. boothe j.g., saponja j.a., parmenter d.l., 1997 molecular 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morton and voss, 1987). the argan tree can be found in a wide array of envi­ ronments where soils are ranging from heavy clay to poor and rocky desert soils, it grows in altitudinal ranges extending from sea level (along the atlantic coast) up to 1500 m (msanda, 1993). moreover, it has the ability to survive in very dry conditions at very hot temperatures up to 50°c and low rainfall levels between 150 and 400 mm/year, shedding foliage and remaining in a state of dormancy for sev­ eral years during prolonged drought (m’hirit et al., 1998). recently, a. spinosa has attracted worldwide attention as a source of highly valued oil (charrouf and guillaume, 2008). it’s becoming the most expen­ sive edible oil in the world, as the present supplies does not meet demand (lybbert et al., 2011). despite the ecological value and local economic importance of this species, its forest density has been divided by two during the twentieth century and the natural regeneration rate is very low and is worsened by cli­ mate changes and the negative impact of over e x p l o i t a ti o n ( k e n n y a n d d e z b o r o w s k i , 2 0 0 7 ) . besides, its cultivation is not sufficiently developed. in part, this is due to excessive utilization of kernels for oil production which hindered the conventional propagation through seed germination (nouaim et al., 2002); on the other hand, it is due to the slow progress in propagation methods, mainly because of the generally low capacity of this species to form adventitious roots (nouaim et al., 2002; justamante et al., 2017). therefore, research is required to devel­ op efficient vegetative propagation techniques for the production of more planting material and conser­ vation of this species. the rooting efficiency and growth performance of cuttings in many species have been found to be dependent of the cutting thickness (foster et al., 2000). the rooting effectiveness in function of cuttings diameter could be explained by different factors (foster et al., 2000). the variation observed among different cutting diameters could be influenced by physiological conditions (nutrients, car­ bohydrates, auxin, phenolics, lignification etc.), their position within the branch or their ontogenetic aging (kaul, 2008; wendling et al., 2014). moreover, leaf retention was also reported to influence rooting behaviour of cuttings (leakey and coutts, 1989). the proportion of leaves retained by the cuttings during propagation is related to the percentage of cuttings t h a t f o r m a d v e n ti ti o u s r o o t s ( t c h o u n d j e u a n d leakey, 1996; mesén et al., 1997a). another factor which creates a suitable environment for rooting is substrate (tchinda et al., 2013). the importance of substrate type on the rooting capacity of cuttings is w i d e l y r e c o g n i z e d a s t h e s t a r ti n g p o i n t t o b e addressed for a successful vegetative propagation using stem cuttings (hartmann et al., 2002; leakey, 2004). cuttings of many species root successfully in a variety of 50 propagation substrates, but the rooting performance may be greatly influenced by the kind of substrate which is linked to the hydromorphic or xeromorphic status of the species (hartmann et al., 2002; leakey, 2004). the present study aims to eval­ uate the effect of three independent factors (cuttings diameter, leaf retention and substrate) on sprouting, adventitious rooting and survival ability of argania spinosa semi­hardwood cuttings to develop an effi­ cient technique for achieving large­scale production of superior clonal stock plants and conservation of genetic resources. it is a continuation of our previous research on argan vegetative propagation, which concerned the nutrient solution influence (benbya et al., 2018), and the effects of auxin type and genotype (benbya et al., 2019) on the multiplication process. 2. materials and methods experimental site and plant materials vegetative propagation experiment was carried out during the vegetative period of the tree (may to september) at non­mist greenhouse of the biotech­ nology unit of the regional center of agricultural research of rabat (inra­morocco), under natural photoperiod and a mean temperature of 32±2°c. the semi­hardwood cuttings of a. spinosa were collected from selected adult mature trees, naturally growing at the arboretum of oued cherat in the province of bouznika, morocco (33° 81’ 96” n; 7° 11’ 03” w; 45 m altitude), which is located within 2 km of the moroccan atlantic coast and with an average annual rainfall of 460 mm/yr. preparation of cuttings and experimental establish‐ ment plant material was placed in cold dark storage at 4°c for 48h before planting. then, semi­hardwood cuttings were screened for uniform length (10±0.5 cm) and were divided into three groups by diameter: 0.1­0.3 cm with 10 to12 nodes; 0.3­0.6 cm with 8 to 10 nodes and 0.6­0.9 cm with 6 to 8 nodes using cali­ brated electronic digital vernier caliper. given cut­ tings were prepared as multiple nodes, without benbya et al. ‐ clonal propagation of argania spinosa 63 leaves (leafless); or with two leaves, four leaves or eight leaves. the tip of shoot was removed and the lower leaves of each cutting were removed. before planting, cuttings were treated with aqueous solution of 0.2% (w/v) fungicide (dithane 750 g/kg mancozeb) for 10 minutes and subsequently washed with dis­ tilled water to remove excess fungicide. then, the basal end (5.0 cm) of the cuttings was soaked for 5 min in a freshly prepared aqueous solution of auxins (17.12 mm of iba). prepared cuttings were immedi­ ately inserted in different polyethylene (pe) pots (1000 cc) containing three different substrates: steril­ ized sieved fine sand (fs) (ph of 7.3, ph­kcl method; water retention of 160 ml/l; organic matter content of 0.2%, walkley & black method); peat moss (pm) ph of 6; water retention of 800 ml/l; organic matter content of 20%); mixture of fine sand and peat moss (1:1 v/v) (fs/pm). the rooting period for the experi­ ment was run for 12 weeks, and then cuttings were uprooted carefully without harming root with run­ ning water. successfully rooted cuttings (at least one root with a length of at least 1 cm long) were trans­ planted into polyethylene pots (4500 cc) and were maintained for hardening at a spacing of 20 cm × 20 cm for 48 weeks. given cuttings were regularly watered twice a week with tap water during the first twelve weeks; after a hoagland & arnon nutrient solution (hoagland and arnon, 1950) was used as irri­ gation source. data collection the morphological characteristics assessments were realized after 12 weeks for the number of sprouts (ns), length of the longest sprout (sl, cm), sprouting percentage (sp, %), number of roots (nr), length of the longest root (rl, cm), and rooting per­ centage (rp, %), whereas the survival rate success (sr, %) was recorded at week 48 of the hardening phase. experimental design and treatments the experiment was conducted in randomized complete block design (rcbd) with four blocks and three independent variables (cutting size, leaf reten­ tion and substrate) (fig. 1a). for each treatment there were 32 cuttings (eight per block), randomly allocated in groups of two cuttings (forming four fig. 1 ­ clonal propagation of argania spinosa semi hardwood cuttings. (a) cuttings planted in peat moss substrate according to a ran­ domized complete block design (rcbd) under non mist greenhouse conditions (24 weeks). (b) rooted plant cutting set in sand substrate (2 years). (c) well rooted plant cutting set in a mixture of sand and peat moss (1:1 v/v) substrate (3 years). (d) cutting with developed root primordia from fine sand substrate (12 weeks). (e) vigorous adventitious roots of semi hardwood cuttings grown in a mixture of sand and peat moss substrate (2 years). (f) argan plantlet in a mixture of fine sand and peat moss substra­ te under non­mist greenhouse conditions. (3 years). scale bars: 10 mm. adv. hort. sci., 2021 35(1): 61­72 64 3. results effect of the cutting diameter, leaf retention and sub‐ strate on number of sprouts and sprouts length of the argania spinosa cuttings cuttings with 0.6­0.9 cm diameter and four leaves set in fs/pm substrate showed the greater mean number of sprouts and the longest sprout of cuttings (1.81±0.10 cm and 17.25±0.96 cm respectively), while the lowest number of sprouts and the shortest sprout length occurred for cuttings with 0.3­0.6 cm diameter and eight leaves planted in fs substrate (1.06±0.09 cm and 8.19±1.11 cm, respectively). however, thinner cuttings (0.1­0.3 cm) and leafless cuttings for all diameters failed to produce any sprouts and are therefore not shown on the graphs (fig. 2). the mean number of sprouts per rooted cutting showed a progressive increase with increasing cut­ ting diameter (r= 0.56***). a similar trend was also observed in sprouts length per rooted cutting (table 1). results showed that pm substrate as well as experimental units for thirty­six treatment combina­ tions). the experiment was established with three cutting diameters (0.1­0.3 cm; 0.3­0.6 cm and 0.6­0.9 cm), four leaf number treatments (leafless, 2, 4, and 8 leaves) and three different substrates (fs, pm and fs/pm). statistical analysis the main effects and interactions of cutting diam­ eter, leaf retention and substrate were determined using a general linear model (glm) procedure in sas program version 9.1 (sas institute, cary, nc) for all the evaluated parameters. the differences between the treatments were tested using duncan’s multiple range test (dmrt) with at least 95% level of statisti­ cal reliance, and as a result, homogenous groups were acquired and interpreted. collected data were subjected to simple linear regression analysis. all data were reported as means ± standard error (se). sprouting, rooting and survival percentage data were arcsine transformed to ensure normal distribution and homogeneity of variances. fig. 2 ­ effects of cutting diameters, four leaf number treatments (leafless, 2, 4, and 8 leaves) and three different substrates (fs, pm and fs/pm) on mean values of number of sprouts (ns) and length of the longest sprout (sl) per rooted cutting of argania spinosa. values followed by a common letter within each column are not significantly different at p<0.05 using the duncan’s multiple range test (dmrt). a value represents mean ± standard error of means (n = 32). table 1 ­ simple linear regression for cutting diameter and number of sprouts or sprout length (cm) of argania spinosa cuttings ***= highly significant (p<0.001). n = 32. dependent variable (y) independent variable (x) correlation coefficient (r) regression equation p­value number of sprouts cutting diameter (cm) 0.56 y= 0.59 x ­ 0.44 0.000 *** sprout length (cm) cutting diameter (cm) 0.58 y= 5.42 x ­ 4.32 0.000 *** benbya et al. ‐ clonal propagation of argania spinosa 65 fs/pm substrate exhibited higher means number of sprouts compared to fs substrate. however, the fs/pm substrate showed the longest sprouts, fol­ lowed with pm substrate, while fs exhibited the shortest sprout per cutting (table 1). over all the combined data for leaf retention treatments, it showed that with cutting diameter increase, there was an increase in the number of sprouts and sprout length (r=0.58***) (table 1). indeed, cuttings with 0.6­0.9 cm diameter showed the greater mean num­ ber of sprouts and sprout length of cuttings, followed by cuttings with 0.3­0.6 cm. among leaf retention tested, cuttings with 4 leaves produced the longest sprouts compared with 2 and 8 leaves (table 2). based on the analysis of variance (anova), cut­ ting diameter and substrate had a significant effect (p<0.05) on the mean number of sprouts, while leaf retention was not found significant on the number of sprouts per cuttings (table 3). moreover, cutting diameter, leaf retention and substrate had a highly significant effect (p<0.001) on the sprout length, whereas leaf retention had only significant effect on sprout length (table 3). neither mean number of the sprouts per cuttings nor the sprout length was signifi­ cantly (p > 0.05) affected by the two­way and three­ way interaction between the three factors tested (table 3). effect of the cutting diameter, leaf retention and sub‐ strate on number of roots and length of the longest root of the a. spinosa cuttings the highest number of roots and root length were observed in cuttings with 4 leaves, followed by 2 leaves though not significantly, while lowest number table 2 ­ main effects of cutting diameter, leaf retention and substrate on the number of sprouts and length of the longest sprout (cm) of argania spinosa cuttings values followed by the same letter(s) are not significantly diffe­ rent at p<0.05 using the duncan’s multiple range test (dmrt). a value represents mean ± standard error of means (n = 32). treatments sprouts number sprout length cutting diameter (cm) 0.1­0.3 ­ ­ 0.3­0.6 1.40 ± 0.05 b 11.65 ± 0.46 b 0.6­0.9 1.58 ± 0.05 a 14.46 ± 0.44 a leaf retention leafless ­ ­ 2 1.48 ± 0.06 a 13.34 ± 0.58 ab 4 1.56 ± 0.05 a 13.98 ± 0.55 a 8 1.43 ± 0.06 a 11.85 ± 0.56 b substrate fine sand (fs) 1.27 ± 0.07 b 11.30 ± 0.63 c peat moss (pm) 1.58 ± 0.06 a 13.13 ± 0.52 b mixture of fs and pm 1.62 ± 0.05 a 14.74 ± 0.51 a table 3 ­ analysis of variance (anova) for the effects of cutting diameter, leaf retention and substrate and their interactions on number of sprouts and length of the longest sprout cm per argania spinosa cuttings ns= not significant (p>0.05); *= significant (p<0.05); ***= highly significant (p<0.001). df= degrees of freedom. ×= interaction between treatments. source of variance dependent variable no. of sprouts sprout length df f­value p­value df f­value p­value cutting diameter 2 7.36 0.0071 * 2 20.36 <0.0001 *** leaf retention 3 1.30 0.2736 ns 3 4.08 0.0179 * substrate 2 10.08 <0.0001 *** 2 10.15 <0.0001 *** cutting diameter × leaf retention 6 0.03 0.9702 ns 6 0.9559 0.9559 ns cutting diameter × substrate 4 0.39 0.6750 ns 4 0.01 0.9896 ns leaf retention × substrate 6 0.35 0.8451 ns 6 0.23 0.9232 ns cutting diameter × leaf retention × substrate 12 0.20 0.9399 ns 12 0.03 0.9983 ns block 1 0.10 0.7534 ns 1 2.43 0.1200 ns of roots and root length were mostly recorded in cut­ tings with 8 leaves. best response in term of number of roots was obtained when cuttings were set in fs substrate with four leaves and diameters of 0.3­0.6 cm for which the mean number of adventitious roots was 45.06±1.14 (fig. 1e), followed by cuttings with four leaves set in fs substrate with diameters of 0.6­ 0.9 cm (44.130±1.21). the greatest value of root length was recorded when cuttings were grown in fs substrate with 0.3­0.6 cm thickness and with four leaves (33.63±2.12), while cuttings set in a pm sub­ cutting, while leaf retention had only a significant effect (p<0.05). root length was significantly (p<0.05) affected by the interaction between cutting diameter and substrate, while the other interactions between the three factors studied were not significant on the number of roots and root length of cuttings (table 5). 66 adv. hort. sci., 2021 35(1): 61­72 strate didn’t exceed 23 cm (fig. 3; fig. 1e). however, the incidence of necrosis was the greatest among thinner cuttings (0.1­0.3 cm) and leafless cuttings, indeed these cuttings failed to produce any adventi­ tious roots and are therefore not on the graphs (fig. 3). the mean number of roots was dependent on type of substrate; cuttings grown in fs and fs/pm substrates rooted significantly better, whereas the lowest mean number of adventitious roots per cut­ tings was recorded in cuttings propagated in pm sub­ strate (table 4). over the entire experimental period, the three substrates showed pronounced differences in the root length: cuttings planted in fs substrate were found to produce significantly longest adventi­ tious roots, followed by fs/pm substrate then cut­ tings set in pm substrate (table 4). among the cutting diameters studied, (0.3­0.6) cm showed the highest number of roots and the longest root, followed by the cutting diameter (0.6­0.9) cm, while cuttings with (0.1­0.3) cm were the least performing and failed to produce roots (table 4). regarding the anova test values, the effects of cutting diameter, leaf retention and substrate were found highly significant (p<0.001) on the number of adventitious roots formed per cutting (table 5). moreover, cutting diameter and substrate had also a highly significant effect (p<0.001) on root length of table 4 ­ main effects of cutting diameter, leaf retention and substrate on the number of roots and the length of the longest root (cm) of argania spinosa cuttings values followed by the same letter(s) are not significantly diffe­ rent at p<0.05 using the duncan’s multiple range test (dmrt). a value represents mean ± standard error of means (n = 32). treatments roots number root length cutting diameter (cm) 0.1­0.3 ­ ­ 0.3­0.6 38.38 ± 0.74 a 27.24 ± 0.73 a 0.6­0.9 35.61 ± 0.90 b 24.42 ±0.69 b leaf retention leafless ­ ­ 2 36.95 ± 0.94 b 25.65 ± 0.93 ab 4 38.97 ± 1.04 a 27.47 ± 0.83 a 8 35.07 ± 1.03 b 24.36 ± 0.85 b substrate fine sand (fs) 42.07 ± 0.66 a 30.76 ± 0.76 a peat moss (pm) 27.44 ± 0.82 b 18.25 ± 0.63 c mixture of fs and pm 41.48 ± 0.75 a 28.47 ± 0.65 b fig. 3 ­ effects of cutting diameters, four leaf number (leafless, 2, 4, and 8 leaves) and three different substrates (fs, pm and fs/ pm) on mean values of number of adventitious roots (nr) and length of the longest root cm (rl) per rooted cutting of argania spinosa. values followed by a common letter within each column are not significantly different at p < 0.05 using the duncan’s multiple range test (dmrt). a value represents mean ± standard error of mean (n = 32). benbya et al. ‐ clonal propagation of argania spinosa 67 effect of the cutting diameter, leaf retention and sub‐ strate on sprouting percentage, rooting percentage and survival rate of the argania spinosa cuttings although the thinner cuttings gave no sprouts, among the other different treatments, the highest sprouting percentage was 85.00 ± 3.76% when cut­ tings with 4 leaves and 0.6­0.9 cm diameter were planted in fs/pm substrate, while cuttings with 8 table 5 ­ analysis of variance (anova) for the effects of cutting diameter, leaf retention and substrate and their interactions on the number of adventitious roots and the length of the longest root (cm) of argania spinosa cuttings ns= not significant (p>0.05); *= significant (p<0.05); ***= highly significant (p<0.001). df= degrees of freedom. ×= interaction between treatments. source of variance dependent variable no. of roots root length df f­value p­value df f­value p­value cutting diameter 2 11.27 0.0009 *** 2 13.29 0.0003 *** leaf retention 3 7.43 0.0007 *** 3 5.42 0.0049 * substrate 2 134.30 <0.0001 *** 2 98.88 <0.0001 *** cutting diameter × leaf retention 6 0.18 0.8318 ns 6 0.46 0.6345 ns cutting diameter × substrate 4 5.81 0.0034 * 4 0.11 0.8990 ns leaf retention × substrate 6 0.72 0.5809 ns 6 0.30 0.8766 ns cutting diameter × leaf retention × substrate 12 0.14 0.9657 ns 12 0.58 0.6775 ns block 1 2.46 0.1178 ns 1 0.26 0.6133 ns fig. 4 ­ effects of cutting diameters, four leaf number treatments (leafless, 2, 4, and 8 leaves) and three different substrates (fs, pm and fs/pm) on mean values of sprouting percentage (sp), rooting percentage (rp), and survival rate (sr) of argania spinosa cuttings. values followed by a common letter within each column are not significantly different at p<0.05 using the duncan’s multiple range test (dmrt). a value represents mean ± standard error of mean (n = 32). leaves and 0.3­0.6 cm diameter propagated in fs sub­ strate gave the poorest sprouting percentage (61.50± 5.68%). however, leafless cuttings performed least and failed to produce any sprouts (fig. 4). the pro­ portion of cuttings forming new shoots during growth varied between the three substrates. cuttings plant­ ed in pm and fs/pm substrates were the most suc­ cessful in sprouting, while cuttings propagated in fs the data revealed that the substrate caused sig­ nificant (p < 0.05) variations in sprouting, rooting and survival percentage of the rooted cuttings, while cut­ ting diameter and leaf retention were not significant on the survival rate (table 7). however, there was no significant effect (p>0.05) of treatment interactions between the three independent factors on the three parameters studied. 4. discussion and conclusions the present investigation revealed that cutting diameter (0.1­0.3, 0.3­0.6 or 0.6­0.9 cm), leaf reten­ tion (0, 2, 4 or 8 leaves) and substrate (fs, pm or fs/pm) were found to be important factors for suc­ cessful adventitious rooting, sprouting and survival ability of argania spinosa semi­hardwood cuttings. this experiment on a. spinosa cuttings concerning cutting diameter indicated a significant effect on the sprouting, rooting and survival performances. among the different cutting diameters tested, cuttings with 0.3­0.6 cm diameter tended to root better and devel­ op more rooting capacity compared with thinner diameter (0.1­0.3 cm), which failed to produce any sprouts or roots. moreover, larger cuttings (0.6­0.9 cm) showed the best sprouting ability but did not result in any significant improvement in root growth and development. the greatest rooting potential of cuttings with 0.3­0.6 cm diameter could be due to their good storage capacity of carbohydrates and adv. hort. sci., 2021 35(1): 61­72 68 substrate showed the lowest sprouting ability (table 6). results showed that cuttings with 0.3­0.6 cm diameter with two and four leaves set in a fs sub­ strate showed maximum rooting: (65.00 ± 5.15% and 65.00 ± 5.87% respectively). compared with the cut­ tings of 0.1­0.3 cm thickness and leafless cuttings which failed to root, all cutting diameters and leaf retention treatments enhanced the rooting percent­ age. moreover, percentage of cuttings developing adventitious roots decreased when the leaf number exceeds 4 leaves (fig. 4). the maximum rooting rates were obtained for cuttings grown in fs substrate, fol­ lowed by fs/pm substrate, while the cuttings grown on pm substrate showed the lowest rooting capacity (table 6). the survival rate ranged from 57.00 ± 4.53% for plantlets with 0.6­0.9 cm diameter and 8 leaves grown in fs substrate, to 92.50 ± 1.94% for plantlets derived from cuttings with 0.3­0.6 cm diam­ eter and 4 leaves potted on substrate containing a mixture of fine sand and peat moss. however, cutting mortality was the greatest in thinner cuttings (0.1­ 0.3) cm and leafless cuttings which showed 100% mortality rate within forty­eight weeks (fig. 4). the rooted plantlets were successfully hardened and acclimatized under non­mist greenhouse conditions (table 6). these plants showed the best survival rate and performed better growth and development when they were grown in a fs/pm substrate com­ pared with cuttings raised in pm and fs substrates (fig. 1f). table 6 ­ main effects of cutting diameter, leaf retention and substrate on sprouting percentage, rooting percentage, and survival rate of argania spinosa cuttings values followed by the same letter(s) are not significantly different at p<0.05 using the duncan’s multiple range test (dmrt). a value represents mean ± standard error of means (n = 32). treatments sprouting (%) rooting (%) survival (%) cutting diameter (cm) 0.1­0.3 ­ ­ ­ 0.3­0.6 70.69 ± 1.58 b 55.56 ± 2.22 a 78.14 ± 1.72 a 0.6­0.9 75.42 ± 2.52 a 49.55 ± 1.75 b 75.25 ± 2.20 a leaf retention leafless ­ ­ ­ 2 72.96 ± 1.87 ab 52.54 ± 2.03 ab 75.75 ± 1.04 ab 4 77.92 ± 1.93 a 56.33 ± 1.82 a 80.04 ± 2.16 a 8 68.29 ± 1.52 b 48.79 ± 1.90 b 74.29 ± 2.03 b substrate fine sand (fs) 67.54 ± 1.94 b 59.21 ± 2.27 a 65.08 ± 2.37 c peat moss (pm) 74.08 ± 2.01 a 44.42 ± 2.19 b 76.96 ± 2.44 b mixture of fs and pm 77.54 ± 2.17 a 54.04 ± 2.41 a 88.04 ± 2.83 a benbya et al. ‐ clonal propagation of argania spinosa 69 other reserves for adventitious root formation (leakey and storeton­west, 1992; tchoundjeu and leakey, 1996). the good sprouting response may be due to the presence of adequate sugar reserves such as fructose, glucose and sucrose (gehlot et al., 2015). moreover, the level of endogenous auxins and other rooting cofactors might be lower in cuttings with a small diameter, which leads to reduced rooting per­ centage or even the absence of rooting (wilson and van staden, 1999). the rooting efficiency of these cuttings could be also due to the lower content of mineral elements, especially nitrogen which is posi­ tively correlated with rooting (budiarto et al., 2006). in addition, the effect of cutting diameter on rooting capacity could also be attributed to the origin of a cutting within shoots, and to its position of the stock­ plant (leakey, 2004). indeed, thin cuttings are pro­ duced from shoots which arise in sub­dominant posi­ tions of the mother plants which contain a low amount of auxin, grow slowly and stop growth early (howard and ridout, 1991). the lower rooting capaci­ ty of cuttings with larger diameters (0.6­0.9 cm) may probably be due to changes in the extent of lignifica­ tion and the degree of secondary thickening along their stems (girouard, 1969; hartmann et al., 1990). these results are consistent with other studies that found a positive effect of cutting diameter on sprout­ ing, rooting and survival success of rooted cuttings. palanisamy and kumar (1997) showed that the high­ er rooting efficiency of picea abies was obtained by cuttings of 0.3­0.4 cm diameter. ouyang et al. (2015) confirmed the highest rooting efficiency of picea abies obtained by cuttings of 0.3­0.4 cm diameter. moreover, foster et al. (2000) observed that the cut­ tings of pinus taeda with an average diameter of 0.2­ table 7 ­ analysis of variance (anova) for the effects of cutting diameter, leaf retention and substrate and their interactions on sprout­ ing percentage, rooting percentage and survival rate of argania spinosa cuttings ns= not significant (p>0.05); *= significant (p<0.05); ***= highly significant (p<0.001). df= degrees of freedom. ×= interaction between treatments. source of variance dependent variable sprouting (%) rooting (%) survival (%) df f­value p­value df f­value p­value df f­value p­value cutting diameter 2 4.28 0.0434 * 2 6.70 0.0124 * 2 1.96 0.1668 ns leaf retention 3 5.93 0.0047 * 3 3.53 0.0363 * 3 2.80 0.0694 ns substrate 2 6.60 0.0027 * 2 13.98 <0.0001 2 41.37 <0.0001 cutting diameter × leaf retention 6 0.02 0.9849 ns 6 0.06 0.9444 ns 6 0.23 0.7918 ns cutting diameter × substrate 4 0.22 0.8072 ns 4 0.17 0.8411 ns 4 0.13 0.8775 ns leaf retention × substrate 6 0.27 0.8937 ns 6 0.12 0.9745 ns 6 0.29 0.8855 ns cutting diameter × leaf retention × substrate 12 0.15 0.9633 ns 12 0.09 0.9860 ns 12 0.14 0.9655 ns 0.3 cm tended to root better and develop more roots. concerning leaf retention, this study showed that successful vegetative propagation of argania spinosa was restricted to leafy stem cuttings. indeed, defoli­ ated cuttings failed to produce any sprouts / roots and showed a mortality rate of 100% within forty­ eight weeks after planting. this result could be explained by the direct influence of the presence of leaves on the primary shoots, because initial growth of shoots depends on assimilates supplied by leaves and also through their influence on the cutting’s water status (newton et al., 1992; van labeke et al., 2001). moreover, the leaf retention could also exert a strong influence on root initiation and development because it allows post­severance carbon assimilation (leakey and coutts 1989; hartmann et al., 1990; thomas and schiefelbein, 2004). leafy cuttings pro­ vide a continuous supply of photosynthates besides their reserves after implementing them into the sub­ strate (tchoundjeu et al., 2002; leakey, 2004). on the other hand, the inability of leafless cuttings to root has been associated with the rapid depletion of stored carbohydrates in stem tissues after excision from the stock plants (hoad and leakey, 1996; druege et al., 2000). in addition, the leaf retention may also influence the endogenous auxins of the cut­ ting. since rooting is stimulated by the level of auxins available, it is expected that adventitious rooting response of a cutting will be proportional to the num­ ber of leaves retained (tchoundjeu and leakey, 1996). in fact, our results showed that the ability of cuttings to develop adventitious roots decreased when the leaf number exceeds 4 leaves. this could be explained by the balance between the positive adv. hort. sci., 2021 35(1): 61­72 70 effects of assimilate production through allowing suf­ ficient photosynthesis and the negative effects of water loss via transpiration (leakey and coutts, 1989; mesén et al., 1997a). however, cuttings with eight leaves exhibited early root growth and delayed sprouting and subsequently a decreased rooting effi­ ciency. indeed, cuttings with a high number of leaves appear to suffer from water deficit and a consequent reduction in photosynthetic activity as reflected by higher transpiration rates and leaf shedding com­ pared to cuttings with an optimal number of leaves (leakey and coutts, 1989; newton et al., 1992; aminah et al., 1997). thus, the balance between pho­ tosynthesis, transpiration and nutrient transport is an important factor influencing rooting (leakey and coutts, 1989). the positive effect of leaf retention on cutting success has been reported across a wide range of species including triplochiton scleroxylon (leakey and coutts, 1989), cordia alliodora (mesén et al., 1997a), vitis vinifera (thomas and schiefelbein, 2004), eucalyptus hybrids (trueman and adkins, 2013), santalum austrocaledonicum (tate and page, 2018). finally, the study of the substrate effect showed that a significant difference in sprouting and rooting as well as in survival ability was found between the three substrates tested. it was observed that the number of roots, root length and rooting percentage reached significantly higher values in cuttings set in the fs substrate compared to the pm substrate and to the mixture of fine sand and peat moss. this effec­ tiveness of adventitious rooting by rooted cuttings set in fs substrate could be related to its optimal vol­ ume of gas­filled pore­space and oxygen diffusion rate which create an adequate aerated environment for increased transpiration and respiration and enhanced adventitious root formation (andersen, 1986). moreover, sand is a porous substrate that lim­ its the development of microbial pathogens and where roots can be settled well without damage (tchinda et al., 2013). though, the poor aeration and high water holding capacity of peat moss substrate is a fundamental problem for adventitious root forma­ tion, leading to enhance the rate of fungal infection and decay of cuttings before root initiation (schmitz et al., 2013). furthermore, the higher water content in the peat moss substrate induces closing of the stomata and inhibits oxygen diffusion. this anoxia caused by oxygen deficiency or by accumulation of toxic substances, including bicarbonate and carbon dioxide, is suspected of being linked to the rooting problems due to tissue death, wilting, severe defolia­ tion, and reduced water absorption and leaf water potential (mesén et al., 1997b; drew, 1983; veen, 1988). the results also showed that cuttings set in a mixture of sand and peat moss performed better than those set in sand or peat moss substrate alone, in terms of sprouting and survival rate. it has been suggested that the addition of peat moss to fine sand improved the water holding capacity and promoted mineral nutrients absorption (leakey et al., 1990). the uptake of water by cuttings is positively related to the water content of the substrate and this may enhance survival rate by reducing water deficits, leaf abscission and cutting necrosis (newton et al., 1992). a number of comparative studies between different substrates have indicated that cuttings set in a sand substrate performed better in term of rooting and survival capacity. atangana et al. (2006) reported that the higher rooting percentages were observed in sand for allanblackia floribunda. gehlot et al. (2014) also recommended that higher rooting percentages of azadirachta indica were recorded for cuttings planted in sand. moreover, adugna et al. (2015) stat­ ed that in vanilla planifolia, the cuttings set in sand performed the highest rooting percentage. in conclusion, the results of the present study clearly indicate that cuttings with thinner diameters (0.1­0.3 cm) and defoliated cuttings have failed to produce any sprouts or roots and showed a 100% mortality rate within forty­eight weeks after planting. the greatest mean number of sprouts, sprout length and sprouting capacity as well as survival success was performed by cuttings with diameter of 0.3­0.6 cm and 4 leaves set in a mixture of sand and peat moss. the best mean number of roots, root length and rooting ability was achieved by cuttings with diame­ ter of 0.3­0.6 cm and 4 leaves in a fine sandy sub­ strate. this study also reveals that argania spinosa is amenable to clonal propagation using sand­based rooting substrate which could provide a promising p r o s p e c t f o r t h e c o n s e r v a ti o n o f t h i s e n d e m i c species. in addition, it is suggested that this low­cost propagation techniques could greatly facilitate the domestication and the development of superior tree crops as a commercial agroforestry species with genetically homogenous plant material. the cuttings used in the experiments were taken during the spring season. it is therefore recommended that further works should be done to determine the optimal peri­ od for cutting collections for successful clonal propa­ gation of a. spinosa trees. benbya et al. ‐ clonal propagation of argania spinosa 71 acknowledgements this work is supported by public funds through the national institute for agronomic research (inra), ministry of agriculture ­ morocco. references adugna m., belew d., tilahun d., 2015 ­ influence of rooting media and number of nodes per stem cutting on nursery performance of vanilla (vanilla planifolia andr. syn. vanilla fragrans). ­ j. hort. for., 7(3): 48­56. aminah h., dick j.m., grace j., 1997 ­ rooting of shorea leprosula stem cuttings decreases with increasing leaf area. ­ forest ecol. manag., 91(2­3): 247­254. andersen a.s., 1986 ­ environmental influences on adven‐ titious rooting in cuttings of non‐woody species, pp. 223­253. ­ in: jackson m.b. 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properties, lactuca sativa l., physical properties, root system morphology, shoot morphology. abstract: a difficulty in the production of lettuce seedlings in containers is to assure the production of shoot biomass with limited portion of roots, restricted to a small volume of substrate. therefore, we investigate if substrates associated to lettuce cultivars interfere in the seedling quality. the treatments, outlined in a two-factorial scheme, were two cultivars of lettuce and four substrates, arranged in a randomized complete block design, with three replications. the results showed that seedlings produced in the substrate with higher water retention capacity had higher performance in relation to shoot morphology and root system morphology. in conclusion, the data show that the seedlings quality of lettuce cultivars associate with the types of substrates studied and that seedlings produced in substrate with higher water retention have better quality. 1. introduction in horticultural crops the supply of quality seedlings to the producers is important to obtain high production after the establishment of the plants in their growth medium. such quality is related to the plants resistance to biotic and abiotic stresses (zhao et al., 2016). among vegetables, lettuce (lactuca sativa l.) is one of the most cultivated (kim et al., 2016), with a world production of approximately 25 million tons (fao, 2014). in order to maintain this production in an upward manner, quality seedlings must be provided to producers. thus, the production of seedlings is one of the most important stages in lettuce cultivation, because this process reflects on the productive performance of the plants (auler et al., 2015). however, a difficulty in the production of seedlings in containers is to ensure the production of shoot biomass with limited portion of root (lemaire, 1995), restricted to a small volume of substrate, in response to the species/cultivars used. as the substrates have a wide variation in their physicochemical pro(*) corresponding author: jose-trevizan@hotmail.com citation: chiomento j.l.t., frizon p., costa r.c., trentin n.s., nardi f.s., calvete e.o., 2019 water retention of substrates potentiates the quality of lettuce seedlings. adv. hort. sci., 33(2): 197-204 copyright: © 2019 chiomento j.l.t., frizon p., costa r.c., trentin n.s., nardi f.s., calvete e.o. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 6 july 2018 accepted for publication 18 february 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(2): 197-204 198 perties (fermino and kämpf, 2012), choosing a suitable material is essential to the development of the plants (mondragón-valero et al., 2017). the substrates used must be low cost and easy to handle (noya et al., 2017), to have porosity around 85% (kämpf et al., 2009) and water retention capacity (graceson et al., 2013). for lettuce, it is necessary to choose material that ensures these physical characteristics, since it is a species with high water demand (nunes et al., 2017). moreover, the choice of the cultivar is also important, because the genotypes can interact with biotic and abiotic factors, influencing the quality of the seedlings produced (martins et al., 2017). knowing that substrates used in seedlings production are essential to plants germination and establishment (auler et al., 2015) and that crop productivity is linked to this input (smiderle et al., 2001), this question arises: how substrates associated to lettuce cultivars affect the seedlings quality? therefore, based on the hypothesis that the quality of lettuce cultivars is dependent of the water retention capacity of the substrate, the objective of the present study was to evaluate if substrates associated to lettuce cultivars interfere in the seedlings quality. this study provides a view of the development of lettuce seedlings using different substrates to improve the seedlings quality (e.g., increase the growth of shoot biomass and root system) grown in greenhouses. 2. materials and methods plant material, treatments description and experiment site the seeds of lettuce used in the work were of the cultivars mimosa roxa salad bowl (purple), of bright greenish purple color, and mônica sf 31 (green), of medium green color, both of the group crisphead. the materials used as substrates were carbonized rice husk (crh), horta 2® (hor), tn gold® (tng) and a mixture (mix) composed of 40% crh, 40% hor and 20% tng. the composition of hor consists of pine bark, vermiculite, acid correction and fertilizers (nitrogen, phosphorus and potassium) in quantities not supplied by the manufacturer. the composition of tng consists of sphagnum peat, expanded vermiculite, dolomitic limestone, agricultural gypsum and fertilizers (nitrogen, phosphorus and potassium) in quantities not supplied by the manufacturer. no fertilizer was added to the substrates. the rice husk used in the work was carbonized (kämpf et al., 2006). the experiment was developed in the brazilian subtropics, in the city of passo fundo/rs (28° 15’ 46” s, 52° 24’ 24” w), from april to may (fall) of 2017. the trial took place on trays kept on metal benches, 1.2 m above the soil surface, in a agricultural greenhouse of 90 m², with semicircular roof, installed in the northeast-southeast direction. the galvanized steel frame was covered with low density polyethylene film, with anti-ultraviolet additive and with a thickness of 150 microns, and the sides were covered with anti-aphid screen. the irrigation used was with sprinklers, in the mechanized system, with a flow rate of 2 l min-¹ per unit. the irrigation regime consisted of four sprinklers per day, with total wetting of seven minutes. the water blade supplied to the seedlings was 4.35 mm day-1. during the execution of the experiment, the photosynthetically active radiation (par) and the mean air temperature inside the greenhouse were monitored, with mean values of 110.5 μmol m-² s-¹ and 17.4°c, respectively. experimental design the treatments, outlined in a two-factorial scheme, consisted of two lettuce cultivars (purple and green) and four substrates (crh, hor, tng and mix). the production of the seedlings was carried out in trays of expanded polystyrene, with dimensions of 0.34 m of width and 0.68 m of length. each tray had 128 cells, with a volume of 35 cm³. the experimental design was randomized blocks with three replicates (n= 3; one replicate per tray, i.e. three trays were used in the experiment). on april 20, the trays were filled with the substrates crh, hor, tng and mix, and after that, five seeds of the lettuce cultivars were sown in each cell. in each tray each treatment was composed of 16 seedlings, that is, a total of 48 seedlings per treatment (16 seedlings/treatment x 3 replicates). considering that we used 8 treatments, our experiment consisted of a total of 384 seedlings (16 seedlings/treatment x 3 replicates x 8 treatments). determination of physicochemical properties of substrates a sample of 1 l of each substrate was collected and analyzed to obtain physicochemical attributes of the materials. the physical attributes determined in the substrates were: density (d), total porosity (tp), aeration space (as), readily available water (raw) and buffer water (bw). the chemical attributes determined in the subschiomento et al. quality of lettuce seedlings 199 trates were nitrogen (n), phosphorus pentoxide (p2o5), potassium oxide (k2o), organic carbon (oc), hydrogenionic potential (ph), electrical conductivity (ec) and cation exchange capacity (cec) (mapa, 2014). regarding the seedlings, the evaluations began one week after sowing. morphological attributes of the shoot and the root system of the seedlings were evaluated. determination of shoot morphology in relation to the shoot morphology, forty-eight seedlings per treatment were evaluated. four days after sowing the percentage of seed germination was evaluated by means of the equation: germination (%)= (seed germinated/total number of seeds) x 100 (1) after the germination, thinning was performed, leaving one plant per cell in each tray. in addition, the date of emergence of the cotyledons and the issuance of the first, second and third leaves were noted. thirty-three days after sowing, the stem base diameter (sbd) and the shoot height (sh) of the seedlings were measured with a digital caliper. the fresh (sfw) and dry (sdw) weight of the shoot was also evaluated. in order to obtain the dry weight, the plants were kept in a drying oven with forced air circulation, at 65°c for 48 hours, until constant weight, and weighed in an electronic analytical balance. determination of root system morphology regarding to the root system morphology, fortyeight seedlings per treatment were evaluated. the roots were collected and washed in water to eliminate the substrate fragments. thus, the roots were scanned and then the images obtained were analyzed by winrhizo® software. the attributes evaluated were the total root length (tl, cm), root surface area (sa, cm²) and root volume (rv, cm³). the roots were grouped by software in different diameter classes in relation to their total length (böhm, 1979): very thin roots (vtr, ø <0.5 mm), fine roots (fr, ø 0.5 to 2 mm) and thick roots (tr, ø > 2 mm). the fresh (rfw) and dry (rdw) weight of the root system was also evaluated, following the methodology described previously. determination of seedlings quality the seedlings quality was obtained by models of plant development. these development models are mathematical models that consider plant growth variables (cournède et al., 2013), such as shoot morphology and root system morphology. thus, the seedling vigor index (svi) was determined according to abdul-baki and anderson (1973), by the equation: svi= germination (%) x (shoot length + root length) (2) it was determined, also, the dickson quality index (dqi), proposed by dickson et al. (1960), by the equation: dqi= (tdw) / (h/sbd + sdw/rdw) (3) where tdw = total dry weight (g); h = shoot height (cm); sbd = stem base diameter (cm); sdw = shoot dry weight (g); rdw = root dry weight (g). in addition, dry matter accumulation (dma) of the shoot and of the root system was determined, according to atif et al. (2016), by the equation: dma = (dw/fw) x 100 (4) where dw = dry weight (g); fw = fresh weight (g). statistical analysis the data were submitted to analysis of variance and the means of the treatments were compared by tukey test, at 5% probability of error, with the aid of the assistat® program (silva and azevedo, 2016). 3. results physicochemical properties of substrates the results of the physical characterization of the substrates used in this experiment (table 1) showed substrates density (kg m-3) total porosity (m3 m-3) aeration space (m3 m-3) readily available water (m3 m-3) buffer water (m3 m-3) carbonized rice husk 170±12.33 c 0.879±0.11 b 0.365±0.02 b 0.395±0.10 a 0.009±0.003 horta 2® 241±05.26 a 0.837±0.10 d 0.303±0.01 d 0.149±0.02 d 0.020±0.001 tn gold® 088±10.98 d 0.916±0.13 a 0.519±0.11 a 0.202±0.08 c 0.007±0.002 mix 183±07.47 b 0.869±0.14 c 0.325±0.08 c 0.259±0.09 b 0.030±0.001 mean 170.50 0.87 0.378 0.251 0.016 coefficient of variation (%) 13.58 14.11 15.26 14.45 12.25 mix= mixture composed of 40% caborized rice husk, 40% horta 2® and 20% tn gold®. data presented as mean ± standard deviation. means followed by the same letter in the column did not differ significantly by the tukey test (p≤0.05, n= 3). table 1 physical properties of the substrates used in the study adv. hort. sci., 2019 33(2): 197-204 200 that, considering the density of the materials, the tng substrate is the lightest. with the values of tp, as, raw and bw of table 1 we elaborated a graph to visualize the relation between air and water in each substrate (fig. 1). we observed that tng material showed an unbalanced air-water relation (fig. 1). in addition, we observed a better balance between airwater in the mix substrate, that is, when the other materials were combined (40% crh, 40% hor and 20% tng). in addition, also with the values of tp, as, raw and bw of table 1 we elaborated a graph to visualize the water retention curve of each substrate (fig. 2), according to de boodt and verdonck (1972). the hor and mix substrates presented higher water retention, requiring volumes of 0.385 m3 m-3 and 0.285 m3 m-3, respectively, to remain in the range of water easily available to plants (10-50 -cm h2o) (fig. 2). on the other hand, the crh substrate had greater drainage of water (fig. 2). t h e f o u r m a t e r i a l s s h o w e d a v a i l a b i l i t y o f nutrients, except for k2o. among the substrates, crh presented 23% more ph than tng. the opposite was obtained for ec and cec, with crh being 58% lower than the hor for ec and 85% lower than tng for cec (table 2). shoot morphology in all treatments there was 100% germination of the seeds. up to the first true leaf, the two cultivars took the same period (11 days) to differentiate. the differences began to be identified from the second true leaf. for this attribute, the two cultivars produced in the crh and the green cultivar produced in the tng took longer to emit the third true leaf (fig. 3). as a consequence, at the time of transplantation the third leaf was poorly expanded. we did not observe effects of the cultivars on the shoot morphology of the seedlings. this means that the seedlings of both cultivars can be produced on any of the substrates. however, we observed only effect in relation to the substrates for the sh, sfw and sdw. the seedlings produced on the hor substrate had higher sh (5.86 cm ± 1.82) than those produced on the crh substrate (3.24 cm ± 0.77), but did not differ fig. 2 w a t e r r e t e n t i o n c u r v e o f t h e s u b s t r a t e s . c r h : carbonized rice hull; hor: horta 2®; tng: tn gold®; mix: mixture. different letters indicate significant differences by the tukey test (p≤0.05, n = 3). fig. 1 physical characterization of the substrates used in the study; n= 3 crh= carbonized rice hull; hor= horta 2®; tng= tn gold®; mix= mixture. table 2 chemical properties of four substrates substrates n % (m/m) p2o5 % (m/m) k2o % (m/m) oc % (m/m) ph ec (ms cm-1) cec (mmolc kg-1) carbonized rice husk 0.69±0.01 a 1.71±0.10 a 0 07.21±03.03 d 7.2±1.0 a 0.19±0.01 c 134.60±12.2 d horta 2® 0.36±0.09 c 0.39±0.06 d 0 12.60±06.22 b 6.1±2.2 c 0.45±0.01 a 278.60±15.4 b tn gold® 0.65±0.01 b 1.37±0.11 c 0 31.16±11.02 a 5.6±1.3 d 0.36±.0.2 b 892.98±13.7 a mix 0.35±0.02 c 1.48±0.03 b 0 10.38±04.33 c 6.4±3.1 b 0.37±0.03 b 230.05±11.2 c mean 0.51 1.23 0 15.33 6.32 0.34 384.05 cofficient of variation (%) 12.25 15.90 0 16.30 11.51 17.91 14.15 mix= mixture composed of 40% caborized rice husk, 40% horta 2® and 20% tn gold®. data presented as mean ± standard deviation. means followed by the same letter in the column did not differ significantly by the tukey test (p≤0.05, n= 3). chiomento et al. quality of lettuce seedlings 201 root system morphology regarding the root system morphology, we observed significant differences for the interaction between substrates and cultivars in relation to rv and tr (table 3). for the interaction between the factors, the best combination corresponded to the seedlings of the green cultivar produced on the hor substrate, both for root volume and for thick roots (table 3). we also observed significant differences for substrates only for rfw, rdw, sa and fr (table 4). in this respect, we observed that the seedlings produced in hor presented a larger sa, but did not differ statistically from the seedlings produced in mix (table 4). the seedlings developed in crh had smaller amount of fr in relation to those obtained in the other substrates (table 4). regarding the rfw, seedlings produced in hor were superior to those obtained in crh, but did not differ from the seedlings produced in tng and mix (table 4). in addition, the seedlings obtained from hor and mix had a higher rdw in comparison to those developed in crh and tng (table 4). in relation to lettuce cultivars the significant differences occurred for rfw, rdw, tl, sa and fr (table 4). thus, we observed that the seedlings of the green cultivar presented superior performance of the root statistically from the seedlings produced in tng and mix (fig. 4 a). in addition, the seedlings produced on the hor substrate showed higher sfw (0.27 g ± 0.17) and sdw (0.012 g ± 0.0033) than those produced on other substrates (fig. 4 b and c, respectively). in general, seedlings obtained on the hor substrate produced 71% more shoot biomass than those grown in crh (fig. 4). fig. 3 combination of two lettuce cultivars produced in four substrates in relation to the period of emergence and expansion of the first true leaves. crh= carbonized rice hull; hor= horta 2®; tng= tn gold®; mix= mixture; purple=mimosa roxa salad bowl cultivar; green= mônica sf 31 cultivar. fig. 4 shoot morphology of lettuce seedlings produced in substrates. (a) shoot height (sh, cm); (b) shoot fresh weight (sfw, g); (c) shoot dry weight (sdw, g). values are means ± standard deviation. different letters above columns indicate significant differences by the tukey test (p≤0.05, n = 3). crh= carbonized rice hull; hor= horta 2®; tng= tn gold®; mix= mixture. substrates root volume (cm³) thick root (cm) purple cv. green cv. purple cv. green cv. carbonized rice husk 0.019 ±0.004 a b 0.043±0.017 a c 0.090±0.15 a a 0.078±0.10 a c horta 2® 0.074 ±0.029 b a 0.185±0.023 a a 0.501±0.41 b a 3.021±0.51 a a tn gold® 0.053 ±0.014 a ab 0.079±0.027 a bc 0.209±0.17 a a 0.660±0.61 a bc mix 0.066 ±0.007 b a 0.111±0.019 a b 0.420±0.22 b a 1.440±0.78 a b mean 0.07 0.80 cofficient of variation (%) 21.77 53.88 mix= mixture composed of 40% caborized rice husk, 40% horta 2® and 20% tn gold®. data presented as mean ± standard deviation. means followed by the same letter in the column did not differ significantly by the tukey test (p≤0.05, n= 3). table 3 root system morphology of two lettuce cultivars (lactuca sativa l.) produced in four substrates 202 adv. hort. sci., 2019 33(2): 197-204 system in relation to the seedlings of the purple cultivar (table 4). seedlings quality regarding the seedlings quality, we observed statistical diferences for the substrates and for the cultivars. analyzing only the substrates, there were significant differences for the dqi. as for the cultivars, the significant differences were observed regarding the svi and dqi. seedlings produced on the hor substrate presented higher dqi (0.000249 ± 0.000089) than those produced in chr (0.00011 ± 0.000071), but did not differ statistically from seedlings produced in mix (fig. 5). this higher quality of the seedlings produced on hor increased by 56% in relation to those produced on chr material (fig. 5). the green cultivar, regardless of the substrate, presented superiority regarding the svi (fig. 6 a) and dqi (fig. 6 b) in relation to the purple cultivar. 4. discussion and conclusions in general, the research showed that the quality of lettuce cultivars was associated with the types of substrates studied. however, our study showed that fig. 5 quality of the development of lettuce seedlings produced in four substrates. values are means ± standard deviation. different letters above columns indicate significant differences by the tukey test (p≤0.05, n= 3). crh= carbonized rice hull; hor= horta 2®; tng= tn gold®; mix= mixture. mix= mixture composed of 40% caborized rice husk, 40% horta 2® and 20% tn gold®. data presented as mean ± standard deviation. means followed by the same letter in the column did not differ significantly by the tukey test (p≤0.05, n= 3). table 4 root system morphology of two lettuce cultivars (lactuca sativa l.) produced in four substrates total root length (cm) root surface area (cm²) fine root (cm) root fresh weight (g) root dry weight (g) substrates carbonized rice husk 21.18±05.89 2.81±1.13 c 5.01±2.52 b 0.030±0.01 b 0.0017±0.0008 b horta 2® 28.42±10.55 6.54±2.48 a 9.87±2.30 a 0.087±0.04 a 0.0036±0.0010 a tn gold® 24.44±13.36 4.39±1.78 bc 8.61±2.58 a 0.060±0.04 ab 0.0023±0.0006 ab mix 28.73±09.55 5.54±1.53 ab 9.09±1.93 a 0.061±0.02 ab 0.0032±0.0006 ab cultivars purple 21.92±08.13 b 3.70±1.54 b 7.20±3.07 b 0.038±0.01 b 0.0021±0.0008 b green 29.46±10.54 a 5.94±2.23 a 9.55±2.52 a 0.080±0.04 a 0.0033±0.0010 a mean 25.69 04.82 08.37 00.05 0.0027 cv (%) 26.28 21.32 20.07 46.77 35.41 fig. 6 development of seedlings of two lettuce cultivars produced in four substrates. (a) seedling vigor index (svi); (b) dickson quality index (dqi). values are means ± standard deviation. different letters above columns indicate significant differences by the tukey test (p≤0.05, n = 3). purple: mimosa roxa salad bowl cultivar; green: mônica sf 31 cultivar. chiomento et al. quality of lettuce seedlings 203 substrates with higher water retention promoted greater development of seedlings, through models of plant development. in this way, the physical characterization of the substrates used in the production of seedlings allows to select materials with greater water availability, in order to increase the seedlings quality. the higher quality of the seedlings produced in the hor substrate was attribute to the higher availability of water of this material, because the water retention capacity of the substrates influences the growth and development of the seedlings (graceson e t a l . , 2 0 1 3 ) , c o v e r i n g t h e s h o o t m o r p h o l o g y (prevedello and armindo, 2015) and the root system morphology (ferraz et al., 2005). in practice, the data referring to the root system morphology indicated that seedlings of the green cultivar produced in the hor substrate have a more structured lump, which improves seedling sustainability after transplanting and increases plant survival. as in crh there is predominance of large particle sizes, this impairs water retention by the material (zorzeto et al., 2014), which explained the lower quality of the seedlings produced in this substrate. in practice, the expansion data of the first true leaves showed that seedlings produced in the crh would not be suitable for transplant because, in addition to lower water retention, this substrate has ph above the ideal range (5.0 to 6.5) (bunt, 1988), which reduces the availability of nutrients to plants (lemaire, 1995). the higher the seedlings quality delivered to the producers the better the development of the plants in their growth medium and the lower their susceptibility to stresses after transplantation. through the dqi, we verified that more robust seedlings were produced in the hor substrate. during the production of the seedlings we observed that the par mean (110.5 μmol m-2 s-1) was below ideal for the lettuce, corresponding to 196.7 μmol m-2 s-1 (ferentinos et al., 2000). this may have reduced the photosynthetic rate of the plants, reducing accumulations of biomass and, therefore, dqi values. as in our study, other studies showed higher growth and development of seedlings produced in substrates with higher water retention (smiderle et al., 2001; costa et al., 2007), as verified in the hor, and lower performance for seedlings produced in substrates with low water availability (freitas et al., 2013), as observed in the crh. considering the physicochemical characterization of the substrates, the chemical properties are less relevant than the physical properties (belda et al., 2016). this is because fertirrigation is provided to the plants, according to the need of the cultivated species. however, the physical quality of the substrates must still be weighed in the choice of materials. the density of the substrates, for example, is linked to plant stability (noya et al., 2017; wisdom et al., 2017). very light substrates (< 100 kg m-3) do not sustain plants and very dense substrates (>300 kg m-3) impair the root growth of seedlings due to mechanical impediment (de boodt and verdonck, 1972; fermino and kämpf, 2012). in addition, a common problem in substrates is insufficient aeration (nemati et al., 2002) and, therefore, the nurseryman should choose materials with higher aeration levels to improve root growth and increase the acquisition of water and nutrients by seedlings (jones and dolan, 2012). thus, in order to maximize the quality of the seedlings produced, nurserymen must obtain the physical characterization of the substrates, selecting materials with greater water retention capacity. in addition, seedling development models, such as svi and dqi, can be used as indicators of the quality of seedlings produced, as we have verified in our study. in conclusion, the data show that the seedlings quality of lettuce cultivars associate with the types of substrates studied. in addition, we proves that substrates with greater water retention promote greater development of the seedlings. we emphasize that the use of development models can be an alternative to analyze the seedlings quality provided to producers in order to increase lettuce production. we suggest to substrate producers to sell materials with a clear label informing the physicochemical characteristics of the substrate so that nurserymen and producers can establish an adequate management to potentiate the lettuce production chain. acknowledgements to the programa de suporte à pós-graduação de instituições comunitárias de ensino particulares (prosuc) of the coordenação de aperfeiçoamento de pessoal de nível superior (capes), for granting the scholarship, and to the programa de pós-graduação em agronomia (ppgagro), of the universidade de passo fundo (upf), for the training of human resources. in addition, we inform that this study was financied in paert by the capes, brazil (finance code 001). adv. hort. sci., 2019 33(2): 197-204 204 references abdul-baki a.a., anderson j.d., 1973 vigor determination in soybean seed by multiple criteria. crop sci., 13: 630-633. atif m.j., jellani g., malik m.h.a., saleem n., ullah h., khan m.z., ikram s., 2016 different growth media effect the germination and growth of tomato seedlings. sci. technol. devel., 35(3): 123-127. auler a.c., galetto s.l., silva a.r., verona r.b., 2015 lettuce seedlings development index in different subs t r a t e s u s i n g m u l t i v a r i a t e a n a l y s i s . c i e n t í f i c a , jaboticabal, 43(1): 50-57. belda r.m., lidón a., fornes f., 2016 biochars and hydrochars as substrate constituents for soilless growth of myrtle and mastic. ind. 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of algeria a. abed 1, 2 (*), m. bonhomme 3, a. lacointe 3, g. bourgeois 4, d. baalicherif 1 1 national school of agronomic sciences, algiers, algeria. 2 laboratory water‐ rock ‐plant, khemis‐miliana university, algeria. 3 clermont auvergne university, inra, piaf, f‐63000 clermont‐ferrand, france. 4 agriculture and agri‐food canada, saint‐jean‐sur‐richelieu research and development centre, saint‐jean‐sur‐richelieu (qc), canada. key words: budburst, flowering, golden delicious, modelling, temperature. abstract: global warming is a strongly felt reality in recent years in algeria. the fruit trees crop is particularly exposed to the impact of this warming, especially apple trees. a comparative study has been realized between a chronological daily temperature series from 1980 to 2016, and phenological data series (budburst and flowering) from 2000 to 2016, regarding the apple tree variety of golden delicious in two zones of northern algeria, sidi lakhdar (town of ain defla, in an altitude of 211 m) and benchicao (town of médéa, in an altitude of 1133 m). some contrasting tendencies according to sites and periods have been demonstrated: very significant warming at sidi lakhdar site in autumn and spring, in particular in october and april, disturbing thus the entrance of the buds in the endodormancy and ecodormancy. the result is a late action of the cold until february, which proved to be insufficient. however, no average warming has been demonstrated at the benchicao site, where the temperatures between november and january were cold enough to satisfy the need of cold units and raise the endodormancy. it seems that the failure to fulfill the need of cold units at sidi lakhdar site has strongly affected the goodness of fit of the classic phenological models, confirming indirectly the existence of more complex physiological processes (not taken in consideration by models), which manifest themselves in limited zones such as sidi lakhdar site. 1. introduction according to the experts of the intergovernmental group of the climate evolution (igce), from now on to the end of the 21st century, the average temperature will be raising from 2 to 6°c in europe following the regions, the climatic models and the socio-economic scenario. the summer droughts will be more intense as well (giannakopoulos et al., 2005; (*) corresponding author: abedlila24@yahoo.fr citation: abed a., bonhomme m., lacointe a., bourgeois g., baali-cherif d., 2019 climate chan‐ ge effect on the bud break and flowering dates of the apple trees in mountainous and plain regions of algeria. adv. hort. sci., 33(3): 417-431 copyright: © 2019 abed a., bonhomme m., lacointe a., bourgeois g., baali-cherif d. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 15 january 2019 accepted for publication 3 september 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(3): 417-431 418 gleizer et al., 2007). according to legave (2009), a worrying acceleration of the global warming has appeared during the 1990 decade and much more during 2000 decade. in occidental europe and the mediterranean basin, on a recent period of 30 years (1973-2002), we can estimate the average increase of the annual temperature at about 1°c since the end of 1980’s. regional differences are noticed though, with a warming relatively marked in the region of meknes in morocco (2.3°c) (balaghi, 2017), definitely higher than the one marked in the south-west of france (+1.3°c in nimes)(legave, 2009). the global warming will affect, and has already notably done, a wide range of physical/biological systems and human activity sectors, among which agriculture (including livestock) and its principal function of producing and nutrition (seguin, 2010). for algeria, the 21st century shall be characterized by temperatures increase, in the order of 1.0 to 1.5°c at the horizon of 2020 (fourth report of the igce in bourchef, 2013) and precipitations decrease in the order of 15 to 20%. extreme climatic phenomena are already affecting the region, like the rain and thunderstorms of november 2001 in algiers and october 2008 in ghardaia, and the cold waves in january 2005 and february 2001 in all algeria. all these events can be qualified as historic at least regionally. some simulations realized for two types of agricultural years in terms of pluviometry (normal and dry), show at the horizon of 2020 a decrease in the yield of winter cereals from 6% to 14% according to the geographical regions and the year type in algeria (tabet-aoul, 2000; tabet-aoul and bessaoud, 2009). phenology is the study of the occurrence of periodic events in animal and plant life in relation with the climate variations. those are characters that interpret the organisms’ adaptation to the climatic variation (chuine, 2005). the task of plant-phenology is to observe and record the periodically recurring growth stages. leaf unfolding, flowering of plants in spring, fruit ripening, colour changing and leaf fall in autumn are all examples of phenological events (koch et al., 2006). it has been designated as a key point to evaluate the global warming impact on the agricultural cultivations (moriondo and bindi, 2008). many studies have pointed an agreement which many species advanced the spring phenology events (budburst and blooming dates) particularly (doi and katano, 2008; gordo and sanz, 2010; malagi, 2014). since distinct phenological stages were defined decades ago (baggiolini, 1952; lichou et al., 1990; meier et al., 1994), a comparison of available definitions of phenological stages in cherry used independently throughout europe showed overlaps and shortcomings; hence, harmonisation was reached in this respect in the cost cherry fa 1104 working group 2 (cherry phenology and climate change) based largely on the acceptance of the bbch scale and agreed standard cultivars for phenology monitoring. cultivars were selected on the basis of early, medium and late flowering and most widely grown throughout europe. this contribution presents the agreed phenology stages in both visual and wording evidence. similarly, this contribution presents the agreed cultivars to be monitored in future for phenology and climate change effects for harmonization (wenden et al., 2017). in this context, the fruit arboriculture seems relatively vulnerable from the fact of some of its characteristics, rather biological (eg: the fruit trees sustainability and their need to many years of growth before fruiting) than economic. compared to other productions (annual cultivations), the fruit arboriculture is particularly exposed to unfavorable climatic impacts from the fact of multiannual consequences (alternation of production after ceasing) and accumulative (repeated impacts on the tree architecture). on the socio-economic level, strong links have been woven during the time between the product and its production place (eg: provence almonds, roussillon apricots…etc). this characteristic developed in france, for commercial valorization reasons, in a regulatory form of origin and quality naming (eg: agen prunes, lorraine plums…etc.). from this fact, the substitution of varieties and much more species for long-term climatic adaptation reasons seems relatively difficult to be implemented, probably risking to encounter regulatory and human obstacles (legave, 2009). these characteristics constitute an obstacle to the fast changes, not only to the variety range but also to the cultivation systems to cope with rising temperatures or other constraints from climate change. this climate vulnerability has already been expressed in t h e 2 0 0 0 s b y s t r o n g p r o d u c ti o n i r r e g u l a r i ti e s . unprecedented accumulations of unfavorable climatic conditions (frost, high temperatures, excessive rainfall) have been observed during key phases of the annual cycle of trees, from flowering to fruiting. thus, in southern france, very significant production losses were provoked, especially in 2007 for cherry trees following stormy episodes in may and june, which strongly penalized the french production, and in 2008 for apricot following episodes of excessive abed et al. ‐ climate change effect in apple flowering 419 heat as blooming approached. sensitive varieties have had abortion rates that strongly penalize the national production. phenological notes on flower buds of fruit trees, collected under contrasting temperature (time and place) conditions in europe, showed a significant advance of the different phenological stages, especially the flowering dates, for all the places. modeling work on spring phenology strongly suggests that warming has two opposite effects: (1) in autumn and early winter, a slowdown in the satisfaction of cold unit needs, delaying endodormancy; (2) at the end of winter and in spring, an acceleration of the satisfaction of the heat needs during the ecodormancy phase. the more pronounced intensity of this latter effect, consistent with the more pronounced increases in temperature at the end of winter-early spring than in autumn, largely explains the advances in flowering (legave et al., 2009). the analysis of flowering dates over long periods in western europe for the golden delicious apple variety reveals more significant progress in the north of the continent (10 days) than in the oceanic west (6-7 days) and a shortening of flowering time in continental regions (legave et al., 2012). these regional differences across western europe led to a decrease in spatial variability, that is to say, smaller differences between the flowering dates in the contrasting regions (decrease of 8-10 days for complete flowering between the mediterranean and continental regions). modeling studies, based in particular on the correlations between the average temperature of the period of ecodormancy and the observed flowering dates, confirm the notion that flowering advances and shortenings are mainly due to a faster satisfaction of the demand for heat units (legave et al., 2015). however, delayed endodormancy has also been noted in the oceanic and mediterranean regions, which may explain the shorter advances in these areas despite similar or greater warming and ultimately lead to delayed flowering. the joint statistical analysis of flowering date series for the golden delicious variety and temperature dynamics reveals a geographical diversity of responses to warming from autumn to spring. temperate climates in europe are characterized by flowering progress, while soft climates are characterized by flowering progress or stationary flowering dates (eg. morocco and brazil), (legave et al., 2015). at the same time, legave et al. (2015) and el yaacoubi et al. (2016) have shown in mild winter conditions, a longer flowering time associated with the high average temperature of the endodormancy period. in the same context, a comparison of dormancy dynamics of vegetative and floral buds of apple and almond trees was recently conducted between southern france, southern brazil, and northern morocco. differences in dormancy intensity and kinetics have been identified in relation to regional differences in the satisfaction of cold needs and different levels of requirements of the genotypes studied. the observed diversity of dormancy patterns suggests that genotypes adapted to mild climates (eg, almond trees, apple trees with low cold needs) are characterized by the ability of vegetative buds to remain in a state of low dormancy and ability of flower blanks to grow rapidly, guaranteeing the absence of phenological anomalies subsequent to foliage and flowering (el yaacoubi et al., 2015). the apple tree is currently an important fruit species in algeria. production is the most important fruit production, but it does not sufficiently cover the demand. the central region (medea blida ain defla) totals about 7400 ha or about a quarter of the total area. apple cultivation has grown considerably, from 30,000 ha in 2003 to 41,000 ha in 2013, with a production reaching 400,000 tons (f.a.o 2013, mentioned by meradi, 2015). due to the levels of yield and quality obtained, the golden delicious variety is one of the three varieties that dominate the algerian market, particularly in the region of medea (golden delicious 70%, starkrimson 20% and granny smith 5%) (hadj sahraoui, 2014). the apple “hanna”, of its real name “anna”, is a new variety of apple trees introduced in algeria. it is planted in less cold areas, in the center of algeria on the perimeter of high chellif in ain defla, in the west on the sebaou valley of telemcen and in the east to khenchela and m’sila. they are among the varieties less demanding in cold and generally give apples of lesser quality, hardly storable (hamdani et al., 2016). however, apart from regionalized studies aimed at predicting climate change through time series of temperature and rainfall and estimating its impact on crops through the increase of yields in all regions of algeria including constantine region in the east of the country (kherief nacereddine and alatou, 2004; tabet, 2008; zekri et al., 2009) and oran region in the west (benabadji and bouazza, 2000; labani et al., 2006), no study on phenological development as a key element to characterize the impact of climate change has been undertaken. we therefore wanted to begin to fill this gap with this study aiming at first, adv. hort. sci., 2019 33(3): 417-431 420 the characterization of climate change via temperature series and the search for a possible impact on the phenology of the apple tree and, in a second step, the determination of the critical periods with regard to the accumulation of cold units and units of heat, by the implementation of the classical phenological models. for this, we analyzed the time series of phenological data of the golden delicious variety in two contrasting zones from the climatic point of view: a zone of plain with a rather warm climate, sidi lakhdar (town of ain defla) and a cooler zone in altitude, benchicao (town of medea). 2. materials and methods sites and climatic data the temperature data recorded for each site are shown in table 1. the two zones selected in this study are: sidi lakhdar (town of ain defla, latitude: 36° 15 ‘50’’ n, longitude: 2° 09’ 39’’ e, altitude 211 m, located in the center of algeria 145 km south-west of algiers) known for its semi-arid climate with a mild winter and a very hot summer, and benchicao (town of médea, latitude: 36° 11 ‘59’’ n, longitude: 2° 50’ 55’’ e, altitude 1133 m, located 80 km south-west of algiers) in a mountainous area with a warm temperate climate. daily maximum and minimum temperature data obtained over a period of 37 years (1980 to 2016) were collected at weather stations near selected sites belonging to the national office of meteorology. average temperatures were calculated using maximum and minimum temperatures. missing daily data were estimated using two methods: a linear interpolation for some values over 1 to 3 days (means to fill the missing values of tmax and tmin were carried out): correlations with another site for the longest periods namely; between the station of sidi lakhdar and the station of chlef (latitude 36° 10’ n, longitude 1° 20’ e, altitude 116 m) for the month of may of the year 2005, and between the station of benchicao (médéa) and the station of bordj bou arreridj (latitude 36° 04 ‘23’’ n, longitude 4° 45’ 39’’ e, altitude 930 m) for the months of january, february, march and april of the year 1980 and the month of may of the year 2001. similarly, a correlation was made between the médéa site and the sétif meteorological station (latitude 36° 11 ‘28’’ n, longitude 5° 24’ 49’’ e, altitude 1038 m) for the months of september, october and november of the year 1981, and february and december of the year 1990. phenological data data collected from 2000 to 2012 were provided by specialized state agencies. these are average dates that represent all the orchards visited. those from 2013 to 2016 were collected directly from the same orchards, which were among the most apple orchards planted at both sites. phenological monitoring 3 to 4 times a week was carried out on adult trees, the number of which sufficiently covered the total area of a given orchard (50%), respecting the two orientations (north-south and west-east). these orchards have not undergone any chemical treatment to break endodormancy or accelerate flowering. phenological stages were described according to the bbch scale (meier et al., 1997, 2001). the phenological stages of bud break (bud burst, baggiolini stage c and stage 51 of the bbch scale) and early flowering (10% open flowers, baggiolini f1 stage and 61 bbch scale) were observed from 2000 to 2016 on the two apple orchards maintained according to conventional horticultural practices. both stages were reported affected when 60% of the trees in the orchard had reached the given stage. table 1 phenological and temperature data collected in climate-contrasting sites for ‘golden delicious’ apple trees site benchicao (md) sidi lakhdar (sd) region (town) medea ain defla latitude/longitude 36°11' 59'' n / 2°50' 55'' e 36°15' 50'' n / 2°09' 39'' e altitude (m) 1133 211 climatic area sub-humid semi-arid period recorded of temperature 1980-2016 1980-2016 bud burst stage and observation period a bbch 51 / 2000-2016 bbch 51 /2000-2016 flowering stage and observation period a bbch 61 / 2000-2016 bbch 61 /2000-2016 a: bbch 51, 61; stages in phenological code bbch (meier, 1997), are respectively swelling buds of inflorescences and 10% of flowers open. abed et al. ‐ climate change effect in apple flowering 421 a, b and c. the parameter a determines the width of the window on which the function is not zero. the larger the value, the larger the temperature range over which the cold units are wide. parameter b determines the sharpness of the response curve and its asymmetry. the more b differs from zero, the s h a r p e r t h e i m a g e ( a n d m o r e a s y m m e t r i c ) . parameter c determines the value of the average response when b is close to zero and represents a limit to the temperature range over which cold units accumulate, when b is significantly different from zero. the wang model was first defined by wang and engel (1998). it is characterized by an optimum and is not symmetrical. this concerns the family of the beta function. it is composed of three parameters, namely tmin, topt and tmax (minimum, optimal and maximum temperatures). the sigmoid model was introduced by hänninen (1990). it consists of two parameters, d and e. the d parameter defines the sharpness of the response. values far from zero induce a sharper response curve. the parameter e is the average response temperature. smooth utah/ wang and smooth utah/ sigmoid t h e s m o o t h u t a h m o d e l w a s i n t r o d u c e d b y bonhomme et al. (2010) and is a smoothed version of the utah function proposed by richardson et al. (1974). this function assumes that cooling can occur only over a range of temperatures and has four parameters: tm1, topt, tn2 and min. negative cooling values can be accumulated on hot days, increasing the amount of cold to reach. tm1: this parameter defines the sharpness of the decrease of the cold effect on the endodormancy of the buds. the lower tm1, the slower is the decrease. topt: this parameter corresponds to the optimum average daily temperature, for which a cooling unit is accumulated each day. tn2: this parameter defines the intermediate response, i.e. the temperature (above topt) that has half of topt’s effectiveness for inducing endodormancy. m i n : t h i s p a r a m e t e r d e fi n e s h o w m u c h t h e impact of high temperatures can be negative. when min = 0, high temperatures do not have a negative impact on endodormancy release. when min = 1, the negative impact of a day that is too hot is equivalent to the positive effect of a day in topt. each model is characterized by efficiency (eff), an estimated time (t0) and a quadratic error (rmse: root mean square error). modeling and data analyses to better explain the phenological behavior of the golden delicious variety in the two sites studied and to highlight the effect of the temperatures on the latter in terms of satisfaction in cold units and heat units, statistical analyses were carried out under r (r development core team 2008), concerning regression curves between the different temperature components (minimum, average and maximum) and the year as well as the two phenological stages (bud burst and flowering). similarly, parametric name correlation tests of spearman were performed between two variables namely annual and monthly temperature (minimum, average and maximum) and year on the one hand and phenological stages on the other hand. calculation and establishment of cold unit accumulation curves were performed using the utah model (richardson et al., 1974). utah model the utah model was designed by richardson et al. (1974). this model combines the cold units for temperatures between 0 and 16°c and associates a negative value with temperatures higher than 16°c. this model is built to use fixed degree-days (independent of cold units) to predict bud break. the utah model (richardson et al., 1974) transforms the hourly temperature into a cold unit from -1 to 1. the cumulative number of utah cold units at time t is expressed as follows: ucutot = with (u= 0 for t≤1.4°c, u= 0.5 for 1.4°c <t≤2.4°c, u= 1 for 2.4°c <t≤9.1°c, u= 0.5 for 9.1°c <t≤ 12.4°c, u= 1 for 12.4°c <t≤15.9°c, u = -0.5 for 15.9°c <t≤18.0°c, u = -1 for t≥18.0°c) (ricard, 2014). phenological modeling platform pmp5.5 the phenological models were adjusted using the phenology modeling platform (pmp5.5) proposed by chuine et al. (2013). pmp5.5 is an environmental-use interface aimed solely at managing the construction of a phenological model, fitting a phenological model to the data and simulating using a phenological model. the best results of the bud burst and flowering date prediction in the two studied sites are obtained by two-phase models (knowing that phase 1 corresponds to the accumulation of cold units and phase 2 corresponds to the accumulation of heat units ) quoted below. chuine/wang and chuine/sigmoid the chuine model has been described in chuine (2000) and is composed of three parameters, namely tu t 1  422 adv. hort. sci., 2019 33(3): 417-431 3. results general climatology. annual tendencies the annual average maximum, medium, and minimum temperatures for both sites are shown in figure 1. the linear regression of mean annual temperatures over the 36 available years revealed a significant warming (p = 0.004) at the sidi lakhdar (sd) site, with a significant increase in the mean annual minimum temperatures (p = 0.001), and less for the average annual maximum temperatures (p = 0.04). there is rather a cooling tendency at mountain site of benchicao, although not significant (p = 0.09), concerning the annual average and where the maximum temperatures experienced some significant regression (p = 0.001). the hottest years were 1990, 2010 and 2007 at the site of sidi lakhdar and 2016, 1997 and 2000 at benchicao site. tendencies for the autumn‐spring period a seasonal analysis from the 36 years available shows marked differences between the two sites. the monthly average temperature tendencies for the months of october to may (minimum, mean and maximum daily temperatures) measured during the period 1980 to 2016 for the two sites (sidi lakhdar and benchicao) are summarized in table 2 and figure table 2 temperature data collected characteristics in the two studied sites in algeria during 36 years * p<0.05, ** p<0.01. fig. 1 average annual temperatures (minimum, average, maximum) for the two sites, sidi lakhdar (sd) and benchicao (md) from 1980 to 2016. *, ** indicate the significance level of the correlation for p <0.05 and for p <0.01, respectively. sidi lakhdar: (sd) tmin: y = 0.0403x 66.906 (r² = 0.33**), taverage : y = 0.0305x 41.183 (r² = 0.21**), tmax: y = 0.0234x 21.077 (r² = 0.0864). médéa:(md) tmin: y= 0.0122x 12.479 (r² = 0.03), taverage: y = -0.019x + 54.187 (r² = 0.090), tmax: y = -0.0503x + 121.02 (r² = 0.31**). month daily temperature mean daily temperature during 36 years in °c correlation coefficient of trends and level of significance sidi lakhdar benchicao sidi lakhdar (sd) benchicao (md) r p r p october average 21.3 17.4 0.40 0.01* 0.07 0.50 maximum 27.0 21.6 0.38 0.018* 0.14 0.40 minimum 15.6 13.3 0.44 0.006** 0.36 0.028* november average 15.7 11.4 -0.004 0.1 0.40 0.007** maximum 20.3 14.7 0.03 0.842 0.20 0.008** minimum 10.8 8.1 0.11 0.48 0.17 0.30 december average 12.0 8.0 0.17 0.28 0.16 0.16 maximum 16.4 10.9 0.10 0.556 0. 17 0.31 minimum 7.6 5.2 0.19 0.26 0.009 0.966 january average 10.9 7.3 0.35 0.03* 0.20 0.08 maximum 15.3 10.2 0.24 0.143 -0.20 0.23 minimum 6.4 4.6 0.32 0.051 0.10 0.54 february average 11.9 8.1 0.11 0.48 -0.32 0.005** maximum 16.8 11.2 0.047 0.783 -0.47 0.003** minimum 6.8 5.0 0.16 0.34 0.22 0.18 march average 14.2 10.5 0.01 0.51 -0.23 0.03* maximum 20.0 14.3 0.008 0.961 -0.47 0.030* minimum 8.5 6.8 0.25 0.13 0.025 0.88 april average 16.6 13.3 0.42 0.008** -0.07 0.51 maximum 23.0 17.5 0.36 0.027* 0.012 0.565 minimum 10.4 9.03 0.49 0.002** 0.18 0.28 may average 21.0 17.9 0.32 0.04* -0.1 0.38 maximum 27.9 22.7 0.21 0.19 0.027 0.403 minimum 14.3 13.0 0.40 0.012* 0.089 0.600 abed et al. ‐ climate change effect in apple flowering 423 2. table 2 generates spearman parametric name correlation values between maximum, average and minimum monthly temperatures and year, and where p is the level of significance and r is the correlation coefficient. we focused on the period from october to may, which is the period that most affects the physiological processes associated with the spring phenology of flower buds of fruit trees in our region. average october temperatures are high and increase significantly at sidi lakhdar site. the month of april is also warming significantly on this site (table 2, fig. 2). no significant tendency is recorded for the other months. correlations on monthly temperature tendencies also clearly showed significant summer warming in july at sidi lakhdar site (data not shown). at mountain site of benchicao, on the other hand, as already indicated above, during the 36 years, no significant warming is recorded in average temperature. on the contrary, average temperatures decreased in november and february (p = 0.007 ** and p = 0.005 *, respectively). regarding average minimum temperatures, the lowest values were recorded during the month of january for both sites with 6.4 and 4.6°c. the highest minimum temperature value of 12.1°c and the lowest value of 8.2°c were recorded by order in 2006 and 1991 at the site of sidi lakhdar for this month of january. at mountain site benchicao, the highest value of 9.7°c is reported in 2000 compared to a lower value of 6.6°c in 1980. the site of sidi lakhdar experienced extreme maximum temperatures during the months of october and april which explains the significance of the increase in average temperatures during these two months (fig. 2a). significant regressions of maximum temperatures were recorded at the benchicao site during the months of november, february and march. phenological development comparisons of the phenological tendencies of the apple tree (in terms of bud burst and flowering) in the two contrasting environments were made from the 16 years available. for budding dates of flower buds at sidi lakhdar site, some variation between years was revealed with marked tardiness during the years 2002, 2007, 2012, 2013 and 2016, when there was a bud break between the end of march and the first days of april and an early fruit maturity in 2000, 2003 and 2006, but the overall tendency for all years is not significant (p = 0.07). the tendency towards the advancement of flowering dates (fig. 3) is also not significant (p = 0.24), the earfig. 2 evolution of average monthly temperatures, on both sites, for months where the trend is significant at sidi lakhdar (october and april) and at benchicao (november and february). *, ** indicate the significance level of the correlation for p <0.05 and for p <0.01, respectively. sidi lakhdar: (sd) average april: y = 0.068x 119.18 (r² = 0.2252**), average october: y = 0.0742x 126.9 (r² = 0.1909**). médéa:(md) average february: y = 0,0799x + 167,72 (r² = 0, 1833), average november: y = 0.0483x + 107.88 (r² = 0.1376). fig. 3 trends in bud burst and flowering dates at the sidi lakhdar (sd) and benchicao (md) sites. *, ** indicate the significance level of the correlation for p<0.05 and for p<0.01, respectively. sidi lakhdar: (sd) bud burst/years: y = 0,1397x + 80, 86 (r² = 0, 0104), flowering/years: y = -0.4412x + 105.03 (r² = 0.0872). médéa: (md) bud burst/years: y = 0.7623x + 83.257 (r² = 0.2496), flowering/years: y = 0.8971x + 100.4 (**r² = 0.3263). adv. hort. sci., 2019 33(3): 417-431 424 liest years being 2003, 2009, 2010 and 2011, and the later years 2001, 2004 and 2005 at benchicao site, a significant tendency (p = 0.010) at the late flowering dates of the apple tree is to be reported (fig. 3b). on the other hand, differences in historical trends were shown in the bud break dates, oscillating between advancement during the years from 2000 to 2003, and a delay in the years 2004 to 2008. accumulation of chilling units in winter the utah cold unit (cu) accumulation curves reveal significant interannual differences at the sidi lakhdar site and show that for the years 2001, 2007, 2010 and 2016, this accumulation was insufficient because at below 600 cu, which is well below the estimated needs of the golden delicious variety (900 cu) (fig. 4). on the other hand, at mountain site of benchicao, the needs are always quickly satisfied. in order to analyze the influence of the different months in terms of cold units, we calculated the correlations between the dates of bud burst or flowering and the monthly temperatures (minimum, average and maximum) (table 3). very schematically, the tend e n c i e s c a n b e s u m m e d u p a s f o l l o w s : a t t h e benchicao site, the month of january and the whole period from november to january and february have a strong influence on meeting the needs in cold units because the correlation is positive (the warmer it is, the more the budding/flowering is late), whereas it is not the case in november and december (october remains quite neutral with a negative correlation). the impact of january is preponderant because if we look at the influence of the period from november to january, we find a negative effect of high temperatures on the precocity (r² = 0.40) while the months of november and december have an inverse effect. at the site of sidi lakhdar, the balance sheet is fig. 4 cumulative cold unit according to the utah model at the sidi lakhdar site. a) years when cumulative cold is less than 500 hrs; b) years when cumulative cold exceeds 800 hrs. table 3 spearman's correlation between bud burst and flowering for ‘golden delicious’ apple tree and mean temperature from october to april * p<0.05, **p<0.01. variable sidi lakhdar (sd) benchicao (md) mean temperature minimum temperature maximum temperature mean temperature minimum temperature maximum temperature bud burst flowering bud burst flowering bud burst flowering bud burst flowering bud burst flowering bud burst flowering october -0.031 0.11 -0.21 0.13 -0.20 0.033 0.015 0.019 -0.106 0.135 0.081 0.14 november -0.41 * -0.12 -0.55 * -0.21 -0.38 -0.036 -0.54 * -0.225 -0.57* -0.232 -0.47* 0.18 december -0.18 -0.43 * -0.11 -0.232 -0.2 -0.50 * -0.40 * 0.054 -0.52* -0.11 -0.322 0.18 january 0.31 * -0.002 0.006 -0.24 0.46 * 0.14 0.46 * 0.3 0.232 0.15 0.54* 0.41 february 0.10 0.01 -0.09 0. 12 0.01 0.13 0.063 0.073 0.021 -0.015 0.13 0.15 march -0.43 * -0.34 * -0.27 -0.147 -0.36 -0.36 * -0.60 * -0.65 * -0.60* -0.64* -0.55* -0.56* april -0.45 * -0.30 -0.41 * -0.145 -0.48 * -0.33 * 0.08 -0.072 -0.101 -0.30 0.12 0.041 november-january 0.30 * -0.26 0.14 -0.45 * 0.30* -0.30* 0.43 * 0.40 * 0.28 0.33 0.52* 0.53* november-february 0.06 -0.052 0.21 -0.14 0.18 -0.14 0.48 * 0.44* 0.233 0.212 0.30 0.21 november-march 0.041 -0.29 0.01 -0.20 0.132 -0.30 0.184 0.27 0.062 0.104 0.33 0.30 march-april -0.56 * -0.45 * 0.08 -0.025 -0.54 * -0.330 -0.26 -0.44* -0.304 -0.54* -0.133 -0.20 abed et al. ‐ climate change effect in apple flowering 425 globally the same. the correlations between mean and maximum temperatures in january on the one hand, and bud break dates on the other, are significantly positive, indicating the importance of this month’s temperatures for the satisfaction of cold unit requirements, the month of october remains little determinant. a hot january is a delay in meeting cold needs and bud break. a negative tendency of the high temperatures of the period from november to january on the precocity (r2 = 0.30) was recorded whereas the other months go rather in the direction of a gain of precocity. significant and negative relationships were observed between maximum temperatures in december and flowering, with a correlation coefficient of -0.50. accumulation of forcing units in spring march-april period (-0.44) show the link between the early flowering period and the average temperature it is rather march that plays the main role for both sites. at the benchicao site, only march temperatures show a significant negative correlation with bud break and flowering dates. negative correlations obtained between average temperatures of the period, with a very high prevalence of march temperatures. at the site of sidi lakhdar, march and april strongly influence the precocity via maximum temperatures and average temperatures. phenology modeling for both sites, the best sequential models selected are given in table 4. they were chosen on the basis of efficiency (eff) and rmse (rmse), but also taking into account the physiological relevance of the temperature response curves for the cold unit stacking phase and the heat unit stacking phase. rmse may appear acceptable (~ 5 days) on a flowering date but the efficiencies are less good. the correlation between the observed values and the predicted values confirms this diagnosis (fig. 5). the efficiency is very slightly improved (reaching a difference of 0.03 to 0.09) by eliminating the years when the cumulative cold units are not satisfactory at sidi lakhdar site. table 4 modeling bud burst and flowering for ‘golden delicious’ apple tree by chuine/wang and smooth utah/sigmoid in the two studied sites with all years and without years where chilling does not satisfied site two sites with all years two sites without years where chilling dose not satisfied, 2001, 2007, 2010, 2016 phenological stage bud burst flowering bud burst flowering model chuine / wang smooth utah / sigmoid chuine / wang smooth utah / sigmoid t0 starting date -107.8 -75.3 110.6 61.4 rmse 5.39 4.86 5.93 4.16 eff 0.49 0.47 0.50 0.56 settings chuine wang smooth sigmoid chuine wang smooth sigmoid a 0.50 topt 20.69 tm1 -37.54 d -40 a 0.68 topt 17.09 tm1 9.72 d -2.72 topt 18.45 b 12.70 tmin 3.30 topt 23.52 e 6.40 b -28.90 tmin 2.40 tn 2 27.52 e -21.80 min -0.86 c 27.04 tmax 43.14 tn 2 24.40 c -15.36 tmax 26.36 min -0.99 fig. 5 comparison between the observed and the predicted flowering date of apple in two sites. a) for all years by the smooth utah/sigmoid model. y = 0.5286x + 49.199; r² = 0.4796; b) without the years where the cumulative cold units are not satisfied by the smooth utah/sigmoid model. y = 0.5041x + 51.647; r² = 0.5555. adv. hort. sci., 2019 33(3): 417-431 426 by examining separately the two sites, and in particular that of sidi lakhdar, which shows years when the accumulation of cold units is not satisfied (fig. 6a), we see that the withdrawal of these years greatly improves the results of modeling (fig. 6b) and in p a r ti c u l a r f o r fl o w e r i n g . t h e r e l e v a n c e o f t h e response curve obtained for the cold unit function is also greatly improved (table 5). at benchicao site (table 6), the efficiency of the two-phase model is medium and figure 7 shows that it is difficult to predict the early years. in fact, the two-phase model is not much better than a onephase model on bud break and not better for flowering. the results of the one-phase model (only the heat-accumulation model: degree-day growth, parabolic, richarsdon, wang, sigmoid, threshold and smooth utah) yielded non-significant results. 4. discussion and conclusions the sensitivity of phenophases to temperature changes is a good indicator of the long-term biological impacts of climate change and terrestrial ecosystems (richardson et al., 2013). several studies have shown that the phenophases most sensitive to temperature variations are those occurring in spring or summer and that there is a relatively linear relationship between the occurrence of these phenophases site sidi lakhdar with all years sidi lakhdar without years where chilling dose not satisfied, 2001, 2007, 2010, 2016 phenological stage bud burst flowering bud burst flowering model chuine/ sigmoid chuine/ sigmoid chuine/ sigmoid chuine/ sigmoid t0 -102.3 61 -119 -59.7 rmse 3.79 5.02 3.30 2.10 eff 0.72 0.44 0.80 0.90 settings chuine sigmoid chuine sigmoid chuine sigmoid chuine sigmoid a 1.61 d -32.51 a 2.95 d -11.93 a 0.96 d -2087 a 3.21 d -40 b 5.67 e -21.66 b -25.47 e 0.50 b -29.7 e -25.47 b 12.47 e 14.93 c 19.42 c 8.60 c -6.72 c 16.93 table 5 modeling bud burst and flowering for ‘golden delicious’ apple tree in sidi lakhdar by chuine/sigmoid model with all years and without years where chilling does not satisfied table 6 modeling bud burst and flowering for ‘golden delicious’ apple tree in benchicao by smooth utah/wang and chuine / sigmoid models with all years fig. 6 comparison between the observed and the predicted bud burst date of the apple tree at the sidi lakhdar site. a) for all years by the chuine/sigmoid model. y = 0.8316x + 13.544; r² = 0.7207; b) without the years when the c u m u l a t i v e c o l d u n i t s a r e n o t s a t i s f i e d b y t h e chuine/sigmoid model. y= 0.8165x + 15.548; r² = 0.8066. site benchicao with all the years phenological stage bud burst flowering model smooth utah / wang chuine / sigmoid t0 -104.9 -78.7 rmse 5.43 4.91 eff 0.48 0.46 settings smooth utah wang chuine sigmoid tm1 -34.85 topt 22.31 a 0.45 d -39.99 topt 21.15 tmin 3.60 b -10.58 e 6.39 tn2 29.42 tmax 49.9 c 1.03 min -0.81 abed et al. ‐ climate change effect in apple flowering 427 and temperature (gordo and sanz, 2010; morin et al., 2010; beaubien and hamann, 2011). our study is a first attempt to evaluate the impact of global warming on the bud break and flowering of the apple tree in northern algeria. the study of the chronological series of temperatures (minimum, average and maximum) from 1980 to 2016 (36 years) in the two important apple production sites, namely: sidi lakhdar and benchicao, showed a very significant warming in october and april at the site of sidi lakhdar (sd) likely to strongly disrupt the entry into endodormancy and ecodormancy. strangely, the benchicao (md) site does not show global warming and even a cooling trend in november and february, which could lead to an endodormancy and a start of satisfaction in cold units and then an acceleration of the recovery of growth leading to greater precocity. establishing the time of endodormancy emergence is important (guerriero et al., 2002), but is hampered by the complexity of the process, including the fact that, under natural conditions, cold and warm temperatures alternate, causing “inversions” in the process of endodormancy emergence (overcash and campbell, 1955; couvillon and erez, 1985; erez and couvillon, 1987). some results suggest that partial dissatisfaction with cold can be compensated by heat unit supplementation for bud break (dantec, 2014). to lift the endodormancy, the bud must accumulate enough cold (campoy et al., 2011). when the plant has reached its cold needs, the endodormancy is lifted and the buds can resume their growth as soon as the conditions become favorable, i.e. under certain (rather high) temperature conditions, soil moisture and nutrients, and for photosensitive species, certain photoperiod conditions (körner and basler, 2010; polgar and primack, 2011). these favorable conditions must last for a certain period of time for the bursting of the buds to appear. for our case, the cold needs were always met to lift the endodormancy of the apple tree at the benchicao site, and this as from the end of december or the first half of january (fig. 4). conversely, at the sidi lakhdar site, values below the threshold for satisfying cold needs estimated at 900cu for the golden delicious apple tree were obtained for the years 2001, 2007, 2010 and 2016. when it takes place, the satisfaction of cold needs is later, around midfebruary (fig. 4) and does not really start until the end of november. everything leads to a dominating importance of the month of january in the course of the endodormancy lifting process and it will be interesting to look at what the future climate scenarios give for this particular period for the choice of future apple varieties. the months of november and december play a precocious role at the benchicao site (negative correlation with the date of bud burst or flowering). this can only be understood if the organogenesis in the buds continues during these two months and therefore there is no endodormancy at this time. the observed increase in average november and october minimum temperatures (table 2) is consistent with this. at the site of benchicao, the month of march is crucial for the precocity, it is the temperatures of this month which allow the growth after the satisfaction of the needs of cold towards the end of december and the beginning of january all the more so as the temperatures of the february remain rather low (and tend towards a cooling). the tendency to tardiness with the site of benchicao is thus coherent with the cooling in the month of february. the october warming at sidi lakhdar site may explain later entry into endodormancy. gentle temperatures (12°c) in february can then accelerate bud burst and flowering, at least for years when cold needs are met. otherwise endodormancy will be greatly delayed or disrupted. according to the study of legave et al. (2015), carried out in three geographically contrasti n g c o u n t r i e s o f t h e m e d i t e r r a n e a n r e g i o n , i n morocco (meknes), france (nimes) and italy (forlì) over the last 40 years in order to understand the impact of climate change, especially the increase in temperature, on the golden delicious apple tree, the forcing period is shorter in meknes. legave et al. (2012) also found a marked trend towards shorter s i m u l a t e d d u r a ti o n o f f o r c i n g p e r i o d a n d l a t e fig. 7 comparison between the observed and the predicted flowering date of the apple tree at the benchicao site by the chuine/sigmoid model. y = 0.4501x + 57.365; r² = 0.4632. adv. hort. sci., 2019 33(3): 417-431 428 endodormancy period. the physiological functioning of the golden delicious apple tree during the dormant and growing season may explain, in part, the regional differences observed in the flowering dates (heide, 1993). in the same context, kauffman and blanke (2018) have reported after a study conducted on three cherry cultivars at different levels of cold needs (minimum, medium and high) that, in optimum chill, the optimum forcing was ca. 8.000 gdh (>12  °c), irrespective of variety, allowing up scaling of the results to possibly other varieties. overall, the results have shown that diminishing chilling as a result of climate change can be compensated for, in part up to 50%, by a larger amount of forcing to obtain natural flowering in the orchard. these results may explain the good progress of flowering on the site of sidi lakhdar, although the cold needs were not often satisfied. el yaacoubi et al. (2014) also reported that spring temperatures appear to be essential for complete flowering in mild climates. in the latter case, early full flowering dates occurred when the average t e m p e r a t u r e d u r i n g t h e f o r c i n g p e r i o d r a p i d l y exceeded 15°c provided adequate satisfaction of the cold requirements. phenological models predicting the occurrence of different phenophases as a function of environmental conditions (mainly temperature and photoperiod), predict that the global increase in temperature during the winter will slow or even jeopardize the endodormancy emergence due to lack of cold (chuine et al., 2016). the onephase and two-phase models for all years do not give good results at the sidi lakhdar site. this is explained by the negative influence of years when cold unit n e e d s h a v e n o t b e e n m e t . i f t h e s e y e a r s a r e removed, the two-phase chuine/sigmoid model for bud burst and flowering gives good results. this may mean that in these cases of partial non-fulfillment of cold unit requirements, the physiological processes involved in bud break-up and flowering are different or that “something” in addition occurs. at benchicao site, the efficiency of the two-phase models is average, since the requirements in cold units are often met; only the forcing period can have an effect on the precocity. this study aimed to show the effects of the anticipated increase in temperature on two phenological phases of the apple tree (golden delicious) in two algerian sites with contrasting climates. we highlighted contrasting trends by site and by period. warming at sidi lakhdar site in autumn and spring, however, the statistical data of temperatures did not raise any average warming at the benchicao site. rather surprising and never described before, there has been a tendency to cool down some months at the benchicao site. critical periods for cold units were identified, concerning the period between november and january at benchicao site, but january t e m p e r a t u r e s w e r e m o r e i m p o r t a n t i n l i ft i n g endodormancy. at the site of sidi lakhdar, buds enter late into endodormancy and the result is a late action of cold that extends until february without always being sufficient. forced side, it is the temperatures of the month of march that have a discriminating effect on bud burst and flowering at the site of benchicao combined with those of april at the site of sidi lakhdar. on this site, despite the warming in april, we do not gain in precocity probably because of a satisfaction of cold needs “to the limits” as describe legave et al. (2012) for the nimes region. we have also highlighted, particularly at the site of s i d i l a k h d a r t h a t m o r e c o m p l e x p h y s i o l o g i c a l processes must be at work especially the years of low cumulative cold units. it is not excluded that other factors, not included in this work, could be involved in the budburst process such as photoperiod or precipitation (vitasse et al., 2009; grab and craparo, 2011; el yaacoubi et al., 2014). except for the twophase chui ne/si gmoi d budburst and floweri ng model, which gave better results at sidi lakhdar site after the elimination of the years when the cold unit requirements were not met, all the models give rather weak efficiencies indirectly confirming the non-taking into account of a complexity of factors associated with physiological functioning for sites like sidi lakhdar’s. the study of the impact of global warming on the apple tree requires a precise determination of the accumulations in cold units necessary for the emergence of endodormancy and budding in various environments. this involves highlighting these two phases by forcing techniques at the laboratory level and anatomical studies of meristematic bud tissues to see their ability to bud. references baggiolini m., 1952 les stades repères dans le dévelop‐ pement annuel de la vigne et leur utilisation pratique. revue romande d’agriculture et d’arboriculture, 8(1): 4-6. balaghi r., 2017 le changement climatique dans la région de fés‐meknés. etat de l’art, vulnérabilité, impact sur les terres et les principales cultures. résumé à l’attention des décideurs. i.n.r.a., rabat, morrocco, abed et al. ‐ climate change effect in apple flowering 429 pp. 1-14. b e a u b i e n e . , h a m a n n a . , 2 0 1 1 s p r i n g fl o w e r i n g response to climate change between 1936 and 2006 in alberta, canada. bioscience, 61: 514-524. benabadji n., bouazza m., 2000 quelques modifica‐ ti o n s c l i m a ti q u e s i n t e r v e n u e s d a n s l e s u d ‐ o u e s t del’oranie (algérie occidentale). rev. energ. ren., 3: 117-125. b o n h o m m e m . , r a g e a u r . , l a c o i n t e a . , 2 0 1 0 optimization of endodormancy release models using series of endodormancy release data collected in france. acta horticulturae, 872: 51-60. bourchef m., 2013 axes stratégiques en matière de changements climatiques sur le nord de l’algérie. ‐ rapport sur l’élaboration de la stratégie nationale de gizc pour l’algérie, pp. 69. campoy j.a., ruiz d., egea j., 2011 dormancy in tem‐ perate fruit trees in a global warming context : a review. sci. hortic., 130: 357-372. chuine i., 2000 a unified model for the budburst of trees. j. theor. biol., 207: 337-347. chuine i., 2005 un reseau d’observations phenologiques pour la gestion du changement climatique, pp. 49-58. in: landmann g., and s. landeau (eds.) de l’observa‐ tion des écosystèmes forestiers à l’information sur la forêt. editions quae, paris, france, pp. 93. chuine i., bonhomme m., legave j.m., de cortazaratauri i.g., charrier g., lacointe a., ameglio t., 2016 can phenological models predict tree phenology accurately in the future? the unrevealed hurdle of end dormancy break. ‐ global change biology, 22: 34443460. chuine i., de cortazar-atauri i.g., kramer k., hänninen h., 2013 plant development models, pp. 275-293. in: schwarz m.d. 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l’impact du changement climatique sur la phé‐ nologie des arbres le long d’un gradient altitudinal dans le pyrénées. ‐ gdr phénologie 2009, 21-22 octobre, montpellier, france. wang e., engel t., 1998 simulation of phenological development of wheat crops. agr. syst., 58: 1-24. wenden b., barreneche t., meland m., blanke m., 2017 harmonisation of phenology stages and selected cherry cultivars as bioindicators for climate change. acta horticulture, 1162: 9-12. zekri d., lehout a., souilah n., alatou d., 2009 étude des variations thermiques saisonnière dans les régions semi‐arides. laboratoire de développement et valorisation des ressources phytogénétiques, faculté des sciences de la nature et de la vie. université mentouri constantine, algeria. impaginato 21 adv. hort. sci., 2021 35(1): 21­32 doi: 10.36253/ahsc­9945 postharvest quality of pepino melon (solanum muricatum aiton) as influ­ enced by npk fertilizer rates, growing environment and storage temperature c. mutua (*), r. gesimba, j. ogweno department of crops, horticulture and soils, egerton university, p.o. box 536, 20115 egerton, kenya. key words: firmness, shelf life, total soluble solids, weight loss. abstract: the present study evaluated the effect of npk fertilizer (17:17:17) rates (0, 100, 200, 300 and 400 kg ha­1) on the postharvest quality of field and greenhouse grown pepino melons (solanum muricatum ait.) stored at room temperature (15­22°c) and at low temperature (7°c). the study was carried out in randomized complete block design with fruits from the field and greenhouse, five npk fertilizer rates as treatments and the two storage temperatures repli­ cated three times. data were collected on percentage fruit weight loss (pwl), total soluble solids (tss), firmness and shelf life. results indicated that green­ house and field grown fruits from the control and plants supplied with 100 kg npk ha­1 had low pwl at both storage temperatures. field grown fruits from the control stored at room temperature had the highest tss and were firmer after 28 days of storage. field grown fruits not supplied with fertilizer and stored at low temperature had a shelf life of 27 and 26 days in trial one and two respectively. application of 100 kg npk ha­1 and storage of pepino melon fruits at low temperature can be used to enhance quality and shelf life. 1. introduction pepino melon (solanum muricatum aiton) is a little­known vegetable crop which belongs to the family solanaceae. it originated from the tropi­ cal and subtropical region of andes and is grown for its edible fruits (heiser, 1964). pepino melon fruits are aromatic, juicy, scented, mild sweet, and vary in size, shape and colour depending on the cultivar (martinez­romero et al., 2003). the fruits mature 30 to 80 days after pol­ lination and the skin is usually golden yellow with purple stripes (nuez and ruiz, 1996). several studies have reported significant losses in horti­ cultural produce after harvest (toktam et al., 2019). such losses are caused by dehydration, decay, and physiological disorders during posthar­ vest handling. fresh fruits and vegetables also undergo rapid transforma­ tion in nutritional and sensory quality after harvest, some of which con­ tribute to loss of market value (ahmad and siddiqui, 2015). the losses can (*) corresponding author: carolmutua713@yahoo.com citation: mutua c., gesimba r., ogweno j., 2021 ­ postharvest quality of pepino melon (solanum muricatum aiton) as influenced by npk fertilizer rates, growing environment and storage tempe‐ rature. ­ adv. hort. sci., 35(1): 21­32 copyright: © 2021 mutua c., gesimba r., ogweno j. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 20 october 2020 accepted for publication 27 january 2021 ahs advances in horticultural science https://doi.org/10.36253/ahsc-9945 http://www.fupress.net/index.php/ahs http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2021 35(1): 21­32 22 be reduced through good management of pre­ and postharvest factors (toktam et al., 2019). postharvest quality is also affected by climatic fac­ tors such as temperature and light intensity, and other pre­harvest factors like soil type, fertilization, irrigation, mulching, and other cultural practices (toktam et al., 2019). temperature affects growth and development of fruits and vegetables as well as cellular compounds, their structure and this in turn affects produce firmness (toktam et al., 2019). f e r ti l i z e r s h a v e a l s o b e e n s h o w n t o i n fl u e n c e postharvest quality of most fruits and vegetables. the type of fertilizer used and the amount applied will dictate the quality of the resulting vegetables (arah et al., 2015). application of potassium fertiliz­ ers on tomato has been shown to improve fruit colour, reduce the occurrence of yellow shoulder and enhance titratable acidity (passam et al., 2007). on the other hand, application of high doses of nitroge­ nous fertilizers to greenhouse grown tomatoes reduces fruit quality by reducing total soluble solids (passam et al., 2007). temperature management between the time of harvesting and consumption has been shown to be effective in maintaining the quality of harvested veg­ etables. high temperatures increase metabolic activi­ ties and ethylene production but this is dependent on other factors like oxygen or carbon dioxide levels, time of exposure and the ripening stage (de wild et al., 2003). storage of vegetables at low temperature slows down metabolic processes and hence extends the shelf life of horticultural produce (arah et al., 2015). the present study sought to investigate the effect of npk fertilizer rates, growing environment and storage temperature on the postharvest quality of pepino melon. 2. materials and methods experimental site description t h e e x p e r i m e n t w a s c o n d u c t e d a t t h e horticulture research and teaching field, egerton university, njoro. the field lies at a latitude of 0° 23’ south, longitudes 35° 35’ east in the lower highland iii agro ecological zone (lh3) at an altitude of approximately 2,238 m above sea level. average maximum and minimum temperatures range from 19°c to 22°c and 5°c to 8°c, respectively, with a total annual rainfall ranging from 1200 to 1400 mm. the soils are predominantly mollic andosols (jaetzold and schimdt, 2006). the greenhouse used was 8 m by 60 m and the covering material was polythene with a thickness of 12×150 microns purchased from amiran kenya ltd. the mean monthly temperatures in the greenhouse and field during the experiment are pre­ sented in table 1. plant material and experimental design pepino seedlings (ecuadorian gold variety) were obtained from garlic and pepino farm, nakuru and planted in the field and greenhouse. the experimen­ tal design was randomized complete block design (rcbd) with fruits from the five npk fertilizer treat­ ments, the two growing environments and two stor­ age temperatures replicated three times. the five npk fertilizer treatments were [0, 100, 200, 300 and 400 npk (17:17:17) kg ha­1], two growing environ­ ments (field and greenhouse) and two storage tem­ peratures (room and low temperature). mature green pepino fruits were harvested from the field and greenhouse experiments and stored at low tem­ perature (7°c) in a refrigerator and at room temper­ ature (15­22°c) in the biotechnology laboratory of table 1 ­ average monthly field and greenhouse temperature (°c) in trial one and two experimental conditions temperature 2018 2019 trial one nov dec jan feb mar apr may field 20.9 19.7 20.9 21.7 22.8 22.6 21.2 greenhouse 30.3 21.0 33.4 30.2 29.4 34.0 35.8 2019 2020 trial two july aug sept oct nov dec jan field 19.1 19.2 20.5 19.3 19.3 18.9 19.1 greenhouse 18.5 29.4 30.0 28.0 32.0 28.0 35.3 mutua et al. ‐ pepino melon postharvest studies 23 egerton university. the experimental design was rcbd consisting of fruits from the five treatments replicated three times. the experiment therefore comprised of five treatments similar to those of the field and greenhouse experiments each replicated three times giving a total of thirty experimental units each represented by a plastic tray. each experimen­ tal unit comprised of twenty pepino fruits randomly selected from the harvest of the individual respec­ tive treatments in the field and greenhouse experi­ ments. data collection and analysis data were collected and recorded on percentage fruit weight loss (pwl), firmness, total soluble solids (tss) and shelf life. to determine pwl, five fruits in each replication for each treatment were marked before storage and weighed using a digital balance (hangping ja 12002, japan). the same fruits were weighed at the beginning of the experiment and weighing continued at an interval of 7 days for 28 days. the results were expressed as the percentage loss of initial weight using the formula: percentage weight loss = initial weight­final weight × 100 initial weight fruit firmness was determined using hand held penetrometer (model 62/dr, uk) from the begin­ ning of the experiment and continued at an interval of 7 days for 28 days. the results were reported in kg force. tss was determined on the same fruits used for determination of firmness using a hand held refractometer (0­30°brix) (rhw refractometer, optoelectronic technology company ltd, uk) was used as per the procedure described by tigchelaar (1986). results were expressed as °brix. this was done at the beginning of the experiment and con­ tinued at an interval of 7 days for 28 days. the shelf life of pepino fruits was determined by counting the number of days at which at least 50% of the fruits had reached senescence and were not marketable (too soft, wrinkled or with fungal rots). quality evaluation was done using a rating scale of 1­5 (miguel and marita, 1996). data collected were subjected to analysis of variance (anova) and significant means separated u s i n g t u k e y ’ s h o n e s t l y s i g n i f i c a n t d i f f e r e n c e (tukey’s hsd) test at p≤0.05. the sas statistical package (sas institute, 2005) was used for data analysis. 3. results percentage weight loss (pwl) npk fertilizer rates, growing environment and storage temperature had a significant effect at p≤ 0.05 on pwl of pepino fruits after 28 days of storage in trial one. during this trial, highest pwl of 10.863% and 15.77% were recorded in greenhouse grown fruits from plants supplied with 300 and 400 kg npk ha­1 during production as well as in field grown fruits from plants supplied with 400 kg npk ha­1 regardless of the storage temperature (table 2). the lowest pwl was, on the other hand, recorded in greenhouse and field grown fruits without npk fertilizer applica­ tion (control) stored at low temperature although the difference in weight loss for this treatment was not significantly different from that of other treatment combinations. in trial two, npk fertilizer rates, grow­ ing environment and storage temperature had a sig­ nificant effect on pwl from day 7 to day 28 of the study. on day 28, greenhouse and field grown fruits from plants supplied with 400 kg npk ha­1, stored at room temperature had the highest pwl of 19.38% and 15.54% respectively (table 2). it was noted that as the fertilizer rates increased the pwl also increased in both growing environ­ ments and storage temperatures in both trials. generally, fruits stored at low temperature had lower pwl compared to those stored at room temperature in both trials. greenhouse grown fruits also had a higher pwl compared to field grown fruits in both trials. total soluble solids (tss) npk fertilizer rates, growing environment and stor­ age temperature had a significant effect at p≥0.05 on tss of pepino fruits from day 7 to day 28 in both tri­ als. in trial one, field grown fruits from plants which were not supplied with fertilizer (control) and stored at room temperature had the highest tss from day 7 to day 28. the highest tss was recorded after 28 days of storage where field grown fruits from plants not supplied with npk fertilizer and stored at room tem­ perature had a tss of 8.67 °brix which was significant­ ly higher than that recorded from fruits from all other treatment combinations. greenhouse grown fruits from plants supplied with the highest fertilizer rate of 400 kg npk ha­1 and stored at low temperature had the lowest tss of 4.40°brix after 28 days (table 3). in trial two, field grown fruits from plants not supplied with npk fertilizer (control) and stored at room tem­ adv. hort. sci., 2021 35(1): 21­32 24 * means followed by the same letters in a given day and trial are not significantly different according to tukey’s honestly significant difference test at p≤0.05. room storage temperature varied between 15 and 22°c. low temperature was 7°c. storage temperature environment fertilizer (kg ha­1) weight loss day 7 day 14 day 21 day 28 trial one room temperature field 0 1.067 2.050 2.837 3.987def 100 1.423 2.800 3.620 5.303 def 200 1.980 3.580 4.713 6.383 cdef 300 2.500 5.147 5.717 9.120 bcd 400 4.417 7.280 11.353 12.457 ab greenhouse 0 1.213 1.840 2.820 3.413 ef 100 2.037 4.317 5.590 7.047 cde 200 2.673 5.540 7.160 8.913 bcd 300 3.167 6.400 8.720 10.863 abc 400 5.810 9.327 11.410 15.770 a low temperature field 0 0.523 1.043 1.283 1.557 f 100 0.917 1.720 1.997 4.300 def 200 1.057 2.170 2.763 2.310 ef 300 1.740 3.883 4.040 7.120 cde 400 2.737 5.497 8.020 15.123 a greenhouse 0 0.543 1.087 1.630 2.173 ef 100 1.063 1.353 2.070 2.633 ef 200 1.543 2.417 2.867 4.012 def 300 2.240 2.977 5.080 3.183 ef 400 3.950 6.107 9.067 8.630 bcd trial two room temperature field 0 0.940 ij 1.173 kl 1.443 kl 4.187 fghi 100 1.893 fghi 2.633 fghijk 3.123 fghij 7.100 defg 200 2.507 efgh 3.130 defghi 4.590 cdefg 8.723 cde 300 3.667 bcd 4.773 cde 5.653 cd 11.690 bc 400 4.550 b 7.363 b 10.883 b 15.543 ab greenhouse 0 1.156 hij 1.670 ijkl 1.943 ijkl 3.360 fghi 100 3.067 cdef 3.077 efghi 4.230 defgh 7.293 def 200 3.703 bcd 4.333 cdef 5.327 cd 9.290 cd 300 4.113 bc 4.813 cd 6.190 c 11.600 bc 400 6.617 a 10.477 a 14.703 a 19.397 a low temperature field 0 0.663 j 0.917 l 1.283 l 1.653 i 100 1.047 ij 1.420 ijkl 1.997 ijkl 2.243 hi 200 1.970 efghi 2.173 hijkl 2.783 hijkl 2.487 hi 300 2.720 defg 2.887 fghij 3.533 efghi 3.017 ghi 400 4.127 bc 3.997 cdefg 4.740 cdef 5.967 defgh greenhouse 0 1.607 ghij 1.337 jkl 1.720 jkl 2.190 hi 100 2.313 efgh 2.473 ghijkl 2.993 ghijk 4.593 efghi 200 3.117 cde 3.520 defgh 4.223 defgh 5.330 defghi 300 3.693 bcd 4.163 cdefg 4.823 cde 4.880 efghi 400 4.473 b 5.487 c 6.20 c 5.930 defgh table 2 ­ effect of npk fertilizer rates, growing environment and storage temperature on percentage weight loss of pepino fruits in trial one and two mutua et al. ‐ pepino melon postharvest studies 25 storage temperature environment fertilizer (kg ha­1) tss (°brix) day 0 day 7 day 14 day 21 day 28 trial one room temperature field 0 4.00 6.00 a 6.93 a 7.83 a 8.67 a 100 4.00 4.37 cde 5.73 b 6.13 c 6.40 bcde 200 4.00 4.53 cde 4.97 c 5.73 cd 6.13 cdef 300 4.00 4.53 cde 4.90 c 5.47 de 5.80 defg 400 4.00 4.17 de 4.67 cde 4.80 ghi 5.20 ghi greenhouse 0 4.00 5.00 b 6.00 b 6.77 b 7.07 b 100 4.00 4.40 cde 4.90 c 5.47 de 5.80 defg 200 4.00 4.47 cde 4.90 c 5.23 ef 5.67 fgh 300 4.00 4.17 de 4.63 cde 4.86 fghi 5.10 ghij 400 4.00 4.10 e 4.30 ef 4.53 hijk 4.80 ij low temperature field 0 4.00 5.00 b 5.63 b 6.00 c 6.83 bc 100 4.00 4.60 bcd 4.83 c 5.13 efg 5.77 efg 200 4.00 4.43 cde 4.63 cde 4.97 fg 5.27 ghi 300 4.00 4.30 de 4.40 def 4.73 ghij 4.97 hij 400 4.00 4.10 e 4.13 f 4.33 jk 4.56 ij greenhouse 0 4.00 4.77 bc 5.00 c 5.90 c 6.50 bcd 100 4.00 4.60 bcd 4.77 cd 4.93 fghi 5.23 ghi 200 4.00 4.47 cde 4.63 cde 4.80 ghi 4.97 hij 300 4.00 4.30 de 4.33 ef 4.50 ijk 4.73 ij 400 4.00 4.17 de 4.10 f 4.20 k 4.40 j trial two room temperature field 0 4.00 5.60 a 6.70 a 7.60 a 8.133 a 100 4.00 4.50 cde 5.36 c 5.67 d 6.10 cd 200 4.00 4.33 defg 4.97 cde 5.26 ef 5.77 def 300 4.00 4.17 efg 4.53 fghi 4.80 ghi 5.00 hij 400 4.00 4.60 g 4.13 ijk 4.27 j 4.50 jkl greenhouse 0 4.00 4.70 bc 5.23 c 6.07 c 6.50 bc 100 4.00 4.50 cde 4.80 def 5.10 efg 5.50 efgh 200 4.00 4.33 defg 4.97 cde 4.87 ghi 5.13 ghi 300 4.00 4.13 fg 4.53 fghi 4.60 ij 4.90 ijk 400 4.00 4.03 g 4.07 k 4.27 j 4.47 kl low temperature field 0 4.00 5.27 a 5.83 b 6.47 b 6.93 b 100 4.00 4.60 bcd 5.00 cde 5.37 de 5.83 def 200 4.00 4.30 defg 4.70 efg 4.93 fghi 5.43 fgh 300 4.00 4.13 fg 4.50 fghij 4.70 hi 5.00 hij 400 4.00 4.07 fg 4.10 jk 4.27 j 4.60 jkl greenhouse 0 4.00 4.87 b 5.17 cd 5.67 d 6.00 cde 100 4.00 4.50 cde 4.77 defg 5.00 efgh 5.53 efg 200 4.00 4.30 defg 4.53 fghi 4.73 ghi 5.00 hij 300 4.00 4.13 fg 4.37 ghijk 4.60 ij 4.83 ijk 400 4.00 4.03 g 4.13 ijk 4.23 j 4.30 l table 3 ­ effect of npk fertilizer rates, growing environment and storage temperature on tss (°brix) of pepino melon fruits in trial one and two * means followed by the same letters in a given day and trial are not significantly different according to tukey’s honestly significant difference test at p ≤ 0.05. room storage temperature varied between 15 and 22°c. low temperature was 7°c. perature had the highest tss from day 14 to day 28. on day 7, field grown fruits from plants not supplied with npk fertilizer and stored at room or under low temperature had the highest tss of 5.6 and 5.27 °brix, respectively (table 3). the highest tss was recorded after 28 days of storage where field grown fruits from plants not supplied with npk fertilizer and stored at room temperature had a tss of 8.13 °brix which was significantly higher than for greenhouse grown fruits from plants supplied with 400 kg npk ha­1 and stored 26 adv. hort. sci., 2021 35(1): 21­32 at low temperature with a tss of 4.3°brix. it was observed that tss increased as the storage time pro­ gressed and decreased as the fertilizer rates increased regardless of the environment under which the fruits were produced and the temperature during storage. generally, field grown fruits had higher tss compared to greenhouse grown fruits regardless of the storage temperatures in both trials. on the other hand, fruits stored at low temperature had lower tss values com­ pared to those stored at room temperature in both trials. firmness npk fertilizer rates, growing environment and storage temperature had a significant effect on firm­ ness of pepino melon fruits on day 7 and day 28 in trial one, and day 21 and 28 in trial two. in day 7 of trial one, field grown fruits from plants which were not supplied with npk fertilizer and stored at either room or low temperature had the highest firmness of 4.67 kg f and 4.83 kg f, respectively. however, this was not significantly different from the firmness of 4.57 kg f recorded for greenhouse grown fruits har­ vested from plants not supplied with npk fertilizer and maintained under low temperature during stor­ age. on day 28, the highest firmness was recorded in field grown fruits from plants not supplied with npk fertilizer and stored at low temperature with a firm­ ness of 3.83 kg f. the lowest firmness of 0.52 kg f was recorded in greenhouse grown fruits from plants supplied with 400 kg npk ha­1 and stored at room temperature after 28 days of storage (table 4). in trial two, npk fertilizer rates, growing environ­ ment and storage temperature had a significant effect on firmness of pepino melon fruits at day 21 and day 28 of storage. in day 21 of storage, field grown fruits from plants not supplied with npk fertilizer and stored at low temperature had the highest firmness of 4.13 kg f but this was not significantly different from the firmness of 3.93 kg f recorded from green­ house fruits from plants not supplied with npk fertiliz­ er and stored at low temperature, field grown fruits from plants not supplied with npk fertilizer stored at room temperature with a firmness of 3.70 kg f and field grown fruits from plants supplied with 100 kg npk ha­1 maintained under low temperature with a firmness of 3.67 kg f (table 4). the lowest firmness of 1.70 kg f was recorded in greenhouse grown fruits from plants supplied with 400 kg npk ha­1 stored at low temperature but this was not significantly differ­ ent from the other treatment combinations. generally, it was observed that field grown fruits were firmer compared to greenhouse grown fruits and fruits stored at low temperature were firmer compared to those stored at room temperature. firmness also decreased as the fertilizer rates and storage days increased. shelf life npk fertilizer rates, growing environment and storage temperature had a significant effect (p≤0.05) on the shelf life of pepino melon fruits in both trials. in trial one, field grown pepino fruits from plants which were not supplied with npk fertilizer (control) and stored at low temperature (7°c) had the longest shelf life of 27 days. field grown fruits from plants supplied with 100 kg npk ha­1 stored at low tempera­ ture had shelf life of 22 days but this was not signifi­ cantly different from the shelf life of greenhouse grown fruits from plants not supplied with npk fertil­ izer and those supplied with 100 kg npk ha­1 and maintained at low temperature with a shelf life of 21 and 19 days respectively (table 5). the lowest shelf life of 11 days was recorded in greenhouse grown fruits from plants supplied with 400 kg npk ha­1 and stored at room temperature although this was not significantly different from that of field grown fruits from plants supplied with 300 and 400 kg npk ha­1 and stored at room temperature, greenhouse grown fruits from plants supplied with 200 and 300 kg npk ha­1 and stored at room temperature and field grown fruits from plants supplied with 400 kg npk ha­1 and stored at low temperature. in trial two, field grown pepino fruits from plants not supplied with npk fertilizer and stored at low temperature had the longest shelf life of 26 days, fol­ lowed by field grown fruits from plants supplied with 100 kg npk ha­1 stored at low temperature with a shelf life of 21 days. the shelf life recorded for fruits from this treatments was, however, not significantly different from that of greenhouse grown fruits from plants not supplied with fertilizer, those supplied with 100 kg npk ha­1 and stored at low temperature and field grown fruits from the control and stored at room temperature (table 5). the lowest shelf life was recorded in greenhouse grown fruits from plants sup­ plied with 400 kg npk ha­1 stored at room tempera­ ture with a shelf life of 10 days but this was not signif­ icantly different from the shelf life recorded for green­ house grown fruits from plants supplied with 200 and 300 kg npk ha­1 stored at room temperature and that of field grown fruits from plants supplied with 400 kg npk ha­1 and stored at room temperature. generally, it was observed that fruits stored at mutua et al. ‐ pepino melon postharvest studies 27 low temperature had a longer shelf life than those stored at room temperature. field grown fruits had a longer shelf life compared to greenhouse grown fruits. the shelf life decreased as the npk fertilizer rates increased. 4. discussion and conclusions there was a progressive increase in percentage fruit weight loss as the storage days advanced. field and greenhouse grown fruits from plants which table 4 ­ effect of npk fertilizer rates, growing environment and storage temperature on firmness (kg) of pepino melon fruits in trial one and two storage temperature environment fertilize (kg ha­1) firmness (kg) day 0 day 7 day 14 day 21 day 28 trial one room temperature field 0 5.00 4.67 a 4.03 3.60 3.03 b 100 5.00 3.90 c 3.27 3.03 2.77 bc 200 5.00 3.57 cde 3.20 2.87 2.37 cdef 300 5.00 3.03 ef 2.57 2.00 1.57 hijk 400 5.00 2.83 f 2.33 1.50 1.02 kj greenhouse 0 5.00 3.97 bc 3.57 2.90 2.47 cdef 100 5.00 3.73 cd 3.13 2.23 2.07 efgh 200 5.00 3.43 cdef 2.83 2.13 1.80 ghij 300 5.00 2.93 ef 2.20 1.83 1.50 ijk 400 5.00 2.17 g 1.87 1.27 0.52 l low temperature field 0 5.00 4.83 a 4.77 4.33 3.83 a 100 5.00 3.97 bc 3.67 3.37 2.70 bcd 200 5.00 3.83 cd 3.40 3.07 2.50 bcde 300 5.00 3.57 cde 3.20 2.83 2.17 defg 400 5.00 3.33 cdef 2.97 2.17 1.50 ijk greenhouse 0 5.00 4.57 ab 4.40 3.30 3.03 b 100 5.00 3.53 cde 3.30 2.73 2.27 cdefg 200 5.00 3.20 def 2.93 2.40 1.93 fghi 300 5.00 3.00 ef 2.57 2.13 1.57 hijk 400 5.00 2.87 f 2.20 1.80 1.27 jk trial two room temperature field 0 5.00 4.63 4.07 3.70 abc 2.73 bc 100 5.00 3.90 3.67 3.10 def 2.60 bc 200 5.00 3.80 3.17 2.87 efgh 2.13 def 300 5.00 3.43 2.87 2.17 ijk 1.87 fg 400 5.00 3.00 2.43 1.90 jk 1.20 ij greenhouse 0 5.00 4.27 3.80 3.07 def 2.47 cde 100 5.00 3.93 3.27 2.80 fgh 2.03 efg 200 5.00 3.60 3.00 2.40 hij 1.70 fgh 300 5.00 3.13 2.63 2.00 jk 1.30 hi 400 5.00 2.90 2.20 1.70 k 0.85 j low temperature field 0 5.00 4.70 4.37 4.13 a 3.83 a 100 5.00 4.40 3.93 3.67 abc 2.83 bc 200 5.00 4.13 3.60 3.33 cde 2.93 b 300 5.00 3.93 3.37 3.07 def 2.63 bc 400 5.00 3.63 3.07 2.77 fgh 2.07 defg greenhouse 0 5.00 4.27 4.07 3.93 ab 3.40 a 100 5.00 4.03 3.87 3.50 bcd 2.83 bc 200 5.00 3.83 3.20 2.97 efg 2.50 bcd 300 5.00 3.43 2.87 2.53 ghi 2.10 def 400 5.00 3.20 2.53 2.17 ijk 1.63 ghi * means followed by the same letters in a given day and trial are not significantly different according to tukey’s honestly significant difference test at p≤0.05. room storage temperature varied between 15 and 22°c. low temperature was 7°c. adv. hort. sci., 2021 35(1): 21­32 28 received 400 kg npk ha­1 and stored at room temper­ ature had the highest pwl. similar results were reported in sweet potato in which excessive applica­ tion of nitrogen led to an increase in percentage weight loss during storage (mark et al., 2003). nitrogen fertilizer rates affect the rate of water loss in fruits and vegetables (warner et al., 2004). transpiration is the main cause of deterioration because it results in direct loss of weight. weight loss is the major cause of softening and shriveling of fruits and vegetables damaging the appearance of fruits and loss of market value (wilson et al., 1999). the quality of most fruits and vegetables is affected by weight loss but this depends on the temperature and humidity during storage (perez et al., 2003). storage of pepino fruits at room temperatures (15­22°c) could also have resulted in production of high levels of ethylene, increased respiration and subsequent weight reduction. high temperatures during storage leads to increased water loss resulting to shriveling and loss of fresh appearance of the fruits (wills et al., 1989). fruits lose weight when metabolic activities increase and this is accelerated by an increase in temperature around the produce resulting in loss of water. weight loss is mainly as a result of water loss and as temperature increases the rate of water loss also increases. in this study, pepino melon fruits were harvested when green mature and as ripening progressed there was an increase in ethylene produc­ tion which led to senescence and shriveling of the fruits during storage (wills et al., 1989). greenhouse fruits had a higher percentage weight loss probably because of the high preharvest temperature (table 1). at room temperature the temperatures were higher than at low temperature (7°c) and this could have resulted to faster ripening, increased respira­ tion rates and hence high pwl. fruits stored at low temperature had lower pwl compared to those stored at room temperature. vanitha and mehalai (2016) also reported that pepino fruits stored at room temperature had a higher weight loss com­ p a r e d t o t h o s e s t o r e d a t l o w t e m p e r a t u r e . temperatures above 20°c can lead to abnormal physiological processes in fresh produce, respiration occurs and water is lost to the surrounding environ­ ment and hence reduction in weight. although there w a s a n i n c r e a s e i n p w l a s t h e s t o r a g e d a y s increased, the rate was much lower in pepino fruits stored at low temperature. loss in weight could also b e d u e t o a c ti v i t y o f p o l y g a l a c t u r o n a s e w h i c h increases cell wall permeability and hence increase in transpiration. low temperature reduces respiration and metabolic processes thereby slowing down the rate of fruit weight loss during storage. low tempera­ ture also reduces the sensitivity of fruits to ethylene and senescence is reduced (wills et al.,1989). in both trials, field and greenhouse grown pepino fruits sup­ plied with the highest npk fertilizer rate had the highest pwl. this is in agreement with the findings of hailu et al. (2008) and mark et al. (2003) where appli­ cation of highest nitrogen fertilizer rates had the highest physiological weight loss of carrots and sweet potatoes during storage. the increased pwl due to increased level of nitrogen supply may be attributed to the higher moisture content in the fruits which may lead to decreased shelf­life due to rapid meta­ bolic activity, moisture loss and shrinkage in storage (el­tantawy and el­beik, 2009). on the other hand, fruits from the control (no npk fertilizer) had the lowest pwl which could be attributed to low mois­ ture content in the fruits, slowed metabolic activities a n d h e n c e r e d u c e d m o i s t u r e l o s s . t h e p w l decreased as the phosphorous and potassium rates table 5 ­ effect of npk fertilizer rates, growing environment and storage temperature on shelf life (days) of pepino melon fruits in trial one and two * means followed by the same letters are not significantly diffe­ rent according to tukey’s honestly significant difference test at p≤0.05. room storage temperature varied between 15 and 22°c. low temperature was 7°c. storage temperature environment fertilizer (kg ha­1) shelf life (days) trial 1 trial 2 room temperature field 0 18 cd * 20 bcd (15­22°c) 100 17 cde 18 cdef 200 15 defg 16 fghij 300 14 fghi 14 ijk 400 12 hi 11 lm greenhouse 0 18 cd 17 efgh 100 16 defg 14 hijk 200 13 ghi 12 klm 300 12 hi 10 lm 400 11 i 10 m low temperature (7°c) field 0 27 a 26 a 100 22 b 21 b 200 18 cd 17 defg 300 16 def 15 ghij 400 14 fghi 13 jkl greenhouse 0 21 b 21 bc 100 19 bc 19 bcde 200 17 cdef 16 efghi 300 16 defg 15 ghij 400 15 efgh 14 ijk mutua et al. ‐ pepino melon postharvest studies 29 in the npk fertilizer increased. this could be due to the fact that potassium plays a role in maintaining fruit firmness but high rates do not result to further increase in firmness. the firmer the fruit the less the pwl and reduction in firmness results to more pwl. on the other hand, high nitrogen levels coupled with high phosphorous levels reduce fruit quality because most of the carbohydrates are translocated to the shoots rather than to the developing fruits resulting to dense vegetative growth. fruits produced by plants which have dense vegetative growth tend to be less firm resulting to high pwl while fruits from plants with less vegetative growth are firmer and hence low pwl. in summary, pwl progressively increased with increase in storage time in both room and low temperatures. as the fruit continues to ripen the rate of respiration also increases and this also leads to increase in weight loss. however, low tem­ p e r a t u r e l e a d s t o d e l a y e d r i p e n i n g a n d h e n c e reduced respiration resulting to low pwl compared to ambient room temperatures. tss increased as the storage days increased. our results are in agreement with harman et al. (1986) and hailu (2016) who reported that as pepino melon and mango fruits mature tss increases significantly during maturation and ripening. the increase in tss might be due to alteration of cell wall structure and the breakdown of complex carbohydrates into simple sugars. at room temperature, the temperatures were high and this led to an increase in metabolic process­ es, respiration and ripening resulting to high tss. increase in tss could also be due to excessive mois­ ture loss of fruits which led to increased concentra­ tion of pepino fruits stored at room temperature (nath et al., 2011). at high temperatures the rate of ripening is higher than at low temperatures and this increases tss. field grown pepino fruits had a higher tss compared to greenhouse grown pepino fruits in both storage temperatures. the high tss recorded in field grown pepino fruits could be due to high light intensity and thus high photosynthesis leading to more accumulation of sugars in the fruit compared to greenhouse grown fruits where the light intensity was low leading to reduced photosynthesis and hence low accumulation of sugars in the fruits (beckmann et al., 2006). any factor that interferes with photosynthesis will affect glucose and sucrose accumulation in the fruit and thus alter tss (rana et al., 2014). high relative humidity in the greenhouse may also have led to reduced transpiration and this enhances flow of water in the xylem vessels and this is good for the fruits because fruits act as drains for high concentrations of organic molecules leading to low water potential (bertin et al., 2000). the low water potential in the fruits promotes absorption of water by the fruits leading to “dilution effect” making the fruits to have low tss compared to those grown in the field (rana et al., 2014). the low tss recorded in greenhouse grown fruits could also be due to the fact that high temperatures during ripening of pepino melon reduce sugar content of the fruits (pluda et al., 1993). fruits stored at low temperature had a lower tss compared to that of fruits stored at room tem­ perature. this could be due to delayed fruit ripening and slow conversion of carbohydrates into simple sugars. during ripening there is breakdown of com­ plex carbohydrates into simple sugars and this increase tss. at high temperatures the conversion of carbohydrates into simple sugars is accelerated and this results to high tss whereas at low temperature ripening is delayed and the hydrolysis of carbohy­ drates to sugars is slower, resulting to low tss. in the present study, tss ranged from 4.00­7.07 and 4.00­ 8.13 °brix in trial one and two respectively. other studies have reported lower tss of pepino melon in the range of 4.91­5.40 °brix (kola, 2010) and 5.04­ 5.46 °brix (maruapey and yuwono, 2016). the low tss could be attributed to high water content in pepino fruits in the range of 90­92% (gonzalez et al., 2000) and the fact that the quality of pepino melon fruits is greatly influenced by the environment in which these studies were conducted which is quite different from the environment in this study. tss decreased as the fertilizer rates increased in both growing environments and storage temperatures in both trials. field grown fruits from control plants had the highest tss and this could be due restriction of vegetative growth because no npk fertilizer was applied and thus the fruits became the only sink for sugars and hence increase in tss (pluda et al., 1993). greenhouse grown fruits from plants supplied with the highest fertilizer rate of 400 kg npk ha­1 had the lowest tss and this might be due to excessive vegeta­ tive growth of both the main and side shoots there­ fore most of the photosynthates were directed to the young developing shoots rather than to the fruits leading to low sugar concentration in the fruits (pluda et al., 1993). excess nitrogen fertilizers make plants be more succulent, thus fruits from plants sup­ plied with 400 kg npk ha­1 had a high water content and this might have led to dilution of sugars in the fruit resulting to low tss. adv. hort. sci., 2021 35(1): 21­32 30 fruit firmness decreased as the storage days increased. decrease in firmness is strongly related to increased weight loss because as the fruits lose weight they become soft hence decreased firmness. in this study, firmness decreased as the npk fertilizer rates increased. fruits from plants which were sup­ plied with 400 kg npk ha­1 had the lowest firmness and this could be due to the fact that plants with dense vegetative growth are less firm than those with low or moderate vegetative growth (toktam et al., 2019). fruits which were not supplied with npk fertilizer (control) were firmer due to decreased veg­ etative growth. loss of moisture and enzymatic changes results to change in firmness (ball, 1997). hemicelluloses and pectins become more soluble and this causes changes and loosening of the cell wall (paul et al., 1999). in the present study, both trials field grown pepino fruits were firmer than green­ house grown fruits. this could be due to the fact that l o w e r t e m p e r a t u r e d u r i n g t h e g r o w i n g s e a s o n increases firmness (anagnostou and vasilakakis, 1995). in the greenhouse the temperatures (table 1) were high and it has been reported that high pre­ harvest temperatures tend to decrease firmness (paul et al., 1999). previous studies reported that loss of firmness in pepino melon is due to softening which is caused by breakdown of structural cell wall carbo­ hydrates and an increase in soluble pectic substances during storage (heyes et al., 1994). increase in pectic substances leads to weakening of cell walls and reduction of cohesive forces binding cells together resulting to loss of firmness (heyes et al., 1994). in summary, fruit softening is caused by structural as well as compositional changes in various components of the cell wall carbohydrates partly as a result of fruit softening enzymes (abbasi et al., 2011). other studies have reported that fruit softening is as a result of cell wall digestion by pectinesterase, poly­ g a l a c t u r o n a s e a n d o t h e r e n z y m e s a n d t h i s i s increased by an increase in storage temperature (ahmed et al., 2009). low temperature storage main­ tained firmness of pepino melon fruits. pepino fruits stored at low temperature had a longer shelf life compared to those stored at room temperature. this could be attributed to reduced ethylene production, respiration, ripening, weight loss, senescence, retention of firmness and reduction of other metabolic activities and this enhances shelf life and quality of produce (lei yi et al., 2019). on the other hand, pepino fruits stored at room tempera­ ture had a shorter shelf life because high tempera­ ture results to increased ethylene production, respi­ ration, ripening, weight loss, senescence, loss of firm­ ness and other metabolic processes and this reduced shelf life (mutari and debbie, 2011). field grown fruits had a longer shelf life compared to greenhouse grown fruits. this could be attributed to lower tem­ perature in the field during the growing season (table 1) as low temperatures have been reported to increase firmness (anagnostou and vasilakakis, 1995). in the greenhouse the temperatures were high and it has been reported that high temperatures t e n d t o d e c r e a s e fi r m n e s s ( p a u l e t a l . , 1 9 9 9 ) . therefore, field grown fruits remained firmer than greenhouse grown fruits and hence the former had a longer shelf life. fruits from the control had the longest shelf life and this could be attributed to low nitrogen levels and low water content in this fruits hence they remained firmer. on the other hand, fruits from plants supplied with high npk fertilizer rates had a short shelf life and this might be due to high water content in the fruits due to excess nitro­ gen which also leads to postharvest decay especially fruits which were stored at room temperature. based on the foregoing results and discussion, we conclude that application of high amounts of npk fer­ tilizer leads to increased weight loss, less firm fruits and low tss of pepino fruits stored at room tempera­ ture. storage of pepino melon fruits at 7°c maintains quality through reduced weight loss and maintaining firmer fruits. we therefore recommend application of 100 kg npk ha­1 for both field and greenhouse grown pepino melon and storage at low temperature (7°c) for enhanced quality and shelf life of the fruits. acknowledgements the authors would like to express their sincere appreciation and thanks to world bank through the centre of excellence in sustainable agriculture and a g r i b u s i n e s s m a n a g e m e n t ( c e s a a m ) , e g e r t o n university for the financial support of this research. thanks to the biotechnology laboratory and department of crops, horticulture and soils of egerton university, kenya for hosting this 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(*) corresponding author: shamili@ut.ac.ir citation: salimpour a., shamili m., dadkhodai a., zare h., hadadinejad m., 2019 ­ evaluating the salt tolerance of seven fig cultivars (ficus carica l.). ­ adv. hort. sci., 33(4): 553­565. copyright: © 2019 salimpour a., shamili m., dadkhodai a., zare h., hadadinejad m. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 19 june 2019 accepted for publication 5 september 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(4): 553­565 554 t h e g r o w i n g d e m a n d f o r t h e fi g w o r l d w i d e (aksoy, 2005) is due to its richness in mineral com­ pounds (i.e. iron and copper) and considerable amounts of vitamins a and c (flaishman et al., 2008). the fig is consumed in fresh, dried, powdered, canned, chocolate­covered forms, and utilized in the preparation of jam, syrup and muscat­halvah (de masi et al., 2005). significant genetic variation in the fig species, due to obligatory outcrossing, has led to the establish­ ment of new genotypes with desirable properties. according to the latest reports, there are currently more than 600 known fruit­producing cultivars and genotypes, which are distinct in leaf morphology, growth vigor, internal and external color of the fruit, taste and quality index of the fruit, shape and thick­ ness of the fruit, the diameter of the ostiole, and pro­ ductivity period (condit, 1955; toribio and montes, 1996; garcía­ruiz et al., 2013). sabz or verde (green), siyah (black), shah anjir (king fig), atabaki, kashki and mati are considered as the most important and marketable cultivars of iranian figs for local markets or export. the ‘bar anjirʼ is the most commonly­used caprifig (condit, 1955; pourghayoumi et al., 2016). several research centers have focused on differ­ ent aspects of physiology and breeding of fig cultivars and genotypes. for example, some researchers emphasized on genetic diversity of fig using morpho­ logical (khadivi et al., 2018) and molecular (cabrita et al., 2001; khadari et al., 2005; giraldo et al., 2008) markers. some others attempted to study the capri­ figs (dalkılıç et al., 2011), while others have consid­ ered the variety of the fruit and its qualitative fea­ tures (solomon et al., 2006; polat and caliskan, 2008; ercisli et al., 2012). in addition, the variation in the behavior of fig cul­ tivars and genotypes to abiotic stress such as chilling (karami et al., 2018), drought (gholami et al., 2012) and salinity (zarei et al., 2016) have attracted the researchers’ attention. salinity is one of the most important environmen­ tal factors which reduces the growth, development and production of plants (sevengor et al., 2011). while some researchers suggested reducing leaf area as the plants’ responses to the salinity stress, others enumerate reduced stem length, root length, fresh and dry weights, and relative leaf water content (yamasaki and dillenburg, 1999; bolat et al., 2006; najafian et al., 2008; adish et al., 2010; khayyat et al., 2014; khoshbahkt et al., 2014; soliman and abd alhady, 2017). other important processes that are negatively affected by salinity are protein synthesis (taylor et al., 2004; murcute et al., 2010) and nitro­ gen metabolism (owais, 2015; ashraf et al., 2017), whereas in other studies the inhibition of plant growth caused by salinity has been attributed to a decrease in photosynthesis (garcia­sanchez et al., 2006). fig is a moderate salt­tolerant crop (golombek and lüdders, 1990). the available studies on the response of fig commercial cultivars to different lev­ els of salinity indicated a significant variation in mor­ phological characteristics, growth parameters, physi­ ological behavior, photosynthetic efficiency, gas exchange ratio, product quality, and productivity (essam et al., 2013; metwali et al., 2014; alswalmeh et al., 2015; zarei et al., 2016, 2017; soliman and abd alhady, 2017). considering the position of iranian cultivars in global fig market, the present study examined the growth and photosynthetic rate of commercial fig cultivars (i.e. ‘sabzʼ, ‘siyahʼ, ‘shah anjirʼ, ‘atabakiʼ, ‘kashkiʼ, ‘matiʼ and ‘bar anjirʼ) exposed to six salt treatments corresponding to the following electrical conductivities (ec): 0.5, 2, 4, 6, 8 and 10 dsm­1 to identify the most salt­tolerant cultivar. 2. materials and methods the present study was conducted in the plant b r e e d i n g d e p a r t m e n t , f a c u l t y o f a g r i c u l t u r e , university of shiraz (36° 29’ n and 32° 52’ e), iran during 2016­2018. plant materials the plant materials were included six 20­years­old edible fig cultivars (sabz, siyah, shah anjir, atabaki, kashki, mati), and a caprifig (‘bar anjirʼ) were located at estahban fig research station (36° 29’ n and 32° 52’ e) (table 1). the hard­wood cuttings, 20 cm in length and one cm in diameter, were collected from one­year­old branches on march 25, 2016. the cut­ tings were treated with a fungicide (benomyl 2000 ppm) and a rooting hormone (iba solution 3000 ppm). then, the upper side of the cutting was cov­ ered to prevent the decay, and each cutting was placed in a dark plastic bag (25 x 18 cm2), which was filled by sand. in the next stage, the bags were locat­ ed in the shade­house conditions (temperature: 28±2°c d/18±2°c n, rh=50%, and 50% shade) and were irrigated twice a day. in june 2017, rooted­cut­ tings were transplanted in pots filled with 500 g of gravel and 20 kg of the media described in table 2. salimpour et al. ‐ evaluating the salt tolerance of seven fig cultivars 555 the media included the mixture of soil, leaf compost and sand (1:1:1), which was steam­disinfected. a pressure plate extractor (model adc, by santa barbara, united states) was used to measure the media water capacity. the pots were kept under s h a d e ­ h o u s e c o n d i ti o n ( t e m p e r a t u r e : 3 0 ± 1 ° c d/18±0.5°c n, rh=50%, and 50% shade). salt treatment the salt treatments were provided through the irrigation water. treatments included low salt treat­ ments (ec= 0.5 and 2 ds m­1, a and b, respectively), intermediate salt treatments (ec= 4 and 5 ds m­1, c and d, respectively) and high salt treatments (ec= 8 and 10 ds m­1, e and f, respectively). in order to avoid osmotic stress, salt treatments were introduced gradually, starting from 1/4 up to the final concentration. then, irrigation frequency was calculated based on the media filed capacity and water requirement (essam et al., 2013; zarei et al., 2016). salt treatment was performed within nine weeks from 23/7/2017­ to 26/9/2017. in addition, all of the plants were irrigated by distilled water for four months (26/1/2018). stem length the length of the stem was recorded at the begin­ ning and end of the experiment. the difference between the two values was recorded as the differ­ ence in stem length (cm). stem diameter the stem diameter was recorded at the beginning and end of the experiment by digital caliper (4 cm above the soil surface). the difference was recorded as the difference in stem diameter (mm). number of leaves the number of expanded leaves was counted at table 1 ­ the growth and bearing habit of studied fig cultivars (sabet sarvestani, 1999; safai 2002; jafari et al., 2016) parameters cultivar ‘sabzʼ ‘siyahʼ ‘shah anjirʼ ‘atabakiʼ ‘kashkiʼ ‘matiʼ ‘bar anjirʼ growth and bearing relatively high high moderate moderate growth and low bearing high growth and moderate bearing moderate growth and low bearing low­ moderate growth and high bearing bud conical terminal buds with curved tip curved and pointed terminal bud terminal pointed bud terminal conical bud terminal conical, with tip terminal conical bud without tip terminal conical bud fruit medium, yellowish, no neck, thick pulp medium, dark purple, no neck, thin skin, flesh uniformly red, low pulp thickness large, necked, yellow, pink pulp, fully seed large, rounded, reddish­purple fruit, reddish pulp, reddish medium, green, , open ostiole necked, white pulp, low seedy large, no neck, open ostiole, dark and thick fruit, white, reddish pulp medium, with long neck, containing blastophagous bees type of consumption dried fruit fresh fruit both fresh and dried fruit fresh fruit fresh and processed fruit fresh fruit caprifig yield very high high high moderate moderate moderate moderate taste excellent sweet­and­sour sweet sweet­and­sour sweet sweet­and­sour sweet bearing period early bearing early bearing mid­season bearing early bearing late bearing early bearing mid­season bearing table 2 ­ physico­chemical properties of the soil ec= electrical conductivity; cec= cation exchange capacity. soil texture san (%) silt (%) clay (%) ec (ds/m) cec (me/100) ph lime (%) sandy clay­loam 58±1.01 26±1 16±0.9 1.45±0.21 10.84±0.81 7.7±0.17 ±351.33 organic c (%) n (%) k (ppm) p (ppm) cu (ppm) mn(ppm) fe (ppm) zn (ppm) 1.17±0.05 0.17±0.002 126±1.3 3.2±0.05 0.26±0.002 3.86±0.04 2.85±0.03 0.056±0.001 adv. hort. sci., 2019 33(4): 553­565 556 the beginning and end of the experiment. the differ­ ence between the two values was recorded as the difference in the number of leaves. specific leaf area (sla), leaf dry matter content (ldmc) and leaf succulence the fourth top leaf was harvested after ending the experiment. the leaf area was recorded using a leaf area meter (ci­202 portable laser leaf area meter). the leaves were then dried in an oven (75°c, 48 hrs.) and the dry weight was recorded (ldw). the specific leaf area (in cm2 g­1) was calculated by using the eq. (1). ldmc and leaf succulence were calculated using eq. (2) and (3). sla = la/ldw (1) where la is leaf area (cm2) and ldw is leaf dry weight (g), according to hunt et al., 2002. ldmc = ldw/lfw (2) where ldw and lfw are leaf dry weight (g) and leaf fresh weight (g) respectively (garnier et al., 2001). leaf succulence = lfw/la (3) where lfw is leaf fresh weight (g) and la is leaf area (cm2), according to agarie et al., 2007. relative water content (rwc) mature leaves were collected nine weeks after salt application at mid­day, and were transferred immediately to the lab. then, five similar leaf discs without any vein were separated from each sample and weighted (w1). further, the disc samples were placed in distilled water (4 hrs) under laboratory con­ ditions (24 ± 1oc). subsequently, the samples were surface dried and re­weighed (w2). furthermore, the discs were placed in an electric furnace (model: memmert, made by karl klob factory, germany) (90°c, 60 min) and reweighted (w3). finally, the rela­ tive water content was calculated using the eq. (4) (barrs and weatherley, 1962). rwc = (w1 ­ w3) x 100 (4) (w2 ­ w3) electrolyte leakage (el) the top expanded leaf was harvested and 5 leaf discs without any vein were prepared. the samples were rinsed three times with distilled water, and incu­ bated in 10 ml of distilled water (at 40°c for 30 min). after cooling, the electrical conductivity was mea­ sured using an electrical conductivity meter (h18633 model) (c1). the samples were then autoclaved (at 120°c for 15 min). after cooling, the electrical conduc­ tivity was re­measured (c2). the electrolyte leakage was calculated via eq. (5) (sairam et al., 1997). el = (c1/c2) x 100 (5) leaf protein content first, the fresh leaf sample (0.2 g) was powdered with liquid nitrogen. then, two ml of potassium phosphate buffer (38.5 ml nah 2po 4, 68.5 ml of na2hpo4, 0.074 g edta and 1 g of pvp), ph = 7.15 was added and well­homogenized. the extract was cen­ trifuged at 13000 rpm for 15 min at 4°c, and the super­ natant was used to measure protein content. about 20 μl of the extract, 80 μl of potassium phosphate buffer (ph= 7.15) and 5 ml of coomassie brilliant blue (c47h48n3nao7s2) was stirred for 2 min. in addition, the absorbance was read at 595 nm by using a spec­ trophotometer (biowave ii model) after incubating 5 min at room temperature. the extraction buffer was used as blank. the protein content (in mg g­1 fw) in the sample was calculated according to the sample absorp­ tion using the bovine albumin serum (c123h193n35o37) standard curve (bradford, 1976). leaf nitrogen content the kjeldahl method was used to determine the nitrogen content in fresh leaf samples (kjeldahl, 1883). photosynthetic indexes the photosynthetic indices were recorded using a compact­portable­photosynthesis­system (lci, uk). the device was put on attached leaf (third expanded leaf) at midday, then transpiration rate (in mol h2o m­2 s­1) and stomata conductance (in mol co2 m⁻² s⁻¹) w e r e r e c o r d e d a ft e r t w o m i n ( e v a n s a n d v o n caemmerer, 1996). experimental design and data analysis the present experiment was conducted in a complete randomized design. the factors included fig cultivars (seven cultivars) and nacl treatments (six concentrations), with five replications. sas version 9.1.3 (sas®, 1990) was used for the statistical analy­ sis. shapiro­wilks test confirmed the normality of the data. in addition, leven's test confirmed the variance homogeneity. further, tukey test was conducted for mean comparisons (p<0.01). finally, pearson coeffi­ c i e n t w a s u s e d f o r a n a l y z i n g t h e r e l a ti o n s h i p between the parameters. 3. results the results of variance analysis indicated that the studied cultivars had a significantly different behavior salimpour et al. ‐ evaluating the salt tolerance of seven fig cultivars 557 3 leaves in the lowest and highest salinity levels in this cultivar. in addition, ‘sabzʼ and ‘atabakiʼ cultivars had the lowest number of leaves under intermediate salt conditions (4 and 6 dsm­1) (table 4). the leaf number of ‘atabakiʼ cultivar under higher salt condi­ tion was significantly lower than 0.5 ds m­1 of ec. specific leaf area, leaf dry matter content and leaf succulence the specific leaf area followed a decreasing trend due to an increase in salt concentration in the ‘sabzʼ, ‘siyahʼ, ‘shah anjirʼ and ‘kashkiʼ cultivars. in ‘atabakiʼ cultivar, under ec of 6 and 10 dsm­1 the reduction was observed. in ‘bar anjirʼ cultivar, there was no sig­ nificant difference between 2­10 dsm­1 salt levels (table 4). except for ‘matiʼ and ‘bar anjirʼ cultivars showing 32.21 and 60.55% increase in sla, the rest of the cultivars had descending trend of sla during the experiment. leaf dry matter content of salt­ exposed fig cultivars followed a descending trend. the difference in ldmc of seven cultivars under low salt concentration (0.5 and 2 dsm­1) was not remark­ able. under moderate salt condition (4 dsm­1), most of the cultivars had similar values but under higher salt (6 dsm­1 and more), ‘shah anjirʼ and ‘siyahʼ culti­ vars showed an ascending trend. leaf succulence dis­ played slight rising trend. ‘bar anjirʼ had the highest value under low salt conditions, while ‘siyahʼ showed the highest value under moderate and high salt con­ dition. the values of this parameter was unchanged under moderate salt conditions and the decrease started when nacl reached 8 dsm­1 and more (table 4). leaf succulence of ‘sabzʼ, ‘shah anjirʼ and ‘siyahʼ under ec of 10 ds m­1 were 1.64, 1.64 and 1.61 times higher than their value under ec of 6 dsm­1. relative water content of leaf salinity had a significant effect on reducing leaf under salt conditions, due to salt concentration and cultivar variation (table 3). stem length w i t h i n c r e a s i n g s a l i n i t y l e v e l , s t e m l e n g t h decreased significantly in all cultivars. the greatest effect was observed in ‘bar anjirʼ cultivar, which decreased from 78.84 cm (under 0.5 ds m­1 salinity) to 28.44 cm (under 10 ds m­1 salinity). the lowest effect was observed in the ‘siyahʼ cultivar, which reduced from 51.3 cm (in 0.5 ds m­1 salinity) to 35.8 cm (in 10 ds m­1 salinity) (table 4). the decrease in stem length for all the tested cultivars was between low (0.5 ds m­1) and high (10 ds m­1) nacl­treated plant. this decrease for most of the cultivars (includ­ ing ‘sabzʼ, ‘shah anjirʼ, ‘matiʼ and ‘bar anjirʼ) was more than 200%. stem diameter by raising salinity, the stem diameter decreased significantly in all cultivars. the highest reduction in stem diameter was observed in the ‘matiʼ cultivar, which decreased from 6.35 mm (under 0.5 dsm­1 salinity level) to 3.41 mm (under 10 dsm­1). the low­ est effect was observed in ‘siyahʼ cultivar, reducing from 5.37 mm at the lowest salinity level to 3.2 mm at the highest salinity level (table 4). in addition, the decrease in stem diameter of low and high salt treat­ ed plants varied between 157.93 to 219.89% (‘kashkiʼ and ‘bar anjirʼ cultivars, respectively). number of leaves with an increase in salinity levels, the number of leaves in all cultivars followed a decreasing trend. the greatest effect of salinity was observed in ‘bar anjirʼ, which difference in the number of the leaves in the lowest and highest salinity levels was 10.2 leaves. the lowest effect of salinity on leaf number was observed in ‘shah anjirʼ, and the difference was table 3 ­ the interaction of cultivar and salinity on growth and physiological parameters of seven fig (the mean square value is given) ns, * and **= not significant, significant at 5 and 1% respectively (by tukey mean comparison test). physiological parameters cultivar salinity cultivar x salinity difference in stem length 1273.11 * 5916.23 * 223.68 ** difference in stem diameter 9.61 ** 30.78 * 1.34 ** difference in leaf number 107.80 ** 220.38 * 11.25 ** specific leaf area 174.37 ** 27.628 ns 11.23 ** relative water content 1540.57 ** 264.34 ** 289.57 ** electrolyte leakage 21.31 ** 239.41 ** 11.23 ** leaf protein 37007542 ** 268172052 ** 4586351 ** leaf nitrogen 0.93 ** 5.94 ** 0.071 ** transpiration rate 4.46 ** 90.58 ** 3.89 ** stomata conductance 0.099 ** 1.025 ** 0.074 ** 558 adv. hort. sci., 2019 33(4): 553­565 relative water content in all cultivars. the highest decrease was observed in the ‘siyahʼ cultivar, which reduced from 90.83% in the lowest salinity level to 53.03% in the highest salinity level. the lowest effect was observed on the ‘sabzʼ cultivar. the intermediate salt condition of 4 dsm­1 did not make a significant difference from 2 dsm­1, except in ‘sabzʼ (table 5). the ‘kashkiʼ and ‘bar anjirʼ cultivars had the lowest decline of rwc during the experiment (15.15 and 15.33%, respectively), while ‘siyahʼ showed the stronger decrease in rwc (41.62%). electrolyte leakage salinity had a significant ascending effect on elec­ trolyte leakage in seven fig cultivars. the highest salini­ ty increased the ionic leakage in ‘siyahʼ and ‘atabakiʼ table 4 ­ the influence of saline water on stem length, stem diameter, leaf number, specific leaf area, ldmc and leaf succulence of fig cultivars genotype treatments stem length (cm) stem diameter (mm) leaf number specific leaf area (cm2 g­1) ldmc leaf succulence sabz a 59.72 b 4.53 c 8.20 c 11.53 b 0.68 b 0.13 i b 48.00 c 3.19 d 6.80 d 13.38 a 0.70 b 0.11 i c 38.64 c 3.29 d 6.00 d 11.60 b 0.52 d 0.17 h d 27.90 d 2.34 e 4.00 e 11.76 b 0.57 c 0.15 h e 30.75 d 2.51 e 3.20 f 10.22 c 0.57 c 0.17 h f 27.30 d 2.39 e 2.60 f 7.85 e 0.52 d 0.24 g siyah a 51.30 c 5.37 b 5.80 d 4.79 f 0.74 a 0.28 e b 38.80 c 4.91 c 4.60 d 3.64 g 0.73 a 0.38 d c 30.24 d 3.24 d 2.50 f 3.94 g 0.61 c 0.41 c d 36.50 d 3.87 d 3.40 f 3.76 g 0.60 c 0.44 c e 28.66 d 3.49 d 1.00 g 3.77 g 0.60 c 0.44 c f 35.80 d 3.20 d 2.50 f 2.04 g 0.69 b 0.71 a shah anjir a 49.72 c 4.72 c 4.00 d 11.27 b 0.70 b 0.13 i b 52.40 c 5.27 b 6.40 d 11.04 b 0.54 d 0.17 h c 29.30 d 3.68 d 5.40 d 10.35 c 0.62 c 0.16 h d 32.90 d 3.60 d 2.60 f 9.73 c 0.74 a 0.14 h e 33.04 d 3.15 d 1.60 f 9.52 c 0.76 a 0.14 h f 23.06 d 2.54 e 1.00 g 6.40 e 0.69 b 0.23 g atabaki a 76.50 a 4.75 c 7.60 c 11.89 b 0.71 b 0.12 i b 59.20 b 5.17 b 7.20 c 8.49 d 0.68 b 0.17 h c 53.74 c 4.56 c 6.60 d 7.76 e 0.61 c 0.21 g d 40.50 c 3.99 d 5.00 e 5.64 f 0.49 d 0.36 e e 25.30 d 1.45 f 3.00 f 8.82 d 0.44 e 0.26 g f 44.08 c 2.46 e 3.60 f 6.01 e 0.49 e 0.34 e kashki a 78.00 a 5.18 b 12.40 a 9.55 c 0.61 c 0.17 h b 53.62 b 5.50 b 7.00 c 9.23 d 0.62 c 0.18 h c 50.14 c 3.77 d 8.00 c 9.08 d 0.52 d 0.21 g d 38.88 c 2.97 d 4.40 e 7.88 e 0.57 c 0.22 f e 46.02 c 3.26 d 3.80 f 6.75 e 0.57 c 0.26 g f 45.46 c 3.28 d 6.60 d 6.65 e 0.52 d 0.29 f mati a 68.22 b 6.35 a 9.80 b 5.95 f 0.68 b 0.25 g b 64.80 b 5.80 b 9.60 b 8.55 d 0.62 c 0.19 h c 52.10 c 4.65 c 5.80 d 5.21 f 0.45 e 0.43 c d 38.30 c 4.19 c 3.20 f 4.64 f 0.45 e 0.48 c e 33.80 d 4.24 c 5.20 e 4.20 f 0.45 e 0.53 b f 30.90 d 3.41 d 2.40 f 7.86 e 0.44 e 0.29 f bar anjir a 78.84 a 3.98 c 13.60 a 5.60 f 0.35 f 0.51 b b 58.00 b 4.02 c 11.00 b 8.36 d 0.29 f 0.41 c c 48.00 c 3.67 d 8.40 c 8.74 d 0.30 f 0.39 d d 43.56 c 3.19 d 7.40 c 8.98 d 0.32 f 0.35 e e 32.20 d 2.90 d 4.20 e 8.68 d 0.28 f 0.42 c f 28.44 d 1.81 e 3.40 f 8.99 d 0.30 f 0.38 e data are average of five replications. in each column means with a common letter have no significant difference at 1% of tukey test. treatments a, b, c, d, e and f are 0.5, 2, 4, 6, 8, and 19 ds m­1 of ec, respectively. salimpour et al. ‐ evaluating the salt tolerance of seven fig cultivars 559 cultivars (table 5). the electrolyte leakage of ‘atabakiʼ, ‘siyahʼ and ‘bar anjirʼ cultivars showed the highest dif­ ference between the moderate and high salt condi­ tions (39.19, 32.89 and 31.65% increases, respectively). leaf protein the results indicated that salinity had a significant e ff e c t o n l e a f p r o t e i n o f s t u d i e d fi g c u l ti v a r s . increasing the stress to 4 ds­1 of ec increased the table 5 ­ the influence of saline water on electrolyte leakage, protein, nitrogen, transpiration rate, stomata conductance and rwc of fig cultivars data are average of five replications. in each column means with a common letter have no significant difference at 1% of tukey test. treatments a, b, c, d, e and f are 0.5, 2, 4, 6, 8, and 19 ds m­1 of ec, respectively. genotype treatments rwc (%) electrolyte leakage (%) protein (mg. gˉ¹. fw) nitrogen content (%) transpiration rate (mol h2o m ­2s­1) stomata conductance (mol co2 m⁻² s⁻¹) sabz a 76.20 c 19.53 c 0.90 c 3.73 a 59.72 b 0.46 d b 74.80 c 20.75 c 0.93 c 3.55 b 48.00 d 0.13 g c 68.20 d 18.76 c 1.21 b 3.28 b 38.64 c 0.20 f d 62.55 d 17.80 d 1.79 b 3.13 b 27.90 c 0.20 f e 62.22 d 19.20 c 2.20 a 2.47 c 30.75 g 0.06 h f 61.88 d 22.01 c 2.45 a 2.31 c 27.30 g 0.05 h siyah a 90.83 a 15.77 e 0.86 c 3.55 b 51.30 b 0.53 c b 76.78 c 16.42 d 0.86 c 3.59 b 38.80 c 0.78 a c 70.15 c 18.61 c 1.22 b 3.43 b 30.24 e 0.16 g d 67.55 d 19.91 c 2.17 a 3.21 b 36.50 e 0.14 g e 65.09 d 22.31 b 2.35 a 2.98 c 28.66 g 0.07 h f 53.03 e 24.73 a 2.60 a 2.97 b 35.80 g 0.04 h shah anjir a 89.23 b 16.23 d 0.76 c 3.28 b 49.72 b 0.45 d b 88.79 b 15.17 e 0.92 c 3.28 b 52.40 b 0.48 d c 81.68 b 18.67 c 0.99 c 3.10 b 29.30 d 0.30 d d 77.66 c 21.24 b 1.01 b 2.50 c 32.90 d 0.15 g e 77.56 c 20.61 c 1.56 b 3.15 b 33.04 f 0.08 h f 72.15 c 22.22 c 1.36 b 2.92 c 23.06 e 0.11 g atabaki a 81.16 b 14.37 e 0.91 c 3.13 b 76.50 a 0.67 b b 77.22 c 15.39 e 1.01 b 3.34 b 59.20 c 0.36 e c 75.33 c 16.56 d 1.15 b 3.16 b 53.74 d 0.38 d d 73.47 c 19.29 c 1.62 b 2.97 c 40.50 d 0.40 d e 71.09 c 22.75 b 2.04 a 2.43 c 25.30 g 0.06 h f 64.65 d 23.05 b 2.31 b 2.29 c 44.08 h 0.03 h kashki a 85.34 b 16.51 d 0.74 c 2.98 c 78.00 b 0.40 a b 78.92 c 20.10 c 0.85 c 3.31 b 53.62 c 0.27 f c 75.33 c 20.04 c 1.08 b 3.13 b 50.14 e 0.13 g d 73.48 c 22.49 b 1.31 b 2.89 c 38.88 e 0.19 g e 73.29 c 23.20 b 1.84 b 2.75 c 46.02 e 0.14 g f 72.41 c 22.75 c 2.19 a 2.33 c 45.46 e 0.11 g mati a 96.32 a 14.46 e 0.84 c 2.97 c 68.22 a 0.75 a b 88.58 b 17.27 d 0.94 c 3.50 b 64.80 b 0.43 d c 83.88 b 19.24 c 1.09 b 3.27 b 52.10 e 0.13 g d 78.39 c 21.22 b 1.23 b 3.09 b 38.30 e 0.23 f e 74.58 c 21.65 c 1.71 b 2.66 c 33.80 f 0.15 g f 70.23 c 22.38 c 2.01 a 2.60 c 30.90 g 0.04 h bar anjir a 95.00 a 14.83 e 0.71 c 3.78 a 78.84 d 0.20 f b 93.68 a 15.30 e 0.75 c 3.32 b 58.00 d 0.34 e c 91.82 a 16.68 d 0.81 c 3.14 b 48.00 b 0.26 f d 88.52 b 19.72 c 1.04 b 2.82 c 43.56 b 0.09 h e 82.81 b 21.26 b 1.76 b 2.75 c 32.20 f 0.08 h f 80.44 b 21.96 c 1.96 b 2.30 c 28.44 g 0.04 h adv. hort. sci., 2019 33(4): 553­565 560 total protein content gradually. the highest amounts were observed in ‘siyahʼ (2.60 mg g­1 fw) and ‘sabzʼ (2.45 mg g­1 fw) cultivars and the lowest in ‘shah anjirʼ (1.36 mg g­1 fw) and ‘bar anjirʼ (1.96 mg g­1 fw) cultivars (table 5). the increase in protein content was expected between moderate (4 ds­1) and high (10 ds­1) nacl treated plant. this increase for most of the cultivars (including ‘sabzʼ, ‘siyahʼ, ‘atabakiʼ, ‘kashkiʼ and ‘bar anjirʼ) was more than 200%. leaf nitrogen by increasing salinity levels in all seven fig culti­ vars, leaf nitrogen content decreased. the highest reduction was observed in ‘bar anjirʼ cultivar at ec of 1 0 d s ­ 1 ( 2 . 3 0 % ) a n d t h e l o w e s t r e d u c ti o n w a s observed in ‘siyahʼ cultivar, which decreased from 3.55% at the lowest salinity level to 2.99% at the ec o f 1 0 d s m ­ 1 ( t a b l e 5 ) . t h e ‘ m a ti ʼ , ‘ k a s h k i ʼ a n d ‘atabakiʼ showed 10.03, 5.04 and 0.88% increase in n content under moderate salt condition (4 dsm­1), while the rest cultivars exhibited a decrease in n con­ tent (table 5). photosynthetic indices the interaction of salinity stress and cultivar on photosynthetic indices (transpiration rate and stoma­ ta conductance) was significant. with increasing nacl levels, the rate of transpiration decreased in seven fig cultivars. the highest reduction was observed in ‘atabakiʼ cultivar (0.85 mol h2o m ­2 s­1 at 10 dsm­1 of ec). the lowest influence was observed in ‘kashkiʼ (3.41 mol h2o m ­2 s­1 at 10 dsm­1 of ec). for all the studied cultivars this parameter did not differ signifi­ cantly between 4 and 6 dsm­1 (intermediate salt con­ ditions). high salt concentrations (8 and 10 dsm­1) caused a noticeable difference among the genotypes, where ‘atabakiʼ, ‘siyahʼ, ‘matiʼ and ‘sabzʼ were fall to 12.18, 19.63, 22.16 and 23.40% of their initial values ( t a b l e 5 ) . s t o m a t a c o n d u c t a n c e d e c r e a s e d b y increasing salt concentration in all cultivars, and reached its lowest level at the highest salinity level (10 dsm­1). the lowest stomata conductance at of ec 10 dsm­1 was observed in ‘atabakiʼ (0.03 mol co2 m⁻² s⁻¹) and the lowest in ‘kashkiʼ (0.11 mol co2 m⁻² s⁻¹) (table 5). the ‘atabakiʼ, ‘matiʼ and ‘siyahʼ cultivars displayed the highest decrease of stomata conduc­ tance from 05. to 10 dsm­1of ec (3.86, 5.91 and 7.95% decrease of their initial values, respectively) correlation analysis table 4 indicates the bivariate pearson correla­ tions among the parameters. the bold faces values indicate high correlated values (higher than 0.5). difference in the number of leaves had high correla­ ti o n w i t h r e l a ti v e w a t e r c o n t e n t ( 0 . 5 8 3 * * ) , transpiration rate (0.899**) and stomata conduc­ tance (0.915**). specific leaf area and relative water content were positively correlated (0.680**). in addi­ tion, both photosynthetic indices showed high corre­ lation (0.877**) (table 6). 4. discussion and conclusions the negative effect of salinity leads to the changes in soil structure, competition in nutrients uptake in different parts of the plant and eventually inhibition of nutrients absorption (gholami et al., 2012). na+ reduces plant biomass by disrupting the protein syn­ thesis, destroying chlorophyll, and decreasing the activity of the enzymes which are involved in biosyn­ physiological parameters leaf nitrogen leaf protein electrolyte leakage difference in stem length difference in the number of leaves difference in stem diameters specific leaf area relative water content transpiration rate stomata conduc­ tance leaf nitrogen 1 leaf protein ­0.170 * 1 electrolyte leakage ­0.225 ** 0.017 1 difference in stem length ­0.375 ** ­0.006 0.068 1 difference in the number of leaves 0.331 ** ­0.009 ­0.055 ­0.108 1 difference in stem diameters ­0.415 ** 0.323 ** 0.179 ** 0.093 ­0.287 ** 1 specific leaf area 0.393 ** ­0.239 ** ­0.103 ­0.079 0.199 ** ­0.480 ** 1 relative water content 0.351 ** ­0.124 ­0.122 ­0.077 0.583 ** ­0.499 ** 0.680 ** 1 transpiration rate 0.362 ** ­0.111 ­0.098 ­0.079 0.899 ** ­0.435 ** 0.227 ** 0.581 ** 1 stomata conductance 0.195 ** 0.025 ­0.033 ­0.036 0.915 ** ­0.149* ­0.020 0.463 ** 0.877 ** 1 table 6 ­ the correlation analysis of physiological parameters of fig cultivars * and **= correlation is significant at the 5 and 1% levels, respectively. salimpour et al. ‐ evaluating the salt tolerance of seven fig cultivars 561 thesis (phosphoenolpyruvate carboxylase, ribulose­ l,5­bisphosphate carboxylase, pentose phosphate pathway enzymes and glycolysis pathway enzymes) (demiral, 2005). the major strategies of plants to overwhelm this stress included: reduction in cl­ and na+ uptake, leaf loss, decrease in leaf specific area and relative leaf water content, synthesis of osmotic compounds, exclusion of toxic ions into vacuole, change in mem­ brane stability, and increase in the activity of antioxi­ dant enzyme (mutsushita and matoch, 1992; sato et al., 2006). based on the results of the present study, the stem length in ‘siyahʼ and ‘sabzʼ was less affected by salt stress. in addition, under intermediate salt condi­ tions ‘shah anjirʼ, ‘siyahʼ and ‘sabzʼ had the lowest stem length. it has already reported that salt­sensi­ tive fig cultivars such as ‘brown turkiʼ and ‘piusʼ dis­ played reduction in stem length under salinity condi­ tions (alswalmeh et al., 2015; zarei et al., 2016). a similar decrease in stem length under salt conditions was reported in almond (najafian et al., 2008) and pistachio (adish et al., 2010). the findings of soliman and abd alhady (2017) and zarei et al. (2016), indicated that the stem diam­ e t e r i n s a l t ­ e x p o s e d fi g c u l ti v a r s h a d a l i n e a r decrease. furthermore, the decrease in stem diame­ ter in salt­treated plums (bolat et al., 2006), citrus (khoshbahkt et al., 2014) and pomegranate (khayyat et al., 2014) showed similar pattern. in the present study, stem diameter of ‘siyahʼ cultivar was less affected by salt. reducing the number of leaves in salt­exposed plants is due to limit leaf production or early leaf aging (yeo et al., 1991; munns and tester, 2008). based on the results, the highest leaf loss was observed in ‘bar anjirʼ cultivar. similar results are available in almond (momenpour et al., 2018), pista­ chio (adish et al., 2010) and fig cultivars (essam et al., 2013; alswalmeh et al., 2015; zarei et al., 2016; soliman and abd alhady, 2017). reduction in the relative leaf water content under salinity stress indicates lower water uptake by plants. limited access to water due to increase in osmotic potential reduces the cell development and decreas­ e s t u r g o r p r e s s u r e o f t h e c e l l s ( y a m a s a k i a n d dillenburg, 1999). in the present study, salinity reduced the relative content of leaf water in fig culti­ vars, except ‘bar anjirʼ. in ‘siyahʼ cultivar, a dramatic decrease was observed in the relative leaf water con­ tent. under various salt concentrations, the trend of this parameter stayed unchanged. under intermediate salt conditions the specific leaf area of ‘siyahʼ and ‘sabzʼ had not evident changes. according to owais (2015), with increasing salt levels, the relative leaf water content in grape genotypes decreased, but this decrease was lower in tolerant cultivars. a similar decrease was observed in the relative leaf water content under salinity stress in fig (zarei et al., 2016; soliman and abd alhady, 2017) and pomegranate (khayyat et al., 2014). the values of sla and ldmc reveal an important exchange in plant function between high sla, low ldmc (cultivars with rapid production of biomass) and low sla, high ldmc (cultivars with an efficient conservation of nutrients) (poorter and garnier, 1999). according to our results, ‘siyahʼ and ‘sabzʼ cul­ tivars had the lowest sla and the highest ldmc value, confirmed its production efficiency under vari­ ous salt concentration. in addition, ‘bar anjirʼ and ‘shah anjirʼ had the highest sla and the lowest lsdm under different nacl concentration, due to their lim­ ited production efficiency under salt conditions. rising the salinity level, increased leaf succulence. this ability characterizes a balance between growth rate and the necessity of osmotic adjustments (flowers and yeo, 1986), which regulate low external w a t e r p o t e n ti a l e n c o u r a g e d b y s a l i n i t y s t r e s s (flowers and colmer, 2008). moreover, it explains the better carbon assimilation capacity per unit area (de vos et al., 2013). based on our findings ‘siyahʼ and ‘sabzʼ cultivars had the highest leaf succulence, which related to the balance between growth ratio and osmotic regulation under salt conditions. peroxidation of lipids is regarded as an extra effect of salinity on plants (demidehik et al., 2002). the findings of the previous researchers on increas­ ing ion leakage under salinity stress in fig (abdoli nejad and shekhafandeh, 2014; zarei et al., 2016) and pomegranate (khayyat et al., 2014) confirms our results. proteins biosynthesis is an important biochemical process which is affected by salt stress. expression of specific genes under nacl stress assistances the plant to adapt to adverse conditions (murcute et al., 2010). according to our findings, protein content increased under high salt condition, but this increase was greater under intermediate salt for ‘siyahʼ and ‘sabzʼ cultivars. it has reported that sodium chloride treatment reduced leaf protein content in salt­sensitive grape (alizadeh et al., 2010) and figs (abdoli nejad and shekhafandeh, 2014). in fact, salt inhibits the synthesis of nitrate reduc­ adv. hort. sci., 2019 33(4): 553­565 562 tase, glutamine synthase and glutamate synthase ( w h i c h a r e i n v o l v e d i n n i t r o g e n m e t a b o l i s m ) , decreases nitrogen metabolism (hossain et al., 2012), changes active forms of nitrogen, reduces amino acids synthesis, and finally increases the activity of degrading enzymes (de souza et al., 2016). in saline conditions, cl­ competes with nitrate (abdelgadir et al., 2005), leading to the decline of nitrogen in differ­ ent parts of the plant (yu et al., 2016; hasan and miyake, 2017). according to the owais (2015) and doulati baneh et al. (2014), salinity had a decreasing effect on the leaf nitrogen content in fruit crops. although the relationship between salinity and nitro­ gen metabolism is very complex, balanced nitrogen metabolism significantly affects salinity tolerance in plants (teh et al., 2016). in the present study, the impact of salinity on leaf nitrogen content of differ­ ent fig cultivars was not the same. photosynthesis is another important plant phe­ nomenon, significantly affected by abiotic stress. reduction of photosynthesis under salinity stress is attributed to stomata factors (reduced co2 perme­ ability, stomata closure, decrease in plants transpira­ tion, stomata conductance reduction), and non­stom­ ata factors (cell membrane dehydration, structural changes in the cytoplasm and chlorophyll degrada­ tion) (brugnoli and lauteri, 1991; reza et al., 2006; tabatabaei, 2006). salinity reduces stomata conduc­ tance in hybrids of fig, by decreasing the photo­con­ ductivity of leaf cells (golombek and lüdders, 1990; zarei et al., 2016), which is in agreement with the results of the present study. similar results were reported in pistachio (adish et al., 2010). according to the results of the present study, ‘atabakiʼ and ‘kashkiʼ cultivars showed the lowest and the highest stomata conductance under high salt condition, respectively. salinity reduces evaporation and transpiration in plants (bhantana and lazarovitch, 2010; dudley et al., 2008). the linear relationship among reduction of plant evapotranspiration, transpiration and increase in salinity levels was reported in pomegranate (shani and ben­gal, 2005; mohamed ibrahim and abd el­samad, 2018) and date palm (tripler et al., 2007), which is coordinated with the results of the current study. in addition, the maximum quantum efficiency of photosystem ii, electron transfer, gas exchange, and carbon dioxide assimilation decrease under salt con­ ditions (joao­correia et al., 2006). in the present study, atabaki cultivar had the highest transpiration rate at the low and mild salinity levels, and with increasing salinity, a greater decrease was observed (table 5). the lowest effect of salinity on transpira­ tion rate was observed in ‘kashkiʼ (table 5), the culti­ var which was able to balance the transpiration level at mild and severe stress levels, probably due to hav­ ing thicker cuticle. the most important physiological process affected by this stress is photosynthesis (sudhir and murthy, 2004; acosta­motos et al., 2017). reduction in pho­ tosynthesis efficiency is followed by a series of mole­ cular events including cell membrane dehydration, stomata closure, decreased co2 entry, reduction in leaf permeability to co2, structural changes in the cytoplasm and subsequent alteration in the activity of enzymes (tabatabaei, 2006). on the other hand, nitrogen plays effective role in plant growth and construction of vital plant struc­ tures such as amino acids and proteins (arghavani et al., 2017). nitrogen is necessary to generate cellular components such as rubisco, which is responsible for assimilating carbon dioxide. therefore, by limiting nitrogen uptake, salinity stress affects the photosyn­ thesis efficiency, leading to a decrease in vegetative and reproductive growth (coruzzi and bush, 2001; marschner, 2012; zarata­valdez et al., 2015). in the present research, salinity had a significant effect on growth parameters and photosynthetic indices in seven cultivars of fig. the differences in stem length, stem diameter and leaf number in all cultivars followed a downward trend. the stomata conductance of all fig cultivars was same up to 4 ds m­1 nacl. moreover, the transpiration rate did not exhibited variation unless in salt concentration higher than 8 dsm­1. the ‘siyahʼ and ‘sabzʼ cultivars had the lowest decreases in stem length and diameter, the lowest leaf water content and transpiration rate, and the maximum leaf abscission. additionally, ‘siyahʼ and ‘sabzʼ cultivars had the highest leaf succulence and ldmc and the lowest sla, which related to the balance between growth ratio and osmotic regula­ tion under salt conditions. the ‘matiʼ, as an interme­ diate salt­tolerance cultivar, had the lowest leaf abscission under severe salinity levels. the ‘shah anjirʼ, as the most salt­sensitive cultivar, could not balance transpiration rate and leaf water content under salt treatment higher than 4 dsm­1. acknowledgements t h e p r o j e c t w a s s u p p o r t e d b y t h e h e a d o f r e s e a r c h a n d t e c h n o l o g y c e n t e r , u n i v e r s i t y o f hormozgan; plant breeding department, faculty of salimpour et al. ‐ evaluating the salt tolerance of seven fig cultivars 563 agriculture, shiraz university; and estahban fig research station. references a b d e l g a d i r e . m . , o k a m . , f u j i y a m a h . , 2 0 0 5 ­ characteristics of nitrate uptake by plants under salini‐ ty. ­ j. plant nut., 28: 33­46. abdoli nejad r., shekafandeh a., 2014 ­ salt stress‐ 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impaginato 283 adv. hort. sci., 2019 33(2): 283-294 doi: 10.13128/ahs-24300 first insight into araucaria araucana (molina) k. koch under its southernmost european growing condition: a proposed descriptor list for morphological characterization m. antonetti, s. nin, g. burchi crea, centro di ricerca orticoltura e florovivaismo,via dei fiori, 8, 51017 pescia (pt), italy. key words: germplasm, monkey puzzle, phenology, phenotyping, climate, pistoia’s nurseries. abstract: araucaria araucana is a south american endemic conifer of conservation concern. after its introduction to europe, this species has been often planted for ornamental purposes in parks and gardens, where its unusual appearance was admired. after the mid-1970s this tree received increasing attention from the nursery growers of pistoia (tuscany) and became of considerable economic importance. however, being one of the iconic threatened trees listed in cites appendix i, the international trade of these species is rigorously regulated. this study was aimed at developing a first morphological descriptor list for further phenotyping of in loco produced plant material. a first step of the research focused on the description and a better understanding of a. araucana phenological phases inferred from mediterranean climate conditions. the second phase regarded the analysis of observed or measured morphological characteristics of the tree, branches, scales, inflorescences, fruits and seeds observed on a subset of 4 selected putative populations over a 3-year period of vegetative growth. the results allowed to select 39 most discriminant descriptors, which are presented together with their range of variability and classes. the achieved descriptor list represents a suitable tool for the selection of genotypes and for the breeding of a. araucana. 1. introduction araucaria araucana (molina) k. koch, commonly known as ‘monkey puzzle tree’ or ‘pehuèn’, is an endangered conifer species native to southcentral chile and south-western argentina, where it has a relatively limited distribution, split between the main area spanning both sides of the andes and two other disjunct small subpopulations in the coastal cordillera of chile (donoso, 1993, 2006; donoso et al., 2008; drake et al., 2009). the present distribution is a remnant of a more extensive former distribution, which has been severely diminished by logging, human-set (*) corresponding author: maurizio.antonetti@crea.gov.it citation: antonetti m., nin s., burchi g., 2019 first insight into araucaria araucana (molina) k. koch under its southernmost european growing condition: a proposed descriptor list for morphological characterization. adv. hort. sci., 33(2): 283-294 copyright: © 2019 antonetti m., nin s., burchi g. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 10 december 2018 accepted for publication 11 march 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(2): 283-294 284 fires and land clearance since european colonization in the mid-19th century (veblen, 1982; burns, 1993; rechene, 2000). in particular, the intense human seed collecting and animal grazing have led to a lack of natural reproduction by seed, and when any regeneration occurs it is principally asexual with tree sprouting from roots (schilling and donoso, 1976; gallo et al., 2004). since 1976, this species has been protected in chile under the status of a chilean national monument and, since 1997, it is also protected internationally under the convention on international trade in endangered species of wild fauna and flores (cites) (farjon and page, 1999; herrmann, 2006). a r a u c a r i a a r a u c a n a w a s f i r s t i n t r o d u c e d i n england by the scottish naturalist archibald menzies in 1795. its unusually straight cylindrical bole and whorled branches as well as its 10-15 cm thick tortoise-shell-like bark made it internationally popular as an ornamental plant. the following decades saw a rapid spread of this impressively large and long-lived conifer throughout all the european continent. it was introduced in italy from paris in 1822 and the first a. araucana tree was planted in the garden of the marquis pucci’s favourite property in florence, tuscany. this italian region represents the southernmost limit of its distribution area. thanks to its perfect adaptability to the tuscan soil and climate conditions, this majestic slow-growing tree was included in the important ornamental horticulture district of pistoia (nw from florence) starting from the ii postworld war period. thereafter, there was a notable and rapid increase of a. araucaria commercial propagation in the pistoia district in line with the steadilygrowing demand. this tree became of considerable economic importance and the period between the 1970s till the early 2003 saw the maximum expansion of commercialization rate and tree planting in private properties and public gardens. due to its constantly declining distribution, together with its slow growth and its limited dispersal ability, in 2003, according to the regulation (ce) n. 1497, the listing of this species was transferred from appendix ii to appendix i of cites (http://www.cites.org/eng/app/appendices. html; valid from october 4th, 2017), which strictly regulates the trade in its timber and seeds, and listed in the 2008 iucn red list of threatened species (http://www.iucnredlist.org; march 2017) as an endangered species currently on risk of extinction. as a consequence, plant nurseries had to adopt a mandatory stock register of alive and dead specimen, where both entries and exits (including origin, quantity, causes of death, etc.) had to be specified. a progressive and ongoing reduction of monkey puzzle tree propagation in the pistoia district has resulted from increased regulation, complex management, high risk of penalties, increased costs for staff training, and risks from plant diseases associated with climate change. ultimately, the number of nurseries holding and propagating araucaria plants conspicuously declined during the last fifteen years. the identification of a new tuscan variety could offer the opportunity to disengage from the procedural constraints imposed by the cites convention. hence, the production of a descriptor list for the characterization of tuscan selected germplasm is a necessary first step towards the definition of genetic diversity based on morphological variation and for varietal identification in araucaria. the descriptor list might represent the first attempt at achieving an unified documentation system thereby enabling through a standardized format an easier exchange of information between researchers and collection curators. although there is a high demand for new descriptor lists to be developed for many forest conifer species, up to our knowledge there is only an upov descript o r l i s t a v a i l a b l e f o r p i c e a a b i e s l . ( https:// www.upov.int/test_guidelines/en/fulltext_tgdocs.jsp?q= picea; copyright © 2011, upov). despite the conservation interest in this species, little is known of its phenotypic and genetic variation. the genetic diversity of monkey puzzle between andean and coastal chilean populations has been investigated in previous studies by delmastro and donoso (1980) and rafii and dodd (1998). more recently, advanced biotechnologies, such as rapds, isozymes, microsatellite and rflp analysis, were used to characterize genetic heterogeneity within and among some south american populations (bekessy et al., 2002; ruiz et al., 2007; marchelli et al., 2010; martín et al., 2012). however, no reports were found in the world literature on morphological traits. t h i s stu d y was p art o f th e c arav iv p ro j ect ‘characterization of araucaria araucana germplasm selected by the nursery industries of the pistoia’s district for commercial development’, supported by the ministero delle politiche agricole alimentari e forestali (mipaaf-oiga), aimed at contributing to a better understanding of araucaria growing and to enhance the commercial exploitation of local genetic resources. this paper provides a brief account of araucaria araucana phenological phases under italian climatic conditions and is focused on the development of a first descriptor lists in order to antonetti et al. auracaria araucana. first insight 285 characterize in loco produced plant material and make information available to other growers in a systematic and unambiguous form. 2. materials and methods plant material the plant material used in this study was the a. araucana germplasm available in the pistoia’ nursery district, covering an area of approx. 965 sq km, ranging from 50 m to 550 m above sea level, located in northern tuscany. phenology throughout three-year growing cycles (20152017), the phenological phases (onset of flowering, full bloom, fertilization, fruit ripening and seed production) of a. araucana trees belonging to 8 putative populations were observed every two weeks from march to june (during the flower maturation), and monthly in the rest of the year. taking into account the peculiar structure of the reproductive buds of this species, we decided to consider as onset of flowering the inflorescence appearance and as full bloom the inflorescence maturity, i.e. the pollination phase. four out of the selected populations belong to pistoia’s hinterland in a plain area (43°53’ n; 10°55’ e; 60 m a.s.l.), while the remaining four are located in high hills (44°0’ n; 10°52’ e; 550 m a.s.l.). the number of plants for each population ranged from 10 to more than 200, varying in age, gender and sexual maturity. morphological descriptor list twenty/twenty-five-year-old specimens belonging to a subset of 4 putative populations were randomly defined in order to perform a morphological description. the considered populations were derived from seeds of different origin (unknown, dutch fair, spanish fair, local selected progeny) and grown in three private nurseries, located very close to each other in a plain area under the same organic regime. local selected progeny refers to seeds collected from a couple of old trees located in villa lodolo (s. marcello pistoiese, 44°03’ n; 10°47’ e; 623 m a.s.l.). these trees were introduced from argentina in 1920 and represent the main genetic source of a. araucana local germplasm. climatic data, i.e. atmospheric pressure (ap), medium (avg t), maximum (max t) and minimum (min t) air temperatures, relative air humidity (rh), wind run (wr), global horizontal irradiation (ghi), rainfall (rain), evaporation (ev), for the same area were collected monthly from january 2015 to december 2017 (table 1). minimum, maximum, average, standard deviation and coefficient of variability (%) values of each morphometric character were calculated for the whole set of plants by using one-way analysis of variance (anova). this in turn led to the definition of classes for all the measured traits by subdividing the total range into intervals 2 times the standard deviation, i.e. whether above or below the average value of each parameter as described in bassi (2003). statistical analysis was performed using spss 20 software (chicago, il, usa). table 1 seasonal averages (2015-2017) of main meteorological data collected in pistoia’s nursery areaa ap= atmospheric pressure; avg t= medium air temperature; max t= maximum air temperature; min t= minimum air temperature; rh= relative air humidity; wr= wind run; ghi= global horizontal irradiation; rain= rainfall; ev= evaporation. a processed from ce.spe.vi database, pistoia (http://www.cespevi.it/meteo.htm; paolo marzialetti © 1996/2018 ce.spe.vi. pistoia). year season ap (mbar) avg t (°c) max t (°c) min t (°c) rh (%) wr (km) tsi (kwh/m²) rain (mm) ev (mm) 2015 winter 1009.33 8.33 14.73 3 68.33 86.7 1.53 2.7 1.6 spring 1011 18.7 26.67 11.03 62.33 73.37 3.93 1.73 4.9 summer 1009 24.77 33.13 16.87 57 69.6 4.17 1.23 6.5 fall 1016.33 11.5 18.07 6.97 78.33 33.73 1.17 3.67 1.07 2016 winter 1007.33 9.1 14.9 4.47 74 77.6 1.37 5.9 1.4 spring 1007 16.83 25.2 9.83 65 77.9 3.7 2 5 summer 1010.67 24.2 33.03 16.37 57.67 66.9 4.2 1.57 6.5 fall 1014.67 10.43 18.3 5.1 78 24 1.3 3.57 1.03 2017 winter 1012.33 8.47 15.77 2.43 67.33 66.4 1.67 3.53 1.77 spring 1009.67 19.5 27.7 11.53 60 76.03 4.07 1.4 6.3 summer 1009.33 23.67 32.47 15.43 57 65.6 4.03 1.7 6.43 fall 1011.33 9.93 18 4.3 76 28.3 1.27 5.13 1.1 adv. hort. sci., 2019 33(2): 283-294 286 3. results phenology the results of our observations throughout the growing cycles of male and female a. araucana trees in the pistoia district are shown in figure 1. the main collected phenological data (flowering onset, flower maturation, fertilization, fruit ripening and seed production) were compared with those found in previous published surveys on native andean populations (table 2). morphological descriptor list starting from field observations over a three-year growing cycle, a total number of 39 descriptors were developed for further germplasm phenotyping, divided into 6 sections: 1) tree, 2) branches, 3) leaves, 4) male inflorescences, 5) female strobiles, 6) seeds and p r o d u c t i v i t y . t h e s e d e s c r i p t o r s a p p l y t o twenty/twenty-five-year-old trees (i.e. approx. the age of first fruit bearing) grown in nurseries as ornamental plants. the descriptor list has been enriched by images and drawings for a better understanding fig. 1 araucaria araucana male (♂) and female (♀) phenological stages under tuscan climate conditions. table 2 differences between the maturation stages of female and male inflorescences and seed production in araucaria araucana populations in their origin area and in italy i = first year; ii second year; iii = third year. a tortorelli, 1956; montaldo, 1974; donoso and cabello, 1978; rodriguez et al., 1983; marticorena, 1995. phenological phases chile/argentinea italy ♀ ♂ ♀ ♂ flowering onset nov. i aug. i / sep. i mar. i jul. /aug. i maturation dec. i dec. i may i may ii fertilization jan. ii apr. ii seed production dec. ii / feb. iii sep. / oct. ii antonetti et al. auracaria araucana. first insight 287 of the less familiar and discernible traits. descriptor list examples a 288 adv. hort. sci., 2019 33(2): 283-294 4. discussion and conclusions very few studies have examined botanical aspects in a. araucaria following individual plants over their lifetimes, and all are rather dated and referred to plants grown under south america climatic conditions (montaldo, 1974; donoso and cabello, 1978; hoffman, 1982; rodríguez et al., 1983). no data concerning the phenology and growing of this species under both european and italian environmental conditions have been reported elsewhere, except for a very old contribution to the understanding of cytology and sexual reproduction in a. araucana plants grown in northern france (favre-duchartre, 1960). søndergaard (2003) reported about new introductions of monkey puzzle to scandinavia and the west coast of norway, while kubus et al. (2014) evaluated hardiness of a. araucana trees grown in open ground in poland. however only data on annual shoot growth, tree height and degree of frost damages were given. on the other hand, phenological observations are some of the most sensitive data in identifying how plant species respond to regional climate conditions and to climatic changes. l i k e a l l g y m n o s p e r m , m o n k e y p u z z l e h a s extremely simple flowers without any ornamental value. the male and female reproductive structures are carried by ‘cones’ and are as a rule separated, in a araucaria draws have been taken and modified from the website http://www.eryprihananto.com (© ery prihananto, indonesia). b mean of 25 seeds. antonetti et al. auracaria araucana. first insight 289 fact monkey puzzle is usually dioecious (martinez, 1957; bekessey et al., 2002). nevertheless, although being basically female, 4 out of 15 flowering trees were found to produce both male and female cones during recording in southwestern of norway; moreover, on completely isolated male and female trees, a couple of cones of the other sex were observed in some years in the northernmost area (søndergaard, 2003). the author suggested a possible correlation between stress (by isolation and climatically exposed situations) and monoecious behavior in a. araucana. in our census, only one monoecious tree was found among all considered specimen (approx. 700 plants) of the pistoia province. the ratio of females to males in the examined populations grown in plain area was 1:4 (on a total of approx. 25% differentiated trees) being biased towards the male sex, while a rather balanced sex ratio was observed in older (approx. forty-five-year-old) totally differentiated trees grown in high hills (fig. 2). this finding is almost in accordance with sex occurrence reported by søndergaard (1975), who determined half female and half male trees in a population with 76% flowering trees in the west coast of norway. no other comparison with literature was possible for sex ratio, which has never been reported in elsewhere published data. since the relationship between tree growth and climate appeared to be sex-dependant, in that male trees were more sensitive to land precipitation and female fig. 3 araucaria araucana mature catkins during pollination. fig. 2 totally differentiated araucaria araucana female (a) and male (b) trees grown in pistoia’s high hills, showing strobiles and catkins formed in consecutive years. trees appeared more sensitive to air surface temperature during the prior period of growth (hadad and roig juñent, 2016), it is probably realistic to assume that gender imbalance in favor of male or female at the beginning of sexual differentiation might vary according to different climatic environments, such as those found in norway and italy. on the contrary, day length did not seem to have a strong influence on the growth and development of the monkey puzzle (søndergaard, 2003); as a matter of fact, day l e n g t h d e p e n d a n t t r e e s w o u l d n e v e r s u r v i v e scandinavian latitudes, since growth would begin too early and cease too late causing extensive frost damage and eventually killing the plant. under the tuscan environmental conditions, male catkins start out erect in july-august, stop growing during the winter season and then become elongated in shape, pendant and reddish-brown at maturity in may of the following year. formed by many small scale-shaped leaflets, called microsporophyllus, they gave rise to a large quantity of pollen. in barely adult trees male caktins are found mostly in groups of 1-3 cones (fig. 3), while older adult trees have groups of 2 up to 7-8 cones. first differences in the apex of potentially vegetative or female flower buds become visible at the end of march (fig. 1d). in flower buds the apex develops into a round dome of 3-4 cm, with more elongated and less tight scales provided with long yelloworange appendixes. female inflorescences, having f e r t i l e s c a l e s w h i c h c o n t a i n t h e o v u l e s , c a l l e d macrosporophylls, are distinguishable in april (fig. 1e), grouped in light green strobiles at the extremity of the new sprouts. greatest frequency of full bloom adv. hort. sci., 2019 33(2): 283-294 290 and pollination was observed in may (fig. 1c and 1f). some differences were found in flower appearance among growing areas. generally, flower buds developed two up to three weeks later when trees were grown at higher altitudes and experienced rigid winters. after fruit set, that occurs in april of year ii (fig. 1g), the sessile and generally solitary and immature female strobiles are erect, globular, with a symmetrical shape, and green colored. they take usually about 4-5 months to develop into ripe globular dark brown mature cones (fig. 1i) and remain closed until the complete maturation of the seeds. scales usually fall off at maturity in august-september of the same year, although an ongoing trend towards the postponement of fruit drop towards november-late december was observed as a consequence of the gradual rise in mean temperatures (fig. 4). as already assessed in previous studies (søndergaard, 2003; sanguinetti, 2014), cones occurrence was found to vary in relation with sun exposure; more in details, cones were most abundant in the south part of the crown. almost all trees showed a marked alternate bearing; especially in males, yearly fluctuation in cone number seemed too large to be imputable only to climatic variations among years, particularly evident for rainfall (fig. 5). alternate bearing was found to be much more pronounced in barely sexually differentiated trees compared to plants older than fifty years, suggesting that in young plants alternate bearing represents a strategic mechanism to save nutrient reserves for significant vegetative growth. approx. 200-250 seeds, reddish to brown and oblong to obconical in shape, were released from each strobile; these range of seeds is fully included in t h o s e f o u n d o u t f ro m t h e l i t era t u re ( 150 3 0 0 ) (montaldo, 1974; donoso and cabello, 1978; salazar et al., 2000). negative effects of heavy rainfall on pollination and seed production have been reported (sanguinetti et al., 2002). fructification began when plants were twentytwenty-five-year-old, partially in agreement with reproductive organs appearance reported by salazar et al. (2000) in chilean a. araucana plants, while trees in neuquén-argentina have been reported to become sexually mature after thirty years of age, once the trunk has reached a diameter larger than 20 cm (muñoz ibáñez, 1984). conversely, according to søndergaard (2003), flowering was not initiated before the trees were forty/fifty-year-old in northern europe and this discrepancy might be related to the altitude and latitude difference. the ontogenetic stages presented here (fig. 6) were in accordance with the a. araucaria biological fig. 4 minimum, maximum and average temperature values a n d t r e n d s i n p i s t o i a ( y e a r s 1 9 5 1 2 0 1 7 ) (http://www.cespevi.it/meteo.htm; paolo marzialetti © 1996/2018). fig. 5 annual rainfall values and trends in pistoia (years 19512 0 1 7 ) ( h t t p : / / w w w . c e s p e v i . i t / m e t e o . h t m ; p a o l o marzialetti © 1996/2018) fig. 6 schematic representation of the ontogenetic cycle of araucaria araucana. solid lines represent diploid phases (female and male gametogenesis) and embryogenesis after fertilization, while dashed lines represent haploid phases (ovules and pollen). (from favre-duchartre, 1960, modified). antonetti et al. auracaria araucana. first insight 291 cycle highlighted by favre-duchartre (1960) in northern france; moreover, differences in springtime temperatures between france and norway corresponded fairly well to the differences in time (about one month) for release of pollen and seedfall at the two sites, as reported by søndergaard (2003). on the contrary, as shown in table 2, a. araucana phenology was found to differ strongly from that observed in its n a t u r a l d i s t r i b u t i o n i n t h e a n d e s m o u n t a i n s . obviously, differences between months were principally due to the reversal of the hemispheres and consequentially of the seasons: when it is spring in europe, it is autumn in the andes and vice versa. however, changes seemed probably to be associated also to other factors, such as altitude, climate changes, growing conditions, etc. the effect of climate on the phenology, and in particular on the timing of reproduction, is well known for plants and extensively documented (beebee, 1995; stenseth and mysterud, 2002). these studies have demonstrated that onset of reproduction in spring may have advanced by a week or two due to recent changes in climate over much of europe, but to a much lesser extend in south america (walther et al., 2002; stenseth et al., 2002). contrary to expectations, it was noticed that in males the appearance of the catkins takes place approximately in the same months (august-september in chile and july-august in italy), which, however, correspond to the end of summer in italy and the end of winter in chile. moreover, in chile the male catkins reach full maturity within 3-4 months, whereas in our environmental conditions full bloom occurred within 9-10 months. this could be explained by the fact that in italy the f u r t h e r d e v e l o p m e n t o f m a l e i n f l o r e s c e n c e i s stopped during the winter, while in chile, where the season turns towards spring, caktins keep growing without interruption. on the other hand, maturation seems to be related to the photoperiod coinciding in both hemispheres with long days in late spring (beginning of december in chile and end of may in italy). similar considerations can be drawn for females as well. the time between female flower appearance (march in italy and november in chile) and maturation (end of may in italy and beginning of december in chile, as for males) is about 3 months in our country and 1 month in chile. in both cases, the entry of the pollen tube into the ovary takes place within a month from the pollination. on the other hand, in our experimental conditions the true fertilization has been reported to occur after approx. 11 months (april of year ii) from pollination (favre-duchartre, 1960), whereas timing of fecundation has never been mentioned in the literature under andean environments. there are contradictory records about the time of seed maturation in chile, as it is unclear if fruits release the seeds after 11-13 months (donoso and cabello, 1978) or 16-18 months (tortorelli, 1956; montaldo, 1974) from pollination. data herein obtained showed that gravity seed dispersal, which u s u a l l y t a k e s p l a c e a t t h e e n d o f t h e s u m m e r (approx. 12-13 months after pollination), moved towards autumn (14-15 months after pollination) in 2016, and then towards early winter (16-18 months after pollination) in 2017. descriptor lists include the basic description of the traits, and the different classes of their expression (characterization) or how to measure the range of their variation (evaluation). most of the descriptors for characterization and evaluation are species-specific, but should be preferentially evaluated under homogeneous growing conditions in order to obtain comparable data and to avoid potential environmental influence on the phenotype. in our study, in order to find the most appropriate descriptors able for distinguishing effectively between individual phenotypes without loss of discriminating power, repeated observations were made on all plant material available in the pistoia’s nursery district. it immediately became clear that there were no evident differences among the expression of growing characters of young potted seedlings (fig. 7), despite the very high number of individuals within the various collections, whereas a high degree of betweenand within-population variability was noted among older trees (fig. 8). a first analysis of the available specimen clearly evidenced that plant habitus as well as various other vegetative traits consistently varied with plant age, fig. 7 araucaria araucana seedlings in pistoia’s nurseries (a: two-year-old seedlings; b: six-year-old seedlings). adv. hort. sci., 2019 33(2): 283-294 292 being this species a long-lived and massive tree up to 50 m tall and 2 m in diameter and attaining maximum ages of at least 1300 years (montaldo, 1974). moreover, different climatic patterns and growing conditions, such as plant density and weed control, might have influenced plant growth and habitus as well. the variability observed here is consistent with the outbreeding dioecious reproductive habit of this species and suggests that these populations should continue to be viable and able to respond to moderate levels of environmental change (hadad and roig juñent, 2016). however, recent phytopathological analysis in tuscany have revealed an increase in mortality, especially of female individuals, without a specific pathogen responsible (rizzo, 2017, personal communication). the most widely accepted theory is that weaker plants, due to climate-related stress, are more vulnerable to damage caused by aspecific pathogens. well as plants grown in nearby locations. with respect to the 39 descriptors detailed in the descriptor list, some very peculiar characters, i.e. insertion angle of the scales on the trunk (n. 8), uniformity of the apparent diameter on the primary and secondary branch (n. 19 and 20), catkin bending (n. 26), were individuated beside some of the most common traits, such as shape and density of canopy (n. 3 and 4), maximum length and width of the leaves (n. 22 and 23). undoubtedly, one of the tree’s most distinguishing feature is its scales, which are stiff, dark green and glossy with a spiny tip and completely cover each branch, closely overlapping each other. but we found surprisingly interesting the size together with the insertion angle of the scales on primary and secondary branches, resulting in evident dissimilar apparent diameters of branches. on the contrary, female inflorescence didn’t show any distinctive feature among populations, therefore it was not taken into account as a descriptor. in fact, while the true flower represents the main distinctive character in magnoliophyta, the pinophyta flowers, that are extremely simple, are not particularly relevant for the description of the species. in our study, morphological traits of a. araucana were observed, measured and documented for the first time under tuscan growing condition. in particular, the research was developed using trees from putative populations growing in the pistoia’s nursery district. the comparison of the observed phenotypic characteristics showed a wide range of variability among and within the considered populations. the resulting data allowed to classify accessions, and to build a catalogue of specific descriptors with embedded biological information that is an essential step towards germplasm phenotyping (in particular new variety description), management or for direct use in agriculture. limitations linked to the potential environmental influence on the phenotype are presented as well. apart from this preliminary study, nothing is known of the patterns of morphological variation within this species. the development of this descriptor list will assist in the systematic and objective recording and exchange of information, which in turn will increase utilization of genetic resources along with a better screening and use of a. araucana biodiversity for breeding programs. in order to analyze relationships between individuals or groups of specimens within locally grown populations, the morphometric characterization of a. araucana trees is in progress based on the defined descriptor list and suitable statistical multivariate approaches. genetic fig. 8 habitus variability in araucaria araucana trees grown in pistoia’s hinterland in a plain area. a correct germplasm characterization should consider adult plants, being necessary the presence of flowers and fruits. however, as the aim of the present study is the phenotyping of young cultivated plants for sale, only trees sufficiently young for trade but at the beginning of sexual differentiation were suitable for characterization. this is why only twenty/twenty-five-year-old specimen belonging to a subset of 4 putative populations grown under similar environmental and agronomic conditions were considered. plants over 25 year-old were discarded as antonetti et al. auracaria araucana. first insight 293 analysis of the same populations are being performed and will be processed in order to validate the discriminating efficiency of the presented descriptor list. acknowledgements authors acknowledge to the vivai bartolini, azienda macchia tommaso and the kind collaboration of andrea tozzi (villa lodolo) for providing plant material and hystorical information. this research was supported by the ministero d e l l e p o l i t i c h e a g r i c o l e a l i m e n t a r i e f o r e s t a l i (mipaaf-oiga), project caraviv “characterization of araucaria araucana germplasm selected by the nursery industries of the pistoia’s district for commercial development”. references bassi d., 2003 growth habits in stone-fruit trees. cnr, roma, italy. beebee t.j.c., 1995 amphibian breeding and climate. nature, 374: 219-220. bekessy s.a., allnutt 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convey p., menzel a., parmesan c., beebee t.j.c., fromentin j.-m., hoeghguldberg o., bairlein f., 2002 ecological responses to recent climate change. nature, 416: 389-395. impaginato 215 adv. hort. sci., 2019 33(2): 215-226 doi: 10.13128/ahs-23924 the use of organic nano-supplements of fertilizer for lily forcing period a. hatamzadeh 1, s.-s. shafiei-masouleh 2 (*) 1 department of horticultural science, faculty of agricultural sciences, university of guilan, rasht, iran. 2 department of genetics and breeding, ornamental plants research center (oprc), horticultural sciences research institute (hsri), agricultural research, education and extension organization (areeo), mahallat, iran. key words: amylases, chitosan, forcing, flowering, magnetite, magnetic supplement of fertilizer, sugars. abstract: lilium is one of the most important ornamental plants after roses, carnations and chrysanthemum in the world that is requested as potted of cut flowers. it is so important to consider its quality along with its production rate in terms of yield (quantity). however, it always needs to intend the product costs aside from quantity and quality. fertilizer utilization is so important and this may be improved by compounds that promote it and also have synergetic effects themselves. we examined both carboxymethylated chitosan (cmc) and magnetic nano-carbocymethylated chitosan (mncc) to produce lilium bulb and advised them especially magnetic chitosan. in this study, these compounds (magnetic and non-magnetic chitosan) at concentrations of zero, 2.5, 5, 10 and 15 mg/l were examined during lily forcing in three cultivars, including cherbourg, navona, brunello, which are from asiatic and oriental lilies. the results showed that highest concentrations (10 and 15 mg/l) among between examined concentrations regardless of compounds types and cultivars did not make toxicity and had significant effects on plants biology and physiology [contents of carbohydrates and enzymes affecting these carbohydrates (amylases)]. however, for observing morphological changes may be need to use higher concentrations of these compounds. note that this needs to examine the non-toxicity of higher concentrations in future studies. 1. introduction flowers have always had a valuable place among different classes of society and various ceremonies and rituals. lilium is one of the most important ornamental plants after roses, carnations and chrysanthemum in the world that is mostly used as cut flowers throughout the year. furthermore, it is used as potted plants in gardens and green landscapes (shafiee-masouleh et al., 2014). it is necessary to consider the nutrition of lilies like other plants in the greenhouse production. and also, optimization and improvement of photosynthetic efficiency of plants can be effective in increasing photosynthetic storages and thus increasing its quanti(*) corresponding author: shafiee.masouleh@areeo.ac.ir citation: hatamzadeh a., shafiei-masouleh s.-s., 2019 the use of organic nano-supplements of fertilizer for lily forcing period. adv. hort. sci., 33(2): 215-226 copyright: © 2019 hatamzadeh a., shafiei-masouleh s.-s. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 14 septmeber 2018 accepted for publication 18 february 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(2): 215-226 216 tative and qualitative yields in terms of flower number, plant size and vase life. different groups of lilies (asiatic, oriental and l. longiflorum) have different nutritional requirements. for example, asiatic lilies has been reported that have the best growth when are fertilized by lower levels during forcing (treder, 2003). chitosan particles in nano-scale can be important in the delivery of drugs in medicine, because these compounds promote the absorption of active molecules or compounds through the cell membrane and allow organs to have bioavailability to molecules. chitosans can be used as encapsulated nanoparticles or used directly and therefore, the surface of these particles with chelating structure of the modified chitosan can play an important role in sustainable agriculture (kashyap et al., 2015). magnetic chitosan nanoparticles with the effect of chelating elements in the experiment conducted by shafiee-masouleh et al. (2014) increased the biomass of lilium and the storage organs (bulbs) during bulb production. the effect of magnetic fluids on living systems has been studied both in medicine and in the world of plants, and it has been shown that magnetic fluids can be effective in the production of calluses and metabolic activities. the effects of magnetism on plants by earth’s magnetic field, pulsed and inductive constant magnetic fields, an electromagnetic field, and effects of magnetic nanoparticles have been studied by researchers and theirs roles have been confirmed to enhance photosynthesis and growth and development of plants (pavel et al., 1999; răcuciu and creangă, 2007 b; răcuciu et al., 2009; shafieemasouleh et al., 2014). it has also been shown that superparamagnetic nanoparticles, which are as permanent magnets effected by external magnetic fields or ambient temperature of their environment, affect membrane systems and membrane ion exchange (pavel and creangă, 2005; faeghi and seyedpour, 2013). many researchers investigated the effects of chitin and the chitosan polymers and oligomers as spraying, soil application or seed treatment and fertilizer treatments, they reported that photosynthesis and plant growth were enhanced (el-tantawy, 2009; dzung et al., 2011; farouk and amany, 2012). the allocation of carbon in plants is important that is affected by various factors such as the level of photosynthetic compounds, the number and size of competing sink (flowers, seeds, fruits, bulbs and tubers, etc.) and their location in plants and the potential for initial storage in the leaves and re-translocation in plants. understanding the components that affect the initiation and development of organs and the balance between source and sink organs are essential to regulate the allocation of assimilates (du toit, 2001). the chl content of the leaf affects photosynthesis, and chl a/b is the best index to understand the photosynthetic capacity and direct information on the activity of the enzymes involved in the photosystem ii in the chloroplast membrane (răcuciu and creangă, 2007 b; răcuciu et al., 2009). iron deficiency reduces the amount of photosynthetic pigments. in addition, the electron transfer in the photosystems i and ii undergoes a change. also, the activity of 1, 5-d-phosphate-carboxylase and the photosynthetic function of plant decrease. therefore, it is necessary to provide iron for the plant, because the biochemical properties of iron and its effect on the metabolic pathway of the plant are important. uptaking iron in alkaline soils is difficult; this is due to the formation o f f erri c h yd ro x i d e i n th e p res en ce o f o x ygen , because plant cannot uptake it (thoiron and briat, 1999). the main component in reducing no3absorption is iron deficiency. ferrous enzymes (nitrite and nitrate reductases) affect the no3absorption. in a d d i t i o n , g r o w t h o f p l a n t s d e c r e a s e s d u e t o decreased no3absorption and the synthesis of metabolites (proteins, nucleic acids, chlorophylls, etc.) (borlotti et al., 2012). the use of chelating agents is useful to remove iron uptake problems by plants (abadía et al., 2011). magnetic fluids at suitable concentrations have a positive effect on the photosynthetic capacity of plant. iron ions in the structure of magnetic fluids can be an important source of iron for the development of plants (răcuciu and creangă, 2007 b). iron is not in the chl structure, but it is one of the essential elements to synthesize chl (răcuciu et al., 2009). in general, magnetic fluids are nanoparticles dispersed in water or in a hydrocarbon fluid such as citrate. biocompatibility of magnetite (fe3o4) has been confirmed (răcuciu et al., 2009). however, treating plants with electromagnetism or any other magnetic field with these particles is destructive and changes the genotype and phenotype of plants and causes chromosomal deviations (pavel et al., 1999; pavel and creangă, 2005; răcuciu and creangă, 2007 a, b; răcuciu et al., 2009). chitosan is one of deacetylated derivatives of chitin. as a natural polymer is abundant and can be degraded by biological agents and it can be used in agriculture. this molecule is environmentally friendly and non-toxic that is used in the formulation of slow release fertilizers (wu and liu, 2008). in vitro use of chitosan in a suitable concentration in vitis vinifera l. hatamzadeh and shafiei-masouleh organic fertilizer for lilium 217 stimulated the photosynthesis and increased plant growth with increasing root and shoot biomasses. it also protected the plant against botrytis cinererea fungus and cytological changes (barka et al., 2004). we utilized the effects of chitosan, as nanoparticle with magnetic properties on plant growth and development in lily bulb production (shafiee-masouleh et al., 2014) and cucumber (the data were not published yet). modern agriculture should look for factors that while having positive effects as well as its use to be easy, affordable and reasonable. it is not possible to use a large magnetic field in farms. also, use of macromolecule of chitosan as foliar application will cause stomata obstruction and reduce gas exchange and photosynthesis. therefore, the achievement of a soluble compound or nano-structured composite with the synergic effects of two compounds (magnetite and chitosan) will be very valuable at the same time. to achieve these purposes, this research studies the following: i) increasing yield and post-harvest quality of cut lily by increasing the efficiency of nutrient uptake and photosynthetic performance of the plant; ii) institutionalization of chitosan use at the nanoparticle scale during forcing of lily flowers; iii) investigating the effect of magnetic nanoparticles on yield and quality of lily flowers; and iv) physiological and morphological understanding of the effect of chitosan nanoparticles on the yield and quality cut flowers of lily. 2. materials and methods plant material and cultivation conditions in this study, bulbs of three cultivars of lilium, including brunello (asiatic, 16-18 cm in circumferences), navona (asiatic, 16-18 cm in circumferences) and cherbourg (oriental, 18-20 cm in circumferences) were used to study effects of two organic supplements of fertilizer (osfs). after melting of vernalizing substrate of bulbs, the bulbs were disinfected with 0.1% benomyl fungicide for 15 min and dried in the air for one day. thirty uniform bulbs in size were selected per cultivars to force. the culture substrate contained cocopeat and perlite (1:1, v/ v) that was completely homogeneous. the bulbs were planted in one-kilogram plastic bags with some pores into 10 cm depth and irrigated with tap water until emergence. after emergence every 23 days depending on the weather and substrate conditions were fertigated. greenhouse experiments in the research greenh o u s e o f t h e f a c u l t y o f a g r i c u l t u r a l s c i e n c e s , university of guilan, rasht, iran in latitude of 37° 12’ 3.94’ n and longitude of 49 38’ 55.78’ e were performed. preparation of fertilizer supplements c a r b o x y m e t h y l a t i o n o f c h i t o s a n . t h e c a r boxymethylated chitosan (cmc) was prepared from chitosan [poly (d-glucosamine), (c 6 h 11 no 4 ) n , deacetylated chitin] with low molecular weight 100,000300,000 (acros, acros organics, geel, belgium) to increase solubility of chitosan by carboxymethylation. the procedure of preparing of cmc was presented by shafiee-masouleh et al. (2014). an ir (ir470, shimadzu, kyoto, japan) (fig. 1) was used to analyze the produced h-form cmc (not salt containing na ion). preparation of nano-particles magnetite according to shafiee-masouleh et al. (2014), nano-particles of fe3o4 were synthesized. fecl3 (merck, germany) and fecl2.4h2o (merck, germany) were used to prepare fe3o4. the whole procedure of preparing fe3o4 can be observed at the mentioned paper. preparation of magnetic nano-carboxymethylated chitosan the magnetic nano-carboxymethylated chitosan (mncc) was prepared with encapsulation of fe3o4 by carboxymethylated chitosan (cmc) according to fig. 1 ir spectrum of carboxymethylated chitosan. this spect r u m s h o w s t h a t t h e c a r b o x y l m e t h y l g r o u p s (-ch3cooh; 1741 cm-1; stretching vibration feature of c=o groups) resulted from monochlroacetic acid salt are linked to o atom in -oh group, and -nh2 exists on carboxymethylated chitosan. adv. hort. sci., 2019 33(2): 215-226 218 shafiee-masouleh et al. (2014). this was carried out by carbodimiide (merck, germany) and saline phosphate buffer in sonication conditions. the details of laboratory procedures can be studied in the mentioned paper. analyses of the size and morphology of nano-particles were performed with sem (fig. 2), and ir and xrd were used to identify the coating and structure of mncc (figs. 3 and 4). statistical design a split plot factorial experiment was conducted in a completely randomized design with 15 main plots and 9 (1 + 8) sub plots. the main factors included three lilium cultivars and sub factors were two types of compounds as organic supplements of fertilizer (osfs); including carboxymethylated chitosan (cmc) and magnetic nano-carboxymethylated chitosan (mncc) both of them at concentrations of 0, 2.5, 5, 10 and 15 mg/l. one group of zero concentration was considered as control treatment for both of compounds. the experiments were carried out by 5 replications and with 135 pots in the research greenh o u s e o f t h e f a c u l t y o f a g r i c u l t u r a l s c i e n c e s , university of guilan, iran. at the end of forcing period, the growth and morphological characteristics were measured for each plant (in puffy bud stage: when first flower bud showed the color and little opening in top of bud) and were harvested at a height of 45 cm from the final flower bud and transferred to the laboratory. after the stems were cut diagonally under water, they were placed into a 1000 ml bottles containing 500 ml distilled water and transferred to the growth chamber to study the postharvest life and biochemical properties. variance analysis of data was measured using sas software (version 9.1, 2003). mean comparisons were performed with tukey’s test at probability levels of 5 or 1% according to variance analysis. 3. results and discussion magnetic nanoparticles (pavel et al., 1999; pavel and creangă, 2005; răcuciu and creangă, 2007 a, b; răcuciu et al., 2009) in maize and barley, constant magnetic field (dhawi et al., 2009) in a palm tree, pulsed magnetic field (radhakrishnan and kumari, 2012) in soybeen, chitosan (górnik et al., 2008) in grape and chitosan oligosaccharides (dzung et al., 2011) in coffee seedlings have been investigated. all of researchers reported an increase in mineral uptakfig. 2 the image of magnetic nano-carboxymethylated chitosan particles by sem microscopy. size and morphology of fe3o4 coated by carboxmethylated chitosan. fig. 3 ir spectrum of magnetic nano-carboxymethylated chitosan. transferring the absorbance band of -nh2 group from 1589 cm-1 to 1602 cm-1 and increasing absorbance intensity in wavelength number 1741 cm-1 show ester bond into some parts of carboxy groups in carboxymethyl chitosan on the surface of fe3o4 nanoparticles. fig. 4 xrd pattern of magnetic nano-carboxymethylated chitosan. the widths of large peaks report crystal properties of magnetic nanoparticles. the average of particles sizes based on debye scherrer equation was calculated 10 nm. hatamzadeh and shafiei-masouleh organic fertilizer for lilium 219 unlike variance analysis, significant differences between three factors were observed on the vegetative length, total length and chl intensity. differences between treatments on vegetative development showed that morphological characteristics are less affected by the experimented osfs and their concentrations or these compounds may have decreasing effects on these characteristics. in cherbourg cultivar, the highest length of vegetative stem was observed in the absence of osfs or at low concentration (5 mg/l) of cmc, which showed a significant difference with application of 2.5 mg/l cmc in navona. of course, these differences were not significant coming by the effects of magnetism or chitosan, separately. based on the results of 3-way anova test (table 1), effects of osfs on vegetative development of three lily cultivars due to the uniform variances of data around the mean and type i error did not show a significant differences for 3-way interactions between cultivars, compounds and their concentrations. only a significant 2-way interaction (between cultivar and concentrations of osfs) was observed on the chl intensity by anova test. and also, it was observed significant differences between cultivars (table 1). n=5. the means with similar letters have not any significant differences at hsd0.01. w hsd0.05 y type i error **, *, ns = significance at p≤0.01 and p≤0.05 and non-significance, respectively. table 1 effects of osfs on vegetative development of three lily cultivars cultivars (a) treatments vegetative length (cm) total length (cm) w chl intensity (spad index)osfs (b) cons of osfs (mg/l) (c) cherbourg cmc 0 56.08±2.39 a 84.16±2.51 a 46.62±1.76 efg 2.5 54.48±5.14 ab 81.36±5.40 ab 48.52±2.43 defg 5 57.70±2.14 a 84.60±3.52 a 42.46±2.74 ef 10 52.30±3.31 ab 79.80±3.98 ab 42.16±2.22 ef 15 54.10±1.49 ab 81.40±3.34 ab 46.70±2.35 efg mncc 0 56.08±2.39 a 84.16±2.51 a 46.62±1.76 efg 2.5 55.30±2.91 ab 82.00±3.29 ab 46.50±3.21 efg 5 53.00±3.41 ab 81.40±4.41 ab 46.32±1.99 efg 10 53.12±1.36 ab 80.60±1.93 ab 39.98±2.83 g 15 50.84±2.73 ab 82.20±2.65 ab 46.02±1.99 efg navona cmc 0 48.30±2.23 ab 81.20±3.01 ab 59.52±0.31 abcd 2.5 47.60±2.01 b 78.00±1.92 ab 60.16±1.54 abcd 5 50.80±2.00 ab 81.80±1.57 ab 61.60±0.58 abc 10 50.30±1.51 ab 81.90±2.65 ab 61.98±1.15 ab 15 46.20±1.83 ab 76.00±2.07 ab 61.40±1.57 abc mncc 0 48.30±2.23 ab 81.20±3.02 ab 59.52±0.31 abcd 2.5 39.60±0.80 ab 69.90±0.48 ab 57.10±3.82 abcde 5 49.30±2.22 ab 80.00±1.25 ab 60.16±1.13 abcd 10 45.60±1.83 ab 75.30±2.33 ab 62.72±1.83 a 15 50.10±1.86 ab 80.00±3.58 ab 57.86±0.97 abcde brunello cmc 0 50.90±2.24 ab 70.70±2.29 ab 53.38±3.54 abcdefg 2.5 49.10±4.14 ab 68.50±5.34 ab 51.10±2.14 abcdefg 5 50.90±3.12 ab 69.20±4.53 ab 50.12±1.74 abcdefg 10 52.60±4.01 ab 72.30±5.27 ab 53.76±1.81 abcdef 15 49.50±1.90 ab 66.80±2.81 b 49.34±0.50 bcdefg mncc 0 50.90±2.24 ab 70.70±2.89 ab 53.38±3.54 abcdef 2.5 54.50±2.23 ab 77.30±1.72 ab 49.70±0.61 bcdefg 5 49.90±2.14 ab 69.80±2.18 ab 51.84±1.78 abcdefg 10 50.90±3.60 ab 69.60±3.14 ab 51.60±1.68 abcdefg 15 52.80±3.18 ab 71.80±3.99 ab 49.22±2.08 cdefg anova (3-way) a ** ** ** b ns ns ns c ns ns ns a×b ns ns ns a×c ns ns * b×c ns ns ns a×b×c ns y ns y ns y cv (%) 10.46 8.68 8.58 220 adv. hort. sci., 2019 33(2): 215-226 pared to other treatments. total length showed similar reaction with vegetative length to treatments but the least value was observed in brunello treated by 15 mg/l cmc. however, chl intensity under effects of treatments showed the most value in navona treated by 10 mg/l mncc and the least were observed in cherbourg treated by 10 mg/l mncc (table 1). it seems that morphological characteristics of cultivars treated by two types of osfs in different concentrations are under dominant effect of cultivar; on the other hand, morphological characteristics of different lily cultivars show the various reactions to osfs and their concentrations. generally, from the above observations on vegetative development, it can be interpreted that firstly, size of the plant is affected by genotype, and secondly, magnetism may increase the synthesis of chl and chl density, but it does not affect the size of the shoots and this needs to be investigated more with higher concentrations. nguyen van et al. (2013) stated that chitosan nanoparticles can easily penetrate into plant cells and increase the biological activity of cells. they pointed to an important effect of chitosan nanoparticles on the biophysical properties of the coffee plant such as increasing chl content and plant growth and development. răcuciu and creangă (2007 b) used low-density (less than 100 μl/l) ferrofluids without electromagnetic treatment or any other magnetic field for corn seedlings. they observed that due to their super-paramagnetic effects (a special effect of magnetic nanoparticles) on the structure of photosynthetic enzymes, growth was enhanced. răcuciu and creangă (2007 a, b) and răcuciu et al. (2009) used two fluids of magnetic nanoparticles of fe3o4 coated with or without tetraethylammonium hydroxide in petri dishes for germination of corn seeds without the use of a magnetic field (50 μl per liter). they observed that plant height increased, but when the concentration increased, toxicity was reported. they referred that magnetic iron is a source for plant growth. in addition, magnetism has a good effect on photosynthesis. el-sayed (2014) reported the increasing effect of magnetic water treatment on plant growth of vicia faba, chl a and b, and carotenoids, and contents of gibberellic acid and kinetin it is known that kinetin (cytokinins) is effective in preservation and prevention of the chlorophylls destruction (taiz and zeiger, 2002). farouk and amany (2012) reported an increase in chl a and total chl by spraying the chitosan (250 mg/ l) on chickpea compared to low concentrations (50 a n d 1 2 5 m g / l ) , a n d t h e i r i n t e r p r e t a t i o n a b o u t increasing the content of photosynthetic pigments was the increase of cytokinin level and stimulation of the synthesis of chlorophyll, and they interpreted this as the role of amino groups in chitosan on the mentioned processes. chlorophyll density below 40 (spad index) indicates a disruption to photosynthetic process (netto et al., 2005). however, health of the photosynthetic apparatus in all treatments, especially high concentrations of two osfs (magnetic and non-magnetic) can be seen. răcuciu et al. (2009) reported that when concentration of fe3o4 magnetic nanofluid was increased up to 300 μl/l, content of chl a decreased by about 20 mg/l of fresh weight. in addition, răcuciu and creangă (2007 b) stated that the magnetic nanofluid of fe3o4 coated with tetramethylammonium hydroxide (50 μl per liter) produced more pigments (chl a and b and carotenoid), and higher concentrations of fluids destroyed the photosynthetic process. limpanavech et al. (2008) reported that chitosan affected the expression of chloroplast genes in dendrobium orchid and altered the chloroplast size. dzung et al. (2011) sprayed chitosan at appropriate concentrations on coffee seedlings and reported an increase in content of chlorophyll and also in uptake of mineral elements (nitrogen, phosphorus, potassium, calcium and magnesium), and enhancement of photosynthesis as well as the synthesis of chlorophyll of leaves. as can be seen in table 2, 3-way interactions between cultivars, osfs and their concentrations in anova test due to type 1 error could not affect reproductive development of lily plants. however, mean comparisons showed significant effects at hsd0.01 on these characteristics. in cherbourg, the most inflorescence lengths were observed in the plants treated with 15 mg/l mncc, but in navona without significant different with cherbourg, the most lengths of inflorescence was observed in control and 10 mg/l cmc. the shortest length of inflorescence was observed in brunello. however, this cultivar with the least concentration of magnetic osf had the most length and generally, it seems that on this characteristic, the effect of genotype was dominant toward osfs and their concentrations. how to make a reaction of a plant as flower bud number affected by 3-way interactions of factors shows dominant effects of genotype toward osfs and their concentrations. we can statistically analyze each cultivar separately to observe 2-way interactions and/or single effects of osfs on the responses of lily plants. cherbourg and navona could not show response to osfs and their concentrations for bud number. hatamzadeh and shafiei-masouleh organic fertilizer for lilium 221 however, brunello reaction to osfs and their concentrations was obvious. it can be said that this cultivar could respond to the osfs compared with two other cultivars, because produced the most bud number with 2.5 mg/l of magnetic compound. in the case of bud length, although 3-way interaction showed significance, it can be seen for this morphological characteristic that the effect of genotype was dominant again, and cherbourg produced larger buds c o m p a r e d w i t h t w o o t h e r s . f u r t h e r m o r e , t h i s response was observed about flowering date, and cherbourg flowered later (table 2). from the observations of the effects of three factors, including cultivar, osfs and concentrations of them, it can be interpreted that this range of the tested concentrations could not affect vegetative and reproductive morphology, and it seems that more concentrations of osfs must be investigated in future research. it is known that one of the factors that contribute to growth of the reproductive part (flower bud length) is potassium. amino groups in the carboxymethylated chitosan and its derivatives can table 2 effects of osfs on reproductive development of three lily cultivars cultivars (a) treatments inflorescence length (cm) bud no. bud length (mm) flowering date (days)osfs (b) concentration of osfs (mg/l) (c) cherbourg cmc 0 28.08±0.56 abc 6.80±0.58 abc 116.63±0.24 a 94.60±0.24 a 2.5 26.88±0.84 abcdef 6.80±0.37 abc 116.31±0.77 a 93.00±0.77 a 5 26.90±1.57 abcdef 6.20±0.20 b 123.27±0.49 a 93.20±0.49 a 10 27.50±1.47 abcd 7.00±0.32 abc 116.14±0.55 a 92.5±0.55 a 15 27.30±1.87 abcde 6.40±0.40 abc 118.64±0.37 a 93.80±0.37 a mncc 0 28.08±0.56 abc 6.80±0.58 abc 116.64±0.24 a 94.60±0.24 a 2.5 26.70±1.09 abcdef 6.40±0.51 abc 122.06±0.51 a 93.60±0.51 a 5 28.40±1.41 ab 6.80±0.37 abc 120.39±0.58 a 93.80±0.58 a 10 27.48±0.88 abcd 6.80±0.37 abc 110.43±0.68 a 93.40±0.68 a 15 31.36±2.06 a 7.00±0.58 abc 119.86±0.81 a 94.40±0.81 a navona cmc 0 32.90±1.61 a 9.00±0.32 a 82.87±0.68 b 59.40±0.68 b 2.5 30.40±0.53 ab 9.50±0.29 ab 80.02±0.73 b 60.20±0.73 b 5 31.00±2.26 ab 9.20±0.49 a 79.80±0.49 b 59.80±0.49 b 10 31.60±1.35 a 9.00±0.63 a 81.16±0.63 b 59.00±0.63 b 15 29.80±1.11 ab 9.40±0.24 a 78.72±0.49 b 58.80±0.49 b mncc 0 32.90±1.61 a 9.00±0.32 a 82.87±0.68 b 59.40±0.68 b 2.5 30.30±0.89 ab 9.20±0.97 a 76.04±0.58 b 59.20±0.58 b 5 30.70±1.07 ab 9.00±0.32 a 77.69±0.60 b 59.60±0.60 b 10 29.70±1.07 ab 10.00±0.71 a 78.95±1.02 b 59.20±1.02 b 15 29.90±2.34 ab 8.40±0.68 a 80.34±0.60 b 59.60±0.60 b brunello cmc 0 19.80±1.41 cdefg 5.40±0.60 c 78.91±0.97 b 60.20±0.97 b 2.5 19.40±1.37 defg 5.80±0.97 b 86.39±1.47 b 61.60±1.47 b 5 18.30±1.52 g 5.80±0.66 b 79.46±1.71 b 62.20±1.71 b 10 19.70±1.38 defg 5.80±0.97 b 80.04±1.56 b 62.80±1.56 b 15 17.30±1.28 g 5.60±1.20 c 84.75±1.38 b 61.00±1.38 b mncc 0 19.80±1.41 cdefg 5.40±0.60 c 78.91±0.97 b 60.20±0.97 b 2.5 22.80±1.23 bcdefg 6.40±0.75 abc 84.40±1.59 b 62.20±1.59 b 5 19.90±0.89 cdef 5.40±0.75 c 78.47±1.17 b 60.60±1.17 b 10 18.70±0.85 fg 4.60±0.24 c 76.75±0.20 b 61.20±0.20 b 15 19.00±0.96 g 5.50±0.96 b 80.59±1.72 b 61.60±1.72 b anova (3-way) a ** ** ** b ns ns ns c ns ns ns a×b ns ns ns a×c ns * ** b×c ns ns ns a×b×c ns y ns y ns y cv (%) 17.61 6.69 1.63 n=5. the means with similar letters have not any significant differences at hsd0.01. w hsd0.05 y type i error **, *, ns = significance at p≤0.01 and p≤0.05 and non-significance, respectively. adv. hort. sci., 2019 33(2): 215-226 222 serve as a place for the absorption of some metal cations. this compound is used to remove heavy metals and water purification and this shows its chelating role (chang and chen, 2005; chang et al., 2006). chelating sites of chitosan may be effective in absorbing essential metals ions such as manganese, iron (fe+2 and fe+3), potassium, magnesium, etc. for plants. the abilities of polymer or oligomer chitosan to stimulate plant growth under in vitro conditions; v i t i s v i n i f e r a l . ( b a r k a e t a l . , 2 0 0 4 ) ; o r c h i d dendrobium phalaenopsis (nge et al., 2006); and the growth of protocorm like bodies in the dendrobium orchid (pornpienpakdee et al., 2010) have already been reported. pornpienpakdee et al. (2010) used chitosan macromolecules, which had been deacetylated up to 70%, at a concentration of 10 mg/l under in vitro culture conditions for dendrobium orchid and produced plants with more length. in addition, limpanavech et al. (2008) when used the polymer or oligomer chitosan (with deacetylated degrees of 70, 80 or 90%) at concentrations of 1-100 mg/l, they observed that polymers at concentrations of 1-10 mg/l and oligomers at concentrations of 50-100 mg/l resulted in more inflorescences production. gornik et al. (2008) applied a type of commercial compound of chitosan, biochikol 02.pc containing 2% chitosan, at concentration of 0.5% on grape cuttings and reported that root system expanded and it increased the n u m b e r o f n e w b r a n c h e s a n d t h e i r l e n g t h s . radhakrishnan and kumari (2012) reported an increase in leaf number and growth of soybean, and an increase in pod length and grain weight under the influence of pulsed magnetic field. el sayed (2014) reported that magnetic water treatment increased the sink yield in bean (seed and number of seeds per plant). he suggested this effect may be due to increased photosynthetic function of the plant under influence of magnetic treatment. ohta et al. (1999) reported an increase in the photosynthetic reservoir (flower number) in lisianthus with plant seed treatment by chitosan acid solution. limpanavech et al. (2008) also demonstrated that mechanism of chitosan effect on increasing the number of plant photosynthesis reservoirs (number of flowers) in the dendrobium orchid may be caused by effect on the development of photosynthetic apparatus and increasing the size of chloroplasts after chitosan spraying. kananont et al. ( 2010) reported that different types of chitosan as polyand oligosaccharides with varying degrees of deacetylation at a concentration of 10 mg/l are effective for in vitro culture medium of seed germination of dendrobium orchids and growth of the protocorm like bodies. they stated that chitosan amino groups may be effective factor o n t h e g r o w t h o f t h e p r o t o c o r m l i k e b o d i e s . pornpinpakdee et al. (2010) described role of concentration of different types of chitosan on growth rate of orchid dendrobium. the results of 3-way anova (table 3) show significant differences in content of soluble carbohydrates of terminal bud of inflorescence in three cultivars of lily treated with osfs and their concentrations. the highest content of glucose was significantly observed in brunello treated with 15 mg/l osfs regardless of their magnetic or non-magnetic properties. navona was in second rank in response to treatments compared to brunello. however, cherbourg under effects of osfs and their levels showed the least glucose content regardless of being magnetism or not compared with two other cultivars. fructose synthesis in the terminal bud had different reaction to the type of osfs based on genotypes. generally, the most concentrations of osfs regardless of their structures had the most effect on fructose content (table 3). sucrose approximately had similar response to glucose toward treatments, but about this carbohydrate after ‘brunello’, ‘cherbourg’ had second score for the most content not ‘navona’. starch and other sugars and proteins (enzymes) are photosynthetic products that are transported to storage organs (flowers, seeds or bulbs). iron acts as a c o f a c t o r f o r m a n y e n z y m e s , f o r m i n g p a r t o f cytochromes and involves in biochemical reactions, including respiration, photosynthetic material transfer, nitrate synthesis, nitrogen fixation, and dna synthesis (he et al., 2011). el sayed (2014) reported that photosynthetic function of bean plant increases with magnetic water treatment. he reported that contents of glucose and sucrose as well as polysaccharides in the leaves, stems and the whole plant of bean were higher toward irrigation without magnetic field. in addition, positive effect of chitosan on plant growth may be due to its effect on increasing the phosphorus content. phosphorus is an essential element in the biosynthesis and carbohydrate transfer for cell division and the formation of dna and rna (farouk and amany, 2012). according to table 4, variance analysis of osfs effects (type and concentration) on the α-amylases activities of three cultivars of lily showed the significance of 3-way interactions. osfs effects on α-amylase in ‘navona’ compared with two other cultivars showed the highest activity of this enzyme in the highest tested concentration of non-magnetic osf. hatamzadeh and shafiei-masouleh organic fertilizer for lilium 223 while, the highest activity recorded for this enzyme in ‘brunello’ was observed in magnetic osf (mncc) unlike ‘navona’. cultivar cherbourg unlike two other cultivars showed the highest activity of α-amylase regardless of being magnetic of osf with the high concentrations of two osfs, in tissue of the last flower bud of inflorescence. therefore, responses types of cultivars toward being magnetic or not of osfs were different. on the other hand, the amount of activity of this enzyme in ‘navona’ was higher regardless of the type of osf and their concentrations. according to 3-way anova, β-amylase activity in tissue of the terminal flower bud of inflorescence was significantly affected by three factors (table 4). the activity of this enzyme unlike α-amylase was more in ‘brunello’, and like α-amylase, the activity of this enzyme was more with the highest concentration of magnetic osf. the β-amylase activity in ‘cherbourg’ was like α-amylase in the same cultivar. however, in ‘navona’, this enzyme showed the highest activity affected by the both of magnetic and table 3 effects of osfs on soluble carbohydrates into the terminal bud tissue of inflorescence in three lily cultivars n=3. the means with similar letters have not any significant differences at hsd0.01. w hsd0.05 y type i error **, *, ns = significance at p≤0.01 and p≤0.05 and non-significance, respectively. cultivar (a) treatments glucose fructose sucrose osfs (b) concentration of osfs (mg/l) (c) cherbourg cmc 0 0.80±0.11 g 2.20±0.13 ghij 3.21±0.19 ghi 2.5 1.95±0.24 fg 2.21±0.18 ghij 5.70±0.28 def 5 3.54±0.18 de 3.34±0.17 efg 8.21±0.35 ab 10 4.50±0.23 bcd 3.33±0.20 efgh 8.19±0.28 abc 15 3.50±0.24 de 4.61±0.21 bcd 5.70±0.23 def mncc 0 0.80±0.11 g 2.20±0.13 ghij 3.21±0.19 gh 2.5 0.89±0.13 g 3.33±0.16 efghi 5.71±0.24 def 5 1.98±0.23 fg 3.27±0.18 efghi 5.67±0.15 def 10 3.50±0.11 de 4.60±0.28 bcd 8.23±0.28 ab 15 2.01±0.29 g 5.51±0.23 ab 8.22±0.44 ab navona cmc 0 2.64±0.22 ef 2.81±0.18 fghi 1.53±0.26 i 2.5 2.66±0.24 ef 4.03±0.24 cde 3.03±0.20 hi 5 3.49±0.22 de 4.03±0.20 cde 3.07±0.19 hi 10 5.03±0.31 abc 4.83±0.26 bc 4.53±0.25 fgh 15 5.02±0.28 abc 6.05±0.31 a 5.81±0.17 de mncc 0 2.64±0.22 ef 2.81±0.18 fghi 1.53±0.26 i 2.5 3.51±0.23 de 1.54±0.13 jk 1.59±0.15 i 5 5.02±0.32 abc 2.60±0.11 fghij 3.10±0.16 ghi 10 5.84±0.23 ab 2.75±0.19 fghi 3.06±0.19 hi 15 5.88±0.23 ab 4.02±0.17 cde 4.49±0.23 fgh brunello cmc 0 1.54±0.17 fg 0.82±0.12 k 4.73±0.23 fgh 2.5 2.72±0.18 ef 0.83±0.14 k 6.80±0.34 bcd 5 4.03±0.25 cde 2.12±0.14 ij 06.80±0.31 bcd 10 4.81±0.23 abcd 3.65±0.19 cdef 9.81±0.41 a 15 6.03±0.30 a 3.57±0.19 def 9.84±0.46 a mncc 0 1.54±0.17 fg 0.82±0.12 k 4.73±0.23 efg 2.5 4.02±0.23 cde 0.88±0.18 k 4.81±0.24 efg 5 4.81±0.25 abcd 2.13±0.18 hij 6.46±0.19 cde 10 4.84±0.21 abcd 3.58±0.20 def 9.82±0.40 a 15 6.02±0.26 a 4.70±0.25 bcd 9.79±0.36 a anova (3-way) a ** ** ** b ns ** ** c ** ** ** a×b ** ** ** a×c ** ** ** b×c ns ns ** a×b×c ** ** ** cv (%) 10.88 10.28 7.96 adv. hort. sci., 2019 33(2): 215-226 224 non-magnetic osfs with different manner toward αamylase in the same cultivar. the earth’s magnetic field affects orientation of the ferromagnetic particles and modulations of reactions. it has been reported that the magnetic field has an effect on the biochemical processes and stimulation of the activity of proteins and enzymes on increase of the seed vigor (dhawi et al., 2009). tham et al. (2001) reported an increase in shoot growth of rice seedling in the hydroponic medium using acid solution of chitosan. magnetic fields have been reported to increase sugar content in sugar beet roots (beta vulgaris) and the content of gluten in wheat (triticum aestivum) (dhawi et al., 2009). radhakrishnan and kumari (2012) in their experiment on soybeans indicated a positive effect of a pulsed magnetic field on the increased activity of the αand β-amylases. in our experiment, mncc with its synergetic effects of magnetism, chitosan, and iron, was remarkably increased the photosynthetic structures, table 4 effects of osfs on amylases (n=3) into the terminal bud tissue of inflorescence and vase life (n=5) of three lily cultivars z the means with similar letters have not any significant differences at hsd0.01. w hsd0.05 and n=5 y type i error **, *, ns = significance at p≤0.01 and p≤0.05 and non-significance, respectively. cultivars (a) treatments α-amylase β-amylase vase life (days)wz osfs (b) concentration of osfs (mg/l) (c) cherbourg cmc 0 4.48±0.21 gh 5.98±0.49 g 7.00±0.00 ab 2.5 4.42±0.21 gh 7.97±0.30 g 8.20±0.97 ab 5 6.61±0.33 ef 12.31±0.90 de 7.00±0.71 ab 10 6.61±0.20 ef 13.49±0.68 d 7.20±0.37 ab 15 9.00±0.32 cd 12.33±0.99 de 6.00±0.63 ab mncc 0 4.48±0.21 gh 5.98±0.49 g 7.00±0.00 ab 2.5 6.56±0.21 ef 6.01±0.43 g 6.60±0.51 ab 5 7.14±0.17 def 8.04±0.30 g 6.67±0.67 ab 10 8.99±0.49 cd 12.04±0.95 def 8.00±0.00 ab 15 11.07±0.44 ab 8.07±0.33 g 6.40±0.51 ab navona cmc 0 5.60±0.27 fg 9.06±0.36 efg 7.80±0.37 ab 2.5 8.09±0.40 cde 9.11±0.45 ef 7.20±0.49 ab 5 8.02±0.41 cde 12.15±0.63 de 7.80±0.20 ab 10 9.63±0.34 bc 15.21±0.65 cd 8.60±0.51 a 15 12.04±0.53 a 15.11±0.73 cd 8.20±0.49 ab mncc 0 5.60±0.27 fg 9.06±0.36 efg 7.80±0.37 ab 2.5 3.07±0.20 hi 12.10±0.50 def 6.80±0.37 ab 5 5.57±0.22 fg 14.92±0.68 cd 8.80±0.58 a 10 5.59±0.27 fg 18.09±0.79 abc 7.80±0.37 ab 15 8.03±0.34 cde 18.13±0.74 abc 9.25±0.48 a brunello cmc 0 2.04±0.14 i 6.52±0.37 g 5.80±0.58 ab 2.5 2.18±0.12 i 8.16±0.44 fg 4.80±0.73 b 5 4.27±0.24 gh 13.38±0.53 d 6.20±0.37 ab 10 6.82±0.32 ef 15.27±0.75 cd 7.00±0.77 ab 15 6.81±0.35 ef 19.30±0.69 ab 7.67±0.67 ab mncc 0 2.04±0.14 i 6.52±0.37 g 5.80±2.58 ab 2.5 2.09±0.07 i 12.99±0.61 de 6.20±0.20 ab 5 4.18±0.22 gh 15.43±0.81 bcd 7.60±0.51 ab 10 6.80±0.24 ef 15.11±0.70 cd 5.80±1.02 ab 15 9.29±0.50 bc 19.41±0.79 a 5.80±1.02 ab anova (3-way) a ** ** * b ** * ns c ** ** ns a×b ** ** ns a×c ** ** ns b×c ** ** ns a×b×c ** ** nsy cv (%) 7.84 7.99 19.18 hatamzadeh and shafiei-masouleh organic fertilizer for lilium 225 the transfer of photosynthetic products and the storage of carbohydrates; so that takeda et al. (1983) stated that the increase of substrate promotes the activity of amylase enzymes, especially α-amylase. according to table 4, the longer vase life was observed in ‘navona’ in the high concentrations of osfs (magnetic and non-magnetic) based on 3-way interactions compared with two other cultivars. hajnorouzi et al. (2011) reported that using a combination of earth’s magnetic field and weak pulsed electromagnetic field on corn seedlings can increase the growth rate and decrease the iron content of the plant and maintain the membrane’s health and decrease oxidative burst. since the magnetic field is the natural property of the earth, plants and other living creatures are permanently responding to the magnetic field during their lives. earth acts as a magnet with its northern and southern poles, and natural effects of the magnetic field can change the growth and yield of plants on the ground. in particular, the electromagnetic spectrum of solar radiation stimulates plant growth through the process of photosynthesis. the possible mechanism is the changing of the electrostatic balance of the plant system at the membrane surface of the cell, which is the primary site for any plant growth restriction or promotion (radhakrishnan and kumari, 2012). therefore, the role of superparamagnetic nanoparticles can be interpreted in our experiment based on the influence of the earth’s magnetic field. it can be stated, instead of the iron salt (which is often difficult to uptake by plants), a chelating agent in the nutrition solution can be used both as the iron source for the plant and also is suitable for the absorption of other elements. between two tested compounds, mncc is a more suitable compound than cmc because it has a positive synergic effect in addition to providing iron in the plant, which is effect of both chitosan and magnetism. in addition, the characteristics of particle size in this type of supplement, i.e. nanosize, affect the biophysical characteristics and biological activities of the plant. 4. conclusions our experiment showed that utilizing the magnetic composite of chitosan (mncc) or modified and chelating macromolecule of chitosan (cmc) in horticulture can significantly affect the growth and development physiology of plants. we introduced this compound, especially magnetic compound, as fertilizer supplement in production period of lily bulb (shafiee-masouleh et al., 2014); now in this experiment for three cultivars forcing period of lily shows that high concentration of both osfs can be used as a supplement in nutrition solution. the highest concentrations (10 and 15 mg/ l) regardless of osf types and the cultivars response caused significant physiological effects on the content of carbohydrates and also the enzymes that are influence on carbohydrates (amylases). it seems that observing the remarkable morphological changes needs to use the higher concentrations of both osfs without toxic effects. however, this subject (the use of higher concentrations) must be examined in horticultural plants. the examined cultivars (cherbourg, navona and brunello) showed almost different responses to osfs, but generally magnetic osf can be advised and for security utilization needs to be examined with higher concentrations at future research. references abadía 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brazil. 2 brasilian agricultural research corporation, embrapa roraima, caixa postal 133, 69301‐970 boa vista, rr, brazil. key words: biometric characterization, camu-camu, class size, fruit growing, rate of emergency, water content. abstract: camu-camu has aroused the interest of various industries like natural preservatives, ice creams, juices, jellies, wines, natural dyes, but there is little technical information about the seeds. the objective of this work was to determine the biometric, physical and vigor characteristics of camu-camu seeds. seeds originating from native populations of roraima were used. the biometry was determined and the data were analyzed in excel spreadsheet and calculated the mean, median, variance, standard deviation and seeds classified as small, medium and large, based on mass. the vigor was determined by electrical conductivity, seedling emergence, emergence velocity, plant height, stem diameter, in a completely causal design, with four replicates of 25 seeds. the average results for width, thickness, length, individual mass, volume, weight of one thousand seeds and number of seeds per kilogram showed large variability. the size of the seed has direct correlation with vigor, large seeds have greater vigor. 1. introduction the camu-camu, myrciaria dubia (kunth) mcvaugh, is a small fruit tree of the myrtaceae family, popularly known as araçá-dágua, azedinho, camocamo and caçari (smiderle and sousa, 2008). it occurs naturally in the amazon region, growing naturally along the banks of rivers, streams, channels and lakes (chagas et al., 2012; souza et al., 2017). the fruit of the camu-camu is used in the preparation of juices, jellies, alcoholic drinks and ice creams, among others (yuyama, 2011; sousa et al., 2013). the main property of the camu-camu is the high vitamin c content, around 6,112 ± 137.5 ascorbic acid 100 g-1 of pulp, better than most cultivated plants (yuyama, 2011). the skin of the camu-camu when fresh contains high levels of anthocyanin and ascorbic acid (villanueva-tiburcio et al., 2010). the interest aroused by the camu-camu is due to its bioactive compounds and biological activity of antioxidant action, which reduce lipid peroxidation, revert high levels of total cholesterol and triacylglycerols, and increase the levels of hdl-cholesterol (gonçalves, 2012). (*) corresponding author: nascimentocr0@gmail.com citation: do nascimento c.r., chagas e.a., smiderle o.j., de andrade souza a., cardoso chagas p., 2019 biometric characteristics of camu‐camu seeds from native populations in the state of roraima, classified by size class. adv. hort. sci., 33(3): 359-364 copyright: © 2019 do nascimento c.r., chagas e.a., smiderle o.j., de andrade souza a., cardoso chagas p. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 24 september 2018 accepted for publication 23 april 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(3): 359-364 360 seed characteristics are important in the study of any species, as they give an understanding of the dispersion and establishment of seedlings. the classification of seeds by size or weight is a strategy to be adopted in the standardisation and emergence of seedlings, and to obtain seedlings of similar size or greater vigour (carvalho and nakagawa, 2012). therefore, the biometry of fruits and seeds have a diagnostic value in differentiating species within a genus, and can contribute to their recognition (pereira and ferreira, 2017). in order to find the ideal size class for the multiplication of different plant species, biometry is used to determine physiological quality, but the results have differed considerably, even when the seeds are from the same species. biruel et al. (2010) found that larger seeds of caesalpinia leiostachya displayed a greater percentage and speed of germination; different results to those reported by queiroga et al. (2011), who studied peanut seeds and found that smaller seeds had a higher germination percentage. seeds of different sizes showed the same high germination percentage, with small seeds being the most viable, and medium and large seeds yielding seedlings that were more vigorous (vendramin and carvalho, 2013; smiderle et al., 2016). this ambiguous situation may be one of the reasons for two or more seed sizes being produced by the same species. variations in size were seen in seeds collected in an area of natural occurrence in the state of roraima, brazil. in view of the above, the aim of this work was to determine the biometry, physical characteristics and vigour of seeds of the camu-camu. 2. materials and methods the experiment was carried out at the seed analysis laboratory of embrapa roraima. the camucamu fruit used in the experiment were harvested from native plants located on the banks of the jatapú river (0° 41.072” n and 59° 18.046” w) at an altitude of 144 m. here the plants develop in rocky soil in the district of caroebe in the state of roraima, and are distributed along the river in various populations and subpopulations. after harvesting, the fruits were packed in plastic bags to prevent damage from crushing, and carefully transported to the laboratory, where they were selected for lack of damage, and then homogenised and sanitised. the mature selected fruit were then depulped and washed in running water, using a sieve for the complete removal of any pulp adhering to the seeds. physical characterisation of the seeds the seeds were evaluated for their physical characteristics. seed biometry. the individual thickness, length and width of 600 seeds were measured using a calliper, with the results expressed in mm. the length was measured along the longitudinal axis of the seed; the width was measured perpendicular to the length, considering the broad face of the seed, which corresponds to the median line; and the thickness, by again measuring perpendicular to the length, but on the smaller seed face, corresponding to the median line including the two cotyledons. the following were then obtained: seed volume (mm3): multiplying the three dimensions (width, length and thickness), without considering the actual shape of the seed. individual seed weight: obtained by weighing each seed. based on these measurements, the seeds were separated into three size classes (large, medium and small). 1000 seed weight (g). six samples of 100 seeds were used for each size class. the samples were weighed on a 0.001 g precision balance and the weight determined by multiplying the result by 10. number of seeds per kilogram. after determining the thousand-seed weight (tsw), the number of seeds per kilogram was calculated using the “rule of three”. water content (%). carried out using the standard oven method at 105±3°c for 24 hours. four replications of 10 seeds were used, where each sample was wrapped in aluminium and placed in an oven. after 24 hours, the samples were removed from the oven and placed in a desiccator for cooling, and then weighed on a 0.001 g precision balance. the water content was calculated based on the difference between the wet and dry weights, applying the formula proposed in the rules for seed analysis (mapa, 2009), with the result expressed as a percentage. the biometric data were analysed in an excel spreadsheet. the mean, median, variance and standard deviation were all calculated. determining the vigour of small, medium and large seeds of the camu‐camu the experiment was carried out in a greenhouse at embrapa roraima, in boa vista. the experimental design was completely randomised (crd), with three treatments comprising the size classes (small, medido nascimento et al. ‐ seeds biometry and vigor of camu‐camu 361 um and large), and four replications of 25 seeds. seeds with a weight between 0.66 g and 0.84 g were considered small, those with a weight between 0.92 g and 1.20 g were considered medium, and those with a weight between 1.27 g and 1.60 g considered large. after classification, the following analyses were carried out: electric conductivity (ec). the seeds were weighed on a 0.0001 g precision balance and then immersed in 75 ml of water for 24 hours at a constant temperature of 25°c. the electrical conductivity was then measured using a digital conductivimeter. the results were expressed in μs cm-1 g-1 of seed. seedling emergence. the test was carried out following procedures described in the rules for seed analysis (mapa, 2009). the tests were conducted in trays containing 50% sand + 50% sawdust as substrate moistened with water up to 60% of its retention capacity, using four replications of 25 seeds distributed at a depth of 2.0 cm. the test was performed in a nursery of 50% sombrite shade screen. to evaluate the non-germinated seeds, they were removed from the substrate, cut down the middle with pruning shears and identified for viability and senescence. the formula used for calculation was: e = (n/a) x 100: where e = percentage emergence; n = number of emerged seedlings; a = total number of seeds placed for emergence. speed of seedling emergence. established by means of a daily count of emerged seedlings, the index being calculated from the expression: se = (e1/n1) + (e2/n2) + ... + (en/nn), where: se = speed of seedling emergence; e1 = number of seedlings emerged at the first count; n1 = number of days elapsed until the first count; e2 = number of seedlings emerged at the second count; n2 = number of days elapsed until the second count; n = last count. plant height. evaluated four months after sowing with the aid of a graduated rule (cm). measured from the cotyledon node to the end of the first pair of leaves in normal seedlings identified at the end of the test for seedling emergence. stem diameter. evaluated four months after sowing using a digital calliper (mm). determined at the insertion of the cotyledon. the data for water content, electrical conductivity, seedling emergence, speed of emergence, plant height and stem diameter were submitted to the lilliefors test for normality. they were then submitted to analysis of variance (anova), using a completely randomised design with four replications, and the mean values compared by tukey’s test at 5% probability using the sisvar software (ferreira, 2014). 3. results and discussion from the results, it was seen that there was a significant difference in the biometric variables of the analysed seeds, and it was possible to separate them into three classes according to size: small, medium and large. the data for variance, standard deviation and coefficient of variation, and the mean results for individual weight, width, thickness, length and volume of the camu-camu seeds are shown in table 1. the biometric data shown indicates that the studied population was accurately sampled, since the values for variance were low (<1) for each of the characteristics under study. the values for standard deviation shown in table 1 indicate a low sample variation for each of the characteristics under evaluation. the values for the coefficient of variation demonstrate the low variation in the variables, considering the mean value of the characteristics. however, variation can be seen when the seeds are sorted by size. table 1 biometric characteristics of camu-camu seeds from native populations in the state of roraima, classified by size class class mean variance standard deviation coefficient of variation (%) weight (g) small 0.79 0.002 0.04 5.65 medium 1.11 0.004 0.06 5.90 large 1.45 0.008 0.08 6.12 width (mm) small 12.04 0.541 0.73 6.11 medium 12.90 0.147 0.38 2.97 large 14.42 0.233 0.48 3.34 thickness (mm) small 5.54 0.065 0.25 4.59 medium 6.11 0.213 0.46 7.56 large 6.74 0.329 0.57 8.51 length (mm) small 15.54 0.620 0.79 5.07 medium 17.42 0.206 0.45 2.61 large 19.14 0.342 0.58 3.05 volume (mm3) small 1,035.77 6,144.378 78.38 7.57 medium 1,371.98 6,104.830 78.13 5.69 large 1,859.47 20,759.298 144.08 7.75 adv. hort. sci., 2019 33(3): 359-364 362 similar results were found in camu-camu seeds from the banks of the anauá river in roraima, with a weight which ranged from 0.80 g to 1.46 g, and greater results than the seeds of populations from the rio urubu, which ranged from 0.56 to 0.78 g (souza et al., 2017). according to gonçalves et al. (2008), species with wide geographic distribution may present differences in their characteristics due to the effects of adaptation and to origin. the variation in seed size may interfere with their physiological quality, still poorly researched in forest species (oliveira et al., 2009; smiderle et al., 2016). the three classes of seed size displayed significant differences for thousand-seed weight (tsw) and the number of seeds per kilo (nsk) with a low coefficient of variation, showing little variation within each size (table 2). the initial water content of the seeds in the different size classes at the time the experiment was set up was greater than 35%, with significant differences between the classes (table 3), however it was lower than that reported by yuyama et al. (2011), who obtained a value for moisture between 45 and 56%, with no effect on germination. according to braga et al. (2012), hydration of the seeds can favour test performance, because seeds that are more humid, within certain limits, germinate more quickly. the different classes of seed displayed variations in the variables under analysis. it was found that values for electrical conductivity depend on the size of the seed, where the large, small and medium seeds presented higher, lower and intermediate physiological quality respectively (table 3). according to vieira and krzyzanowski (1999), the lower physiological potential in small seeds is probably due to the lower organisational intensity of the cell-membrane systems. the data for electrical conductivity in the small seeds showed a negative correlation with the other variables under study, demonstrating that the highest value for electrical conductivity corresponded to reductions in the percentage and speed of seedling emergence. these results confirm those reported by vinhal-freitas et al. (2011), and demonstrate that tests of vigour differentiate between seed classes, indicating significant differences between the larger and smaller size classes, where larger seeds showed greater vigour with the smallest values for electrical conductivity. with the large seeds, the lower value for electrical conductivity was due to a greater organisation of the cell components, since, despite field emergence not differing statistically from that of the small seeds, they showed greater speed of emergence in addition to viability, even when not germinated, as the small seeds that did not emerge had all died. it is important to point out that despite the differences seen in electrical conductivity between the seed sizes, each seed class had low values for ec, and values for emergence that were higher than the minitable 2 summary of the analysis of variance and comparison of the mean values for thousand-seed weight (tsw) and number of seeds per kilo (nsk) in camu-camu seeds from native populations in the state of roraima, classified by size * significant at 5% probability by f-test. mean values followed by the same letter do not differ by tukey’s test at 5% probability. * significant at 5% probability by f-test. table 3 summary of the analysis of variance and comparison of mean values for water content (wc, %), electrical conductivity (ec, μs cm-1 g-1), emergence (emerg, %), speed of emergence, (se, index), seedling height (ht s, cm) and stem diameter (diam s, mm) in camu-camu seeds classified by size source of variation df mean square tsw nsk treatment 2 346,622.68* 435,142.53* replication 5 633.44 672.57 error 10 1,610,651.55 1,318.23 cv (%) 4.16 3.35 overall mean value 963.88 1,082.76 class mean value small 734.27g 1.362 medium 943.67 g 1.061 large 1,213.70 g 825 source of variation df mean square wc (%) ec μs cm-1 g-1 emerg (%) se ht s (cm) diam s (mm) treatment 2 14.67* 1.83* 433.33* 0.021* 79.12* 0.599* replication 3 0.08 0.002 5.55 0.000008 0.131 0.0012 error 6 0.29 0.001 5.55 0.000008 0.049 0.0008 mean 37.17 3.27 88.33 0.056 15.61 2.13 cv (%) 1.46 1.11 2.67 5.17 1.42 1.36 class mean values small 35.35 c 3.95 c 80 b 0.01 c 11.30 c 1.71 c medium 36.99 b 3.22 b 100 a 0.03 b 15.34 b 2.20 b large 39.17 a 2.61 a 85 b 0.14 a 20.18 a 2.48 a do nascimento et al. ‐ seeds biometry and vigor of camu‐camu 363 dência de sementes de ‘mimosa caesalpiniifolia’ benth., sobre a germinação e vigor. ‐ revista árvore, 29(6): 877-885. biruel r.p., paula r.c., aguiar i.b., 2010 germinação de sementes de ‘caesalpinia leiostachya’ (benth.) ducke (pau‐ferro) classificadas pelo tamanho e pela forma. ‐ revista árvore, 34(2): 197-204. braga n.s., morais c.s.b., rossetto c.a.v., 2012 hidratação controlada de sementes de pinhão manso. revista ciência agronômica, 43(3): 589-597. carvalho n.m., nakagawa j., 2012 sementes: ciência, tecnologia e produção. 5th edition. ‐ jaboticabal: funep, pp. 590. chagas e.a., lima c.g.b., carvalho a.s., ribeiro m.i.g., sakazaki r.t., neves l.c., 2012 propagação do camu‐camu (‘myrciaria dubia’) (h.b.k.) mcvaugh). revista agro@mbiente on-line, 6(1): 67-72. ferreira d.f., 2014 sisvar: a guide for its bootstrap pro‐ c e d u r e s i n m u l ti p l e c o m p a r i s o n s . ‐ c i ê n c i a e agrotecnologia, 38(2): 109-112. gonçalves a.e.s.s., 2012 compostos bioativos do camu‐ camu (‘myrciaria dubia’ mcvaugh): caracterização e atividade biologica. tese doutorado, universidade de são paulo, brazil, pp. 114. gonçalves j.v.s., albrecht j.m.f., soares t.s., titon m., 2008 caracterização física e avaliação da pré‐ embebição na germinação de sementes de sucupira‐ preta (‘bowdichia virgilioides’ kunth). ‐ cerne, 14(4): 330-334. m a p a , 2 0 0 9 r e g r a s p a r a a n á l i s e s d e s e m e n t e s . ministério da agricultura, pecuária e abastecimento, secretaria de defesa agropecuária, brasília, df, brazil, pp. 399. oliveira a.b., medeiros-filho s., bezerra a.m.e., bruno r.l.a., 2009 emergência de plântulas de ‘copernicia hospita’ martius em função do tamanho da s e m e n t e , d o s u b s t r a t o e d o a m b i e n t e . ‐ r e v i s t a brasileira de sementes, 31(1): 281-287. pereira s.a., ferreira s.a.n., 2017 fruit and seed biom‐ etry and seedling morphology of ‘parkia discolor’ (spruce ex benth.). ‐ revista árvore, 41(2): 1-8. queiroga v p., freire r.m.m., araujo m.e.r., lima v.i., queiroga d.a.n., 2011 influência do tamanho da semente de amendoim sobre sua qualidade fisiológica. ‐ revista agro@mbiente on-line, 5(1): 30-34. silva a.c.d., smiderle o.j., oliveira j.m.f., silva t.j., 2017 tamanho da semente e substratos na produção de mudas de açaí. adv. for. sci., 4: 151-156. smiderle o.j., sousa r.c.p., 2008 teor de vitamina c e características físicas do camu‐camu em dois estádios de maturação ‐ revista agro@mbiente on-line, 2(2): 61-63. smiderle o.j., souza a.g., almeida m.s., souza a.a., 2016 caracterização biometrica e superação de dor‐ mencia de sementes de biribá no crescimento inicial de seedlings. revista congrega urcamp (cd-rom), 1: 23-34 mum established by the seed standards (mapa, 2009). as for plant height and stem diameter, the larger seeds promoted the best results, followed by the medium and small seeds respectively. there was a positive correlation between seed size and plant height and stem diameter, i.e. larger seeds gave rise to larger, more vigorous plants. these results differ from those obtained by souza et al. (2017), who reported that seeds from a population of the anauá river considered small, displayed better results for these characteristics. wagner junior et al. (2011) demonstrated that seed size has an effect on the emergence and initial development of jabuticaba seedlings (plinia cauliflo‐ ra), and that large seeds gave seedlings that were more vigorous. in açaí silva et al. (2017) in organic substrate obtained higher values for plant height and stem diameter when large seeds were used, as well a s . l a r g e s e e d s p r o d u c e m o r e v i g o r o u s p l a n t s independent of the substrate in euterpe oleracea. alves et al. (2005) pointed out that in general, larger seeds are correlated with higher rates of initial seedling growth, which increases the probability of success during their establishment, since the rapid growth of roots and shoots allows the plant to take advantage of the nutrient and water reserves of the soil and carry out photosynthesis. wagner junior et al. (2011) stated that the germination process in m a n y s p e c i e s i s i n fl u e n c e d b y s e e d s i z e . consequently, within the same batch, small seeds present lower seedling emergence and less vigour than the medium and large seeds. 4. conclusions the physical characterisation of camu-camu seeds shows great variability in weight, width, thickness, length and volume. the thousand-seed weight and the water content are influenced by size, with values increasing in direct proportion to the size of the seed, while the number of seeds per kilo decreases in inverse proportion. medium and large seeds give rise to plants that are more vigorous. references alves e.u., bruno r.l.a., oliveira a.p., alves a.u., paula r.c., 2005 influência do tamanho e da proce‐ 364 adv. hort. sci., 2019 33(3): 359-364 sousa r.c.p., santos d.c., neves l.t.b.c., chagas e.a., 2013 tecnologia de bioprocesso para produção de ali‐ mentos funcionais. revista agro@mbiente on-line, 7(3): 366-372. souza o.m., smiderle o.j., souza a.g., chagas e.a., chagas p.c., bacelarlima c.g., morais b.s., 2017 influência do tamanho da semente na germinação e vigor de plântulas de populações de camu‐camu ‐ scientia agropecuaria, 8(2): 119-125. vendramin d.w., carvalho r.i.n., 2013 qualidade fisiológica de sementes de pitangueira (‘eugenia uniflora’ (l.) (myrtaceae). ‐ estudos biologicos, 35(84): 59-65. vieira r.d., krzyzanoswski f.c., 1999 teste de conduti‐ vidade elétrica, pp. 1-26. in: krzyzanoswski f.c., r.d. vieira, and j.b. frança neto, (eds.) vigor de sementes: conceitos e testes. abrates, londrina, brasil, pp. 218. v i l l a n u e v a t i b u r c i o j . e . , c o n d e z o h o y o s l . a . , asquieri e.r., 2010 antocianinas, ácido ascórbico, polifenoles totales y actividad antioxidante, en la cás‐ c a r a d e c a m u ‐ c a m u ( ‘ m y r c i a r i a d u b i a ’ ( h . b . k ) mcvaugh). ciências e tecnologia de alimentos, 30(supl.1): 151-160. vinhal-freitas i.c., garcia junior j.e., segundo j.p., vilarinho m.s., 2011 germinação e vigor de semen‐ tes de soja classificadas em diferentes tamanhos. agropecuaria técnica, 32(1): 108-114 wagner júnior a., silva j.o.c., pimentel l.d., santos c.e.m., bruckner c.h., 2011 germinação e desenvol‐ vimento inicial de duas espécies de jabuticabeira em função do tamanho de sementes. acta scientiarum agronomy, 33(1): 105-109. yuyama k., 2011 a cultura de camu‐camu no brasil. ‐ revista brasileira de fruticultura, 33(2): 335-690. y u y a m a k . , m e n d e s n . b . , v a l e n t e j . p . , 2 0 1 1 longevidade de sementes de camu‐camu submetidas a diferentes ambientes e formas de conservação. ‐ revista brasileira de fruticultura, 33(2): 601-607. impaginato 61 adv. hort. sci., 2020 34(1s): 61­69 doi: 10.13128/ahsc­8395 different growing conditions can modu­ late metabolites content during post­ harvest of viola cornuta l. edible flowers i. marchioni 1, 2, l. colla 1, 3, l. pistelli 1, 4 (*), b. ruffoni 2, f. tinivella 3, g. minuto 3 1 dipartimento di scienze agrarie, alimentari e agro‐ambientali (dis‐ aaa‐a), università di pisa, via del borghetto, 80, 56124 pisa, italy. 2 crea, centro di ricerca orticoltura e florovivaismo, corso inglesi, 508, 18038 sanremo (im), italy. 3 centro di sperimentazione e assistenza agricola, regione rollo, 98, 17031 albenga (sv), italy. 4 centro interdipartimentale di ricerca nutraceutica e alimentazione per la salute (nutrafood), università di pisa, via del borghetto, 80, 56124 pisa, italy. key words: cold storage, greenhouse cultivation, horned pansy, secondary metabolites. abstract: edible flowers are inflorescences traditionally used in various part of the world to enrich sweet and savoury recipes. the flowers of viola spp. were appreciated since the romans, and today the fresh products are now incorpo­ rated as ingredients in different culinary preparations. in this work, cultivation of potted viola cornuta l. cv. penny lane was performed in greenhouse with different environmental conditions (basal heating, additional led lighting and moisture management) and therefore the biomass production (number of flow­ ers per square meter and plant dimension per pot) was assessed. the plants are characterised by flowers with dark purple and orange petals in the same corolla. the shelf­life of detached flowers was studied in post­harvest condi­ tions at 0 and 4 days of cold storage at 4°c (polyethylene boxes, 12/12 h light/dark condition) to simulate the condition of i gamma products. sugars and secondary metabolites were analysed. basal heating seems not to increase flower number but could contribute to reach a well­balanced simultaneous presence of different antioxidant molecules (polyphenols, anthocyanins, carotenoids). our data highlight that the short cold storage under light condi­ tion lead to an increase in the content of total polyphenols and antioxidant activity, although a general reduction in pigments and sugars is observed. 1. introduction edible flowers are currently part of a niche market and perceived as a culinary novelty, even if their consumption is known for thousands of years. in fact, there are several historical evidences that highlight the use of the inflorescences to prepare and garnish dishes, from some ancient (*) corresponding author: laura.pistelli@unipi.it citation: marchioni i., colla l., pistelli l., ruffoni b., tinivella f., minuto g., 2020 ­ different growing conditions can modulate metabolites content during post‐harvest of viola cornuta l. edible flowers. ­ adv. hort. sci., 34(1s): 61­69 copyright: © 2020 marchioni i., colla l., pistelli l., ruffoni b., tinivella f., minuto g. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 28 december 2019 accepted for publication 26 february 2020 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(1s): 61­69 62 civilisation as greeks and romans, to more recent times, e.g. the victorian period in england (mlcek and rop, 2011; cunningham 2015). most appreciated species were roses (rosa spp.), calendula (calendula officinalis l.), saffron (crocus sativus l.), dandelion (taraxacum officinale l.), and elder inflorescences (sambucus nigra l.) (mlcek and rop, 2011). in indian and chinese cultures, edible flowers are used as com­ ponents of medicines based on herbs, in addition to culinary purposes (wongwattanasathien et al., 2010). several edible flowers are beneficial to human health showing anti­infiammatory effects and antioxidant and ros scavenging activities (mlcek and rop, 2011). today, around 180 specie are known to produce edible flowers (lu et al., 2016), and viola spp. are among the most common and currently consumed. these flowers are characterized by a sweet and refreshing taste, in addition to a pleasant velvety tex­ ture (neumann and o’connor, 2009; koike et al., 2015). edible violas belong to 3 different species, namely viola cornuta l. (horned pansy), viola tricolor l. (johnny jumpup), and viola × wittrockiana gams (garden pansy) (neumann and o’connor, 2009). the plants are similar to each other except for flower size, which its diameter is in garden pansies (up to around 11.5 cm) > horned pansies (up to around 2.5 cm) > johnny jump ups (less than 2.5 cm in diameter) (bailey, 1998; kessler et al., 1998). over the years, intensive breeding programs selected new varieties with unique flower colours (pure­colour or multi­ coloured flowers), greater flowers number, and plant temperature tolerance (bailey, 1998). the cultivation of v. cornuta is similar to the one of v. × wittrock‐ iana. these speciesare grown as autumn and spring bedding plants, although they are also raised for the summer and winter markets (pearson et al., 1995). in order to produce edible flowers safe for human con­ sumption, chemical products, such as synthetic fertil­ izers and pesticides, has to be avoided during plants production; for this reason, only organic cultivation is allowed (fernandes et al., 2017). no special needs for cultivation are required, indeed well drained com­ mercial potting soil can be used. viola flowers are often cultivated in greenhouse, and properly defined environmental factors, such as temperature, pho­ toperiod and irradiance are fundamental for the quality of the flowers (gandolfo et al., 2016). pansies should be grown between 4 and 13°c, in order to reduce plant growth rate, internode elongation and to ensure high quality flowers (cavins et al., 2000). in fact, flower size (mm 2) decreased linearly with increasing temperature between 9 and 31°c (pearson et al., 1995). the ideal temperature for growth and flowering ranges from about 14°c to 21°c (kessler et al., 1998). moreover, pansies are obligate fr (far red)­dependent long­day plants and, for this reason, fr radiation are required to promote the flowering process, in addition to red (r) radiation (kozai et al., 2016). blue light is able to reduce the time required to produce flower buds in v. × wittrockiana (rashidi et al., 2018). full­bloomed, edible flowers can be sold in pots or, mainly, in small and medium rigid plastic pack­ ages to avoid their rapid drying and to preserve their fragile texture (whitman, 1991; kelley et al., 2001). however, flowers are high perishable so that differ­ ent approaches were performed to prolong their shelf­life. cold storage is documented for v. tricolor and v. x wittrockiana, using sealed low­density polyethylene film bags. these two species were able to preserve their commercial attractiveness up to 2 weeks of storage, when kept between 0 and 2.5 °c (kelley et al., 2003). more recently, different new post­harvest technologies were applied on viola spp. edible coatings (e.g. alginate), crystallization and osmotic dehydration improved violas shelf­life, as shown by a good visual quality for prolonged period (fernandes et al., 2018 a, b, 2019 a, b). coated pan­ sies contained higher level of polyphenols and antioxidant activity than uncoated ones, on all assayed storage times (up to 14 days). gamma irradi­ ation are also tested, and this methodology increased polyphenols content and antioxidant activity in v. tri‐ color flowers, compared to no irradiated controls (koike et al., 2015). edible flowers are selected and perceived by their fragrance, appearance, size and colour. consumers prefer yellow and orange flowers rather than blue (kelley et al., 2001, 2002). within this regard, v. cornuta cv. ‘penny lane’ with orange­ violet flowers have been selected for this work. the aim of this work was to cultivate v. cornuta l. ‘penny lane’ and test the effect of different cultiva­ tion strategies for a higher production of flowers with good quantities of nutritional compounds. moreover, the post­harvest treatment has been performed to analyze the change in bioactive compounds. storage temperature was maintained around 4­6°c (cold stor­ age) and flowers were also exposed to artificial light to simulate the refrigerated sector of the grocery market. metabolites (polyphenols, anthocyanins, carotenoids, sugars) were analysed to determine the shelf­life of packaged flowers as i gamma products. to the best of our knowledge, any investigation of metabolites during post­harvest cold storage studies marchioni et al. ‐ cultivation and post‐harvest strategies on viola cornuta flowers 63 were performed on v. cornuta. 2. materials and methods plant cultivation, greenhouse condition and flower blooming plants of viola cornuta l. ‘penny lane’ with orange­violet flowers (fig. 1) were purchased by gruppo padana ­ ortifloricoltura dei fratelli gazzola s.s. società agricola (paese, tv, italy) and planted in 420 pots with a diameter of 14 cm (1 l volume). they were placed on 4 benches of an iron­glass green­ house (called sam­lab) equipped with a climatic control system at the “centro di sperimentazione e assistenza agricola” (cersaa) in albenga (sv) 43° 3’ 14’’ north and 8° 13’ 1’’ east). at the beginning of the experiment, plants were 3 cm height with a diameter of 2.5 cm. the substrate used was “ts4” soil from “turco silvestro” company (albenga, sv, italy), char­ acterized by ph 6.5, electrical conductivity 0.56 ds/m, dry bulk density 250 kg/m3 total porosity 90% v/v. the experimental design foresees 7 treatments (60 plants each) in the samlab and reported in table 1. the presence or absence of basal heating was guaranteed by either electric mat warmset at 50°c or water at 35°c (by hydraulic coil) and the addition of 1 or 2 hours of light after the astronomical sunset to extend the photoperiod, as reported in table 1. in consideration of the number and the layout of the benches of the greenhouse and the possibility to sub­ divide the lighting of led lamps used for the experi­ mentation, only the selected treatments were tested (table 1). each bench was equipped with 4 led lamps placed at a distance of 1.50 meters from the surface of the pallet depending on the type of lamp and cul­ ture. for the tests, specifically, valoya b200 led lamps with ap673l spectrum were used (blue 12% ­ green 19% ­ red 61% ­ far red 8% ­ par 92%). each lamp has a total power consumption of 192 w, a photon flux in the range 400­700 nm of 284 mmol s­1 and a photon flux in the range 300­900 nm of 311 mmol s­1). during the trial in the greenhouse the reg­ istered average temperature was 18°c and the aver­ a g e h u m i d i t y w a s 6 4 % . f r o m 0 1 / 1 4 / 2 0 1 9 t o 02/28/2019 evaluations were carried out to observe whether different kind of basal heating and supple­ mentary light could affect plant growth. at the end of the trials, the investigations included the measure­ ment of plant diameter (cm) per each pot and the number of flowers per meter square. thus, flowers were picked by hand in the morning for further analyses and cold treated. flowers storage conditions fresh picked flowers were stored in polyethylene boxes at 4 °c with a 12 hours photoperiod in order to simulate better the condition of supermarket fridge fig. 1 ­ flowers of viola cornuta cv. penny lane grown in pot in samlab greenhouse. table 1 ­ greenhouse treatments (basal heating of benches and/or additional light) of viola cornuta cv. penny lane. treatments are car­ ried out for 3 months, from transplantation through flowering period until the end of trials treatment basal heating additional light electric mat 50°c hot water 35°c absent 1 hour 2 hours absent 1 x x 2 x x 3 x x 4 x x 5 x x 6 x x 7 x x adv. hort. sci., 2020 34(1s): 61­69 64 counter. the refrigerated cells were equipped with led lamps (valoya, finland) having the following spectrum: blue 21% ­ green 38% ­ red 35% ­ far red 6% ­ par 94%. flower storage was evaluated after 4 days post­harvest (time 4) performing the following biochemical analyses: total phenolics content, antiox­ idant activity (dpph assay), total anthocyanins con­ tent, total carotenoids content and total soluble sug­ ars content. for each biochemical analysis three homogeneous biological replica were used. each replica was stored at ­20°c until further analyses. fresh flowers (time 0) are used as control. polyphenol content p o l y p h e n o l s w e r e e x t r a c t e d a s r e p o r t e d b y bretzel et al. (2013). fresh flowers (200 mg) were homogenized in 2 ml of methanol 70 %, and of total phenolic content was determined using the folin­ ciocalteau assay (singleton and rossi, 1965). the absorbance was read at 765 nm in a uv­1800 spec­ trophotometer (shimadzu corp., kyoto, japan) and the total phenolic concentration was expressed as catechin equivalents per gram of fresh weight. dpph scavenging activity the antioxidant activity of each sample was deter­ mined through the 2,2­diphenyl­1­picrylhydrazyl radi­ cal (dpph) free radical scavenging assay, as described by brand­williams et al. (1995). the absorbance was read at 517 nm. antioxidant activity was expressed in ic50, which represent the concentration of the sample able to reduce the initial amount of radical dpph by 50%. consequently, lower ic50 value of sample corre­ sponds to greater antioxidant activity. anthocyanin content total anthocyanins were extracted as reported by bretzel et al. (2013). 200 mg of sample were homog­ enized in 750 µl of acidified methanol (meoh/hcl 10:0.1). the absorbance was read at 535 nm and the total anthocyanin concentration was expressed as malvidin equivalents per gram of fresh weight. carotenoids content determination of total carotenoids content was determined using lichtenthaler’s formula (1987). f r e s h fl o w e r s ( 1 0 0 m g ) w e r e a d d e d t o 5 m l o f methanol 99 % and it was incubated for 24 hours at 4°c. the absorbance was read at 665.2 nm, 652.4 nm and 470 nm. total soluble carbohydrates content total soluble carbohydrates content was estimat­ ed from dried flowers (20 mg) using anthrone proto­ c o l a c c o r d i n g t o y e m m a n d w i l l i s ( 1 9 5 4 ) . t h e absorbance was read at 630 nm, using glucose as external standard. statistical analysis the normal distribution of the residuals and the homogeneity of variance was determined and then data were statistically analysed. the results of bio­ mass (number of flowers and growth in pot) were expressed as mean values and analyzed using one­ w ay an alys is of var ian ce (a n ova ) followed by tukey’s hssd test with p=0,05. the results of post­ harvest treatments have been performed using anova student’s t­test to determine the significant difference of each treatment between fresh samples (time 0) and samples after 4 days (time 4), with p < 0.05. biochemical results were analysed by one­way anova followed by fisher’s probable least­squares difference test with cut­off significance at p ≤ 0.05 (statview®, version 5.0, sas® institute corporation). the dependent variables were analysed using two­ way anova, with the factors ‘‘treatment” and ‘‘post harvest days’’ (phd). 3. results and discussion horned pansy (viola cornuta) is a biannual plant with long flowering period through different seasons. it is also considered as cold­tolerant plant, since the minimum temperature for flowering is around 4°c, w h i l e t h e o p ti m a l t e m p e r a t u r e i s a r o u n d 2 6 ° c (blanchard and runkle, 2011). winter temperature and light are very important factors to determine a good production of flowers (boldt and altland, 2019), and heating and supplemental lighting are often pro­ vided in greenhouse cultivation to improve the quan­ tity and quality of flowers (dieleman and meinen, 2007; oh et al., 2010). for this reason, horned pansy plants were subjected to different treatments (table 1) to evaluate their effect on the growth and flowers number (table 2) and thus the yield of edible flowers. the results indicated that the treatment n. 7 (con­ trol, no basal heating, no additional light hours) and treatment n. 3 (no basal heating, 2 h of supplemen­ tary light) determined the larger diameter of plants (table 2). however, there is no statistically significant difference (t student analysis) between treatment n. 3 and the control n. 7 (18.0 and 17.1 cm/plant respectively), probably due to the effect of light towards vegetative growth. the addition of basal temperature of the benches by hot water (treatment marchioni et al. ‐ cultivation and post‐harvest strategies on viola cornuta flowers 65 the visible characteristics and nutraceutical compo­ nents (benvenuti et al., 2016). thus, the influence of light and temperature for the production of different metabolites was also determined. edible flowers are considered a good source of antioxidant molecules (rop et al., 2012; loizzo et al., 2015) and polyphenols (including phenolic acids and anthocyanins) are considered the main antioxidant compounds. a first detail of phenolic composition and properties of v. cornuta edible flowers highlight­ ed that their polyphenols content is lower than v. × wittrockiana (moliner et al., 2019). the metabolites were analyzed at the time of harvest (time 0), and after short period of post­harvest in a chamber at lower temperature and in the presence of light (12h). the post­harvest treatment was chosen to mimic the condition of the benches of grocery stores. pigments, as carotenoids and anthocyanins are the important compounds for evaluating the visual quality of flow­ ers. at time of harvest (time 0), the worst treatment resulted the n. 3 (addition of 2h light), since the recorded amount of both pigments were the lowest, 0.14 and 5.63 mg/g fw for carotenoids and antho­ cyanins, respectively (table 3). instead, the highest values were determined with the treatment n.1 (temperature 50°c by electric mat and light 1h (0.32 and 10.42 mg/g fw for carotenoids and antho­ cyanins). the increased temperature, either by elec­ tric mat (n. 2) or by hot water (n. 5), did not support a n y i n c r e a s e i n fl o w e r p i g m e n t a ti o n , b o t h f o r c a r o t e n o i d a n d a n t h o c y a n i n s . c o n t r o l fl o w e r s showed good carotenoid values (0.30 mg/g fw), while anthocyanins suffered without addition of light or temperature (6.93 mg/g fw). of our knowledge only few papers have been published so far on the 5) corresponded to a decrease of the growth (15,6 cm/plant), while when also additional lighting was performed the decrease was not significant (treat­ ments n. 1, 4, 6). the growth of the plants seems to be affected when the additional light is added, in the absence or with higher temperature of the benches (table 2). the effect of supplemental led lighting is known to affect positively many plant growth para­ meters of several plants, including pansy (koksal et al., 2015), so these results are in agreement with pre­ vious reports. the treatment 2 (electric mat 50°c) highlighted the lowest value of number of flowers (604.44 flowers/m 2) followed by treatment n.6 (636.94 flowers/m2), while the other trials showed similar higher amounts (table 2). taken together, the biometric parameters suggest that the single elonga­ tion of photoperiod (2h) plays a positive role to increase the biomass and to produce more flowers, and the temperature is a secondary effect. the quali­ ty of plants in relation to light and temperature is debated since long time (liu and heins, 1997; adams et al., 1998), and the lower temperatures and higher irradiance seems to produce higher quality of flow­ ers, including pansy (pearson et al., 1995; boldt and altland, 2019). the results presented here are in agreement of the observed influence of the exposure duration, intensity and combinations of light to the growth and flowering of v. × wittrockiana (oh et al., 2010). the lengthening of the photoperiod has been confirmed as important factor in v. × wittrockiana ’rose’, during experiments aimed to the determine the influence of photoperiod and phytochrome (rashidi et al., 2018). in that research, the night inter­ ruption decreased the plant dimension. the quality of flowers, especially the edible ones, are related to table 2 ­ effect of different cultivation (basal heating and/or supplementary light) on biomass production of viola cornuta cv. penny lane plant diameter (cm) and the number of flowers (per meter square) were detected at the end of flowering period. data are expressed as mean value (n=60) and analyzed using one­way analysis of variance (anova) followed by fisher’s probable least­ square difference test with p=0.05. treatment diameter (cm) number of flower per m2 1 electric mat 50°c + light 1 h 15.8±0.40 ab 838.42±42.9 a 2 electric mat 50°c 16.2±0.46 ab 604.44±48.75 b 3 light 2 h 18.0±0.40 a 864.42±40 a 4 hot water 35°c + light 1 h 16.6±0.33 ab 851.42±33.8 a 5 hot water 35°c 15.6±0.38 b 812.43±44.85 a 6 hot water 35°c + light 2 h 16.1±0.24 ab 636.94±43.55 ab 7 control 17.1±0.50 a 793.93±38.35 a 66 adv. hort. sci., 2020 34(1s): 61­69 influence of light and temperature on the content of pigments in viola spp. (rashidi et al., 2018), so these results will contribute to define the effect of these factors and their contribution to the pigmentation. other metabolites of horned pansy were determined at time of harvest, such as polyphenols andsugars, that are expected as fundamental nutraceutical com­ ponents, as well as the scavenger reducing power (by dpph assay). with regards to total soluble carbohy­ drates (tss) statistically significant differences were observed among the various treatments: higher sug­ ars amounts were observed in the treatments n. 1 and 4, characterized by the addition of light (1 h) and higher temperature. although the other treatments showed the similar less quantities of sugars, the low­ est amount is observed in treatment n. 3 (tss 179.57 mg/g fw). the highest amount of total polyphenols (12.42 mg/g fw) was measured in treatment n. 1 (basal electric heating at 50 °c with 1 hour of addi­ tional light), and the lowest value in the control and n. 6 (7.66 and 7.36 mg/g fw, respectively). the antioxidant activity is higher in the treatments of addition of temperature by electric mat (n. 1 and 2) with ic50 (dpph assay) values of 0.80 and 0.82 mg/ml respectively, followed by the trials n.3 and 4. flowers of the control and treatment n.6 showed the lower scavenger reducing activity. in the present work the concentration of total polyphenols ranged 7.36 and 12.42 mg gae/g fresh weight. these values agree with those found in v. × wittrockiana, reported by other authors (rop et al., 2012). however, the polyphenol values could be underestimated by the method of extraction, as already shown in gonzàles­ barrio et al. (2018). in fact, they reported different polyphenol amounts in v. × wittrockiana by using either acidic hydrolysis or maceration instead of the method adopted in this work (bretzel et al., 2014). other reports showed the influence of storage at different temperature in different flowers (kelley et al., 2003). moreover, different packages used for the storage conditions can affect the quality of flowers (landi et al., 2018). changes of appearance, and aes­ thetic value were performed on v. × wittrockiana (kelley et al., 2003). the results obtained at the time table 3 ­ determination of carotenoids, anthocyanins, polyphenols, radical scavenging activity (dpph assay), and soluble sugars of viola cornuta flowers grown under different greenhouse conditions (av1­7, see table 1) and cold stored for 0 (time 0) o 4 (time 4) days postharvest data are expressed as means (n=3, ± se.) anova followed by fisher’s probable least­square difference test was used, with a cut­off signi­ ficance at p=0.05. smalls letter indicate comparisons between treatments at the same postharvest day (phd); capital letters indicate comparisons between the two phd for the same treatment. interaction between treatments and phd were analysed by two­way anova. treatment carotenoids (mg/g fw) anthocyanins (mg/g fw) polyphenols (mg/g fw) dpph assay (ic50 mg/ml) soluble sugars (mg/g fw) time 0 1 electric mat 50°c+ light 1 h 0.32 ± 0.00 a a 10.42 ± 0.32 a a 12.42 ± 0.11 a a 0.82 ± 0.00 a a 209.69 ± 3.91 a a 2 electric mat 50°c 0.27 ± 0.00 c b 8.05 ± 0.20 c a 9.93 ± 0.68 b b 0.80 ± 0.01 a a 183.84 ± 6.97 b a 3 light 2 h 0.14 ± 0.01e b 5.63 ± 0.14 e b 11.00 ± 0.09 ab a 0.84 ± 0.02 aba 179.57 ± 3.98 b a 4 (hot water 35°c + light 1 h) 0.29 ± 0.01 bc a 9.28 ± 0.49 bc a 11.85 ± 0.40 ab a 0.86 ± 0.02 ab a 207.45 ± 4.85 a a 5 hot water 35°c) 0.28 ± 0.01 c a 10.29 ± 0.25 ab a 9.35 ± 1.06 b a 0.89 ± 0.01 b b 181.29 ± 3.84 b a 6 hot water 35°c + light 2 h 0.26 ± 0.00 d a 6.93 ± 0.42 da 7.36 ± 0.16 c b 1.19 ± 0.05 d b 183.90 ± 10.96 b a 7 control 0.30 ± 0.00 b a 8.71 ± 0.57 cd a 7.66 ± 0.25 c b 1.09 ± 0.01 c b 179.90 ± 1.44 b a time 4 1 electric mat 50°c+ light 1 h 0.33 ± 0.01 b a 4.81 ± 0.19 d b 12.46 ± 0.25 a a 0.78 ± 0.01 a a 203.61 ± 2.03 a a 2 electric mat 50°c 0.36 ± 0.01 a a 7.63 ± 0.18 c a 11.62 ± 0.16 b a 0.82 ± 0.02 aa 158.91 ± 2.90 d a 3 light 2 h 0.21 ± 0.00 e a 9.22 ± 0.19 b a 10.88 ± 0.21 bc a 0.84 ± 0.01 b a 161.09 ± 0.36 d b 4 hot water 35°c + light 1 h 0.28 ± 0.01 c a 10.73 ± 0.56 a a 11.65 ± 0.32 ab a 0.90 ± 0.02 c a 175.50 ± 1.30 c b 5 hot water 35°c 0.23 ± 0.00 d a 6.65 ± 0.44 c b 10.43 ± 0.45c a 0.77 ± 0.02 a a 164.42 ± 3.41 d a 6 hot water 35°c + light 2 h 0.25 ± 0.01 d a 4.75 ± 0.49 d b 9.32 ± 0.04 d a 0.94 ± 0.02 c a 159.48 ± 2.99 d b 7 control 0.27 ± 0.01 c b 7.49 ± 0.79 c a 10.37 ± 0.27 c a 0.78 ± 0.01 a a 192.31 ± 2.56 b a anova p‐value treatment < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 phd 0.0014 < 0.0001 < 0.0001 < 0.0001 < 0.0001 treatment × phd < 0.0001 < 0.0001 0.0041 < 0.0001 0.0060 marchioni et al. ‐ marchioni et al. ‐ cultivation and post‐harvest strategies on viola cornuta flowers 67 of cold storage (time 4) were compared to those at the time of harvest (time 0). the data reported here indicated that the purple­pink flowers maintained the carotenoids content after 4 days of cold storage in the treatments n. 1, 4 and 6, whereas in the treat­ ments n. 2 and 3 values of carotenoids increased. meanwhile, in flowers of treatment n. 5 and 7 (con­ trol) the amount of carotenoids decreased (table 3). after 4 days of postharvest treatment, anthocyanins are the most affected metabolites by cold storage. in fact, the amount of anthocyanins decreased in the treatments n. 1, 5 and 6, whereas in the treatment n. 3 values increased (9.22 mg/g fw). the treatment n. 1 showed the largest decrease, 10.42 mg/g fw at time 0 and 4.81 mg/g fw at time 4. however, the loss of pigmentation is not always documented, but it is peculiar of each species and variety, as already demonstrated in other species as acmella oleracea, salvia discolor, begonia semperflorens, tropaeolum majus (landi et al., 2018). the total polyphenols con­ tent after cold storage maintained the same values of that detected at time 0, with the exception of treat­ m e n t s n . 2 , 6 a n d c o n t r o l , w h e r e p o l y p h e n o l s increased, 11.62, 9.32 and 10.37 mg/g fw at time 4, respectively (table 3). the antioxidant activity increased in the treatments with hot water (n.4, 5, 6). different susceptibility to the storage process was observed in other edible flowers, with different changes (increase or decrease) on nutraceutical val­ ues up to 8 days of postharvest (landi et al., 2018). the soluble sugars dropped significantly in the treat­ ment n. 4, since the values was the highest at time 0 but reduced at 80% at time 4 (207.45 and 175.5 mg/g fw). other decrease in the content of sugar is observed for the treatments n.3 and 6. soluble sug­ ars are important nutritional components of the flowers and represent a good characteristic for the choice of edible flowers (mlcek and rop, 2011). however, there are few works on the sugar profile of edible flowers, e.g. rosa micrantha (guimarães et al., 2010). in experiment done with cut lily flowers was discussed the role of reducing sugars, as a typical reaction of plants that defend themselves against injury due to chilling or frost (van doorn and han, 2011). 4. conclusions the different cultivation treatments used in this work are differently correlated with the analyzed metabolites. in order to obtain flowers with high quality of brilliant color, one hour of supplementary lighting and a basal heating of 50°c seem to be the r i g h t c o m b i n a ti o n o f f a c t o r s . t h e c o l d s t o r a g e imposed to the flowers as the post­harvest treatment indicated that the flowers treated with additional 2 h of light (treatment n. 3) retained values of the metabolites during the post­harvest, with the excep­ tion of sugars. however, even if the additional light­ ing seems to preserve the flowers from depigmenta­ tion and to maintain the nutraceutical compounds, the decreased content of the observed sugars could be a consequence of the phenomenon of senes­ cence. further studies on the influence of illumina­ tion on plastic bags and the evaluation of ethylene production can be useful for the definition of the post­harvest process in v. cornuta. in addition, the investigation of the other minor nutritional compo­ nents can be crucial to define a more detailed condi­ tion of storage. acknowledgements t h i s w o r k w a s s u p p o r t e d b y a g r a n t f r o m european union in the frame of interreg alcotra v­a france­italy antea project n. 1139 ­ attività innovative per lo sviluppo della filiera del fiore edule/fleurs comestibles: innovations pour le devel­ opment d’une filière transfrontalière. references adams s.r., pearson s., hadley p., 1997 ­ the effects of temperature, 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gammon, engenharia agronômica, rua prefeito jayme monteiro, 791, 19700‐000 paraguaçu paulista, são paulo, brazil. key words: elicitation, fruit quality, phenolic compounds, secondary metabolites, solanum lycopersicum. abstract: salicylic acid (sa) and ferulic acid (fa) are considered phenolic com­ pounds that act as elicitors due to their regulatory functions on plant growth, development, metabolic and physiological responses in plants. the aim of this research was to evaluate the effect of sa and fa on growth, fruit quality and synthesis of secondary metabolites in tomato (solanum lycopersicum cultivar santa clara). the experiment was conducted in pots in a greenhouse. the appli­ cation of sa and fa was performed at concentration of 1.0 mmol l­1 alone and in combination, with water treated plants as control. exogenous application of sa and fa either alone or in combination (sa + fa) resulted in increases in bio­ mass accumulation and chlorophyll contents in tomato plant; and soluble sugar, total polyphenol, flavonoids, lycopene and β­carotene contents in fruits. it was concluded that application of sa and fa resulted in higher production and concentration of secondary compounds in tomato. 1. introduction tomato (solanum lycopersicum l.) belongs to the solanaceae family and it is considered as low­calorie food. it is a good source of vitamins a, c and e, and mineral salts (adalid et al., 2010; kazemi, 2014). in addition to vitamins and minerals, tomato has a high lycopene and β­carotene content, which has antioxidant and anticancer properties. carotenoids is naturally present in many fruits and vegetables and plays an important role as a functional food when consumed as part of the diet, producing specific health benefits such as reducing the risk of various diseases (martínez­hernández et al., 2016; mehta et al., 2018) and polyphenols (flavonoids, flavanones and flavones) are also present in significant amount in tomato acting as antioxidant, anti­mutagenic, anti­proliferati­ ve, anti­inflammatory and anti­atherogenic activities (martí et al., 2016; chaudhary et al., 2018). salicylic acid (sa), a natural plant hormone, act as an important sig­ nalling molecule triggering tolerance against abiotic and biotic stresses (*) corresponding author: pgorni@gmail.com citation: gorni p.h., da silva cornelissen b., pereira a.a., 2021 ­ exogenous salicylic acid and ferulic acid improve growth, phenolic and carotenoid content in tomato. ­ adv. hort. sci., 35(4): 335­ 341. copyright: © 2021 gorni p.h., da silva cornelissen b., pereira a.a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 3 march 2020 accepted for publication 3 september 2021 ahs advances in horticultural science https://doi.org/10.36253/ahsc-8295 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2021 35(4): 335­341 336 (hernández­ruiz and arnao, 2018; gorni et al., 2020; gorni et al., 2021). sa plays a significant role in many physiological and biochemical processes of the plant, being able to act as growth regulators and also as an abiotic elicitor capable of increasing the synthesis of secondary compounds beneficial to human health (javanmardi and akbari, 2016). ferulic acid (fa) is a phenolic compound syn­ thetized from the metabolism of phenylalanine and tyrosine in plants. it is an important biological and structural component of the plant cell wall and it accumulates in soil and influences plant growth (li et al., 2013; paiva et al., 2013). fa is considered as non­ enzymatic antioxidant and acts under stress to elimi­ nate free radicals produced in plants (andreasen et al., 2001; engwa, 2018). studies on the exogenous application of fa in plants have demonstrated that this compound acts as an important regulator of sev­ eral physiological processes related to plant growth and mitigating stress, such as, stomatal closure, cell division, membrane permeability, photosynthesis, respiration and many other metabolic processes (santos et al., 2008; li et al., 2013; singh and deen, 2014; hussain et al., 2017; cheng et al., 2018). however, exogenous sa (kazemi, 2014) and fa (singh and deen, 2014) applications were effective in inducing the growth and formation of secondary metabolites in tomato plants. therefore, it is suggest­ ed that the application of sa and fa may result in combined effects of growth promotion and induction of biosynthetic pathways of secondary metabolism. previous studies have revealed the hormonal and eliciting action of sa and fa (1.0 mmol l­1) in tomato plants (hussain et al., 2017; kumar et al., 2017). in t h i s c o n t e x t , t h e a p p l i c a ti o n o f s a a n d f a c a n improve the productive performance and the biosyn­ thesis of secondary compounds in tomato, making possible an increase in the commercial value of this crop, reaching greater market competitiveness. in this study we evaluated the effect of leaf spray of sa and fa either alone and in combination on the growth, yield and secondary compounds in tomato cultivar santa clara. 2. materials and methods the experiment was conducted under greenhouse (without temperature and humidity control) covered with a 50% solar radiation shade located in gammon colleges, paraguaçu paulista (22°41’76” s, 50°58’33” w, 517 a.s.l.), sao paulo, brazil. trademark tomato cultivar santa clara seeds were placed in a germination tray and after the training period (30 days) they were planted in 18 l pots con­ taining soil. soil samples were collected and submit­ ted to chemical analysis according to van raij et al. (2001) (table 1) and was corrected by applying lime­ stone to increase the saturation of bases to 80% (40.0 g pots­1), potassium chloride (3.0 g pots­1) and simple super phosphate (20.0 g pots­1). for fertiliza­ tion with micronutrients it was added yoorin master 1 si® (granulated) (si: 10%, b: 0.1%, mn: 0.3%, cu: 0.05% and zn: 0.55%) (1.5 g pots­1), according to the recommendations of bulletin 100 (iac) for tomato species. the pots were irrigated by sprinklers per day at 8 a.m. and 5 p.m. in order to keep the soil mois­ ture and ensure the availability of water throughout the experimental period. the application of salicylic acid (sa: 138.121 g mol­ 1) and ferulic acid (fa: 194.18 g mol­1) was made for 3 consecutive days after having reached 20 days from transplant. foliar applications of sa and fa at a dose of 1.0 mmol l­1 alone and in combination, and water­ treated plants were used as control. sa and fa treat­ ments were carried out by spraying the shoots of the plants with water­based solutions supplemented with agral® (50 μl l­1 of solution) until the drop point (10 ml per plant), as follows: t1 ­ plants sprayed only with water (control); t2 ­ applications of 1.0 mmol l­1 sa (sa); t3 ­ applications of 1.0 mmol l­1 fa (fa); and t4 ­ applications of 1.0 mmol l­1 sa + 1.0 mmol l­1 fa (sa + fa) by spraying the plants for three consecutive days. plants were harvested 90 days after transplanting the seedlings by collecting leaves, roots and fruits. plant growth the effect of sa and fa on the plants were evalu­ table 1 ­ chemical analysis of the soil used in the experiment ph organic matter g dm­3 h + al mmolc dm­3 ca mmolc dm­3 mg mmolc dm­3 k mmolc dm­3 p mg dm­3 4.4 6.0 20.0 7.0 2.0 0.8 6.0 gorni et al. ‐ salicylic acid and ferulic acid elicitation in tomato 337 ated based on the fallowing: leaf area (cm2), number of leaves per plant, number of fruits per plant, plant height (cm), dry weight of shoot and roots (g plant­1) and mean fruit weight (g plant­1). the leaf area was assessed using imagej® software (powerful image analysis) with 5 plants per treatment. the number of leaves and fruit per plant was determined by manual counting, considering fully expanded leaves and fruits at harvest point. the plant height was deter­ mined by measuring tape. the shoot and root dry weight were determined after drying in an oven with air circulation at 70°c until constant weight. chlorophyll content chlorophyll content (μg ml­1) was determined spectrophotometrically following extraction in aceto­ ne, according to the method of lichtenthaler (1987). fresh leaf tissue (0.6 g) was added in 7 ml of 80% acetone, the tubes were shaken and left to stand for 30 minutes. the readings were performed in a spec­ trophotometer at λ 663 and λ 645 nm. total soluble sugar content total soluble sugar content in fruit was determi­ ned according to the method described by dubois et al. (1956) using glucose as the standard. fresh tissue (0.1 g) was added in 10 ml of 80% ethanol, and mac­ erated in a mortar and left to rest for 30 min. the analyzes followed with the addition of phenol (5%) and sulfuric acid (98 n). the readings were per­ formed in a spectrophotometer at λ 490 nm. preparation of fruit tomato extract determinations of total polyphenolic compounds and total flavonoid were obtained from the tomato juice. the fruits were ground in a low speed food pro­ cessor (3000 rpm) for two minutes and passed in 2 mm sieves. total polyphenols contents total polyphenols contents were determined spectrophotometrically (λ 765 nm) by folin­ciocalteu method with modification described by stagos et al. (2012). the total polyphenols contents were report­ ed based on micrograms of gallic acid equivalents per milliliters (μg gae ml­1). gallic acid solutions were prepared with concentrations of 25 to 500 μg ml­1 in absolute ethyl alcohol. the assays were performed in triplicate. total flavonoid content total flavonoid content was determined spec­ trophotometrically (λ 510 nm) by the method of yao et al. (2013). the total flavonoids contents were reported based on micrograms of rutin equivalents per milliliters (μg re ml­1). rutin solutions were pre­ pared with concentrations of 25 to 500 μg ml­1 in absolute ethyl alcohol. the assays were performed in triplicate. carotenoid content carotenoid content were extracted from fresh tomato samples after homogenisation of whole fruits. the homogenised sample was mixtured with chloroform/acetone/ethanol (2:1:1, v/v/v) (sadler et al., 1990). for the determination of lycopene and β­ carotene, the absorbance was read at λ 470 and λ 4 5 0 n m . c a r o t e n o i d c o n t e n t w e r e d e t e r m i n e d according to the equation described by craft and soares (1992), using the molar extinction coefficient of 3450 for lycopene, and 2592 for β­carotene and expressed in micrograms per grams (µg g­1). the assays were performed in triplicate. statistical analysis the experiment was arranged in a completely ran­ domized design with four treatments and five repli­ cations per treatment. the shapiro­wilk test was used to ensure the normality assumption and the homogeneity of variances. the data were submitted to analysis of variance (p≤0.05) and tukey test (p≤0.01) using the sisvar software. the results were presented by means of the treatments and standard error. 3. results the results indicated that sa and fa both caused a significant increase of growth in tomato (table 2). the exogenous application of 1.0 mmol l­1 sa resul­ ted in increasing in the accumulation of root dry weight by 61% and total dry weight by 41.3% compa­ red to control plants. the application of 1.0 mmol l­1 fa resulted in increasing in shoot dry mass by 52.3% and total dry mass by 52% compared to control plants. however, the interaction between sa + fa resulted in increasing in shoot dry mass by 51.5%, root dry mass by 146.37% and total dry mass by 66.6%, when compared to control plants. fruit weight increased by 16.5% in plant treated with 1.0 mmol l­1 fa. the exogenous application of sa + fa resulted in increasing the leaf area by 47% and number of leaves by 204.3% compared to control plants (table 3). application of sa and fa showed increasing of 101.8 adv. hort. sci., 2021 35(4): 335­341 338 and 167.5% for the number of leaves, 70.2 and 68.3% for the number of fruits, when compared with con­ trol plants (table 3). the plant height treated with sa and fa was not changed in relation to control plants. chlorophyll a content (table 4) observed in toma­ to plants treated with sa, fa and the interaction with sa + fa were significantly higher by 26, 12 and 27.6%, respectively compared to control. chlorophyll b and total chlorophyll decreased with the applica­ tion the elicitors (table 4). the total soluble sugar content in fruits, increased up to 36% in 1.0 mmol l­1 sa and 163% in 1.0 mmol l­1 fa treated plants when compared to the control. the exogenous application of sa, fa and the inter­ action with sa + fa resulted in increasing in the accu­ mulation of total polyphenols of 20.4, 110.3 and 12%, in relation to control plants (table 5). total flavonoid content in tomato were 81, 107 and 37% higher in sa, fa and the combined with sa + fa treatments when compared to control plants, respectively. in relation to carotenoid content plants treated with sa also presented increases of 157.3% (lycopene), and 157.7% (β­carotene). the combination of sa+fa showed increases of 65.25% (lycopene), and 65.3% (β­carotene), respectively, in comparison to the con­ trol (table 5). table 3 ­ effect of salicylic acid and ferulic acid on leaf area, number of leaves per plant, number of fruits per plant and plant height in tomato table 4 ­ effect of salicylic acid and ferulic acid on the chlorophyll and fruit total soluble sugar contents in tomato table 2 ­ effect of salicylic acid and ferulic acid on the shoot, root, total dry weight and fruit weight in tomato different letters indicate significant differences by tukey’s test (p≤0.01). treatments shoot dry weight (g plant­1) root dry weight (g plant­1) total dry weight (g plant­1) fruit weight (g plant­1) control 24.01±1.11 c 4.55±0.83 c 28.57±1.60 c 524.46±2.07 b sa 33.06±1.59 abc 7.32±0.36 b 40.38±1.60 b 554.29±7.44 b fa 36.57±2.35 a** 6.84±0.42 bc 43.41±1.86 ab 610.78±11.94 a** sa + fa 36.38±3.99 a 14.02±1.72 a** 47.59±4.57 a** 525.40±20.23 b cv (%) 15.78 15.08 12.04 5.39 treatments leaf area (cm²) number of leaves number of fruits plant height (cm) control 7955.4±466.9 b 65.2±3.69 c 20.8±0.86 b 77.4±2.67 ns sa 8739.6±1054.1 ab 131.6±16.02 b 35.4±3.81 a ** 79.6±2.75 fa 10209.8±850.6 ab 174.4±10.49 ab 35.0±3.20 a 88.2±7.20 sa + fa 11691.6±577.1 a * 198.4±10.09 a ** 28.6±0.40 ab 77.0±1.04 cv (%) 16.11 16.71 19.89 10.75 different letters indicate significant differences by tukey’s test (p≤0.01). treatments chlorophyll a (µg ml­1) chlorophyll b (µg ml­1) total chlorophyll (µg ml­1) fruit total soluble sugar (mg g­1) control 9.66±0.23 c 21.02±0.13 a ** 30.68±0.14 a ** 22.63±0.96 c sa 12.13±0.07 a 15.69±0.02 c 27.88±0.04 c 30.77±1.38 b fa 10.82±0.04 b 13.03±0.08 d 23.90±0.05 d 59.47±1.92 a ** sa + fa 12.33±0.07 a ** 16.05±0.02 b 28.45±0.04 b 26.89±0.21 bc cv (%) 1.73 0.73 0.59 7.30 different letters indicate significant differences by tukey’s test (p≤0.01). gorni et al. ‐ salicylic acid and ferulic acid elicitation in tomato 339 4. discussion and conclusions tomato plants treated with sa and fa presented higher total biomass (table 2). the effect of elicitors plays a key role in growth regulation, plant develop­ ment and these findings can be associated with the changes in hormone functions or the improvement of photosynthesis and carbohydrate accumulation in plants (hussain et al., 2017; gorni et al., 2020). the foliar application of sa and fa trigger a greater cellu­ lar activity that result in a larger number of leaves and leaf area, thus presenting a larger photosynthetic surface, improving the physiological processes (gorni and pahceco, 2016; hussain et al., 2017). regarding the increase of leaf area, number of leaves, number of fruits and fruit weight, our results showed that the application of sa and fa can alter the growing pat­ tern, in tomato plants (table 3). the application of sa and fa increased the growth of leaves and roots, wit­ hout changing the plant height (khan et al., 2003; de carvalho et al., 2020). biomass gains as an effect of sa application were also observed in achillea millefo‐ lium (gorni and pacheco, 2016), foeniculum vulgare (gorni et al., 2017) and mentha spicata (kundu et al., 2018). increase in biomass, were also observed after fa application in plants of arabidopsis thaliana (reigosa and pazos­malvido, 2007), pisum sativum (orcaray et al., 2011) and cucumis sativus (li et al., 2013). p i g m e n t s a r e t h e e s s e n ti a l c o m p o n e n t s f o r growth and development and they can determine the health status of plants (hussain et al., 2017; gorni et al., 2020). based on our results, the applica­ tion the elicitors sa and fa alone or in combination suggest an increase in chlorophyll a (table 4). on the other hand, the application of sa and fa did not affect the chlorophyll b and total chlorophyll content, which directly reflected in the plant height values table 5 ­ pair­wise genetic distance estimates based on observed phenotypes of 21 amaranthus accessions treatments polyphenols (µg gae ml­1) flavonoid (µg re ml­1) lycopene (µg g­1) β­carotene (µg g­1) control 245.58±7.21 c 272.33±4.81 d 7.54±0.16 c 10.03±0.2 c sa 295.58±12.02 b 493.16±2.40 b 19.4±0.16 a** 25.85±0.2 a** fa 516.42±0.00 a** 564.00±4.81 a** 6.96±0.16 c 9.25±0.2 c sa + fa 274.75±4.81 b 372.33±4.81 c 12.46±0.00 b 16.58±0.0 b cv (%) 2.60 1.85 2.39 2.17 different letters indicate significant differences by tukey’s test (p≤0.01). (table 3). anway, other studies conducted on diffe­ rent species reported increases in chlorophyll a, chlo­ rophyll b and total chlorophyll contents consequen­ ces of sa treatment (kazemi, 2014; chakraborty et al., 2016) and in plants sprayed with fa (zhu and wakisaka, 2018). positive effects of sa and fa on the physiological processes of tomato suggest that these elicitors may be associated with the regulation of several essential primary metabolic processes, inclu­ ding synthesis of chlorophylls (zhu and wakisaka, 2018; gorni et al., 2020). the positive effects of the application of sa and fa on the physiological processes of tomato plants were also reflected in higher soluble sugar content (table 4). sugar is one of the ingredients in tomato (hafeznia et al., 2014), and it is an important organic solute with low molecular weight in higher plants. the sugars which are accumulated under stress con­ ditions or as consequence of treatments with sa or fa, keeping osmotic regulation and turgor inside the plant (orcaray et al., 2011; gorni et al., 2020). results show that application of sa also resulted in increa­ sing the total soluble sugar content in tomato plants (kazemi, 2014), and chamomile (zarinkamar et al., 2013); similarly to application of fa in pea (orcaray et al., 2011) and soybean (ferrarese et al., 2001). polyphenols and flavonoids provide many physio­ logical functions for plant survival and are of funda­ mental importance for plants adaptation (verma and shukla, 2015; mehta et al., 2018). our results suggest that the application of sa and fa may modulate and alter the concentration of polyphenolic compounds (table 5) in tomato plants. studies report that the application of sa and fa stimulates the phenyl­ propanoid pathway, increasing the accumulation of polyphenols and flavonoids in plants (salvador et al., 2013; gorni et al., 2021). tomato plants have a good free radical scaven­ ging capacity due to the high concentration of lyco­ zhu m.x., zhao c.f., bai j.g., 2018 ­ ferulic acid pre‐ treatment alleviates heat stress in blueberry seedlings by inducing antioxidant enzymes, proline, and soluble sugars. ­ biol. plant., 62(3): 534­542. craft n.e., soares jr. j.h., 1992 ­ relative solubility, sta‐ bility and absorptivity of lutein and beta‐carotene in organic solvents. ­ j. agr. food chem., 40(3): 431­434. de carvalho j.s.b., da silva j.p.r., batista r.d.c.m., 2020 ­ use of salicylic acid as an attenuator to the effects of water deficit on basil plants. ­ diversitas j., 5(3): 1561­1574. dubois m., gilles k.a., hamilton j.k., rebers p.t., smith f., 1956 ­ colorimetric method for determina‐ tion of sugars and related substances. ­ anal. chem., 28(3): 350­356. engwa g.a., 2018 ­ free radicals and the role of plant phytochemicals as antioxidants against oxidative stress‐related diseases. phytochemicals: source of anti‐ oxidants and role in disease prevention. ‐ bod­books on demand, 7: 49­74. ferrarese m.l.l., souza n.e., rodrigues j.d., ferrare­ se­filho o., 2001 ­ carbohydrate and lipid status in soybean roots influenced by ferulic acid uptake. ­ acta physiol. plant., 23: 421­427. gorni p.h., brozulato m.o., lourenção r.s., konrad e.c.g., 2017 ­ increased biomass and salicylic acid elici‐ tor activity in fennel (foeniculum vulgare miller). braz. j. food technol., 20: e2016172. gorni p.h., pacheco a.c., 2016 ­ growth promotion and elicitor activity of salicylic acid in achillea millefolium l. ­ afr. j. biotechnol., 15(16): 657­665. gorni p.h., pacheco a.c., lima moro a., silva j.f.a., moreli r.r., de miranda g.r., pelegrini j.m., spera k.d., bronzel jr j.l., silva r.m.g., 2020 ­ salicylic acid foliar application increases biomass, nutrient assimila‐ tion, primary metabolites and essential oil content in achillea millefolium l. ­ sci. hort., 270:109436. gorni p.h., pacheco a.c., lima moro a., silva j.f.a., moreli r.r., de miranda g.r., pelegrini j.m., zani­ boni c.b., spera k.d., bronzel jr j.l., silva r.m.g., 2021 ­ elicitation improves the leaf area, enzymatic activities, antioxidant activity and content of secondary metabolites in achillea millefolium l. grown in the field. ­ j. plant growth, 40(4): 1652­1666. hafeznia m., mashayekhi k., ghaderifar f., mousavi­ zadeh s.j., 2014 ­ tomato morphological and bioche‐ mical characteristics in response to foliar applying of salicylic acid. ­ int. j. biosci., 5(9): 237­243. hernández­ruiz j., arnao m., 2018 ­ relationship of melatonin and salicylic acid in biotic/abiotic plant stress responses. ­ agronomy, 8(4): 33. hussain i., singh n.b., singh a., singh h., singh s.c., yadav v., 2017 ­ exogenous application of phytosynt‐ hesized nanoceria to alleviate ferulic acid stress in solanum lycopersicum. ­ sci. hortic., 214: 158­164. javanmardi j., akbari n., 2016 ­ salicylic acid at differ‐ 340 adv. hort. sci., 2021 35(4): 335­341 pene and β­carotene. this pigment, it is the most effective antioxidant which gives color to tomato fruit as maturity progresses (kumar et al., 2017). polyphenolic compounds and carotenoid act as anti­ oxidants because they remove singlet oxygen and other free radicals in cells (shahidi and ambigaipalan, 2015) due to their ability to donate hydrogen from hydroxyl groups positioned along the aromatic ring to prevent oxidation by radicals free from lipids and other biomolecules (sousa et al., 2007). researchers showed an increase in carotenoid content during ripening due to the gradual degrada­ tion of chlorophyll (adalid et al., 2010). our results showed that the application of sa and fa may modu­ late and alter the concentration of lycopene and β­ carotene (table 5) and chlorophyll a contents (table 4) in tomato plants. thus, our study evidenced that foliar application of sa and fa in tomato crop could enhance its antioxidant activity due to the higher concentration of these bioactive compounds. our results corroborate with those found by kumar et al. 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federal university of goiás, goiania, goiás, brazil. 2 federal university of uberlândia, uberlândia, minas gerais, brazil. key words: genetic parameters, processed tomato, solanum lycopersicum. abstract: source of important vitamins, fibers, and minerals, the tomato (solanum lycopersicum l.) stands out in the world agricultural scenario for its economic and social relevance and versatility. the brazilian market is dominat­ ed by multinationals companies, and this market segment obtains cultivars from other countries, with genetics accurate to climatic conditions and cultiva­ tion method very different from those used in brazil. as a result, the local culti­ vation of tomatoes plants becomes dependent on market variations and has required a material that has limited production efficiency. this study aimed to estimate genetic parameters from agronomic traits and to select industrial tomato lines using the selection index. a randomized block experimental design with three replications was used. eighty­five industrial tomato lines from the germplasm bank of the vivati plant breeding ltda were evaluated. each plot had 12 plants. the two central plants of each plot were evaluated. the evaluations were carried out using adapted morphological descriptors described in the guidelines for carrying out the distinguishability, homogeneity, and stability (dhe) tests of the ministry of agriculture, livestock, and supply of brazil (mapa). the genotypic determination coefficient (h2) of the traits related to fruit pericarp thickness, fruit firmness, fruit yield, average cycle, average number of fruits per plant, and soluble solids was high. the base index and the classic index presented the largest gain from selection for the fruit yield trait. rank summation index and genotype­ideotype distance index had the highest total selection gain values. the tomato lines pxt­601 and pxt­610 stood out as superior genotypes by the methods of direct selection and by selection indexes. 1. introduction tomato is grown in different regions of the world and stands out as the most produced vegetable in the world, second only to potatoes in the cultivated area (geraldini et al., 2018). part of the success of tomatoes comes from its diversity in food and nutritional aspects that help human health. the fruit is rich in vitamins a and c and lycopene, substances that help prevent cancer of the gastrointestinal tract (simão and rodriguez, (*) corresponding author: nascimento_mariana1@hotmail.com citation: viera nascimento m., ribeiro ávila m.c., fiori de abreu­tarazi m., oliveia nogueira a.p., cardoso campos l.f., dos reis nasci­ miento a., 2020 ­ identification of promising tomato breeding lines with determinate growth by selection index. ­ adv. hort. sci., 34(3): 337­ 347 copyright: © 2020 viera nascimento m., ribeiro ávila m.c., fiori de abreu­tarazi m., oliveia nogueira a.p., cardoso campos l.f., dos reis nascimiento a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 11 january 2020 accepted for publication 18 february 2020 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(3): 337­347 338 2008). brazil is in ninth place in the world tomato pro­ duction ranking. at the top is china, accounting for 31% of production, followed by india with 11%, and the united states with 8% of global production (dossa and fuchs, 2017). in 2017, 36.688 hectares of tomato were grown in brazil, 47.40% of the produc­ tion was destined for fresh consumption, and 52.60% for processing industries (marcomini and molena, 2018). the brazilian national market is dominated by multinationals and acquires imported cultivars, with genetic characteristics adapted to climatic conditions and cultivation systems very different from those found in brazil. as a result, the brazilian cultivation of tomato becomes dependent on market swings. it obtains cultivars with productive potential restricted if compared to the yields reached in the environment that they were developed. also, the plants may suffer losses by climate intolerances and plant diseases, when facing the brazilian growing conditions. due to the economic importance of the crop, tomatoes produced for processing industries have been the focus of research, especially in genetic breeding with the aim of produce cultivars that pos­ sess genes able to assist in the adaptation and toler­ ance to biotic and abiotic stresses, which can result in important contributions to the sector (parmar et al., 2017). in a breeding program, the objective is to enhance the main phenotypic trait and conserve or improve the expression of secondary traits at the same time (nogueira et al., 2012). however, the direct selection of quantitative traits is influenced by the environ­ ment, which may cause unfavorable changes in other traits (vasconcelos et al., 2010). one way to improve this process is to use the simultaneous selection of a group of important agro­ nomic traits, that is, to use the selection indexes. these indexes relate information of different traits and make it possible to perform a selection effective­ ly, which increases the probability of success in a plant breeding program (cruz et al., 2012; vianna et al., 2013; rezende et al., 2014). considering the importance of the industrial pro­ cessing of tomatoes and the market demand for cul­ tivars that meet the requirements of this industrial chain, it is indispensable to know the relationship between agronomic traits and the study of the index­ es. this makes it possible to obtain the best predic­ tion of gains and yields and greater efficiency in the selection process. given the above, this aimed to estimate genetic parameters for agronomic traits and to select industrial tomato lines using the selection index. 2. materials and methods the study was conducted in the experimental area of vivati plant breeding ltda, in abadia de goiás unit, goiás, brazil, at 16°45’26” s, 49°26’15” w, and 898 m of altitude. the climate, according to koppen, is classified as tropical humid, characterized by rainy summer with high temperatures and dry winter, with an average annual rainfall of 1.575 mm. the genotypes analyzed in this study are owned by vivati plant breeding ltda, which use their own selection and maintain methods. the seeds were sown in 450­cell polystyrene trays, filled with a sub­ strate composed of coconut fiber, rice husk, and peat and covered with vermiculite. the trays were kept in a greenhouse for 35 days when the seedlings had from two to three true leaves, and they were able to transplant to the field. the soil preparation was carried out with a tractor and rotary tiller. seedbeds were prepared with 1.0 m wide, 0.20 m high, with 1.0 m spacing between beds. at the transplanting, 1.500 kg ha­1 of the npk formu­ lation 04­30­10 was applied. as topdressing fertiliza­ tion, 20 kg ha­1 of map, 75 kg ha­1 of ammonium sul­ fate, 100 kg ha­1 of ammonium nitrate, and 200 kg ha­ 1 of potassium chloride were divided into four appli­ cations with a 20­day interval after transplanting. the seedlings were manually transplanted to the fi e l d 3 5 d a y s a ft e r s o w i n g ( d a s ) , w i t h 0 . 4 0 m between plants and 1.0 m between rows. irrigation was performed by a drip system, supplying the water requirement based on the parameters for crop irriga­ tion management. weed control was performed weekly to avoid competition. insecticide baits were placed through­ out the field to identify the insect infestation rate and help the decision of pesticide application. phytosanitary control was carried out whenever nec­ essary, to maximize fruit production (fao, 2006). the tomato lines were characterized by morpho­ logical traits contemplated in the guidelines for per­ forming the distinguishability, homogeneity, and sta­ bility (dhe) assays by the mapa, which were modi­ fied by the authors. a randomized block experimen­ tal design with three replications was used. eighty­ vieira nascimento et al. ‐ identification of tomato strains in brazil 339 five industrial tomato lines were evaluated. each plot had 12 plants. the two central plants of each plot were evaluated. the descriptors analyzed are shown in table 1. it was estimated the genotypic determination coefficient (h2), according to the estimator below: where: h2 = genotypic determination coefficient; ∅̂ = quadratic genetic component; qmt = mean square of genotypes; qmr = mean square of the residue; and ϒ = number of replications. genotypes were grouped based on the scott­ k n o tt t e s t a t t h e 1 % a n d 5 % p r o b a b i l i t y l e v e l . subsequently, the selection gains estimates were reached by the aid of the selection index methodolo­ gies cited by cruz (2006): direct and indirect selec­ tion; classic index proposed by smith (1936) and hazel (1943); rank summation index of mulamba and mock (1978); base index of williams (1962); and genotype­ideotype distance index (gid). the selec­ tion criterion applied was to increase the traits: fruit pericarp thickness (fpt), fruit firmness (ff), yield (yld), average number of fruits per plant (nfp), and soluble solids (ss). the index proposed by smith (1936) and hazel (1943) was established by the selection index (i) and the genotypic aggregate (h) described below: where: n = number of traits evaluated; b = vector of dimension 1 xn of the selection index weighting coefficients to be estimated; table 1 ­ descriptors for industrial tomatoes (adapted from mapa, 2005) and details on their analysis traits trait description description code comments 01. fruit pericarp thickness slim s the analysis was performed using a digital caliper, measuring the diameter (mm) from the outer wall to the inner wall of the pericarp average a thick t 02. fruit: firmness soft s the analysis was performed by subjecting the fruits to pressure at one point in the middle region, measuring the resistance of the pulp to penetration, using instrutherm model ptr­300 digital penetrometer, and obtaining the values expressed in newton (n) medium m firm f 03. maturation cycle precocious p it was evaluated from the transplanting of seedlings medium m late l 04. yield low l it was determined by the weight and number of fruits per plant average a high h 05. number of fruits per low l it was counted all fruits of each plant, including the green and damaged ones average a high h 06. soluble solids low l the analysis was performed by transferring a drop of the fruit juice to the hanna instruments model hi 96801 digital refractometer prism and then reading it, expressed in °brix average a high h adv. hort. sci., 2020 34(3): 337­347 340 y = nxp dimension matrix (plants) of phenotypic val­ ues of traits; a = is the 1 xn dimension vector of previously estab­ lished economic weights; g = nxp dimension matrix of unknown genetic values of the n traits considered. the vector b = p ­1 ga, where p ­1 is the inverse of the matrix, of dimension nxn of phenotypic variance and covariance between traits. g is the nxn dimen­ sion matrix of genetic variance and covariance between traits. the expected gain for trait j was expressed by: where: ag j(i) = gj (i): expected gain for trait j, with selection based on index i; ds j(i) = selection differential of trait j, with selection based on index i; h2j = heritability of trait j. in the rank summation index of mulamba and mock (1978), the orders of each genotype were s u m m e d , r e s u l ti n g i n t h e s e l e c ti o n i n d e x , a s described below: i= r1+r2+....+rn where: i = index value for a given individual or family; rn = an individual’s rank (or rank) from the j th trait; n = number of traits considered in the index. the weights were given by: l=p1r1+p2r2+....+pnrn where: pj = economic weight attributed to the jth trait. for the base index of williams (1962), the follow­ ing index was used as selection criteria: where: y = are the means; a = are the economic weights of the traits studied. for the index of genotype­ideotype distance (cruz, 2006), the mean and maximum and minimum values for each variable were calculated. xij was con­ sidered as the mean phenotypic value of the ith genotype concerning the ith trait. as well, we consid­ ered the value yij representing the transformed mean phenotypic value and cj as a constant relative to the average genotype depreciation. thus, we had: lij as the lower limit to be presented by the geno­ type, relative to the characteristic j, lsj as the upper limit to be presented by the genotype and voj as the optimal value to be presented by the genotype, under selection. if lij <xij <lsj, then yij = xij; if xij <lij, yij = xij + voj ­ lij ­ cj; if xij> lsj, yij = xij + voj ­ lsj + cj. in the methodology, it was considered cj = lsj ­ lij. the cj value ensured that any value of xij within the range of variation around the optimum resulted in a value of yij of magnitude close to the optimal value (voj), as opposed to the values of xij outside this range. thus, the xij transformation was per­ formed to ensure the depreciation of phenotypic val­ ues out of range. the yij values obtained by transfor­ mation were later standardized and weighted by the weights assigned to each characteristic, obtaining the yij values, as described below: where: s (yj) = standard deviation of the mean phenotypic values obtained by the transformation; aj = weight or economic value of the characteristic. t h e n , w e c a l c u l a t e d t h e g i d i n d e x v a l u e s expressed by the distances between the genotypes and the ideotype, as illustrated: from these indexes, the best genotypes were identified, and the selection gains were calculated. all genetic and statistical analyzes were processed through the computational program in genetics and statistics ­ genes program (cruz, 2016). 3. results and discussion genetic variability was found for all traits by the f­ test at 1% or 5% probability level, which evidenced the ability to perform the selection of superior toma­ to lines. it was verified by values of coefficient of vari­ ation (cv) ranging from 1.36% to 29.03% for mc and n f p , r e s p e c ti v e l y . t h e h i g h e s t c v v a l u e s w e r e observed in trait nfp (29.03%), ss (18.28%), and yld (18.05%) (table 2). the genotypic coefficient of determination (h2) allows us to define the estimate of genetic gain to be achieved and to establish the most appropriate strat­ egy to be used in the breeding program (baldissera et al., 2014). h2 values change according to each charac­ vieira nascimento et al. ‐ identification of tomato strains in brazil 341 teristic and are classified as high when they are high­ er than 0.7 (alvares et al., 2016). the highest h2 values were found for the matura­ tion cycle (71.93%), fruit firmness (58.30%), and yield (51.48%). these values allow us to reach success by the phenotypic selection, which can be proven by the results found in the cvg/cve ratio, which were close to 1.0 for these traits. the lowest h2 values were observed for the number of fruits per plant (29.38%) and soluble solids (33.29%). the medium and high results of the heritability coef­ ficient and coefficient of genetic variation are related to higher selective accuracy, higher genetic variability, and the probability of successfully choosing genotypes with optimal agronomic traits (storck and ribeiro, 2011). the cvg/cve ratio was close to 1.0 only for the table 2 ­ mean square, coefficient of variation, and genetic parameters of agronomic traits and yield of 85 industrial tomato lines fpt= fruit pericarp thickness, ff= fruit firmness, yld= yield, mc= maturation cycle, nfp= number of fruits per plant, ss= soluble solids, h2= genotypic coefficient determination, cv= coefficient of variation, cvg= coefficient of genetic variation, cve= coefficient of experi­ mental variation. ** and * significant by f­test at 1% and 5% probability, respectively. source of variation df mean square fpt ff yld cm nfp ss blocks 2 0.65 0.59 488.22 2.89 1663.12 37.75 lines 84 2.08 ** 0.56 ** 698.53 ** 8.16 ** 1664.94 * 0.82 * residue 168 1.35 0.23 338.89 2.29 1175.83 0.55 cv (%) ­ 15.96 16.69 18.05 1.36 29.03 18.28 cvg/cve ­ 0.42 0.68 0.59 0.92 0.37 0.41 h2 ­ 34.84 58.30 51.48 71.93 29.38 33.29 medium cycle. the cvg/cve ratio can be accepted as an indicator of the obtaining of more relevant genet­ ic gains in the selection of superior genotypes (cruz et al., 2012). the constitution of tomato fruits for the industry has been remodeled through genetic improvement, to select cultivars with desirable characteristics for processing. as a general rule, the desired tomato lines are those that combine higher yield with quali­ ty, and that meet the needs of the industry, which currently are firm fruits, with a high content of solu­ ble solids, a shorter cycle, a higher number of fruits per plant and higher fruit pericarp thickness (iglesias et al., 2015; peixoto et al., 2017). fruit pericarp thickness ranged from 5.36 to 9.04 mm (table 3). only 3.7% of the tomato lines had a table 3 ­ fruit pericarp thickness (fpt), fruit firmness (ff), yield (yld), maturation cycle (mc), number of fruits per plant (nfp), and solu­ ble solids (ss) of 85 industrial tomato lines lines traits fpt mm ff n yld t ha­1 mc days nfp n° plant­1 ss °brix pxt­102 5.5 b 1.79 b 103.79 a 109 b 123.67 a 4.17 a pxt­104 6.55 b 1.98 b 93.96 b 107 b 111 b 4.57 a pxt­106 7.59 a 2.39 b 108.19 a 111 a 130.83 a 3.07 b pxt­107 6.11 b 2.31 b 111.50 a 109 b 109 b 4.07 a pxt­108 7.20 a 2.67 b 123.94 a 110 b 96.33 b 4.13 a pxt­109 7.91 a 2.30 b 85.28 b 110 b 92.33 b 3.77 b pxt­111 7.75 a 2.58 b 68.98 b 107 b 74.83 b 4.43 a pxt­113 7.15 a 3.15 a 85.76 b 112 a 99.17 b 4.77 a pxt­114 7.40 a 2.38 b 72.87 b 110 b 112.83 b 3.03 b pxt­115 8.08 a 2.55 b 86.27 b 107 b 106.00 b 4.20 a pxt­116 7.36 a 1.94 b 117.45 a 113 a 107.17 b 3.67 b pxt­117 8.08 a 2.65 b 119.54 a 112 a 130.83 a 4.40 a pxt­118 6.90 b 2.70 b 110.33 a 110 b 141.17 a 3.77 b pxt­120 7.17 a 2.13 b 114.81 a 113 a 128.17 a 4.73 a pxt­121 6.00 b 2.63 b 115.12 a 112 a 135.17 a 3.17 b pxt­122 7.39 a 2.40 b 106.26 a 113 a 129.33 a 4.10 a pxt­123 8.58 a 2.68 b 103.88 a 112 a 143.33 a 4.30 a pxt­124 6.15 b 2.41 b 113.87 a 113 a 77.67 b 3.87 b pxt­125 5.69 b 3.14 a 93.54 b 113 a 159.00 a 4.80 a pxt­126 6.15 b 3.05 a 115.37 a 109 b 155.83 a 4.83 a to be continued... means followed by the same letters belong to the same group by the scott­knott test at 5% probability level. 342 adv. hort. sci., 2020 34(3): 337­347 table 3 ­ fruit pericarp thickness (fpt), fruit firmness (ff), yield (yld), maturation cycle (mc), number of fruits per plant (nfp), and solu­ ble solids (ss) of 85 industrial tomato lines lines traits fpt mm ff n yld t ha­1 mc days nfp n° plant­1 ss °brix pxt­401 5.37 b 3.08 a 95.68 b 113 a 173.83 a 3.60 b pxt­402 5.90 b 2.74 b 107.19 a 112 a 139.00 a 4.57 a pxt­403 6.37 b 3.33 a 122.89 a 112 a 146.00 a 4.00 b pxt­404 6.74 b 2.11 b 118.68 a 113 a 93.67 b 3.37 b pxt­405 5.50 b 2.88 b 117.25 a 112 a 115.50 b 4.30 a pxt­406 6.37 b 3.30 a 100.62 a 110 b 156.17 a 4.40 a pxt­407 6.43 b 2.85 b 103.61 a 111 a 99.33 b 3.80 b pxt­408 6.01 b 2.62 b 90.30 b 112 a 126.00 a 4.87 a pxt­409 6.03 b 3.22 a 99.00 b 111 a 100.33 b 4.87 a pxt­410 7.42 a 2.80 b 106.20 a 112 a 112.00 b 4.63 a pxt­411 7.81 a 2.46 b 101.26 a 113 a 96.83 b 4.00 b pxt­412 7.58 a 2.65 b 110.89 a 113 a 80.92 b 4.63 a pxt­413 7.95 a 3.12 a 111.25 a 113 a 116.83 b 3.10 b pxt­501 9.04 a 2.85 b 90.56 b 110 b 120.00 a 4.63 a pxt­502 8.44 a 3.35 a 116.72 a 110 b 101.67 b 3.57 b pxt­503 7.72 a 3.35 a 100.34 a 113 a 124.67 a 4.17 a pxt­504 7.10 a 3.48 a 95.05 b 112 a 122.83 a 4.17 a pxt­505 7.44 a 2.84 b 113.92 a 108 b 127.00 a 4.37 a pxt­506 6.79 b 3.06 a 114.68 a 111 a 110.83 b 3.97 b pxt­551 6.66 b 2.97 a 95.56 b 112 a 128.33 a 4.07 a pxt­552 7.16 a 2.68 b 112.40 a 111 a 109.00 b 3.47 b pxt­553 6.92 b 2.99 a 112.46 a 111 a 146.83 a 3.63 b pxt­554 8.69 a 2.67 b 83.15 b 112 a 69.50 b 4.47 a pxt­555 6.96 b 2.65 b 104.99 a 108 b 154.17 a 4.10 a pxt­556 8.49 b 3.06 a 101.46 a 109 b 91.83 b 4.63 a pxt­557 6.92 b 3.08 a 104.24 a 112 a 96.67 b 3.60 b pxt­558 7.53 a 3.69 a 102.49 a 109 b 164.00 a 4.23 a pxt­559 7.31 a 3.77 a 103.30 a 112 a 98.76 b 3.26 b pxt­560 6.88 b 2.50 b 109.27 a 109 b 93.00 b 4.50 a pxt­561 8.22 a 2.70 b 85.59 b 109 b 102.50 b 3.63 b pxt­562 7.73 a 2.88 b 102.51 a 110 b 94.00 b 5.03 a pxt­563 7.22 a 3.05 a 111.74 a 113 a 90.50 b 4.07 a pxt­564 6.95 b 2.50 b 131.20 a 113 a 98.33 b 3.26 b pxt­565 7.12 a 3.07 a 104.29 a 111 a 114.83 b 4.10 a pxt­566 8.44 a 3.38 a 114.83 a 111 a 128.17 a 4.43 a pxt­567 8.35 a 2.84 b 91.36 b 112 a 148.83 a 3.83 b pxt­568 7.23 a 3.42 a 105.30 a 111 a 122.50 a 3.80 b pxt­569 8.06 a 2.87 b 76.93 b 112 a 121.83 a 4.13 a pxt­570 8.02 a 3.34 a 88.84 b 113 a 139.33 a 3.40 b pxt­571 7.23 a 2.80 b 90.29 b 112 a 94.17 b 3.96 b pxt­572 8.14 a 2.76 b 83.28 b 110 b 104.33 b 4.76 a pxt­601 6.4a b 3.31 a 137.92 a 112 a 154.66 a 4.26 a pxt­602 7.59 a 2.76 b 122.97 a 110 b 114.17 b 5.26 a pxt­603 6.19 b 3.08 a 98.68 b 112 a 136.67 a 3.66 b pxt­604 7.60 a 2.98 a 117.62 a 109 b 105.33 b 3.37 b pxt­605 7.82 a 2.94 a 77.11 b 111 a 116.17 b 3.60 b pxt­606 7.67 a 2.82 b 99.15 b 112 a 144.50 a 4.03 a pxt­608 8.82 a 2.79 b 93.17 b 110 b 84.33 b 3.03 b pxt­609 7.29 a 2.78 b 97.48 b 109 b 104.17 b 3.77 b pxt­610 7.69 a 3.08 a 145.98 a 113 a 132.67 a 4.27 a means followed by the same letters belong to the same group by the scott­knott test at 5% probability level. to be continued... vieira nascimento et al. ‐ identification of tomato strains in brazil 343 high thickness of the pericarp. according to vieira et al. (2019), the thickness of the pericarp, together with the resistance of the epidermis and the texture of the placenta tissue, influences the firmness of the fruit (the relationship between the volume of the pericarp and volume of the locular material). only 3.7% of the tomato lines had high values of fruit firmness. firmer fruits present less degradation of the cell wall and increase the resistance of the fruits during the transport process. the fruit firmness ensures resistance to mechanical damage during mechanized harvesting and bulk transport. fruits that are not firm are more susceptible to the transforma­ tion and breakage of the skin, releasing cellular juice and causing fermentation and deterioration of the fruits before the arrival in the industry (vieira et al., 2019). the fruit firmness is extremely important for the industry, because, between the harvest and the unloading process in the industry, there are many losses, due to a large number of disintegrated fruits, r e l a t e d t o e x c e s s i v e c o m p r e s s i o n ( m o u r a a n d golynski, 2018). one of the main characteristics to be used in the selection of the ideal genotype for the tomato pro­ cessing industry and mainly for the producers is fruit yield. among the tomato lines evaluated, again, 3.7% of them obtained high values, above 131 t ha­1. the average yield of the state of goiás, where the tomato lines were evaluated, were 85 and 94 t ha­1 in the 2017 and 2018 harvests, respectively (globo rural, 2018). the average cycle ranged from 106 to 113 days. only 5.88% of tomato lines evaluated had a short cycle. most cultivars marketed by seed companies have a cycle between 95 and 125 days (kelley et al., 2010), which demonstrates that all tomato lines eval­ uated are classified between the short and middle cycles. the use of short­cycle genotypes is desirable in breeding programs, as it allows for a shorter stay in the field, where they will be subject for a shorter time to effects of biotic and abiotic factors such as disease and drought stress (gatut­wahyu et al., 2014). the number of fruits per plant ranged from 69.50 for pxt­554 to 173.83 for pxt­401. cultivars with a low number of fruits per plant are not recommended because they have lower yield during the harvesting process (santos, 2015). high soluble solids content is one of the main characteristics that an industrial tomato material must­have. according to figueiredo et al. (2015), the higher the soluble solids content, the higher the effi­ ciency of industrial production, and the lower the energy expenditure during the pulp concentration procedure. in practice, for each addition of a °brix in the pulp, there is a 20% increase in industrial produc­ tion. values above 4.5°brix are higher than the brazilian average. among 85 tomato lines evaluated, 23.17% is above this value, reaching the maximum value of 5.23 °brix. table 3 ­ fruit pericarp thickness (fpt), fruit firmness (ff), yield (yld), maturation cycle (mc), number of fruits per plant (nfp), and solu­ ble solids (ss) of 85 industrial tomato lines lines traits fpt mm ff n yld t ha­1 mc days nfp n° plant­1 ss °brix pxt­611 7.94 a 2.84 b 104.42 a 113 a 110.67 b 4.00 b pxt­613 7.75 a 3.03 a 92.45 b 109 b 106.50 b 4.40 a pxt­614 7.46 a 3.31 a 99.55 b 112 a 107.00 b 3.50 b pxt­615 7.37 a 2.67 b 83.69 b 110 b 79.33 b 3.73 b pxt­616 7.69 a 3.91 a 98.75 b 111 a 122.50 a 4.87 a pxt­617 8.27 a 3.04 a 85.38 b 113 a 92.83 b 3.97 b pxt­618 8.68 a 3.04 a 114.23 a 110 b 96.33 b 4.57 a pxt­619 6.61 b 3.57 a 97.11 b 111 a 152.00 a 4.17 a pxt­651 7.45 a 3.37 a 56.63 b 109 b 125.83 b 3.60 b pxt­652 8.16 a 4.10 a 74.64 b 109 b 157.67 a 3.57 b pxt­653 8.39 a 3.31 a 82.29 b 112 a 103.83 b 2.93 b pxt­654 6.84 b 3.43 a 84.09 b 111 a 166.17 a 3.87 b pxt­655 7.55 a 2.35 b 108.04 a 109 b 124.17 a 4.27 a pxt­656 6.90 b 3.43 a 114.19 a 110 b 149.50 a 3.90 b pxt­687 6.70 b 2.95 a 98.99 b 109 b 113.83 b 4.77 a means followed by the same letters belong to the same group by the scott­knott test at 5% probability level. adv. hort. sci., 2020 34(3): 337­347 344 direct selection resulted in higher individual gains (table 4). this selection is directed only for one trait of interest and comprises the obtention of maximum gains of a single trait for which selection is practiced. according to how this trait is associated with others, favorable or unfavorable results may occur in traits of secondary importance (cruz, 2016). direct selection for fpt, nfp, and ss resulted in direct gains for fruit firmness, with values of 1.97%, 8.69%, and 2.93%, respectively. noteworthy was the direct selection for the number of fruits per plant, which resulted in the largest indirect gain for fruit firmness. the indexes of selection consist of an alternative that allows the simultaneous selection to perform effectively by combining different traits (rosado et al., 2012). in general, the index of the rank summa­ tion index of mulamba and mock (1978) showed the largest gain of yield (7.89%) and soluble solids (4.02%), followed by the smith (1936) and hazel ( 1 9 4 3 ) i n d e x , w i t h 7 . 2 0 % o f t h e g a i n o f y i e l d . however, these two indexes had low selection gain values for the other traits (table 5). the rank summation index of mulamba and mock (1978) had the highest gain for all the traits and the highest total gain, with values of 22.92%. the geno­ type­ideotype distance index obtained the second­ highest total gain value, with 22.54%. these indices presented a balanced distribution of selection gains. in the research carried out by rosado et al. (2012), the authors reported that the rank summation index of mulamba and mock (1978) was the most appropri­ ate, allowing for a balanced distribution of selection gains for a larger number of yellow passion fruit progenies. the top ten genotypes, selected by all selection methods used in this study and their values of fruit pericarp thickness (table 6), fruit firmness (table 7), yield (table 8), number of fruits per plant (table 9), and soluble solids (table 10) are shown in the tables below. the lines pxt­601 and pxt­610 were selected in all selection methods applied, verifying the superi­ ority of these genotypes. 4. conclusions the rank summation index of mulamba and mock (1978) and the classical index proposed by smith (1936) and hazel (1943) applied to agronomic traits of eighty­five industrial tomato lines turned out to the largest selection gain for the yield trait. rank summation index of mulamba and mock (1978) has the highest total genetic gain values. the lines of tomato pxt­601 and pxt­610 stand out as superior genotypes by the direct selection method and selection indexes. acknowledgements to vivati plant breeding ltda, for support in con­ ducting the project and providing access to the germplasm bank, and to cnpq, for the master’s scholarship granted to the first author. table 4 ­ genetic gain estimates obtained for five traits evaluat­ ed by direct and indirect selection for 85 industrial tomato lines traits genetic gain (%) fpt ff yld nfp ss fpt 6.21 0.43 ­0.43 ­3.25 ­1.94 ff 1.97 14.41 ­3.34 8.69 2.93 yld ­1.41 ­1.51 12.05 ­0.67 ­1.47 nfp ­2.32 5.05 ­0.05 10.26 0.2 ss ­0.12 ­0.23 ­0.44 1.06 6.81 total 4.33 18.15 7.79 16.09 6.53 fpt= fruit pericarp thickness, ff= fruit firmness, yld= yield, nfp= number of fruits per plant, ss= soluble solids. table 5 ­ genetic gain estimates obtained for five traits by selection by the classical index proposed by smith (1936) and hazel (1943), rank summation index of mulamba and mock (1978), base index of williams (1962), and genotype­ideotype distance index for 85 industrial tomato lines fpt= fruit pericarp thickness, ff= fruit firmness, yld= yield, nfp= number of fruits per plant, ss= soluble solids. traits genetic gains (%) smith (1936) and hazel (1943) mulamba and mock (1978) williams (1962) genotype­ ideotype distance fpt ­4.55 1.88 ­2.98 2.31 ff 5.34 5.12 5.83 7.07 yld 7.20 7.89 6.71 6.69 nfp 7.22 4.01 8.77 4.12 ss 0.29 4.02 0.57 2.35 total 15.50 22.92 18.90 22.54 vieira nascimento et al. ‐ identification of tomato strains in brazil 345 table 6 ­ fruit pericarp thickness (fpt) in mm from ten superior genotypes selected by direct selection for fruit pericarp thickness, and classic index proposed by smith (1936) and hazel (1943), rank summation index of mulamba and mock (1978), base index of williams (1962), and genotype­ideotype distance index (gid) selection indexes williams (1962) and direct selection of fruit pericarp thickness smith (1936) and hazel (1943) mulamba and mock (1978) genotype­ideotype distance lines fpt lines fpt lines fpt lines fpt pxt­601 6.44 pxt­601 6.44 pxt­566 8.44 pxt­566 8.44 pxt­610 7.69 pxt­403 6.37 pxt­610 7.69 pxt­558 7.53 pxt­126 6.15 pxt­610 7.69 pxt­558 7.53 pxt­616 7.69 pxt­403 6.37 pxt­126 6.15 pxt­616 7.69 pxt­601 6.40 pxt­558 7.53 pxt­401 5.37 pxt­601 6.44 pxt­117 8.08 pxt­401 5.37 pxt­656 6.90 pxt­117 8.08 pxt­656 6.90 pxt­656 6.90 pxt­405 5.50 pxt­126 6.15 pxt­610 7.69 pxt­555 6.96 pxt­121 6.00 pxt­602 7.59 pxt­123 8.58 pxt­553 6.92 pxt­406 6.37 pxt­618 8.68 pxt­503 7.72 pxt­406 6.37 pxt­619 6.61 pxt­123 8.58 pxt­618 8.68 table 7 ­ fruit firmness (ff) in newton from ten superior genotypes selected by the direct selection for fruit firmness, and classic index proposed by smith (1936) and hazel (1943), rank index of mulamba and mock (1978), base index of williams (1962), and genotype­ideotype distance index (gid) selection indexes williams (1962) and direct selection of fruit firmness smith (1936) and hazel (1943) mulamba and mock (1978) genotype­ideotype distance lines ff lines ff lines ff lines ff pxt­601 3.31 pxt­601 3.31 pxt­566 3.38 pxt­566 3.38 pxt­610 3.08 pxt­403 3.33 pxt­610 3.08 pxt­558 3.69 pxt­126 3.05 pxt­610 3.08 pxt­558 3.69 pxt­616 3.91 pxt­403 3.33 pxt­126 3.05 pxt­616 3.91 pxt­601 3.31 pxt­558 3.69 pxt­401 3.08 pxt­601 3.31 pxt­117 2.65 pxt­401 3.08 pxt­656 3.43 pxt­117 2.65 pxt­656 3.43 pxt­656 3.43 pxt­405 2.88 pxt­126 3.05 pxt­610 3.08 pxt­555 2.65 pxt­121 2.63 pxt­602 2.76 pxt­123 2.68 pxt­553 2.99 pxt­406 3.30 pxt­618 3.04 pxt­503 3.35 pxt­406 3.30 pxt­619 3.57 pxt­123 2.68 pxt­618 3.04 table 8 ­ yield (yld), in mg ha­1, of ten superior genotypes selected by direct selection for yield, and classic index proposed by smith (1936) and hazel (1943), rank summation index of mulamba and mock (1978), base index of williams (1962), and genotype­ ideotype distance index (gid) selection indexes williams (1962) and direct selection of yield smith (1936) and hazel (1943) mulamba and mock (1978) genotype­ideotype distance lines yld lines yld lines yld lines yld pxt­601 137.92 pxt­601 137.92 pxt­566 114.83 pxt­566 114.83 pxt­610 145.98 pxt­403 122.89 pxt­610 145.98 pxt­558 102.49 pxt­126 115.37 pxt­610 145.98 pxt­558 102.49 pxt­616 98.75 pxt­403 122.89 pxt­126 115.37 pxt­616 98.75 pxt­601 137.92 pxt­558 102.49 pxt­401 95.68 pxt­601 137.92 pxt­117 119.54 pxt­401 95.68 pxt­656 114.19 pxt­117 119.54 pxt­656 114.19 pxt­656 114.19 pxt­405 117.25 pxt­126 115.37 pxt­610 145.98 pxt­555 104.99 pxt­121 115.12 pxt­602 122.97 pxt­123 103.88 pxt­553 112.46 pxt­406 100.62 pxt­618 114.23 pxt­503 100.34 pxt­406 100.62 pxt­619 97.11 pxt­123 103.88 pxt­618 114.23 adv. hort. sci., 2020 34(3): 337­347 346 references alvares r.c., silva f.c., melo l.c., melo p.g.s., pereira h.s., 2016 ­ estimation of genetic parameters and selection of high‐yielding, upright common bean lines with slow seed‐coat darkening. ­ genet. mol. res., 15(4), 1­10. b a l d i s s e r a j . n . c . , v a l e n t i n i g . , c 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stored and non-stored (fresh) conditions. the results indicated that there was a significant interaction between pollen viability of pistachio cultivars and storage period. this study showed that the non-stored (fresh) pollen of cultivars batouri and lazaourdi had significantly the highest viability (87%) and in vitro pollen germination (69.7%), respectively; indicating that such cultivars could be used as best pollinators. on the other hand, cultivar nab-el jamal had the lowest viability (43.7%) and in vitro pollen germination (40.3%). it was found that pollen viability for all stored pollen cultivars were significantly reduced by 30% when compared with non-stored (fresh) pollen. however, stored pollen germination for one month was zero for all pistachio cultivars. in addition, the results of viability for all fresh pollen cultivars were poorly linearly correlated (r2=0.149) with the results of in vitro germination of fresh pollen. further research is required to examine both pollen viability and in vitro pollen germination under different short and long-term storage periods and methods. 1. introduction pistachio (pistacia vera l.) is a member of the family anacardiaceae (ak et al., 2016). pistachio trees are dioecious, meaning that the pistillate and staminate flowers are formed on different trees. pistachio trees are wind pollinated as flowers have no petals to attract insects (hosseini et al., 2015). one male tree is required for every eight female trees, but this ratio is usually not observed in orchards (bahramabadi et al., 2018). therefore, the amount of pollen produced in each cluster and germination rate of pollen must be high in male trees (ak et al., 2016). pistachio is mainly cultivated in warm-temperate to subtropical parts of the world for its commercially valuable and edible seeds (tilkat and onay, 2009). the center of origin of pistachio species in the near east includes the central asia and turkey. jordan ranks number 13 for pistachio production in the world and has a world share of 0.1 %. production quantity of pistachio in jordan increased from 10 tons in 1975 to 967 tons in 2016 (fao, 2016), (*) corresponding author: abdallah.aldahadha@narc.gov.jo citation: aldahadha a., al sane k., bataineh a., abu alloush a., hamouri z., 2019 pollen viability and in vitro germination of six pistachio (pistacia vera l.) cultivars grown in northern jordan. adv. hort. sci., 33(3): 441-446 copyright: © 2019 aldahadha a., al sane k., bataineh a., abu alloush a., hamouri z. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 26 september 2018 accepted for publication 24 april 2019 ahs advances in horticultural science short note http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(3): 441-446 442 with an increase in harvested area up to 301 hectares in 2016. in vitro pollen germination is a very useful technique because it can unravel the physiological and biochemical conditions required for the successful pollen germination and pollen tube development (sanjay et al., 2016). in addition, in vitro pollen germination is one of the most convenient and reliable methods used to test the viability of fresh or stored pollen. however, pollen grains of pistacia vera l. have been considered to be difficult to germinate in vitro (golan-goldhirsh et al., 1991). on the other hand, pollen viability usually refers to the ability to distribute functional sperm cells to the embryo sac following compatible pollination (shivanna and ram, 1993). the quality of pollen is evaluated on the basis of viability and vigor. actually, pollen represents a critical stage in the life cycle of plants because viable pollen is essential for effective reproduction of sexual plants. there has been recurrent interest in developing reliable methods for short and long-term storage of pistachio pollen (ateyyeh, 2012). such methods would be useful in storing pollen to be used in pistachio breeding programs (vithanage and alexander, 1985) and supplemental pollination programs in pistachio production (crane and iwakiri, 1981) which requires collection of sufficient amounts of pollen and its storage for short (hours to weeks) or long (months to years) periods, while maintaining viability (shivanna and sawhney, 1997). in addition, storing pollen is very important for cross-pollination, crop breeding, physiology, biotechnology, plant biodiversity and its conservation (polito and luza, 1988). in this study, six pistachio cultivars were grown in maru agricultural research station, irbid, jordan since 1984 (all cultivars were introduced from syria). the percentages of pollen viability and in vitro pollen germination have never been tested in our orchard and these percentages may be varied among these tested cultivars. thus, the overall aim of this experiment was to investigate in vitro germination and pollen viability in six pistachio cultivars under non-stored (fresh) and stored pollen in refrigerator for one month. in addition, this experiment was performed to find a correlation between in vitro pollen germination and viability, to check efficiency of stored pollen and to determine which pistachio cultivar could be recommended as best pollinator. 2. materials and methods plant material and location this research was carried out on six pistachio cultivars: lazaourdi, nab-el jamal, boundiki, batouri, marawhi and aschouri from pistachio orchard at maru agricultural research station. this station is located in irbid governorate at 32° 33’ mn latitude, 35° 51’ e longitude and 589 m above mean sea level ( a l g h z a w i e t a l . , 2 0 1 8 ) . m a r u h a s t y p i c a l mediterranean climate conditions with hot and dry summer and an average annual precipitation of about 380 mm and represents an intermediate drought area. in vitro pollen germination and viability tests were carried out at the biotechnology laboratory of the national agricultural research center (narc), baqa’a, jordan. pollen collection pollen collection took place during the flowering period from 3rd to 6th of april, 2018. pollen was collected from six pistachio cultivars. upon anthesis, pistachio clusters of each cultivar were detached and shacked to a glass square. care was taken through this process to prevent contamination. afterwards, pollen of each cultivar was placed in a closed vial to be used later on. pollen storage for each pistachio cultivar, samples of pollen were placed in small glass vials and these samples of non-stored (fresh) pollen were immediately taken to laboratory for in vitro germination and viability tests. other samples were stored in refrigerator at 4°c for 1 month to compare in vitro pollen germination and viability with those of non-stored pollen. in vitro pollen germination test the medium used for in vitro germination testing as recently described by ateyyeh (2012) contained 1 % a g a r , 1 5 % s u c r o s e a n d 1 0 0 p p m b o r i c a c i d (h3bo3). pollen grains were placed on medium and incubated at 24°c for 24 hours. after this time, 100 pollens from each cultivar were counted using light microscope to estimate the percentage of in vitro pollen germination. for each cultivar, 3 replicates (petri-dishes) were used. pollen is considered to be germinated if the developed pollen tube is exceed (23 times) of its diameter. pollen viability test pollen viability was estimated as described by ateyyeh (2012) by using 1% ttc (2, 3, 5-triphenyl aldahadha et al. ‐ pollen viability and in vitro germination of pistacia vera l 443 tetrazolium chloride) and 60% sucrose. ttc-sucrose solution was stored in brown glass bottle in a refrigerator. one drop of solution was placed onto microslide then a small amount of pollen was suspended in the drop and cover glass was placed onto the microslide, wrapped with aluminum foil and incubated in chamber room at 28°c for 60 minutes. after incubation, 100 pollens from each cultivar were counted using light microscope to estimate the percentage of pollen viability. for each cultivar, 3 replicates (microslides) were used. pollen grains stained orange or bright red color were considered viable. experimental design and statistical analysis the experiment was performed in a factorial design with six pistachio cultivars and two storage treatments (non-stored and stored pollen for 1 month) to investigate pollen viability and in vitro pollen germination separately. there were three replicates for each cultivar and storage treatment. data were analyzed by factorial anova. when there were significant interactions, one-way anova was used and means were separated using least significant difference (lsd). 3. results and discussion in vitro pollen germination both proper pollination and pollen vigor are essential for pistachio productivity since the marketable portion is the seed. to obtain a good fruit s e t , p o l l i n a t i o n a n d f e r t i l i z a t i o n a r e r e q u i r e d . previous studies reported that the yield and quality of nuts were influenced by pollen performance in pistachio (acar and kakani, 2010). it has been indicated that the validity of the in vitro evaluation of pollen germination is a predictor of in vivo behavior (acar and kakani, 2010). the percentages of in vitro pollen germination for six pistachio cultivars are summarized in table 1. the results indicated that the germinability of pistachio pollen varies according to the cultivar under nonstored (fresh) condition (table 1). in particular, fresh pollen of cultivar lazaourdi had significantly the highest in vitro germination percentage (69.7%), followed by cultivars aschouri and boundiki, then cultivars batouri and marawhi. however, fresh pollen of cultivar nab-el jamal had significantly (p < 0.01) the lowest germination percentage (40.3%). in addition, acar et al. (2010) found that under in vitro conditions, pollen germination showed that atli, uygur and kaska male pistachio cvs were generally better than their f1 hybrids. contrary to the reports of polito and luza (1988) who found that pistachio pollen lost its germinability after several days, preliminary tests of vaknin and eisikowitch (2000) revealed that fresh pollen lost most of its germinability within several hours. results of vaknin and eisikowitch (2000) indicated that freshly collected pollen showed the highest germination rate (76.7%). in addition, günver-dalkılıç and dayıdoğru (2011) found that pollen grain germination ratio for pistachio was changed between 78.22% and 63.29% under room conditions at initial day (day 1) in pistachio. however, pollen grain germination ratios were found between 55.83% and 43.26% in pistachio at the 2nd day storage in refrigerator. after one month of pollen storage in refrigerator, in vitro pollen germination was zero for all pollen pistachio cultivars (table 1). this is supported precisely with findings of ateyyeh (2012), suggesting that the table 1 in vitro pollen germination and pollen viability percentage of six pistachio cultivars grown in maru agricultural research station under non-stored (fresh) and stored pollen for one month at 4°c data with the same letter in each column are not significantly different (least significant difference at p<0.05). cultivars in vitro pollen germination (%) pollen viability (%) non-stored pollen (fresh) stored pollen (1 month) non-stored pollen (fresh) stored pollen (1 month) lazaourdi 69.7 a 0 66.7 b 43.0 d nab-el jamal 40.3 d 0 43.7 d 34.3 e boundiki 60.0 b 0 55.0 c 35.7 e batouri 51.0 c 0 87.0 a 53.7 c marawhi 47.3 c 0 65.0 b 46.0 d aschouri 62.3 b 0 85.0 a 67.0 b mean 55 0 67 47 standard error 1.64 0 1.64 3.24 lsd (0.05) 5.142 0 7.547 7.547 adv. hort. sci., 2019 33(3): 441-446 444 stored pollen in refrigerator for one month is not effective method for in vitro pollen germination. furthermore, the pollen grain germination ratios for pistachio were dramatically decreased and reached to about zero starting from the 4th day of storage under room conditions and at 10th day of storage under refrigerated conditions (günver-dalkılıç and dayı-doğru, 2011). pistachio pollen could be stored in refrigerator just for two weeks, which is enough for artificial cross pollination purpose, if the difference in flowering period between males and females didn’t exceed two weeks. vaknin and eisikowitch (2000) found that germinability of pollen kept in the refrigerator for six days was reduced but it retained about 60%. pollen viability the results showed that the percentages of pollen viability are significantly different in regard to pistachio cultivar under non-stored (fresh) and stored conditions (table 1). specifically, non-stored pollen of cultivars batouri (87%) and cv. aschouri (85%) had significantly the highest pollen viability percentage, followed by cultivars (lazaourdi and marawhi), and then cultivar boundiki. nevertheless, cultivar nab-el jamal had significantly (p<0.01) the lowest pollen viability percentage (43.7%). on the other hand, stored pollen for one month significantly (p<0.01) reduced pollen viability percentage in all pistachio cultivars when compared with non-stored pollen. for example, the highest and lowest pollen viability were in cultivars aschouri (67%) and nab-el jamal (34.3%); respectively under stored conditions for one month (table 1). the mean percentage value for fresh pollen viability for all pistachio cultivars was 67%, while for those of stored pollen viability was 47%. thus, all pistachio cultivars lost approximately 30% of their pollen viability when pollen stored at 4°c in refrigerator. however, the mean value for fresh in vitro pollen germination for all pistachio cultivars was 55%. therefore, the percentage of pollen germination was 18% less than those for pollen viability under nonstored conditions. günver-dalkılıç and dayı-doğru (2011) found that the highest and lowest pollen grain viability ratios were obtained as 88.24% (in safranin test) and 70.18% (in ttc test), respectively, in pistachio types (4 male pistachios (pistacia vera l.) grafted on terebinth). ateyyeh (2012) found that fresh pollen viability of pistachio was 87.4% during 2006/2007 season. ateyyeh (2012) found that pollen viability of pistachio under refrigerated conditions for 4 weeks was 0 % a n d 3 8 . 7 % f o r s e a s o n s 2 0 0 6 / 2 0 0 7 a n d 2007/2008; respectively. in fact, many factors affect pollen viability and longevity such as genetic variation between species, abiotic environmental conditions, temperature, moisture content, oxygen pressure, nutritional and physiological conditions under which the plants are grown, and the methods of pollen collection and storage (barnabas and kovacs, 1997). it was suggested that the loss of pollen viability in the course of short-term storage is directly related to changes in the water content of the pollen grains, rather than to a deficiency of essential metabolites (barnabas and kovacs, 1997). correlation between tests there was a significant (p<0.05) regression of in vitro pollen germination on viability (fig. 1), meaning that there is a relationship (linear correlation) between in vitro germination and viability for the f r e s h o r n o n s t o r e d p o l l e n b u t w i t h p o o r f i t (r2=0.149). particularly, in vitro pollen germination and viability percentages were similar for cultivars (lazaourdi, nab-el jamal and boundiki), however, for cultivars (batouri, marawhi and aschouri), in vitro pollen germination was less than viability percentages. this is why the overall correlation between pollen viability and in vitro germination is weak under non-stored pollen condition. on the other hand, there was no correlation between in vitro pollen germination and viability when pollen stored in refrigerator at 4°c as pollen germination was zero for all tested pistachio cultivars. pollen viability has been correlated with in vitro pollen germination in banksia and some other proteaceae plants (schori et al., 1992). a positive and highly significance correlation between different pollen viability stains and p o l l e n g e r m i n a t i o n t e s t i n m o m o r d i c a s p e c i e s fig. 1 correlation between viability and in vitro germination of fresh pollen in six pistachio cultivars grown in maru agricultural research station. significant regression at p<0.05. n=3. aldahadha et al. ‐ pollen viability and in vitro germination of pistacia vera l 445 (rathod et al., 2018), even though, very high percent pollen viability was reported but when the germination test was performed the pollen germination was not showed more than 70 %. it has been pointed out that in vitro pollen germination rates are considered the best indicator of pollen viability (shivanna et al., 1991). furthermore, a good correlation was revealed between in vitro pollen germination with fruit and seed setting in three ornamental tropical tree species (sanjay et al., 2016). 4. conclusions a significant interaction between pollen viability of pistachio cultivars and storage period was found in this experiment. pollen viability in all pistachio cultivars was reduced under storage conditions. pistachio cvs. batouri and lazaourdi might be used as best pollinators. based on our results, it is not recommended to store pollen in refrigerator for one month for germination purposes. further research is necessary to test pollen germination under different conditions and periods of storage. acknowledgements authors would like to warmly thank the director general of the national agricultural research center (narc), jordan for supporting and facilitating this study. thanks are also extended to the staff of maru agricultural research station /irbid. references acar i., ak b.e., sarpkaya k., 2010 effects of boron and gibberellic acid on in vitro pollen germination of pista‐ chio (pistacia vera l.). afr. j. biotechnol., 9: 51265130. acar i., kakani v.g., 2010 the effects of temperature on in vitro pollen germination and pollen tube growth of pistacia spp. sci. hort., 125(4): 569-572. ak b.e., acar i., sakar e., goursoz s., 2016 the impor‐ tance of pistacia species for pistachio production in turkey. acta horticulturae, 1139: 183-188. al-ghzawi a.a., bani khalaf y., al-ajlouni z.i., alquraan n.a., musallam i., bani hani n., 2018 the effect of supplemental irrigation on canopy tempera‐ ture depression, chlorophyll content, and water use efficiency in three wheat (triticum aestivum l. and t. durum desf.) varieties grown in dry regions of jordan. agriculture, 8(5): 1-23. ateyyeh a.f., 2012 effect of storage method on date palm and pistachio pollen viability. jor. j. agric. sci., 8: 573-582. bahramabadi e.z., jonoubi p., rezanejad f., 2018 some cytological and physiochemical features relating to non‐storability of pistachio (pistacia vera l.) pollen. grana, 57(6): 1-8. barnabas b., kovacs g., 1997 storage of pollen, pp. 293-314. in: shivanna k.r., and v.k. sawhney (eds.). pollen biotechnology for crop production and improvement. cambridge university press, uk, pp. 448. crane j.c., iwakiri b.t., 1981 morphology and reproduc‐ tion of pistachio. hort. rev., 3: 376-393. fao, 2016 faostat. agricultural statistics database. fao. food and agriculture organization of the united nations, rome, italy. golan-goldhirsh a., schmidhalter u., müller m., oertli j.j., 1991 germination of pistacia vera l. pollen in liquid medium. ‐ sex. plant reprod., 4(3): 182-187. g ü n v e r d a l k i l i ç g . , d a y i d o g r u ö . , 2 0 1 1 determination of pollen grain viability and germination levels for pistachio and terebinth in aydin/turkey ecolo‐ gy. pak. j. bot., 43(2): 841-848. hosseini n., bahramabadi e.z., rezanejad f., 2015 study of morphological and anatomical traits of male flower, developmental stages of anther and pollen grain of pistachio (pistacia vera l.). iran. j. biol., 28(1): 116-125. polito v.s., luza j.g., 1988 low temperature storage of pistachio pollen. euphytica, 39: 265-269. rathod v., behera t.k., munshi a.d., durgesh k., jat g.s., krishnan b.g., sharma n., 2018 pollen viability and in vitro pollen germination studies in momordica species and their intra and interspecific hybrids. ‐ inter. j. chem. studies, 6(6): 32-40. sanjay k.k., khanduri v.p., kar k., mohan sharma ch., riyal m.k., 2016 effect of growth regulators and time on in vitro pollen germination in three ornamental tropical tree species. j. agr. sci. tech., 18(5): 12471255. schori y., goren t., ben-jacov j., 1992 pollen germina‐ tion and storage in banksia and some other proteaceae plants. acta horticulturae, 316: 19-20. shivanna k.r., linskens h.f., cresti m., 1991 pollen viability and pollen vigor. theor. appl. genet., 81(1): 38-42. shivanna k.r., ram h.y.m., 1993 pollen biology: contribution to fundamental and applied aspects. current sci., 65(3): 226-233. shivanna k.r., sawhney v.k., 1997 pollen biology and pollen biotechnology: an introduction, pp. 1-12. in: shivanna k.r., and v.k. sawhney (eds.) pollen biotechnology for crop production and improvement. cambridge university press, uk, pp. 448. tilkat e., onay a., 2009 direct shoot organogenesis from in vitro derived mature leaf explants of pistachio. 446 adv. hort. sci., 2019 33(3): 441-446 in vitro cell. dev. biol. plant, 45: 92-98. vaknin y., eisikowitch d., 2000 effects of short term storage on germinability of pistachio pollen. plant breed., 119: 347-350. v i t h a n a g e h . i . m . v . , a l e x a n d e r d . m c . e . , 1 9 8 5 synchronous flowering and pollen storage techniques as aids to artificial hybridization in pistachio (pistacia spp.). ‐ j. hort. sci., 60: 107-113. impaginato 449 adv. hort. sci., 2019 33(4): 449­455 doi: 10.13128/ahsc­8098 economic analysis of crisp lettuce production in different planting spacing and soil cover e.p. vendruscolo 1 (*), a.h. alcântara rodrigues 2, s.r. correia 2, p.r. oliveira 2, l.f. cardoso campos 3, a. seleguini 4 1 mato grosso do sul state university, rod. ms 306, km 6.4, cep 79540‐ 000, cassilândia, mato grosso do sul, brazil. 2 agronomy school, goiás federal university, samambaia campus, esperança avenue, cep 74690‐900, goiânia, goiás, brazil. 3 goiás state university, saudade street, 56, vila eduarda, cep 76100‐ 000, sâo luis dos montes belos, goiás, brazil. 4 triângulo mineiro federal university, rio paranaíba avenue, cep 38280‐000, iturama, minas gerais, brazil. key words: economic indicators, lactuca sativa, production costs, vegetables. abstract: the objective of this study was to estimate and evaluate the econom­ ic indicators of lettuce production, cultivated using different soil cover and plant spacing. the experiment was conducted in subdivided parcels, with four replications. the treatments were composed by a combination of three soil cover (uncovered soil, straw and plastic cover) and three planting spaces (0.25 x 0.20, 0.25x0.25 and 0.25x0.30 m). the productivity and economic indicators were evaluated for a production area of 1000 m2. for the different treatments, a total operating cost of usd 781.80 to usd 663.30 1000 m­2 was obtained. it was observed that for the cultivation of lettuce in soil covered by straw, from the rubbing, and spacing of 0.25x0.25 m the economic indicators were raised. with a productivity of 687.70 boxes 1000 m­2, for this treatment was obtained gross revenue of usd 1,828.99, operating profit of usd 1,135.41 and a prof­ itability index of 62.08%. thus, lettuce cultivation provides positive profitability regardless of the spacing or type of cover used and the combination between the 0.25 x 0.25 m planting spacing and the use of straw as a soil cover culmi­ nates in higher monetary gains. 1. introduction the consumption of vegetables has been increasing continuously due to the dietary habits adopted by the population (maziero et al., 2017), which consequently influences the demand for higher yields (silveira et al., 2015). among the products sought by consumers, lettuce is the world leader in terms of acceptance, increasing its importance to the productive sector due to the large volume of commercialization (vieira and barreto, 2006). considering the participation of family farms in the vegetable produc­ (*) corresponding author: agrovendruscolo@gmail.com citation: vendruscolo e.p., alcântara rodrigues a.h., correia s.r., oliveira p.r., cardoso campos l.f., seleguini a., 2019 ­ economic analysis of crisp lettuce production in different planting spacing and soil cover. ­ adv. hort. sci., 33(4): 449­455 copyright: © 2019 vendruscolo e.p., alcântara rodrigues a.h., correia s.r., oliveira p.r., cardoso campos l.f., seleguini a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 5 february 2019 accepted for publication 21 june 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2019 33(4): 449­455 450 tion scenario, one of the main productive obstacles is the investment required. most of the costs are relat­ ed to inputs purchase, as fertilizers, seedlings and seeds (rezende et al., 2005, 2009; batista et al., 2013). this burden of the production process is nec­ essary, in order to obtain higher quality products, increasingly demanded by the consumer market. through the research, it is seeking the application of techniques that assist the rural producers in obtaining greater monetary returns, encouraging them to continue in the agricultural activity. simple changes in management can be effective strategies for reducing production costs. in this sense, for the lettuce crop, it is observed that the use of straw as a cover is an effective way of preserving the soil physic­ ochemical characteristics, favoring plants develop­ ment and increasing the profitability (vendruscolo et al., 2017 a). in addition, due to intense soil move­ ment applied to vegetable production (ziech et al., 2014), the cover contributes to the preservation of organic matter and erosion reduction (cividanes, 2002; souza and resende, 2006). materials that are easily accessible to the rural producer, such as straw from grazing, can be used as mulch, replacing other materials such as plastic, for example, which has an effective participation in the costs of producing vegetables (vendruscolo et al., 2017 b), also representing a source for environmen­ tal contamination (chang et al., 2013). in this sense, it is important to generate information on the eco­ nomic feasibility of techniques applied to agriculture, based on data with high reliability. the presence of a superficial straw layer is benefi­ cial from the point of view of soil quality mainte­ nance (cardoso et al., 2012), favoring the develop­ ment of plants of economic interest (torres et al., 2015; vendruscolo et al., 2017 a). it also favors the maintenance of soil moisture by controlling the direct evaporation of the surface (carneiro et al., 2014), reducing the need for large volumes of water in irrigation. the decrease in weed competition due to the suppression of spontaneous plants is another advantage of soil cover (moraes et al., 2013), which implies the less need for herbicides or even manual weeding. in this sense, it was verified, for the radish culture, that the maintenance of the cultural remains of silk flower on the soil surface increased the num­ ber of commercial roots produced (oliveira et al., 2015). in addition to the soil cover, other management techniques can assist producers in obtaining higher yields and superior quality of their product. the plant population used in agricultural crops is decisive in creating an environment conducive to its develop­ ment and should be established for the specific con­ ditions of a given locality, in order to avoid excessive competition for resources such as water, light, nutri­ ents and carbon dioxide essential to their develop­ ment (taiz et al., 2017). in addition, the unnecessary expense of purchasing seedlings, one of the inputs w i t h t h e l a r g e s t s h a r e i n p r o d u c ti o n c o s t s (vendruscolo et al., 2017 a). specifically for lettuce, it is observed that larger plant spacings can generate plants with higher weight (vasconcelos et al., 2017), which may be related to the broad development of the aerial part observed in commercial genotypes. it is also verified that population density variation can be an effective tool for controlling weeds in different crops (carvalho and guzzo, 2008; bajwa et al., 2017; li et al., 2018), decreasing spending on hand and acquisition of agro­ chemicals. in view of the information above, the objective of this study was to estimate and evaluate the econom­ ic indicators of lettuce cultivation, using different soil cover and plant spacing. 2. materials and methods the study was conducted in an experimental area located at the goiás federal university, in goiânia, goiás, brazil. for the locality the following average climatic indicators are verified: annual precipitation of 1,575 mm and average monthly temperature of 22.9°c, the predominance of aw climate, character­ ized by a tropical climate with rainy season of october to april and a period with precipitations b e l o w 1 0 0 m m m o n t h l y b e t w e e n m a y a n d september. the average climatic parameters of air temperature and humidity were obtained at an evap­ orimetric station at 300 m distance from the experi­ mental area (fig. 1). the soil was classified as latossolo vermelho, fol­ lowing the methodology proposed by santos et al. (2013). the soil chemical analysis (depth of 0­0.2 m), before the implantation of the experiment revealed the following nutrient content: ca2+: 2.8 cmolc dm ­3, mg2+: 1.8 cmolc dm ­3, k+: 0.37 cmolc dm ­3, p (mehlich i): 25.8 mg dm­3, organic matter: 3.0 g dm­3, al3+: 0.0 cmolc dm ­3, h+al: 2.8 cmolc dm ­3, ph (cacl2): 5.3, 7.8 c m o l c d m ­ 3 o f c t c , 6 4 . 0 % o f v , a c c o r d i n g t o donagemma et al. (2011). the soil granulometric analysis, according to da silva (2009), presented 44 g vendruscolo et al. ‐ economic analysis of crisp lettuce 451 kg­1 of clay in 0­0.2 m layer (da silva, 2009). previously to the planting, an initial fertilization was carried out, which consisted of the equivalent application of 320 kg ha­1 of simple superphosphate and liming in order to raise the bases saturation to 80%. the limestone was incorporated and the beds were confectioned with 1.00 m wide and spaced 0.50 m apart. the experiment was conducted in a subdivided plots design, with four replications for each treat­ ment. the treatments were composed by a combina­ tion of three soil cover (uncovered soil, straw and plastic cover) and three planting spaces (0.25x0.20, 0.25x0.25 and 0.25x0.30 m), making a total of nine treatments. each plot had dimensions of 1.0x1.25 m (1.25 m2). for evaluation, the central plants of the two internal lines were used and the remaining plants were used as a border. irrigation was carried out by drippers spaced twenty centimeters apart in three polyethylene tapes suitable for this purpose, positioned between the planting lines. the acquisition of the drip tapes was included in the calculation of the costs, considering the implantation of this system in the production area and the useful life of this material. however, the amounts spent on the purchase of other materials, such as pumps, pipes and others irrigation materials were not considered. after the irrigation tapes placement, the soil cov­ ers were installed according to the treatments. for that, a straw from the grass (zoysia japonica) was dis­ tributed over the plots until a layer of 5 cm was obtained. the plastic cover consisted of the place­ ment of double­sided polyethylene canvas (black and white), with the white face facing upwards. the seedlings of crisp lettuce, cv. vanda, were purchased in a commercial seedling producer, with a surplus of 10% of the quantity required for the replacement of dead plants. the seedlings trans­ planting proceeded on may 1, 2016. for this purpose, pits were opened amidst the covers, with sufficient size for seedling insertion. cover fertilization was carried out in three appli­ cations during the cycle, based on the recommenda­ tions for the crop (trani et al., 2014), applying 60 kg ha­1 of urea (45% n) and 50 kg ha­1 kcl (60% k2o). during the lettuce cultivation, there was no applica­ tion of fungicides, insecticides or herbicides. plants were harvested at 45 days after transplanting. treatments were considered as commercial crops with the purpose of determining the production costs of a productive cycle of crisp lettuce. in this way, the total operational cost (toc) structure was obtained as proposed by martin et al. (1998) by adding up the effective operating cost (eoc), which is composed of the expenses of the operations and inputs used, other expenses involved (oe), and cost­ ing interest per year (ciy). a rate of 5% of total eoc expenses was considered for other expenses (oe) that involves costs with administration, technical assistance and other fees to be paid for the activity, while costing interest (ciy) used was 6.5% per year, over 50% of eoc, estimated as an annual interest rate (martin et al., 1998). before the calculation of toc, the base total oper­ ating cost (toc base) was obtained, for which the costs related to the experimental variables were not considered, remaining constant for all treatments. for monetary amounts, the presentation was made in reais (r$) and us dollars (usd). the conversion was made considering the quotation on june 15, 2018 (usd 1.00 = r$ 3.76). each economic data was based on a single production mean, which was com­ posed by the four replications. the average prices, received by the producers, were obtained from the goiás state supply centers (2018) website. the average price paid to producers in the first half of june of 2018 was usd 2.66 per box of 4.8 kg, for calculation purposes, the same was used in the present study. the average region labor force in 2018 was usd 18.62 day­1. thus, labor costs were obtained through the index generated by the need for manual opera­ tions for each operation, multiplied by the daily value. for inputs, the cost was calculated based on the average product value in the region, obtained in the first half of 2018, and the amount of material used. fig. 1 ­ summary of climatic conditions of relative air humidity and maximum, average and minimum temperature during the period of conduction of the study. adv. hort. sci., 2019 33(4): 449­455 452 the profitability of each treatment was obtained through estimates of gross revenue, obtained multi­ plying the quantity produced (4.8 kg boxes) for the average price received by the producers, the differ­ ence between gross revenue and total operating cost represents the profitability index: the proportion of the gross revenue that represents the final amount after covering the production total operational cost. equilibrium price was also obtained as the minimum price necessary to be obtained to cover the toc at a given level of production total operating cost, consid­ ering the average productivity obtained by the pro­ ducer, and the equilibrium productivity, given the minimum productivity required to cover toc at a given level of production total operating cost. 3. results and discussion the production of crisp lettuce in an area equiva­ lent to 1000 m2 presented a base total operating cost (toc base) of usd 454.64 (table 1), which was f o r m e d b y t h e c o s t o f m e c h a n i z e d o p e r a ti o n s ( 6 8 . 0 2 % ) , m a n u a l o p e r a ti o n s ( 2 0 . 4 7 % ) , i n p u t s (3.88%), other expenses (4.62%) and interest expens­ es (3.00%). the effective participation of mechanized operations was due to the acquisition of dripping tapes, which had a 96.75% participation in this item and 65.81% over toc base. in addition, manual oper­ ations and inputs had a share of 20.47% and 3.88%, respectively, in the toc base. the small inputs participation is due to the non­ insertion of the seedlings acquisition value. however, when this cost is added to the inputs amount, the participation on the toc and the toc itself increases (table 2). the seedlings value also have a participa­ tion of 42.47%, 38.84% and 36.47% for treatments c o m p o s e d o f 0 . 2 5 x 0 . 2 0 m , 0 . 2 5 x 0 . 2 5 m a n d 0.25x0.30 m of planting spacing, respectively. these results related to the seedlings acquisition corrobo­ rate with those obtained by rezende et al. (2005), who verified a participation of 55.20% of the inputs on toc and by rezende et al. (2009), which obtained a burden of approximately 49.30% on toc due to expenditures on inputs and other materials. when the plastic cover was used, it was verified that the acquisition of the cover added to its place­ ment, participated in an average of 9.63%, while the straw had a participation around 2.66%, relative to its distribution on the beds. during the lettuce cycle, weed control burdened in 7.76% and 2.66%, respec­ tively, the treatments without cover and straw cover, table 1 ­ estimated base total operational cost for crispy lettuce in 1,000 m2 description specification quantity unit cost r$ cost r$ unit cost usd cost usd a ‐ mechanized operations tillage hm tp 65cv. 4x2 + grade aradora 14 x 26" 0.30 52.95 15.89 14.08 4.22 disk harrow hm tp 65cv. 4x2 + grade niveladora 28x22" 0.10 52.43 5.24 13.94 1.39 beds preparation hm tp 65cv. 4x2 + roto­encanteirador 0.35 47.75 16.71 12.70 4.44 irrigation irrigation equipment 1.00 1,125.00 1,125.00 299.20 299.20 subtotal a 1,162.84 0.00 309.27 b ‐ manual operations beds preparation man­day 0.50 70.00 35.00 18.62 9.31 seedling transplant man­day 1.00 70.00 70.00 18.62 18.62 fertirrigation man­day 3.00 70.00 210.00 18.62 55.85 harvest man­day 0.50 70.00 35.00 18.62 9.31 subtotal b 350.00 0.00 93.09 c ‐ inputs limestone kg 13.00 0.09 1.16 0.02 0.31 single superphosphate kg 32.00 1.34 42.88 0.36 11.40 kcl (60% k2o) kg 5.00 2.00 10.00 0.53 2.66 urea (45% n) kg 6.00 2.05 12.30 0.55 3.27 subtotal c (r$) 66.34 0.00 17.64 effective operational cost (a+b+c) 1,579.10 419.99 d ‐ other expenses 78.96 21.00 e ‐ costing interest per year 51.32 13.65 base total operating cost (a+b+c+d+e) 1,709.46 454.64 vendruscolo et al. ‐ economic analysis of crisp lettuce 453 ment (moraes et al., 2013). in contrast, the lowest profitability index was obtained using the plastic cover and the planting spacing of 0.25x0.20 m. under these conditions, the low productivity in addition to the cover value were the main factors that con­ tributed to the variables decrease in 23.44%, 45.53% and 28.85% of gross revenue, operating profit and profitability index, respectively, compared with the best treatment. the lower equilibrium yield was obtained for the treatment composed by 0.25x0.30 m planting spac­ ing and straw cover. this result was 15% below the treatment composed of a plastic cover and 0.25x0.20 m planting spacing, for which the highest equilibrium production was verified (table 4). for this treatment, the highest equilibrium price was also obtained with an increase of 47.23%, compared to the lowest equi­ librium price, obtained with the use of straw and planting spacing of 0.25x0.25 m. in general, the use of straw as soil cover favors the economic return with the lettuce crop. in addi­ respectively (table 2). the acquisition costs and placement of the plastic cover, together with the greater seedlings acquisition costs caused by the smaller planting spacing, resulted in the higher toc. this was 15.15% higher than the lowest toc, obtained with the combination of straw coverage and larger planting spacing (0.25x0.30 m) (table 2). according to the average price received by pro­ ducers during the first half of june 2018 (usd 2.66 box 4.8 kg­1), it was found that gross revenue, operat­ ing profit and profitability index obtained varied according to the planting spacing and cover used (table 3). treatment composed of the straw cover and planting spacing of 0.25x0.25 m resulted in higher values for the three variables. this result is due to the lower straw cost and its action on the mainte­ nance of the physical and chemical quality of the soil (collier et al., 2011; cardoso et al., 2012), as well as weed control through suppression of their develop­ table 2 ­ participation of the cost variation factors over the total operational cost, in 1,000 m2, for the cultivation of crisp lettuce in dif­ ferent planting spacing and soil cover table 3 ­ productivity, gross revenue, operating profit and profitability index, obtained with the cultivation of crisp lettuce in different planting spacings and soil cover, in 1,000 m2 cover planting spacing (m) cost in r$ cost in usd seedlings cost cover cost cover placement weed control oe + ciy toc seedlings cost cover cost cover placement weed control oe + ciy toc control 0.25x0.20 870.00 0.0 0.00 210.00 89.10 2,878.50 231.40 0.00 0.00 55.90 23.70 765.60 control 0.25x0.25 690.00 0.0 0.00 210.00 74.30 2,683.60 183.50 0.00 0.00 55.90 19.70 713.70 control 0.25x0.30 585.00 0.0 0.00 210.00 65.60 2,570.00 155.60 0.00 0.00 55.90 17.40 683.50 straw 0.25x0.20 870.00 0.0 70.00 70.00 83.30 2,802.70 231.40 0.00 18.60 18.60 22.20 745.40 straw 0.25x0.25 690.00 0.0 70.00 70.00 68.50 2,607.90 183.50 0.00 18.60 18.60 18.20 693.60 straw 0.25x0.30 585.00 0.0 70.00 70.00 59.80 2,494.20 155.60 0.00 18.60 18.60 15.90 663.30 plastic 0.25x0.20 870.00 196.40 70.00 0.00 93.80 2,939.60 231.40 52.20 18.60 0.00 24.90 781.80 plastic 0.25x0.25 690.00 196.40 70.00 0.00 78.90 2,744.70 183.50 52.20 18.60 0.00 21.00 730.00 plastic 0.25x0.30 585.00 196.40 70.00 0.00 70.20 2,631.10 155.60 52.20 18.60 0.00 18.70 699.70 cover planting spacing (m) produtivity (boxes 4.8 kg) gross revenue operation profit profitability index (%)r$ usd r$ usd control 0.25x0.20 592.96 5,929.57 1,577.01 3,051.09 811.46 51.46 control 0.25x0.25 640.17 6,401.70 1,702.58 3,718.07 988.85 58.08 control 0.25x0.30 641.27 6,412.66 1,705.49 3,842.69 1,021.99 59.92 straw 0.25x0.20 663.33 6,633.27 1,764.17 3,830.57 1,018.77 57.75 straw 0.25x0.25 687.70 6,877.01 1,828.99 4,269.15 1,135.41 62.08 straw 0.25x0.30 553.92 5,539.20 1,473.19 3,045.01 809.84 54.97 plastic 0.25x0.20 526.50 5,265.02 1,400.27 2,325.46 618.47 44.17 plastic 0.25x0.25 564.50 5,644.95 1,501.32 2,900.25 771.34 51.38 plastic 0.25x0.30 557.81 5,578.11 1,483.54 2,947.06 783.79 52.83 454 adv. hort. sci., 2019 33(4): 449­455 tion, it can be easily acquired in the rural property by scrubbing areas with grasses and acts in the preven­ tion of physical and chemical soil erosion (cardoso et al., 2012), as well as acting on inhibition of weed development (moraes et al., 2013), favoring the development of the culture of interest. these facts are supported by a study about the lettuce cultivation on the vegetal residue of different species, which demonstrates that plants cultivated on sorghum and millet straw favor the crop prof­ itability indexes. in this study, it was also observed the importance of choosing the species that will com­ pose the straw cover, since there are effects of allelopathy (vendruscolo et al., 2017 a, b). the straw deposition on the soil surface, in the cultivation of american lettuce and cabbage, also favored the development of these crops, especially when using brachiaria or millet straw (torres et al., 2015). 4. conclusions the insertion of these techniques, with the use of vegetal soil cover, in addition to culminating in higher financial returns, due to lower production costs and increased productivity, also represents a technique of greater environmental viability. it is observed that crops with high demand for technologies tend to g e n e r a t e l a r g e a m o u n t s o f s l o w d e g r a d a ti o n residues, such as plastics, inferring in environmental contamination (chang et al., 2013). thus, we con­ cluded that the crisp lettuce crop provides positive profitability, regardless of the planting spacing or cover type used. for the conditions observed during this study, the combination of the 0.25x0.25 m plant­ ing spacing and the straw as a soil cover culminates in higher financial returns. references bajwa a.a., walsh m., chauhan b.s., 2017 ­ weed man‐ agement using crop competition in australia. ­ crop prot., 95: 8­13. batista m.a., bezerra neto f., ambrósio m.m., gui­ m a r ã e s l . , s a r a i v a j . p . b . , s i l v a m . l . , 2 0 1 3 ­ atributos microbiológicos do solo e produtividade de rabanete influenciados pelo uso de espécies espontâ‐ neas. ­ hortic. bras., 31(4): 587­594. cardoso d.p., silva m.l., carvalho g.j., freitas d.a., avanzi j.c., 2012 ­ plantas de cobertura no controle das perdas de solo, água e nutrientes por erosão hídri‐ ca. ­ rev. bras. eng. agríc. amb., 16(6): 632­638. carneiro r.g., moura m.a.l., silva p.r., silva júnior r . s . , a n d r a d e a . m . d . , s a n t o s a . b . , 2 0 1 4 ­ variabilidade da temperatura do solo em função da liteira em fragmento remanescente da mata atlântica. ­ rev. bras. eng. agríc. amb., 1: 99­108. carvalho l.b., guzzo c.d., 2008 ­ adensamento da beterraba no manejo de plantas daninhas. ­ planta daninha, 26(1): 73­82. chang j., wu x., wang y., meyerson l.a., gu b., min y., xue h., peng c., ge y., 2013 ­ does growing vegeta‐ bles in plastic greenhouses enhance regional ecosystem services beyond the food supply? ­ front. ecol. environ., 11(1): 43­49. cividanes f.j., 2002 ­ efeitos do sistema de plantio e da consorciação soja‐milho sobre artrópodes capturados no solo. ­ pesq. agropec. bras., 37(1): 15­23. collier l.s., kikuchi f.y., benício l.p.f., sousa s.a., 2011 ­ consócio e sucessão de milho e feijão‐de‐porco como alternativa de cultivo sob plantio direto. ­ pesqui. agropecu. trop., 41(3): 306­313. table 4 ­ production and equilibrium price obtained with lettuce cultivation in different spacing and soil cover, in an area of 1,000 m2 * in each variable, data followed by the same letters (small letters for interactions and capital letters for means) are not significantly dif­ ferent using lsd at 5% level. data represent the mean value ± s.d. the mean of four replicates. cover planting spacing (m) production (boxes 4.8 kg) equilibrium price boxes 4.8 kg­1 (r$) boxes 4.8 kg­1 (usd) control 0.25x0.20 287.85 4.85 1.29 control 0.25x0.25 268.36 4.19 1.11 control 0.25x0.30 257.00 4.01 1.07 straw 0.25x0.20 280.27 4.23 1.12 straw 0.25x0.25 260.79 3.79 1.01 straw 0.25x0.30 249.42 4.50 1.20 plastic 0.25x0.20 293.96 5.58 1.48 plastic 0.25x0.25 274.47 4.86 1.29 plastic 0.25x0.30 263.10 4.72 1.25 vendruscolo et al. ‐ economic analysis of crisp lettuce 455 da silva f.c., 2009 ­ manual de análises químicas de solos, plantas e fertilizantes. ­ embrapa solos, brasília federal district, brazil, pp. 623. donagemma g.k., campos v.d.b, calderano s.b., tei­ xeira w.g., viana j.h.m., 2011 ­ manual de métodos de análise de solo. 2nd ed. ­ embrapa solos, rio de janeiro, brazil, pp. 230. li p., mo f., li d., ma b.l., yan w., xiong y., 2018 ­ exploring agronomic strategies to improve oat produc‐ tivity and control weeds: leaf type, row spacing, and planting density. ­ can. j. plant sci., 98(5): 1084­1093. martin n.b., serra r., oliveira m.d.m., angelo j.a., okawa h., 1998 ­ sistema integrado de custos agrope‐ cuários. ­ informações econômicas, 28(1): 7­28. maziero c.c.s., jaime, p.c., duran a.c., 2017 ­ a influên‐ cia dos locais de refeição e de aquisição de alimentos no consumo de frutas e hortaliças por adultos no muni‐ cípio de são paulo. ­ rev. bras. epidemiol., 20(1): 611­ 623. 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oliveira v.a., lumbreiras j.f., coelho m.r., almeida j.a., cunha t.j.f., oliveira j.b., 2013 ­ sistema brasileiro de classificação de solos, 3rd. embrapa solos, brasília federal district, brazil, pp. 353. silveira a.l., neto a.p., de oliveira a.r.c., souza l.n., de oliveira charlo h.c., 2015 ­ doses de fósforo para a produção de alface americana com e sem apli‐ cação foliar de zinco. ­ biotemas, 28(1): 31­35. souza j.l., resende p.l., 2006 ­ manual de horticultura orgânica, 2nd ed. ­ aprenda fácil, viçosa, minas gerais, brazil, pp. 834. taiz l., zeiger e., moller i.m., murphy a., 2017 ­ plant physiology and development. ­ sixth ed. sinauer associates, sunderland, usa, pp. 761. torres j.l.r., araújo a.s., gasparini b.d.n., barreto a.c., tamburús a.y., silva v.r., vieira d.m.s., 2015 ­ desempenho da alface americana e do repolho sobre diferentes resíduos vegetais. ­ global sci. technol., 8(2): 87­95. trani p.e., purquéiro l.f.v., figueiredo g.j.b., tivelli s.w., blat s.f., 2014 ­ calagem e adubação da alface, almeirão, agrião d’água, chicória, coentro, espinafre e rúcula. ­ iac, campinas, são paulo, brazil, pp. 16. vasconcelos u.a.a., costa c.c., oliveira m.n., silva j.w., 2017 ­ residual effect of sheep manure on lettuce c u l ti v a ti o n a t d i ff e r e n t s p a c i n g s . ­ r e v . v e r d e agroecologia desenvolv. sustent., 12(3): 508­511. vendruscolo e.p., campos l.f.c., arruda e.m., sele­ guini a., 2017 a ­ análise econômica da produção de alface crespa em cultivo sucessivo de plantas de cober‐ tura em sistema de plantio direto. ­ rev. bras. ciênc. agrár., 12(4): 458­463. vendruscolo e.p., campos l.f.c., seleguini a., mar­ tins a.p.b., lima s.f., 2017 b ­ economic viability of muskmelon cultivation in different planting spacing in brazil central region. ­ revista facultad nacional de agronomía, 70(3): 8319­8325. vieira b.s., barreto r.w., 2006 ­ first record of bremia lactucae infecting sonchus oleraceus and sonchus a s p e r i n b r a z i l a n d i t s i n f e c ti v i t y t o l e tt u c e . ­ j . phytopathol., 154(1): 84­87. ziech a.r.d., conceição p.c., luchese a.v., paulus d., ziech m.f., 2014 ­ cultivo de alface em diferentes manejos de cobertura do solo e fontes de adubação. ­ rev. bras. eng. agríc. amb., 18(9): 948­954. impaginato 191 adv. hort. sci., 2020 34(2): 191­204 doi: 10.13128/ahsc­7904 the first report: the effect of garlic extract on rooting of cuttings of some ornamental plants and fruit trees a. abbasifar (*), b. valizadehkaji, m. karimi, h. bagheri department of horticultural sciences, faculty of agriculture and natural resources, arak university, 38156‐8‐8349 arak, iran. key words: antimicrobial property, callus, culture substrate, grape, hormone, sycamore, wild privet. abstract: one of the problems with the use of cuttings, particularly woody and semi­woody ones, is to root them for propagation of various plant species. the use of rooting hormones involves a high cost of foreign exchange and creates many environmental problems. garlic extract has a lot of antimicrobial proper­ ties and can prevent the release of microorganisms in the culture medium of plants. in this study, garlic extract at three concentrations (0, 25, and 50 g/l) was investigated on the rooting of three different species include easy­to­root cuttings (rose, wild privet, and poplar), moderate­root cuttings (sycamore, berry, and grape), and hard­to­root cuttings (apple, sour cherry, and cherry) in three applications of the culture substrate, on the cuttings, and in the callus stage. results showed that garlic extract can be used at a concentration of 25 g/l at the callus formation stage to improve the quantity and quality of grape cuttings in terms of shooting and rooting. the quantity and quality of shoots and roots for wild privet cuttings can be improved with garlic extract at 25 g/l and be used on the cuttings. garlic extract at concentrations of 50 and 25 g/l can be used in the callus stage to enhance the quantity and quality of sycamore and berry cuttings, respectively. no positive effects were observed in the other plants, or positive effects were found only either on shooting or on rooting traits. 1. introduction since the uniformity of plants used in gardens and green spaces is important, one of the most effective methods of non­sexual propagation is now the use of various cuttings worldwide. one of the important prob­ lems of using cuttings, particularly hardwood and semi­hardwood ones is to root them. to overcome this critical problem, manufacturers apply dif­ ferent methods, one of which is the use of different rooting hormones, particularly auxins (guan et al., 2015). although the use of hormones for rooting is very effective, these substances are mostly imported goods that require a large amount of foreign money. in addition, the use of plant­ derived substances instead of chemicals and synthetic auxins to deal with adverse environmental effects is an issue that has been considered in the (*) corresponding author: abbasifar1965@yahoo.com citation: abbasifar a., valizadehkaji b., karimi m., bagheri h., 2020 ­ the first report: the effect of garlic extract on rooting of cuttings of some orna‐ mental plants and fruit trees. ‐ adv. hort. sci., 34(2): 191­204. copyright: © 2020 abbasifar a., valizadehkaji b., karimi m., bagheri h. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 19 september 2019 accepted for publication 23 april 2020 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(2): 191­204 192 european union in recent years. this has led to an increasing interest in the production of substances that can help propagate a variety of plants and, at the same time, are environment friendly (wojdyła, 2004; pacholcza et al., 2016). also, one of the prob­ lems during rooting of cuttings is the substrate conta­ mination with pathogens, and consequently the cont­ amination of cuttings and their failure in rooting (hartmann et al., 2002). according to a previous studies (wojdyła, 2004; pacholczak et al., 2016), the use of natural substances can reduce production costs and provide better accessibility compared to chemicals. in addition to various chemical com­ pounds, the use of natural substances in the cultiva­ tion of commercial plants is today gaining interest among manufacturers and producers, which has b e e n a c c o m p a n i e d b y p o s i ti v e a n d i n t e r e s ti n g results. in some cases, such substances have even been appropriately replaced chemical compounds due to better results. if plant extracts can be used to root the cuttings, a new step will be taken towards this branch of agricultural science in addition to sav­ ing costs and non­dependency on the imports of hor­ mones as well as avoiding environmental contami­ nants. plant essential oils play an important role in disinfecting, stimulating, and improving growth, increasing dry matter, and protecting the plant against environmental damages and stresses (bai et al., 2007). garlic extract is a substance with antioxidant, antimicrobial, and antibacterial properties (harris et al., 2001). garlic active compounds often accumulate in its underground organ (garlic bulb). garlic contains sulfide compounds such as allyn, diallyl sulfides, and allicin, with allicin being the most abundant com­ pound in the garlic extract, accounting for approxi­ mately 70% of these compounds (sadaqa et al., 2016). it has been shown that garlic planting in veg­ etable hydroponic substrates prevents the develop­ ment of many diseases and reduces microbial popu­ lation in the substrate (liu et al., 2014). garlic extract has many allelopathic chemicals (wang et al., 2015). the most important chemicals and minerals found in garlic extract are lipids, carbohydrates, fibers, man­ ganese, potassium, sulfur, calcium, phosphorus, mag­ nesium, sodium, vitamin b6, vitamin c, glutamic acid, arginine, aspartic acid, leucine, and lysine (al mayahi and fayadh, 2015). given the importance of ornamental trees and flowers and also fruit trees in human health and eco­ nomics, this study aimed to investigate the possible effects of garlic extract at different concentrations on rooting of cuttings and propagation of rose, wild priv­ et, poplar, sycamore , berry, grape, apple, sour cher­ ry, and cherry cuttings to achieve the best treatment. there is a large body of literature concerning the effect of using hormones on the rooting of cuttings and its optimization (guan et al., 2015), but no research has so far been conducted on the effect of plant extracts on plant rooting. in the design of this research, therefore, attempts were made to treat the cuttings of different plants with garlic extract in terms of rooting to obtain acceptable results at the beginning of the experiment. this research also tried to use an extract that is suitable for disinfection of the substrate. this was the first report on the use of garlic extract on the rooting of different plants, and it is hoped that the results could pave the ground for further research in this field. 2. materials and methods this research was conducted at the research greenhouse of the faculty of agriculture and natural resources, university of arak. to investigate the effect of garlic extract on the rooting of different cut­ tings, a factorial experiment (cuttings as the first fac­ tor and garlic extract concentrations as the second factor) was carried out in a completely randomized design with three replications each with 10 cuttings. plant materials three types of cuttings including easy­to­root cut­ tings (rose, wild privet, and poplar), moderate­root cuttings (plane, berries, and grapes), and hard­to­ root cuttings (apple, sour cherry, and cherry) were prepared from healthy and genetically uniform native plants in the planting season (december). the cut­ tings were 10­15 cm in length with a thickness of 5­7 mm. extraction of garlic cloves the garlic cloves were first peeled and grated for each independent experiment. then, 10 g of the grat­ ed garlic was placed in 10 ml of water at room tem­ perature for 24 h. the garlic extract was obtained after passing through filter papers. preparation of seedbed, planting of cuttings, and application of treatments the sandstone culture substrate was similar for all cuttings. each experiment was consisted of three replicates each with 10 cuttings. cuttings were grown in 9 rows at 10 cm spacing on a row and 2 cm spacing between the rows in the substrate. the extract con­ abbasifar et al. ‐ garlic extract effects on root cuttings 193 centrations were 0 (control), 25, and 50 g/l of dis­ tilled water for 2 seconds. the extract was used at three times: 1) extract dispersion on the culture sub­ strate during substrate preparation and before plant­ ing the cuttings, 2) at planting the cuttings by insert­ ing the bottom half of the cuttings in the extract to disinfect the cuttings, and 3) dispersion on the sub­ strate at the tips of the cuttings in the callus forma­ tion stage. after planting, the cuttings were kept and irrigated regularly under greenhouse conditions for 3 months. measuring morphological traits immediately after the collection of root and aerial samples, their fresh weight was measured using a digital scale. to determine the dry matter content, the samples were placed in an oven at 70°c for 48 h. finally, the dry weights of shoots and roots were determined with a digital scale. further measured traits were the number of roots and shoots, root and shoot length, and longest roots and shoots. during the experiment and the growth of cuttings, the aver­ age temperature and relative humidity were 22°c and 35%, respectively, in the greenhouse. data were independently normalized with such methods as the removal of outliers (data higher and lower than twice the root mean square error) using the equation x = y + 0.5, and were then analyzed using sas 9.4 software. mean values were compared with duncan’s multiple range test at p<0.05. 3. results the use of different concentrations of garlic extract in the substrates of cuttings shoot‐related characteristics. the interaction effect of garlic extract treatment × the substrate and plant type significantly affected the number of green cuttings, total shoot length, number of shoots, mean shoot length, length of the longest shoots, and fresh and dry weights of the shoots. increasing the extract concentration elevated the number of green cuttings, total shoot length, average shoot length, length of the longest shoots, and fresh and dry weights of shoots in rose cuttings. elevated number of green cuttings, total shoot length, average shoot length, and length of the longest shoots were observed in wild privet with the extract levels. in poplar cuttings, all the traits improved markedly at a moderate con­ centration (25 g/l) (table 1). the use of garlic extract in sycamore cuttings had no positive effects on shoot­related traits. in berry cuttings, the moderate concentration had positive impacts on the number of green cuttings and total shoot length, but it was not significant on the other traits (table 1). in grape cuttings, moderate concen­ tration of garlic extract positively affected the num­ ber of green cuttings, average shoot length, and shoot fresh and dry weights, while the high concen­ tration (50 g/l) was not significantly different from the moderate one or had a negative effect. in apple cuttings, the moderate concentration significantly increased the number of green cuttings, and both moderate and high concentrations led to increased number of shoots, but the use of garlic extract had no significant effects on the other traits. in sour cher­ ry cuttings, no positive effects were observed on all the traits. in cherry cuttings, the use of garlic extract at both concentrations had slight positive effects on the number of green cuttings, total shoot length, number of shoots, mean shoot length, and length of the longest shoots (table 1). in this experiment, the highest number of green cuttings, total shoot length, and the longest shoot length were obtained in wild privet with 50 g/l, with the highest number of shoots and fresh shoots at 25 g/l of garlic extract. the highest number of green cuttings belonging to wild privet cuttings was not sig­ nificantly different from berry cuttings treated with a moderate concentration. also, the average shoot length was higher in poplar cuttings treated with a moderate concentration than the other cuttings (table 1). root‐related traits. in this experiment, the inter­ action of garlic extract concentrations × substrate and plant type was significant on total length of roots, root number, mean root length, longest root length, and root fresh and dry weights. garlic extract at a concentration of 50 g/l had positive effects on total length of roots, root number, mean root length, longest root length, and root fresh and dry weights of rose roots. total root length and root number were uppermost in wild privet plant at a high concentra­ tion (50 g/l). also, the moderate concentration (25 g/l) produced the longest roots and the highest root fresh and dry weights in this plant (table 2). in poplar cuttings, a concentration of 25 g/l result­ ed in significant increases in total length of roots, root number, mean root length, longest root length, and root fresh and dry weights. there were no signifi­ cant increases in root traits of sycamore cuttings with the extract application. the extract application was not useful on rooting traits in berry cuttings. extract adv. hort. sci., 2020 34(2): 191­204 194 table 1 ­ effects of different concentration of garlic extract (the culture substrate) on shoot­related characteristics mean values followed by the similar letters within a column are not significantly different from each other at p≤0.05 (duncan’s multiple range test). treatments number of green cuttings total length of shoots number of shoots shoot length mean (cm) the highest shoot length (cm) fresh weight (gr) dry weight (gr) plant rose 0.56 fg 0.89 e 0.67 ef 0.67 ef 0.78 de 0.02 d 0.00 d wild privet 7.44 b 181.50 a 33.00 a 5.63 a 19.62 a 5.27 a 0.58 a poplar 1.78 de 21.39 d 2.78 e 4.96 b 8.17 b 0.62 c 0.03 c sycamore 2.33 d 2.78 e 2.78 e 0.99 e 1.72 d 0.01 d 0.01 cd berry 9.11 a 43.04 b 23.71 a 1.78 d 5.44 c 0.05 d 0.01 cd grape 7.33 b 33.22 c 11.55 c 2.62 c 8.28 b 2.58 b 0.06 b apple 3.89 c 2.89 e 5.78 d 0.50 ef 0.50 de 0.00 d 0.00 d sour cherry 1.33 ef 3.89 e 2.22 ef 0.86 e 1.17 de 0.00 d 0.00 d cherry 0.22 g 0.11 e 0.22 f 0.11 f 0.11 e 0.00 d 0.00 d garlic extract concentration (g∙l) control (0 g.l) 3.41 b 23.80 b 7.23 b 1.71 b 4.17 b 0.77 b 0.09 a 25 g·l 4.26 a 34.98 a 9.46 a 2.88 a 6.35 a 1.01 a 0.06 b 50 g·l 3.67 b 20.44 b 7.88 b 1.11 c 4.17 b 0.70 b 0.02 c plant × garlic extract concentration (g∙l) rose × 0 0.00 j 0.00 g 0.00 i 0.00 j h00.0 g00.0 f00.0 rose × 25 0.67 hij 0.67 g 1.00 hi 0.50 ghij 0.50 fgh g00.0 00.0 f rose × 50 1.00 ghij 2.00 g 1.00 hi 1.50 efghi 1,83 fgh g08.0 ef 01.0 wild privet × 0 5.00 cd 128.17 c 29.00 bc 5.47 c 17.00 c b82.5 a72.0 wild privet × 25 7.67 b 224.75 b 37.50 a 5.37 c 17.50 c a47.7 b63.0 wild privet × 50 9.67 a 255.00 a 32.67 b 6.89 b 26.75 a d00.4 c10.0 poplar × 0 1.00 ghij 4.67 g 1.00 hi 1.33 efghij 1­33 fgh g05.0 f00.0 poplar × 25 3.67 def 59.50 d 4.67 gh 13.55 a 22.67 b e42.1 cd07.0 poplar × 50 0.67 hij 0.00 g 2.67 ghi 0.00 j 0.50 fgh g38.0 ef02.0 sycamore × 0 2.67 efg 5.17 g 3.00 ghi 1.57 defgh 2.67 fg g00.0 f00.0 sycamore × 25 2.33 fgh 3.00 g 2.33 ghi 1.02 fghij 2.00 fgh g00.0 f00.0 sycamore × 50 2.00 fghi 0.17 g 3.00 ghi 0.37 hij 0.50 fgh g05.0 def03.0 berry × 0 8.33 ab 36.67 ef 19.67 d 1.38 defg 5.67 e g17.0 ef02.0 berry × 25 9.67 a 47.47 de 27.00 c 1.67 defgh 5.33 e g00.0 f00.0 berry × 50 9.33 ab 45.00 e 26.00 c 1.58 defg 5.33 e g00.0 f00.0 grape × 0 7.67 b 27.00 f 11.00 e 2.61 de 7.50 de f91.0 cde06.0 grape × 25 8.67 ab 38.33 ef 13.33 e 2.84 d 8.17 d c49.4 cd08.0 grape × 50 5.67 c 34.33 ef 10.33 ef 2.42 de 9.17 d e78.1 cde06.0 apple × 0 3.67 def 2.33 g 4.67 gh 0.50 ghij 0.50 fgh 0.00 g 0.00 f apple × 25 4.33 cde 3.17 g 6.33 fg 0.50 ghij 0.50 fgh 0.00 g 0.00 f apple × 50 3.67 def 3.17 g 6.33 fg 0.50 ghij 0.50 fgh 0.00 g 0.00 f sour cherry × 0 2.33 fgh 10.17 g 4.00 ghi 2.08 def 2.83 f 0.00 g 0.00 f sour cherry × 25 1.00 ghij 1.00 g 3.00 ghi 0.33 hij 0.33 fgh 0.00 g 0.00 f sour cherry × 50 67.0 hij 0.50 g 0.67 hi 0.17 ij 0.33 fgh 0.00 g 0.00 f cherry × 0 0.00 j 0.00 g 0.00 i 0.00 j 0.00 h 0.00 g 0.00 f cherry × 25 0.33 ij 0.17 g 0.33 hi 0.17 ij 0.17 gh 0.00 g 0.00 f cherry × 50 0.33 ij 0.17 g 0.33 hi 0.17 ij 0.17 gh 0.00 g 0.00 f p­value plant <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 garlic extract conc. 0.0072 <0.0001 0.0001 <0.0001 <0.0001 0.0215 <0.0001 plant × garlic extract concentration (g·l) <0.0001 <0.0001 0.0072 <0.0001 <0.0001 <0.0001 <0.0001 abbasifar et al. ‐ garlic extract effects on root cuttings 195 table 2 ­ effects of different concentration of garlic extract (the culture substrate) on root­related traits mean values followed by the similar letters within a column are not significantly different from each other at p≤0.05 (duncan’s multiple range test). treatments total length of roots number of roots root length mean (cm) the highest root length (cm) fresh weight (gr) dry weight (gr) plant rose 2.61 d 0.55 d 0.36 c 0.33 d 0.2 d 0.00 d wild privet 318.75 a 42.57 a 8.43 a 21.22 a 5.27 a 0.58 a poplar 29.61 c 3.33 c 4.14 b 7.12 c 0.62 c 0.03 c sycamore 0.83 d 0.33 d 0.17 c 0.33 d 0.01 d 0.01 cd berry 2.61 d 0.78 d 0.18 c 1.00 d 0.05 d 0.01 cd grape 73.06 b 10.12 b 4.84 b 11.00 b 2.58 b 0.06 b apple 0.00 d 0.00 g 0.00 c 0.00 d 0.00 d 0.00 d sour cherry 0.00 d 0.00 g 0.00 c 0.00 d 0.00 d 0.00 d cherry 0.00 d 0.00 g 0.00 c 0.00 d 0.00 d 0.00 d garlic extract concentration (g∙l) control (0 g·l) 28.67 b 3.44 c 1.92 b 3.48 b 0.77 b 0.09 a 25 g·l 52.83 a 8.27 a 2.43 a 6.00 a 1.01 a 0.06 b 50 g·l 29.74 b 4.64 b 1.41 c 3.48 b 0.70 b 0.02 c plant × garlic extract concentration (g∙l) rose × 0 0.00 g 0.00 f 0.00 f 0.00 e 0.00 g 0.00 f rose × 25 0.00 g 0.00 f 0.00 f 0.00 e 0.00 g 0.00 f rose × 50 7.83 g 1.67 f 1.07 f 1.00 e 0.08 g 0.01 ef wild privet × 0 202.67 c 21.00 c 9.91 a 19.00 b 5.82 b 0.72 a wild privet × 25 418.25 b 56.00 b 8.68 b 28.00 a 7.47 a 0.63 b wild privet × 50 462.00 a a00.67 6.71 c 16.67 b 4.00 d 0.10 c poplar × 0 4.33 g f67.0 0.87 f 1.67 e 0.05 g 0.00 f poplar × 25 82.50 e e33.7 10.18 a 17.33 b 1.42 e 0.07 cd poplar × 50 2.00 g f00.2 0.00 f 0.00 e 0.38 g 0.02 ef sycamore × 0 0.00 g 0.00 f 0.00 f 0.00 e 0.00 g 0.00 f sycamore × 25 0.00 g 0.00 f 0.00 f 0.00 e 0.00 g 0.00 f sycamore × 50 1.00 g f00.1 0.50 f 1.00 e 0.05 g 0.03 def berry × 0 6.50 g f33.1 25.0 f 2.50 e 0.17 g 0.02 ef berry × 25 0.00 g 0.00 f 0.00 f 0.00 e 0.00 g 0.00 f berry × 50 0.00 g 1.00 f 0.31 f 0.50 e 0.00 g 0.00 f grape × 0 42.50 f 8.00 e 5.72 cd 9.50 d 0.91 f 0.06 cde grape × 25 96.50 d 12.50 d 3.06 e 14.00 c 4.49 c 0.08 cd grape × 50 80.17 e 10.67 d 4.75 d 11.00 d 1.78 e 0.05 cde apple × 0 0.00 g 0.00 f 0.00 f 0.00 e 0.00 g 0.00 f apple × 25 0.00 g 0.00 f 0.00 f 0.00 e 0.00 g 0.00 f apple × 50 0.00 g 0.00 f 0.00 f 0.00 e 0.00 g 0.00 f sour cherry × 0 0.00 g 0.00 f 0.00 f 0.00 e 0.00 g 0.00 f sour cherry × 25 0.00 g 0.00 f 0.00 f 0.00 e 0.00 g 0.00 f sour cherry × 50 0.00 g 0.00 f 0.00 f 0.00 e 0.00 g 0.00 f cherry × 0 0.00 g 0.00 f 0.00 f 0.00 e 0.00 g 0.00 f cherry × 25 0.00 g 0.00 f 0.00 f 0.00 e 0.00 g 0.00 f cherry × 50 0.00 g 0.00 f 0.00 f 0.00 e 0.00 g 0.00 f p‐value plant <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 garlic extract conc. <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 plant × garlic extract concentration (g·l) <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 196 adv. hort. sci., 2020 34(2): 191­204 application in grape cuttings had significant impacts on all rooting traits. root growing was not observed in the cuttings of apple, sour cherry, and cherry. in this experiment, the highest total root length and number of roots were observed in wild privet cut­ tings at a high concentration and the highest mean root length was recorded in poplar cuttings at a mod­ erate concentration. the root length and fresh weight were uppermost in wild privet cuttings with a moderate concentration of garlic extract, and the highest root dry weight was measured in the cuttings of this plant in the control treatment (table 2). the effects of different concentrations of garlic extract on the cuttings shoot‐related characteristics. the interaction of plant type and extract concentration resulted in sig­ nificant impacts on the number of green cuttings, total shoot length, number of shoots, mean shoot length, longest shoot length, and fresh and dry weights of shoots. the application of garlic extract did not have positive effects on all the traits in rose cuttings. in wild privet cuttings, moderate concentra­ tion (25 g/l) could further elevate the number of green cuttings, total shoot length, number of shoots, and shoot fresh weight. the longest shoot length and shoot dry weight were observed in wild privet cut­ tings at high concentration treatment (50 g/l) (table 3). the application of garlic extract in poplar cuttings had positive effects on average shoot length and longest shoot length, with no positive impacts on the other traits. garlic extract at moderate concentration significantly affected all shooting traits in sycamore cuttings. in berry cuttings, the use of this extract at both concentrations led to fairly positive influences on the longest shoot length and fresh and dry weights of the shoots, but no positive effects were observed on the other traits. in grape cuttings, no positive effects were observed on the traits except for the number of green cuttings at 25 g/l and dry weight of shoots at 50 g/l. there was no positive effects on the traits in garlic extract treatments of apple cuttings. moreover, in sour cherry cuttings, only the mean shoot length was positively influenced by the moderate concentration (table 3). in cherry, cuttings treated with a moderate con­ centration had positive effects on the number of green cuttings, total shoot length, number of shoots, mean shoot length, longest shoot length, and shoot fresh weight. the highest number of green cuttings was found in berry cuttings at all concentrations. total shoot length and number of shoots were uppermost in wild privet cuttings at moderate­level treatment. in wild privet cuttings, maximum mean shoot length occurred at a concentration of 50 g/l and the longest shoot length was obtained at both moderate and high concentrations. also, the highest fresh and dry weight of shoots was measured in sycamore cuttings in the control (table 3). root‐related traits. analysis of the experimental data showed that garlic extract yielded significant effects on total length of roots, number of root, mean root length, root length, and root fresh and dry weights of different cuttings. rose plant cuttings were not rooted in any of the treatments. in wild privet cuttings, an extract concentration of 25 mg/l increased the total root length and both high and moderate concentrations produced the highest roots (table 4). garlic extract treatments had no positive effects on all root traits in poplar cuttings. the application of different extract concentrations in sycamore cuttings led to no significant differences with the control. the berry, apple, sour cherry, and cherry cuttings were not rooted in this experiment. in grape cuttings, the use of garlic extract at both moderate and high con­ centrations produced positive impacts on the total root length, number of roots, the longest root length, and root fresh and dry weights (table 4). using different concentrations of garlic extract in the callus stage shoot‐related characteristics. according to the results of anova, the interaction of plant type and garlic extract concentrations led to significant differ­ ences in all shoot­related traits at 1% level. the high­ est number of green cuttings was found in wild priv­ et, berry, and grape cuttings, but there were no sig­ nificant differences between the control and treat­ ments. a high concentration of garlic extract had a negative effect on the number of green cuttings in poplar, while it had positive effects on rose and sycamore cuttings at both garlic extract concentra­ tions. in apple cuttings, a high concentration of garlic extract positively affected the number of green cut­ tings. garlic extract had no effects on sour cherry cut­ tings and produced negative consequences in cherry cuttings. the highest shoot length was obtained in poplar cuttings treated with 50 g/l of garlic extract. the highest shoots were measured in poplar cuttings treated with garlic extract. there were no significant differences in the shoot length and the longest shoots in wild privet and rose cuttings with garlic extract treatments. the moderate concentration in sycamore cuttings, both concentrations in berry cut­ abbasifar et al. ‐ garlic extract effects on root cuttings 197 table 3 ­ effects of different concentration of garlic extract (on the cuttings) on shoot­related characteristics mean values followed by the similar letters within a column are not significantly different from each other at p≤0.05 (duncan’s multiple range test). treatments number of green cuttings total length of shoots number of shoots shoot length mean (cm) the highest shoot length (cm) fresh weight (gr) dry weight (gr) plant rose 0.33 f 0.50 e 0.44 e 0.50 d 0.44 f 0.15 d 0.01 f wild privet 7.78 b 111.00 a 33.25 a 3.46 b 12.78 b 8.34 b 2.23 b poplar 6.67 c 71.50 b 9.44 c 8.99 a 18.00 a 11.50 a 2.92 a sycamore 1.50 c 3.72 de 2.00 e 1.19 c 1.83 e 1.08 d 0.10 ef berry 10.00 a 27.11 c 20.89 b 1.34 c 3.22 d 3.77 c 1.02 d grape 6.55 c 30.00 c 9.78 c 3.11 b 7.05 c 8.61 b 1.19 c apple 1.78 e 0.69 e 1.78 e 0.28 d 0.33 f 0.16 d 0.03 ef sour cherry 2.89 d 6.61 d 4.67 d 1.61 c 3.31 d 0.89 d 0.20 e cherry 0.67 ef 0.33 e 0.67 e 0.22 d 0.22 f 0.07 d 0.01 f garlic extract concentration (g∙l) control (0 g·l) 4.46 29.61 a 8.63 2.31 a 5.04 3.72 0.84 a 25 g·l 4.41 25.37 b 9.78 2.25 a 4.56 3.44 0.64 b 50 g·l 3.96 16.37 c 8.31 1.46 b 5.21 3.15 0.91 a plant × garlic extract concentration (g∙l) rose × 0 1.00 efg 1.50 g 1.33 i 1.50 gh 1.33 gfh 0.45 f 0.04 h rose × 25 0.00 g 0.00 g 0.00 i 0.00 j 0.00 h 0.00 f 0.00 h rose × 50 0.00 g 0.00 g 0.00 i 0.00 j 0.00 h 0.00 f 0.00 h wild privet × 0 7.33 bc 95.50 bc 23.67 c 3.81 d 11.00 c 6.77 c 1.83 d wild privet × 25 8.67 ab 127.83 a 41.33 a 3.08 de 13.33 bc 10.07 b 2.36 c wild privet × 50 7.33 bc 104.00 b 35.50 b 3.52 d 14.00 b 8.18 bc 2.49 bc poplar × 0 7.67 bc 92.00 c 12.67 d 7.52 c 15.56 b 13.87 a 3.53 a poplar × 25 5.67 c 35.50 e 8.00 def 10.10 b 19.00 a 6.93 c 1.53 de poplar × 50 6.67 bc 58.75 d 7.67 efg 21.10 a 20.00 a 8.95 b 2.77 b sycamore × 0 0.50 efg 0.67 g 2.00 hi 10.1 ghi 1.50 gfh 1.22 ef 0.04 h sycamore × 25 2.67 de 7.33 g 3.00 ghi 1.96 fg 3.50 ef 1.08 ef 0.24 h sycamore × 50 1.00 efg 0.17 g 1.00 i 0.50ij 0.50 gh 0.23 f 0.03 h berry × 0 10.00 a 28.67 ef 20.00 c 1.44 gh 3.00 gf 3.03 de 0.84 g berry × 25 10.00 a 30.17 ef 19.00 c 1.70 fgh 3.67 ef 4.31 d 1.01 fg berry × 50 10.00 a 22.50 f 23.67 c 0.87 hij 3.00 gf 3.98 d 1.20 f grape × 0 6.33 c 33.00 e 8.68 de 3.49 d 6.33 d 6.85 c 1.13 fg grape × 25 7.00 bc 29.33 ef 9.39 de 3.40 d 8.38 d 9.17 b 1.08 fg grape × 50 6.33 c 34.33 ef 11.33 de 2.42 ef 6.50 d 9.80 b 1.35 ef apple × 0 2.33 def 1.17 g 2.33 hi 0.33 ij 0.33 gh 0.10 f 0.01 h apple × 25 2.33 def 0.57 g 2.33 hi 0.35 i 0.50 gh 0.37 f 0.08 h apple × 50 0.67 efg 0.33 g 0.67 i 0.17 j 17.0 gh 0.02 f 0.01 h sour cherry × 0 3.33 d 10.33 g 7.67 efg 1.44 gh 6.00 de 1.14 ef 0.26 h sour cherry × 25 2.00 defg 3.67 g 3.67 fghi 1.92 fg 2.00 fgh 0.49 f 0.04 h sour cherry × 50 3.33 d 5.83 g 3.67 fghi 1.47 gh 1.25 fgh 1.04 ef 0.32 h cherry × 0 0.33 fg 0.17 g 0.33 i 0.17 j 17.0 gh 0.04 f 0.00 h cherry × 25 1.33 def 0.67 g 1.33 i 0.33 ij 0.33 gh 0.17 f 0.01 h cherry × 50 0.33 fg 0.17 g 0.33 i 0.17 j 17.0 gh 0.01 f 0.01 h p‐value plant <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 garlic extract concentration 0.3226 0.0032 0.2786 0.0819 0.2726 0.8816 0.0007 plant × garlic extract concen­ tration (g·l) <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 adv. hort. sci., 2020 34(2): 191­204 198 table 4 ­ effects of different concentration of garlic extract (on the cuttings) on root­related traits mean values followed by the similar letters within a column are not significantly different from each other at p≤0.05 (duncan’s multiple range test). treatments total length of roots number of roots root length mean (cm) the highest root length (cm) fresh weight (gr) dry weight (gr) plant rose 0.00 d 0.00 c 0.00 c 0.00 d 0.00 d 0.00 d wild privet 292.70 a 42.40 a 6.53 b 18.83 b 4.91 a 0.48 a poplar 171.61 b 13.87 b 11.83 a 20.87 a 1.95 c 0.16 b sycamore 0.33 d 0.22 c 0.15 c 0.22 d 0.03 d 0.00 d berry 0.00 d 0.00 c 0.00 c 0.00 d 0.00 d 0.00 d grape 98.39 c 13.11 b 7.17 b 17.00 c 3.77 b 0.12 c apple 0.00 d 0.00 c 0.00 c 0.00 d 0.00 d 0.00 d sour cherry 0.00 d 0.00 c 0.00 c 0.00 d 0.00 d 0.00 d cherry 0.00 d 0.00 c 0.00 c 0.00 d 0.00 d 0.00 d garlic extract concentration (g∙l) control (0 g·l) 46.68 b 4.40 c 3.30 a 5.54 b 1.18 a 0.08 a 25 g·l 63.30 a 7.42 a 2.60 ab 5.00 b 1.25 a 0.06 b 50 g·l 41.18 b 5.60 b 2.28 ab 6.54 a 0.83 b 0.07 b plant × garlic extract concentration (g∙l) rose × 0 0.00 g 0.00 e 0.00 f 0.00 f 0.00 e 0.00 g rose × 25 0.00 g 0.00 e 0.00 f 0.00 f 0.00 e 0.00 g rose × 50 0.00 g 0.00 e 0.00 f 0.00 f 0.00 e 0.00 g wild privet× 0 265.50 b 42.00 a 6.33 e 16.00 d 6.99 a 0.57 a wild privet× 25 331.50 a 44.33 a 6.25 e 21.00 b 5.34 b 0.45 b wild privet× 50 203.50 c 37.00 b 6.92 e 19.50 bc 2.19 cd 0.41 c poplar× 0 241.00 b 15.33 c 14.24 a 24.00 a 3.15 c 0.29 d poplar × 25 117.67 e 4.50 d 9.82 c 18.00 cd 1.27 d 0.03 g poplar × 50 159.17 d 18.67 c 11.23 b 19.67 bc 1.43 d 0.18 e sycamore× 0 0.00 g 0.00 e 0.00 f 0.00 f 0.00 e 0.00 g sycamore × 25 1.00 g 0.00 e 0.44 f 0.67 f 0.00 e 0.00 g sycamore × 50 0.00 g 0.00 e 0.00 f 0.00 f 0.00 e 0.00 g berry × 0 0.00 g 0.00 e 0.00 f 0.00 f 0.00 e 0.00 g berry × 25 0.00 g 0.00 e 0.00 f 0.00 f 0.00 e 0.00 g berry × 50 0.00 g 0.00 e 0.00 f 0.00 f 0.00 e 0.00 g grape × 0 59.50 f 7.33 d 8.33 d 12.00 e 2.45 c 0.03 g grape × 25 119.50 e 16.33 c 6.23 e 19.00 bc 4.51 b 0.06 f grape × 50 116.17 e 15.67 c 7.34 de 19.67 bc 4.34 b 0.26 d apple × 0 0.00 g 0.00 e 0.00 f 0.00 f 0.00 e 0.00 g apple × 25 0.00 g 0.00 e 0.00 f 0.00 f 0.00 e 0.00 g apple × 50 0.00 g 0.00 e 0.00 f 0.00 f 0.00 e 0.00 g sour cherry × 0 0.00 g 0.00 e 0.00 f 0.00 f 0.00 e 0.00 g sour cherry × 25 0.00 g 0.00 e 0.00 f 0.00 f 0.00 e 0.00 g sour cherry × 50 0.00 g 0.00 e 0.00 f 0.00 f 0.00 e 0.00 g cherry × 0 0.00 g 0.00 e 0.00 f 0.00 f 0.00 e 0.00 g cherry × 25 0.00 g 0.00 e 0.00 f 0.00 f 0.00 e 0.00 g cherry × 50 0.00 g 0.00 e 0.00 f 0.00 f 0.00 e 0.00 g p­value plant <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 garlic extract concentration 0.0658 0.3573 0.0213 0.1200 0.0068 <0.0001 plant × garlic extract concen­ tration (g·l) <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 abbasifar et al. ‐ garlic extract effects on root cuttings 199 moderate concentration of garlic extract, which did not differ significantly with the grape and control wild privet cuttings at the same concentration. the highest root length was obtained in wild privet cut­ tings treated with moderate concentration, which did not show significant differences with the cuttings treated with the high concentration. the longest roots and highest root dry weight were recorded in wild privet cuttings at moderate concentration. poplar control cuttings gained the highest fresh weight (table 6). 4. discussion and conclusions according to the results of three experimental sections, it seems that the effects of garlic extract on the traits related to shoots and roots of different cuttings depend on the time of use, species type, and the extract concentration. in the first section, the use of high garlic extract concentration (50 g/l) in rose cuttings and moderate concentration (25 g/l) in wild privet, poplar, berry, grape, apple, and cherry cuttings had positive impacts. garlic extract had no positive effects on shoot traits in sycamore and sour cherry culture substrates. in schefflera arboricola, garlic extract application increased plant height, leaf number, and fresh and dry weights of leaves (hanafy et al., 2012). in leguminous plants, the extract espe­ cially at high concentrations caused negative effects on growth and decreased plant height (adeleke, 2015). also, using a high concentration of garlic extract in the culture substrates of rose and wild privet, and moderate concentration in poplar and grape substrates had positive effects on their rooting traits. however, a high concentration of garlic extract was not effective in the rooting of sycamore, b e r r y , a p p l e , s o u r c h e r r y , a n d c h e r r y c u tti n g s . considering the evaluated shoot and root traits, the use of 50 g/l and 25 g/l of garlic extract was useful in rose cuttings and poplar and grape cuttings, respectively. garlic extract contains many allelopathic chemi­ cals (wang et al., 2015) with a known antimicrobial and antibacterial agent (harris et al., 2001). garlic planting in vegetable hydroponic substrates has been shown to prevent them from many diseases and to reduce the microbial population in the substrate (liu et al., 2014). the positive effects of garlic extract are more evident at low and moderate concentrations. high concentrations of garlic extract led to decreased growth of shoots and roots due to increased allelo­ tings, and a high concentration in apple cuttings resulted in desirable effects on this trait. the effect of this extract was positive in sour cherry cuttings, and there was a negative effect on the length of shoots produced in cherry cuttings (table 5). the fresh weight of shoots was uppermost in wild privet cuttings treated with 25 g/l of garlic extract, which was not significantly different from that of grape cuttings at 50 g/l. dry weight of wild privet cuttings was the highest in all treatments. the use of garlic extract increased fresh and dry weights of shoots in poplar, sycamore, and berry cuttings. in grape and apple cuttings, a high concentration of gar­ lic extract yielded useful impacts on the fresh and dry weights of shoots while it led to negative conse­ quences in sour cherry and cherry cuttings (table 5). overall, the use of garlic extract at the time of callus formation was evaluated to be positive on the shoot­ related traits of sycamore, berry, and apple cuttings. root‐related traits. analysis of experimental data showed that different concentrations of garlic extract significantly influenced rooting characteristics of dif­ ferent cuttings in the callus stage at 1% level. the use of garlic extract could significantly affect the root number and length at moderate concentration and root dry weight at both concentrations in the callus stage of rose cuttings. the use of moderate concen­ tration had a significant effect on the increase of all root traits in wild privet cuttings in the callus stage. the high concentration caused a decrease in the number of roots and an increase in root length, and the moderate concentration increased the longest root length of poplar cuttings. the use of garlic extract at this stage caused a significant decrease in fresh and dry weights of roots in poplar cuttings. sycamore cuttings showed some increases in the root traits at the moderate concentration. berry cuttings increased the number of roots to some extent at both concentrations. in the cuttings of this plant, the length of roots was the greatest at the moderate concentration of garlic extract and the longest root length, and root fresh and dry weights were greater at the concentration of 50 g/l. in grape cuttings, the moderate concentration increased the number of roots and the high concentration elevated root length. in the cuttings of this plant, both concentra­ tions increased the length of the longest root and reduced root fresh and dry weights. root growing did not occur in apple, sour cherry, and cherry cuttings (table 6). t h e h i g h e s t n u m b e r o f r o o t e d c u tti n g s w a s observed in wild privet cuttings treated with the adv. hort. sci., 2020 34(2): 191­204 200 table 5 ­ effects of different concentration of garlic extract (in the callus stage) on shoot­related characteristics mean values followed by the similar letters within a column are not significantly different from each other at p≤0.05 (duncan’s multiple range test). treatments number of green cuttings shoot length mean (cm) the highest shoot length (cm) fresh weight (gr) dry weight (gr) plant rose 1.22 cd 5.46 c 10.44 d 2.27 d 0.70 e wild privet 9.67 a 8.54 b 29.78 b 23.08 a 7.09 a poplar 4.89 b 18.14 a 35.14 a 13.08 b 4.76 b sycamore 0.78 d 2.14 de 2.44 e 0.40 de 0.08 ef berry 8.78 a 3.66 cd 10.33 d 5.26 c 1.43 d grape 8.67 a 8.41 b 15.39 c 21.81 a 2.98 c apple 1.22 cd 1.45 e 1.61 e 0.17 e 0.03 ef sour cherry 2.33 c 2.46 de 3.83 e 0.53 de 0.09 ef cherry 0.33 d 0.78 e 0.50 e 0.03 e 0.01 f garlic extract concentration (g∙l) control (0 g·l) 4.00 5.08 9.94 b 6.47 b 1.65 b 25 g·l 4.59 5.36 12.50 a 7.82 a 2.08 a 50 g·l 4.04 6.10 12.27 a 6.06 b 1.88 a plant × garlic extract concentration (g∙l) rose × 0 0.67 def 5.73 cdef 11.67 cdef 1.84 fgh 0.33 hi rose × 25 2.00 cdef 4.98 defg 9.00 efgh 3.19 efgh 0.92 ghi rose × 50 1.00 cdef 5.66 def 10.67 defg 1.79 fgh 0.84 ghi wild privet × 0 9.67 a 8.54 cd 30.17 b 21.34 b 6.95 a wild privet × 25 10.00 a 8.81 c 27.83 b 26.27 a 7.41 a wild privet × 50 9.33 a 8.26 cd 31.33 b 20.89 b 6.92 a poplar × 0 5.33 b 17.27 b 26.50 b 10.19 d 3.78 cd poplar × 25 6.33 b 15.03 b 37.67 a 14.92 c 5.51 b poplar × 50 3.00 c 21.09 a 40.00 a 13.79 c 4.66 bc sycamore × 0 0.00 f 0.00 i 0.00 j 0.00 h 0.00 i sycamore × 25 1.33 cdef 5.00 defg 5.67 ghij 0.48 gh 0.15 hi sycamore × 50 1.00 cdef 1.43 ghi 1.67 ji 0.73 gh 0.00 i berry × 0 8.33 a 3.08 efghi 5.00 fghi 3.03 de 1.14 ghi berry × 25 9.00 a 4.08 efgh 11.83 cdef 5.10 ef 1.35 fgh berry × 50 9.00 a 3.82 fghi 12.17 cdef 6.66 e 1.81 fg grape × 0 8.67 a 8.45 cd 17.00 c 21.71 b 3.31 de grape × 25 9.00 a 7.66 cde 13.67 cde 21.88 b 3.18 de grape × 50 8.33 a 9.13 c 15.50 cd 25.87 a 2.46 ef apple × 0 0.67 def 1.00 hi 0.83 ji 0.03 h 0.03 i apple × 25 0.67 def 1.00 hi 0.83 ji 0.06 h 0.01 i apple × 50 2.33 cde 3.42 fghi 3.17 hij 0.41 gh 0.06 i sour cherry × 0 2.00 cdef 0.67 hi 1.00 ji 0.28 gh 0.06 i sour cherry × 25 2.67 cd 3.54 fghi 5.33 ghij 0.49 f 0.17 hi sour cherry × 50 2.33 cde 3.17 fghi 5.17 ghij 0.48 gh 0.05 i cherry × 0 0.67 def 1.00 hi 0.83 ji 0.07 h 0.01 i cherry × 25 0.33 ef 1.33 ghi 0.67 j 0.03 h 0.01 i cherry × 50 0.00 f 0.00 i 0.00 j 0.00 h 0.01 i p­value plant <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 garlic extract concentration 0.1060 0.1614 0.1060 0.0159 0.1672 plant × garlic extract concentra­ tion (g·l) <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 abbasifar et al. ‐ garlic extract effects on root cuttings 201 table 6 ­ effects of different concentration of garlic extract (in the callus stage) on root­related traits mean values followed by the similar letters within a column are not significantly different from each other at p≤0.05 (duncan’s multiple range test). treatments number of roots root length mean (cm) the highest root length (cm) fresh weight (gr) dry weight (gr) plant rose 1.22 d 5.60 c 7.83 c 0.40 d 0.04 d wild privet 9.00 a 16.59 a 32.79 a 13.04 b 4.40 a poplar 3.87 c 14.91 a 24.74 b 6.31 c 0.80 c sycamore 0.44 ef 1.28 d 2.28 d 0.04 d 0.01 d berry 1.00 de 8.82 b 8.07 c 0.35 d 0.06 d grape b14.8 10.09 b 26.78 b 14.65 a 2.59 b apple 0.00 f 0.00 d 0.00 d 0.00 d 0.00 d sour cherry 0.00 f 0.00 d 0.00 d 0.00 d 0.00 d cherry 0.00 f 0.00 d 0.00 d 0.00 d 0.00 d garlic extract concentration (g∙l) control (0 g·l) 2.08 b 4.22 b 7.19 b 4.45 a 0.64 b 25 g·l 3.08 a 7.09 a 12.86 a 3.57 b 0.87 a 50 g·l 2.26 b 7.01 a 12.35 a 2.27 c 0.64 b plant × garlic extract concentration (g·l) rose × 0 0.67 fg 4.78 g 7.33 e 0.25 e 0.00 g rose × 25 2.33 e 6.62 fg 8.33 e 0.30 e 0.03 f rose × 50 0.67 fg 5.39 g 7.83 e 0.65 e 0.09 f wild privet× 0 9.00 ab 14.33 bc 26.50 bc 12.89 bc 3.63 b wild privet× 25 10.00 a 19.26 a 35.67 a 13.96 b 5.06 a wild privet× 50 8.00 bc 16.31 ab 32.00 ab 11.89 bc 3.80 b poplar× 0 5.00 d 11.24 cde 17.07 d 17.24 a 1.18 d poplar × 25 4.50 d 14.80 b 30.67 ab 3.05 d 0.50 ef poplar × 50 2.33 e 18.68 a 26.50 bc 2.29 de 0.73 de sycamore× 0 0.00 g 0.00 f 0.00 f 0.00 e 0.00 f sycamore × 25 1.00 fg 3.42 gh 3.83 ef 0.09 e 0.01 f sycamore × 50 0.33 fg 0.00 h 3.00 ef 0.02 e 0.00 g berry × 0 0.33 fg 0.00 h 0.00 f 0.03 e 0.03 f berry × 25 1.33 ef 13.90 bcd 7.50 e 0.24 e 0.03 f berry × 50 1.33 ef 9.62 ef 13.83 d 0.78 e 0.13 f grape × 0 7.00 c 9.64 ef 24.33 c 18.68 a 3.99 b grape × 25 9.00 ab 9.84 ef 28.00 bc 14.47 b 2.18 c grape × 50 7.67 c 10.80 de 28.00 bc 10.89 c 2.06 c apple × 0 0.00 g 0.00 h 0.00 f 0.00 e 0.00 f apple × 25 0.00 g 0.00 h 0.00 f 0.00 e 0.00 f apple × 50 0.00 g 0.00 h 0.00 f 0.00 e 0.00 f sour cherry × 0 0.00 g 0.00 h 0.00 f 0.00 e 0.00 f sour cherry × 25 0.00 g 0.00 h 0.00 f 0.00 e 0.00 f sour cherry × 50 0.00 g 0.00 h 0.00 f 0.00 e 0.00 f cherry × 0 0.00 g 0.00 h 0.00 f 0.00 e 0.00 f cherry × 25 0.00 g 0.00 h 0.00 f 0.00 e 0.00 f cherry × 50 0.00 g h00.0 0.00 f 0.00 e 0.00 f p‐value plant <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 garlic extract concentration <0.0001 <0.0001 <0.0001 <0.0001 0.0474 plant × garlic extract concentration (g·l) <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 adv. hort. sci., 2019 33(3): 191­204 202 pathic properties (adeleke, 2015). one of the reasons for the failure of rooting in the cuttings is the sub­ strate contamination to pathogenic agents and their entry into the stem, and as a result, the contamina­ tion of cuttings and failure in rooting (hartmann et al., 2002). garlic extract is a strong disinfectant that resulted in increased rooting and improved shooting. in the second part of the experiment, using the moderate concentration of garlic extract had positive impacts on the shoot and root traits of wild privet cuttings. garlic extract contains auxin hormone, and immersing the tips of olive cuttings in garlic extract in combination with other natural auxins­containing substances (e.g., algae) produced the longest roots and increased the number of shoots, leaf number, mean shoot length, and dry weight of shoots (gad and ibrahim, 2018). the same authors reported that the compound was able to compete with iba and no significant differences were found in these traits with those of iba­treated olive cuttings. in the third part of the experiment, the use of moderate­level garlic extract in the callus stage of rose and sycamore cuttings and the high concentra­ tion in the callus stage of the berry cuttings had posi­ tive effects on the shoot and root traits. there is ample evidence that garlic extract has an allelochem­ ical property that affects cell division, absorption of water and minerals, phytohormone metabolism, res­ piration and photosynthesis, enzyme function, and expression of genes (portales­reyes et al., 2015; sadaqa et al., 2016). biostimulators are a group of naturally occurring substances that can be applied either as spraying or at the tips of cuttings, and some of these substances increase water absorption and nutrient transfer, and stimulate growth processes, with possible hormonal properties (dobranszki and teixeira da silva, 2010); a few of them had positive effects on ornamental pine propagation and rooting of ornamental plant cuttings ( s h e v c h e n k o , 2 0 0 8 ; s z a b ó a n d h r o t k ó , 2 0 0 9 ; pacholczak et al., 2016). these substances stimulate the processes occurring in plants to increase growth. khan et al. (2009) and borowski (2009) reported that natural substances alter the growth and develop­ ment of cells in the root system and the concentra­ tion of many substances in the plant. the mechanism of the physiological and biochemical processes of these substances remains unknown, and their infor­ mation and application in horticulture and plant propagation are very limited and require further study and attention (du jardin, 2015; ertani et al., 2015). many plant extracts such as coconut juice, banana pulp, potato puree, date sap, corn extract, papaya extract, and beef extract have been used in micro­ propagation to enhance plant growth (islam et al., 2003; murdad et al., 2010; nambiar et al., 2012; sudipta et al., 2013). the accumulation of carbohy­ drates, particularly simple ones (monomers), in the rooting zone is essential for rooting onset at early stages, and carbohydrate concentrations of applied natural substances had high influences on the rooting (costa et al., 2007). active oxygen species (ross) are oxygen species that destruct cells and destroy membranes and nucleic acids during such tensions as cutting and mechanical damage to cuttings. ross, therefore, is always present at rooting event. biostimulators and natural substances used to increase the growth of different plants reduce the effects of various stresses on plants (dobranszki and teixeira da silva, 2010). phenolic compounds are classified into simple phe­ nols, phenolic acids, hydroxycinnamic derivatives, and flavonoids. researchers have reported that many phenolic compounds and flavonoids function as strong antioxidant compounds that are found abun­ dantly in many plant extracts, including medicinal plants. the active ingredients of medicinal plants include complex chemical compounds produced and stored in the organs of medicinal plants and protect cells from oxidative damage (lin and harnly, 2010). the extracts of medicinal plants have many antimi­ crobial properties and can prevent the release of microorganisms in the culture medium and plant growth media (radwan et al., 2015). considering the shoot and root traits evaluated in the three experiments, the use of 50 g/l and 25 g/l of garlic extract was useful in rose cuttings and poplar and grape cuttings, respectively. moderate concentration (25 g/l) of garlic extract revealed posi­ tive effects on shoot and root traits of wild privet cut­ tings. also, the use of moderate garlic extract had positive effects on rose, and at a high concentration (50 g/l) on sycamore cuttings and on berry cuttings in the callus stage. as a result, to improve the quantity and quality of shooting and rooting in rose cuttings, garlic extract can be used at a concentration of 50 g/l in the substrate or 25 50 g/l in the callus formation stage. a comparison of data from these two experi­ ments indicated that the use of 25 g/l was more effective in the callous formation stage. also, to improve the quality and quantity of shoots and roots in wild privet cuttings, garlic extract can be used at 25 g/l on the cuttings. the quality and quantity of abbasifar et al. ‐ garlic extract effects on root cuttings 203 poplar and berry cuttings can be improved by con­ centrations of 50 g/l and 25 g/l of garlic extract in the callus stage. no positive impacts were noticed on the other plants, or positive effects were seen only on shooting or rooting traits. references adeleke m.t.v., 2015 ­ effect of allium sativum (garlic) extract on the growth and nodulation of cowpea (vigna unguiculata) and groundnut (arachis hypogea). ­ afr. j. agric. res., 11(43): 4304­4312. al mayahi m.z., fayadh m.h., 2015 ­ the effects of garlic extract, its application methods and their interaction on growth and yield of potato, solanum tuberosum (l.) cv. latonia. ‐ adv. agric. bot. int. j. bioflux soc., 7(1): 59­69. bai n.r., banu n.r.l., prakash j.w., goldi s.j., 2007 ­ effects of asparagopsis taxiformis extract on the growth and yield of phaseolus aureus. ­ int. j. basic 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(1) hababouk (cell division and elongation), (2) kimri (firm full colored), (3) khalal (physiolog­ ically mature with a hard and crisp texture and a moisture content between 50­85%), (4) rutab (partially browned, reduced moisture con­ tent 30­45%, fibres softened, perishable), and (5) tamar (fruit colour from amber to dark brown with a moisture content further reduced ­ below 25% down to 10% and less, and the texture is soft pliable to firm to hard) (*) corresponding author: babakmadani2010@gmail.com mirzaalian@hormozgan.ac.ir citation: madani b., dastjerdy a.m., shahriyari a., 2021 ­ improving ‘piyarom’ date palm fruit qua‐ lity with fruit thinning and bunch covering treat‐ ments. ­ adv. hort. sci., 35(1): 11­19 copyright: © 2021 madani b., dastjerdy a.m., shahriyari a. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 16 april 2020 accepted for publication 24 december 2020 ahs advances in horticultural science https://doi.org/10.36253/ahsc-9548 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2021 35(1): 11­19 12 (awad and al­qurashi, 2012). date palm (phoenix dactylifera l.) cv. piyarom is one of the most impor­ tant commercial semi­dry dates in iran which is con­ sumed at tamar ripening stage and fruit reaches full maturity. it is widely grown in hormozgan province of iran with increasing export production. in com­ mercial production, larger fruit with higher quality are more marketable and therefore fruit thinning is one of the main agronomic methods to improve fruit size and quality. fruit thinning is widely used in horti­ cultural production which increases fruit quality, reduces bi­annual bearing and ensures a physiologi­ cal balance between vegetative and reproductive parts due to reduced competition for water and food absorption (slatnar et al., 2020). there are different times and methods for date palm thinning. for exam­ ple, thinning 1/3 of the bunch at pollination period or 1/3 terminal tips of central strands in early kimri stage of fruit development, as has been described by el­badawy et al. (2018) and moustafa et al. (2019). however some date palm cultivars can react contrari­ ly to the different treatments of thinning for exam­ ple, awad and al­qurashi (2015) reported that thin­ ning 5­10 cm bunch four weeks after pollination in ‘barhee’ date did not affect the qualitative characters of fruit. however, ahmed et al. (2019) showed that thinning 1/3 terminal tips of central strands in early k i m r i s t a g e o f f r u i t d e v e l o p m e n t o f ‘ z a g h l o o l ’ decreased titrable acidity and increased soluble solid content. however there has been no research on the effects of different thinning options on the quality of ‘piyarom’ date where most farmers in southern iran currently use bunch thinning only at pollination peri­ od. pre­harvest covering of fruit is practiced in many crops and its usage is increasing in many countries to improve fruit quality. however, the effects of fruit covering on final fruit quality is variable and reflect differences in the covering type, fruit age at covering and cultivar response (sharma et al., 2014). for example kassem et al. (2010) stated that ‘zaghlool’ dates covering with dark polyethylene at kimri stage did not affect total acidity (ta) and total soluble solid content (tss). however, awad and al­qurashi (2012) concluded that bunch craft bagging after pollination increased bunch weight, ta and tss, ascorbic acid and decreased total phenols and rutab percentage of ‘barhee’ date. harhash and al­obeed (2010) showed that bunch bagging increased fruit and bunch weight and quality of ‘succary’ and ‘khalas’ dates. harhash et al. (2020) further showed that bunch weight was greatly increased by covering bunches with white paper and white and brown cloth as compared to control. fruit weight was increased remarkably by using white or kraft paper and white or brown cloth in covering bunches of ‘barhee’ date palm while tss was increased in the fruit which were produced from bunch covering with white or kraft paper and white or brown cloth over control. due to its high consumer appeal, ‘piyarom’ date is a promising cultivar for export but a more consistent high fruit quality is required. therefore, this study assessed the effects of both date fruit thinning and the use of bunch covers on the bunch weight and quality of ‘piyarom’ dates over two seasons. 2. materials and methods experimental procedure fruits selected for this trial were from ‘piyarom’ date palms of the same age (18 years old) and size which were grown on sandy loam soil and drip irri­ gated at the hajiabad agricultural research station in hormozgan province in iran for two years during 2018­2019. all regular cultural practices were con­ ducted according to the normal commercial sched­ ules. pollination was conducted using a local ‘green’ male cultivar to manually pollinate the female flow­ ers in april each year with a total of eight 8 bunches were left on each experimental tree. this trial was a factorial trial with fruit thinning and bunch covers as the two factors. within the fruit thinning treatment, three levels of thinning were examined; (1) removal of one third of the end of total strands from terminal tips of bunch at the time of pollination, (2) removal of one third of terminal tips of central strands in early kimri (performed in june), and (3) untreated flowers (control). the second fac­ tor of experiment was bunch covering at two levels (uncovered bunches and bunch covering with poly­ ethylene mesh which consisted on a 100 cm long and 80 cm wide bag with mesh holes (diameter of each hole is about 2 mm) which were applied in early stage of khalal. the thinning treatments and bunch coverings were performed on four bunches in each palm tree, and fruit sampling of these bunches was performed at harvest. in november on each tree, all bunches were harvested and bunch weight per palm was recorded. the tamar percentage was also calcu­ lated. fruit samples were collected at the tamar stage for physicochemical characteristics measure­ madani et al. ‐ preharvest treatments and ‘piyarom’ date palm fruit quality 13 ments. physico‐chemical characters at harvest time (tamar stage), 100 fruit subsam­ ples from each replicate were sampled from palm trees and the factors related to the colour of the fruit measured by minolta (cr­300, minolta corp, osaka, japan) colorimeter and expressed as l*, c* and h◦. fruit weight, length and diameter of each fruit and seed were measured on 25 fruit per replicate. fruit and seed length and diameter were measured by a digital caliper and the weight of the fruit and seed were measured by a digital scale. fruit total sol­ uble solid content (tss), ascorbic acid, moisture con­ tent, total phenolic compounds and total antioxidant activity were measured on a composite sample of 20 fruit per replicate. tss was measured as brix % with a digital refractometer (dbr95, taiwan). the spec­ trophotometric method was used for measuring ascorbic acid content (etemadipoor et al., 2019). ten ml of distilled water used for homogenizing one gram of fruit tissue. then, the solution filtered and 0.1 ml of it was added to 1% metaphosphoric acid. then, s o l u ti o n c o m b i n e d w i t h 9 m l o f 2 , 6 ­ d i c h l o r o i n d o p h e n o l ( 0 . 0 0 2 5 % ) . a ft e r t h a t , t h e m i x t u r e absorbance was evaluated by spectrophotometer (cecil, ce2501, england) at 517 nm. the results were reported accordingly (mg/100 g fresh fruit tissue). for moisture content 20 g of fruit sample from each replicate was cut into small pieces by a sterilized knife. then, the fresh weight of each sample was measured. then, dried to a constant weight in oven at 70◦c. dry weight was measured and fruit moisture expressed as percentage (marzouk and kassem, 2011). total antioxidant activity (taa), total phenolic con‐ tent (tpc) m e a s u r i n g t a a i n v o l v e d a d d i n g 1 m l 2 , 2 ­ diphenyl­1­picryl­hydrazyl (dpph) (0.1 mm) to 0.1 ml methanolic extract. this solution was subsequently mixed with 1 ml of tris­hcl (ph 7.5). the absorption value of each sample was determined at 517 nm wavelength via spectrophotometer (cecil, ce2501, england) (etemadipoor et al., 2020). the antioxidant activity was determined as follows: antioxidant activity (%) = [1 ­ (abs sample /abs control) × 100] the folin­ciocalteu reagent was used for measur­ ing tpc (ehteshami et al., 2019). briefly, fruit tissue (0.5 g) was squashed and mixed into methanol (3 ml, 85%). the resultant solution was centrifuged at 12,000 × g at 4°c for 20 min. the extract was consid­ ered as supernatant. then, 300 μl of extract was combined with sodium carbonate (1.2 ml, 7%). this mixture was stored at ambient temperature for 5 min. after that folin­ciocalteu (10%, 1.5 ml) was added. the absorbance was determined at 760 nm and tpc was presented as mg gae 100 g­1 fw. statistical analysis of data the experimental was as factorial in a randomized complete block design with three replicates (block) and each replicate consisted of two palm tree. two factors included bunch thinning and bunch covering. bunch thinning consisted of 3 levels and bunch cov­ ering consisted of 2 levels. the treatment unit con­ sisted of four bunches on each of the two tree treat­ ment blocks. data were analyzed by statistical analysis system version 9.1 (sas institute inc., cary, nc, usa). the means were compared using lsd at a significance level of p= 0.05. 3. results and discussion bunch weight and tamar percentage the results of this experiment showed that the coverage did not have a significant effect on the per­ centage of tamar, but date palm fruit thinning in the kimri and pollination stages significantly increased tamar percentage of both years (table 1). the tamar percentage was approximately 94.25­113.75% higher than the control group and occurred as a result of thinning in the kimri stage during both years of experiment. also, the bunch weight was significantly higher in the control as compared to bunch weights that occurred after date palm fruit thinning at the pollination and kimri stages, for both years (table 1). in this research, the tamar percentage was not affected by covering. in the available literature, there are inconsistent results on the use of covering for fruit ripening. for example, awad and al­qurashi (2012) stated that bunch bagging of ‘barhee’ date can eventually reduce the percentage of rutab in comparison with the control. however, kassem et al. (2011) stated that spathe­bagging increased fruit ripening in ‘zaghlool’ date, as compared to the con­ trol. tamar is the last stage of ‘piyarom’ date fruit maturity. however, date fruit do not ripen evenly, a n d e v e n i n a b u n c h r u t a b a n d k h a l a l c a n b e observed at harvest. this causes economic loss of fruit. therefore, increasing tamar percentage in the bunch can rise the commercial value of product. the adv. hort. sci., 2021 35(1): 11­19 14 increase in the percentage of tamar, as a result of the thinning treatment, can be due to a lowered level of competition between fruit for the absorption of water and nutrients, thereby accelerating fruit matu­ rity and increasing the percentage of tamar (radwan, 2017). the results of this study on increasing bunch weight of the control, as compared to the date palm fruit thinning treatment, are consistent with previous results (moustafa et al., 2019) on the khadravi culti­ var. however, the primary goal is to determine an optimum method of date palm fruit thinning so as to improve fruit quality, obtain a reasonable bunch weight and thus enhance marketability. taa, tpc the antioxidant properties of date fruit differ depending on the amount of phenolic compounds in the fruit (hussain et al., 2016). phenolic compounds account for most of the antioxidant properties of dates. they exhibit a range of biological effects such as the prevention of nucleic acid damage. there has been growing interest in the topic of antioxidants, regarding the ability of antioxidants to scavenge free radicals associated with various diseases (aleid, 2014). thus, dates can be used as antioxidative func­ tional food ingredients (aleid, 2014). fruit of differ­ ent date palm cultivars have different total phenolic contents and antioxidant activities (al­turki et al., 2010). in the current study, comparison of the main effects of date palm fruit thinning on total antioxi­ dant activity (taa) showed that date palm fruit thin­ ning during the kimri phase increased the taa as compared to the control (table 2). also, date palm fruit thinning during the kimri phase increased the tpc as compared to the control for both years. moreover, bunch covering increased taa and tpc table 1 ­ main effects of bunch date palm fruit thinning and covering on bunch weight and tamar percentage of ‘piyarom’ date z means within each column for each treatment followed by the same letter are not significantly different at p=0.05. y ns= non significant. treatment bunch weight (kg) tamar (%) 2018 thinning control 15.49 az 29.16 c removal of one third of total strands from terminal tips during pollination 13.44 b 49.33 b removal of one third of terminal tips of central strands in early kimri 11.37 c 62.33 a lsd 4.89 3.82 bunch covering control 13.09 b 47.66 a bunch covering 13.77 a 46.22 a lsd 3.99 3.12 thinning x bunch covering ns y ns 2019 thinning control 15.51 a 32.00 c removal of one third of total strands from terminal tips during pollination 14.05 b 45.66 b removal of one third of terminal tips of central strands in early kimri 11.00 c 62.16 a lsd 3.23 5.10 bunch covering control 13.29 b 46.33 a bunch covering 13.75 a 46.88 a lsd 2.64 4.16 thinning x bunch covering ns ns table 2 ­ effect of fresh organic amendments on nematode population, gall number and galling index treatments taa (%) tpc (mg gae 100 g fw) 2018 thinning control 49.66 c z 130.16 b removal of one third of total strands from terminal tips during pollination 67.26 b 135.45 ab removal of one third of terminal tips of central strands in early kimri 70.43 a 138.21 a lsd 2.89 5.73 bunch covering control 58.73 b 131.23 b bunch covering 66.17 a 137.98 a lsd 2.36 4.68 thinning x bunch covering ns y ns 2019 thinning control 48.18 c 126.51 b removal of one third of total strands from terminal tips during pollination 67.83 b 141.16 a removal of one third of terminal tips of central strands in early kimri 71.10 a 140.06 a lsd 2.21 4.15 bunch covering control 57.31 b 134.28 b bunch covering 67.43 a 138.21 a lsd 1.80 3.39 thinning x bunch covering ns ns z means within each column for each treatment followed by the same letter are not significantly different at p=0.05. y ns= non significant. madani et al. ‐ preharvest treatments and ‘piyarom’ date palm fruit quality 15 compared to the control for both years (table 2). little research exists on the effects of bunch thinning and covering on taa and tpc of date. hussain et al. (2016) stated that thinning treatments of ‘hillawi’ and ‘khadrawi’ increased tpc in comparison with the control, probably because of a greater light exposure to the fruit in response to less fruit density in each bunch. indeed, light plays an important role in processes that are responsible for the accumulation of phenolic compounds. on the other hand, in fruit of the control group, there were lower amounts of light and air circulation available to the fruit, thereby affecting the rate of photosynthetic carbon assimila­ tion rate and tpc (hussain et al., 2016). the contra­ dictory effects of covering on phenolic­compound might be due to differences in the covering material, exact time and period of covering, cultivars and cli­ matic conditions (sharma and sanikommu, 2018). chen et al. (2012) stated that fruit bagging reduced the concentration of phenolic compounds in ‘golden delicious’ apple, whereas griñán et al. (2019) report­ ed that pomegranate fruit bagging increased the antioxidant content. physiochemical characteristics comparison of main effects of coverage on color lightness, chroma and hue showed that coverage sig­ nificantly reduced the lightness and chroma, while at the same time increased the hue in comparison with the control for both years (table 3). also, the com­ parison of main effects of thinning on lightness, chro­ ma and hue showed that thinning in pollination and the early kimri stage significantly reduced the light­ ness and chroma, compared to the control. in addi­ tion, this treatment increased the hue in fruit, com­ pared to the control for both years (table 3). the colour of date fruit is one of the most important fac­ tors that can largely influence customers and can determine prices. so far, the available literature does not include measurements of lightness, chroma and hue when bunch covering and thinning are used as treatments for date palm. the positive correlation table 3 ­ main effects of bunch date palm fruit thinning and covering on colour, tss, ascorbic acid and moisture of ‘piyarom’ date z means within each column for each treatment followed by the same letter are not significantly different at p=0.05. y ns= non significant. treatment l (*) c (*) h (◦) tss brix (%) ascorbic acid (mg 100 g­1) moisture (%) 2018 thinning control 30.31 a z 11.99 a 40.83 c 61.11 b 6.06 c 12.13 a removal of one third of total strands from terminal tips during pollination 25.66 b 9.45 b 46.98 b 63.11 b 11.25 b 11.25 a removal of one third of terminal tips of central strands in early kimri 22.58 c 7.12 c 51.80 a 66.05 a 13.46 a 11.86 a lsd 2.19 1.49 2.60 2.41 1.11 1.19 bunch covering control 27.50 a 10.19 a 45.21 b 63.01 a 10.68 a 11.95 a bunch covering 24.84 b 8.85 b 47.86 a 63.84 a 9.83 a 11.54 a lsd 1.79 1.22 2.13 1.97 0.90 0.97 thinning x bunch covering ns y ns ns ns ns ns 2019 thinning control 28.75 a 11.14 a 44.71 c 62.01 c 6.81 c 10.76 a removal of one third of total strands from terminal tips during pollination 24.06 b 8.05 b 50.30 b 64.88 b 12.13 b 11.60 a removal of one third of terminal tips of central strands in early kimri 21.90 b 6.36 c 55.73 a 67.45 a 13.66 a 10.41 a lsd 2.41 1.25 3.34 2.26 1.28 1.61 bunch covering control 25.90 a 9.17 a 47.87 b 64.96 a 11.34 a 11.09 a bunch covering 23.91 b 7.86 b 52.62 a 64.60 a 10.40 a 10.75 a lsd 1.96 1.02 2.72 1.85 1.04 1.31 thinning x bunch covering ns ns ns ns ns ns 16 adv. hort. sci., 2021 35(1): 11­19 between taa and hue has been shown in table 4. based on our results, bunch covering increased taa which can be effective in improving the colour of the fruit (siddiq et al., 2013). the mechanism involves the formation of dark pigments on fruit, thereby reducing the brightness of the fruit and increasing hue in fruit (siddiq et al., 2013). on similar accounts, covering has reportedly improved the colour of apple (sharma and pal, 2012). a more pronounced fruit colouring is one of the most important goals of thin­ ning. the increase in fruit colour, as a result of the date palm fruit thinning treatment, is due to the absorption of more nutrients in the remaining fruit and also due to the release of more sugars that lead to antioxidant compounds and dark pigments in fruit (hussain et al., 2016). these results are comparable with previous results reported by ahmed et al. (2019) where date palm fruit thinning increased the colour index in ‘zaghlool’ date fruit. the results of statistical compound analysis showed that bunch covering did not affect significantly on soluble solid content (table 3). comparison of mean values of the main effects of thinning on tss showed that thinning in the early kimri caused a significant increase in tss compared to thinning at the pollination stage and in the control group (table 3). with the softening of the fruit, astringency is lost and tss increases, thereby sweet­ ening the fruit (serrano et al., 2001). the increase in tss due to thinning usually occurs because of the fact that fruit use more leaf area for the production of photosynthetic materials, soluble carbohydrates and soluble solids as the fruit ripens (moustafa et al., 2019). the main effects of bunch covering on ascor­ bic acid content showed that bunch covering did not a ff e c t t h i s v a r i a b l e c o m p a r e d t o t h e c o n t r o l . moreover, comparison of the mean values of the main effects of date palm fruit thinning on ascorbic acid content showed that date palm fruit thinning in the early kimri caused a significant increase in ascor­ bic acid compared to the control and also compared t o t h i n n i n g a t t h e p o l l i n a ti o n s t a g e ( t a b l e 3 ) . moreover, ahmed et al. (2019) reported that date p a l m f r u i t t h i n n i n g i n c r e a s e d a s c o r b i c a c i d i n “zaghlool” dates. also, awad and al­qurashi (2012) reported that bagging ‘barhee’ dates increased ascorbic acid content. however, the mechanism by which bunch covering affects ascorbic acid is not clear. the main effects of bunch date palm fruit thin­ ning and covering on the percentage of fruit moisture were not significant (table 3). the percentage of fruit moisture is usually affected by many environmental factors and tree management. in some cultivars, thinning of dates reduces the percentage of moisture in the ‘saidy’ date palms fruit (samouni et al., 2016). however moustafa et al. (2019) showed that date palm fruit thinning does not affect the moisture con­ tent of ‘khadrawi’ date fruit. the results of statistical compound analysis showed significant effects of bunch date palm fruit thinning on fruit length, fruit weight and seed length. comparison of mean values on the main effects of covering bunches revealed that fruit length increased as compared to the control for both years (table 5). mean comparison of the main effects showed that date palm fruit thinning caused a significant increase in the length and weight of fruit in comparison with the control for both years. also, date palm fruit thin­ ning at the time of pollination significantly increased the diameter of fruit and the length of the seeds compared to the control and thinning in the early kimri stage for both years. thinning did not affect the weight and diameter of the seeds for both years (table 5). the effects of coverage on different fruit have been investigated. however, there are contra­ dictory reports on the effects of covering on fruit size. for example, in mango, covering increased the size of the fruit (chonhenchob et al., 2011). in anoth­ er study, bagging reduced fruit size in bananas (hasan et al., 2001). fruit weight and size usually affect the marketing of dates. larger sizes have bet­ ter marketability (al­qurashi and awad, 2011). thinning can be an important treatment in the orchard which improves the quantitative and qualita­ tive properties of fruit for export. in dates, date palm fruit thinning can be performed until the middle of the kimri phase. growth occurs during the cell divi­ sion and enlargement stages. cell division occurs after fertilization and continues until the end of the table 4 ­ pearson correlation between taa, lightness, chroma and hue **= correlation is significant at the 0.01 level. correlation taa lightness chroma hue taa 1 ­0.88** ­0.84** 0.82** madani et al. ‐ preharvest treatments and ‘piyarom’ date palm fruit quality 17 kimri phase. then, cellular growth ensues. a proper distribution of nutrients during cell division and enlargement increases the size of the fruit and caus­ es better marketability. in the process of fruit growth and development, there is competition for water and nutrients. thinning reduces competition between fruit for their absorption of water and nutrients, while providing adequate conditions for growth. this reduces the number of fruit and increases the length of the fruit. these results agree with the results of ahmed et al. (2019) that thinning in ‘zaghlool’ dates at an early stage of fruit development can have a greater effect on fruit size due to a more comprehen­ sive allocation of water and nutrients. 4. conclusions the results of this study showed that covering the date bunch reduced the brightness of the fruit, but increased the bunch weight, fruit length, total antioxi­ dant activity and total phenolic compounds. thinning at the pollination stage increased the weight of the fruit compared to the control. thinning at the kimri stage increased the percentage of tamar, ascorbic acid content, and total antioxidant activity. we con­ clude that bunch covering at the pre­harvest stage is a simple, grower­friendly method. it is safe to use and has several beneficial effects on the physiochemical traits of fruit. this approach can be an integral part of fruit production in orchards. moreover, fruit thinning and covering increased total antioxidant activity and total phenolic compounds of fruit which are important quality factors. also, thinning is recommended for ‘piyarom’ dates at both pollination and kimri stages. acknowledgements the authors would like to thank prof. john b. golding for critical review and revising the manu­ script. table 5 ­ main effects of bunch date palm fruit thinning and covering on fruit and seed length, diameter and weight of ‘piyarom’ date treatments fruits length (cm) fruits diameter (cm) fruits weight (g) seed length (cm) seed diameter (cm) seed weight (g) 2018 thinning control 3.42 c z 1.78 b 6.75 c 2.30 b 1.06 a 0.99 a removal of one third of total strands from terminal tips 4.95 a 2.76 a 10.46 a 3.10 a 1.08 a 1.05 a removal of one third of terminal tips of central strands in 4.30 b 1.95 b 8.77 b 2.10 b 1.00 a 1.11 a lsd 0.42 0.52 1.45 0.32 0.21 0.18 bunch covering control 3.97 b 2.33 a 8.34 a 2.50 a 1.11 a 1.06 a bunch covering 4.48 a 2.00 a 8.98 a 2.50 a 0.98 a 1.04 a lsd 0.34 0.42 1.18 0.26 0.17 0.15 thinning x bunch covering ns y ns ns ns ns ns 2019 thinning control 3.75 b 1.96 b 7.08 c 2.23 b 0.75 a 0.98 a removal of one third of total strands from terminal tips 4.75 a 3.31 a 11.50 a 3.25 a 0.81 a 0.99 a removal of one third of terminal tips of central strands in 4.66 a 2.26 b 8.73 b 2.06 b 0.78 a 1.05 a lsd 0.31 0.45 1.04 0.36 0.20 0.18 bunch covering control 4.05 b 2.41 a 8.90 a 2.62 a 0.82 a 0.95 a bunch covering 4.72 a 2.62 a 9.31 a 2.41 a 0.74 a 1.06 a lsd 0.25 0.37 8.85 0.29 0.16 0.14 thinning x bunch covering ns ns ns ns ns ns z means within each column for each treatment followed by the same letter are not significantly different at p=0.05. y ns= non significant. adv. hort. sci., 2021 35(1): 11­19 18 references ahmed b.h., mehana s.a., zagzog o.a., qauod e.m., 2019 ­ improvements of yield and fruits quality of zaghlool date palm by using the different thinning treatments. ­ j. product. dev., 4: 743­756. aleid s.m., 2014 ­ date fruits processing and processed products, pp. 171­202. ­ in: siddiq m., aleid s.m., k a d e r a . a . 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(ed.) pre‐harvest modulation of postharvest fruits and vegetable quality. academic press, london, uk, pp. 516. siddiq m., aleid s.m., kader a.a., 2013 ­ dates: posthar‐ vest science, processing technology and health benefits. ­ john wiley & sons, usa, pp. 326. slatnar a., mikulic­petkovsek m., stampar f., veber­ ic r., marsic n.k., 2020 ­ influence of cluster thinning on quantitative and qualitative parameters of cherry tomato. ­ eur. j. hort. sci., 1: 30­33. impaginato 213 adv. hort. sci., 2020 34(2): 213­221 doi: 10.13128/ahsc­7770 effect of spread and shallow irrigation wetted area and application of organic mulch on citrus decline amelioration m.s. tadayon (*), s.m. hosseini soil and water research department, agricultural research, education and extension organization (areeo), shiraz, iran. key words: citrus decline, fibrous root, irrigation, orange, root decay. abstract: citrus decline threatens the orchards in the southern part of iran. roots begin to die­out before citrus decline deterioration. in this study, the effect of expanding the irrigation wetted area and decreasing the irrigation depth, and application of compost as organic mulch on root development and amelioration of citrus decline in valencia orange trees (citrus sinensis l. osbeck) was investigated. experimental factors contained the different per­ centage of irrigation wetted area and decreasing irrigation effective root depth by drip irrigation system at three levels ­ w0 (control): 30­40% with 60 cm effective root depth; w1: 50­60% with 45 cm effective root depth; w2: 70­80% with 30 cm effective root depth under tree canopy area and the factor of annu­ al application of compost as organic mulch at two levels ­ m0 (control): means no application of compost and m1: application of 80 kg compost under tree canopy area with 10 cm thickness as ground cover, after rotating the top soil at 10 cm depth for all treatments. the results showed that, annual application of compost as organic mulch under tree canopy area and expanding the irrigation wetted area with decreasing irrigation effective root depth, significantly improved fibrous root length and density at a lower soil depth and decreased the indices of citrus decline such as root rot percentage, leaf and fruit drop and shoot die back. also these treatments increased the water productivity and fruit quality of in declining valencia orange trees. 1. introduction citrus decline, commonly known as ‘dieback’, ‘chlorosis’ or neglecto­ sis, is not a specific disease but a syndrome expressing many disorders in the plant. such syndrome leads to decline in productivity, reduced pro­ ductive life and poor fruit quality. the symptoms of citrus decline contain root rot and blackening, shoot die back, growth stunt, fruit drop, reduc­ tion of canopy, smaller leaf number and size and leaf blotchy mottle (meena et al., 2018). stress­sensitive trees fail to maintain sufficient car­ bohydrate availability resulting in the dieback of the stressed tissues (kreuzwieser and rennenberg, 2014). also, the percentage of total solu­ ble solids and fruit juices in healthy trees is more than declined trees (mauk and shea, 2002). declining trees have more water stress and more (*) corresponding author: m.tadayon@areeo.ac.ir citation: tadayon m.s., hosseini s.m., 2020 ­ effect of spread and shallow irrigation wetted area and application of organic mulch on citrus decline amelioration. ‐ adv. hort. sci., 34(2): 213­221. copyright: © 2020 tadayon m.s., hosseini s.m. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 1 january 2020 accepted for publication 5 may 2020 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(2): 213­221 214 affected trees tend to have higher percentage leaf and fruit drop rates than healthy appearing trees. soil condition and water status (water stress and water logging) significantly reduce the citrus fibrous root density and increase the severity of citrus decline (kozlowski, 1997; graham et al., 2013; morgan, 2015; graham, 2017). citrus decline is an issue that threatens the econ­ omy of various regions of the world, including south­ ern iran. there are several factors that affect the inci­ dence of this complication. the most important influ­ ential factors on citrus decline are environmental stresses such as soil physicochemical conditions (compaction, high ph, bicarbonate, salinity) and soil nutritional status (srivastava and singh, 2009), soil moisture content (water stress and water logging with deficit or over irrigation), and biological stresses including rootstock, nematodes, greening disease (huanglongbing), tristesa virus disease, phytophtho­ ra fungal infection and root fusarium (johnson and graham, 2015; graham, 2017; meena et al., 2018; dewdney et al., 2019). according to the usda, since 2004­2005 total florida citrus production declined by greening disease from 169.1 to 94.2 million boxes in 2015­2016, down 44.4% (usda, 2017). the ideal environment for citrus root develop­ ment is a porous, medium­textured, well­drained soil, where water is easily available but not in excess (dewdney et al., 2019). mulching with organic matter helps retain moisture in the top soil by reducing sur­ face evaporation, as well as moderating soil surface temperatures. it also enhances the decomposition process of soil organic matter and improve the soil aeration and availability of nutrients in the soil (gong et al., 2006; fao, 2011). increasing root water uptake efficiency and life span is possible by irrigation man­ agement such as time and duration (decrease water stress and water logging), also improving the growth environment and drainage, soil compaction, soil ther­ mal stress, bicarbonate and osmosis stress (huber a n d h a n e k l a u s , 2 0 0 7 ; d e w d n e y e t a l . , 2 0 1 9 ) . increased root density also increases water uptake (morgan et al., 2006). in arid and semi­arid regions, the application of drip irrigation system results in root accumulation under emitter (fernandez et al., 1991; tanasescu and paltineanu, 2004; ruiz­sanchez et al., 2005), so the expansion of irrigation wetted area could enhance the fibrous root density under declining condition. increasing irrigation frequency and decreasing irriga­ tion depth stimulate root length density and increase water uptake and under these conditions, irrigation at the field capacity increases root density by 50% (kadyampakeni et al., 2014 a). in citrus decline new root growth did not stop, but the root survival time was reduced from 9 to 12 months to 4 months and the root decay and dieback were increased (dewdney et al., 2019). citrus decline cause water stress and under such condition root growth is preferable to shoot growth (hsiao and xu, 2000). root development dependent on soil aeration and moisture as two fundamental parameters for root healthiness, growth and distribution. root growth in sandy and loamy soils with higher organic matter content is stronger, and increment in soil clay content has a negative relationship with the density of citrus roots (koudounas, 1994) and the slope of clay content in soil profile is directly related to the effect of citrus decline deterioration (srivastava and singh, 2009). in citrus, the highest fibrous length den­ sities were observed in the humid part of the soil at a depth of 0 to 15 cm with higher root activity until 2 m horizontal distance from the trunk (alves et al., 2012). the rate of soil water depletion is directly related to the abundance of fibrous roots and in gen­ eral pattern of water uptake in citrus fruits indicates that water is drained by surface roots with higher amounts of available soil water (noling, 2003). lack of soil aeration and excessive soil moisture causes more severe damage than lack of moisture in declining trees, due to stop breathing and the break­ down of root cells. there is also the growth of anaero­ bic microenvironments and the production of sub­ stances such as nitrite that is toxic for the roots. centralize wetted area and excessive irrigation results in water logging, oxygen deficiency and root fungal infestationin the root zone (johnson and graham, 2015). deficit irrigation and soil water stress stimu­ lates longitudinal and singular growth without lateral roots and repeated moisture stress causes thicker fibrous roots in citrus that could decrease the water uptake efficiency by the citrus roots (castle, 1978). increment of soil aeration and soil moisture distribu­ tion and retention could provide the better condition for root development and amelioration of citrus decline. so in this experiment, the effects of spreading irrigation wetted area and decreasing irrigation depth, and annual application of compost as organic mulch on root development and diminishing of citrus decline indices in valencia orange was investigated. tadayon and hosseini ‐ citrus decline amelioration in valencia orange trees 215 2. materials and methods the experiment was conducted in a commercial orchard (28°38’13.12” n, 54°40’29.69” e and altitude 1138 m) of darab region,with very hot and dry cli­ mate (table 1), located in south west of fars province in iran, over three consecutive years 2016 to 2018. ninty­six uniform 12­year­old valencia orange trees (citrus sinensis l. osbeck), on lime (citrus aurantifo‐ lia) rootstock with citrus decline symptoms (sparse foliage, chlorotic leaves, twig drying, premature leaf fall, reduced productivity and fruit size and die­back canopy complication) were selected based on canopy diameter and labeled based on experimental plan. the distance between the trees was 4×5 m and irri­ gated by drip irrigation system with a loop contained 6 emitters (4 liters per hour). before the application of experimental treat­ ments, a composite soil sample were taken from 0­30 and 30­60 cm depths in oct. 2014 (table 2). soil sam­ ples characteristics were determined at analytical laboratory of soil and water research department, fars agricultural and natural resources research and education center, zarghan, iran. the soil of the experimental field site was calcareous with high ph, high total neutralizing value, and low amount of organic carbon, available p and mn (table 2). the fer­ tilizer application was conducted based on soil test and contained fertigation of ammonium sulfate (21­ 0­0­24s, 450 g tree­1), potassium sulfate (0­0­53+17s, 150 g tree­1) and triple superphosphate (450 g tree­1), manganese sulfate (32mn, 18s, 250 g tree­1) and iron chelate (200 g tree­1 sequestrene 138­fe eddha 6%, 150 g tree­1). a factorial (3×2) experiment was conducted in a randomized complete block design with four replica­ tions and each plot with four declining trees over three years. experimental factors contained the dif­ ferent percentage of irrigation wetted area and decreasing irrigation effective root depth by drip irri­ gation system at three levels as follows: • w0 (control)= 30­40% with 60 cm effective root depth; • w1= 50­60% with 45 cm effective root depth; • w2= 70­80% with 30 cm effective root depth under tree canopy area. also, the factor of annual application of compost as organic mulch at two levels: • m0 (control)= means no application of compost; • m1= application of 80 kg compost with 10 cm thickness as ground cover under tree canopy area. table 1 ­ long­term mean of climatic elements of the study area (darab) synoptic meteorological station statistical period (1997­2018) table 2 ­ soil characteristics in the experimental orchard soil depth bulk density (g cm­³) field capacity (%) wilting point (%) ec (ds.m­1) ph tnv (%) oc (%) p (mg kg­1) k (mg kg­1) zn (mg kg­1) mn (mg kg­1) cu (mg kg­¹) soil texture 0­30 1.25 19.5 10.3 1.32 8.15 35.2 0.76 7.3 264.5 0.76 3.12 0.57 sandy loam 30­60 1.28 19.3 10.4 1.36 8.16 37.7 0.62 7.2 252.4 0.62 3.19 0.56 sandy loam temperature (°c) relative humidity (%) precipitation (mm) evapo. (mm) sunny day no. day max wind (m s­¹) min max ave. abs. min abs. max min max ave. amount no. rainy max daily (montly sum) (montly sum) frost direc. degree velo. m/s april 11.5 26.4 19.0 3.8 34.7 26 70 48 32.50 6 43.70 182.0 276.0 0 253 13 may 16.7 33.6 25.2 9.4 41.6 16 51 34 6.46 2 24.80 286.6 331.4 0 240 15 june 21.6 39.3 30.4 15.6 44.4 11 37 24 1.10 1 9.60 382.8 356.6 0 200 14 july 25.5 41.7 33.6 15.4 46.5 12 38 25 0.92 1 5.80 428.3 341.3 0 171 15 august 26.2 40.9 33.6 19.2 45.2 14 40 27 5.76 2 20.50 422.3 339.8 0 207 16 september 21.9 38.5 30.2 16.2 42.6 14 44 29 0.82 1 4.70 336.0 327.5 0 180 10 october 16.0 33.6 24.8 7.6 38.8 16 48 32 0.32 1 2.60 228.4 308.0 0 226 10 november 10.4 26.5 18.5 2.0 33.5 22 62 42 8.48 2 50.20 138.2 269.6 0 249 8 december 5.7 20.1 12.9 ­2.2 30.0 32 76 54 40.25 5 51.80 79.1 234.9 0 207 7 january 3.8 17.0 10.4 ­2.6 25.6 36 81 58 64.77 6 65.40 64.4 226.1 3 205 8 february 5.0 17.7 11.3 ­2.6 25.8 35 81 58 49.88 6 68.20 79.3 238.4 1 240 10 march 8.0 21.8 14.9 0.2 31.6 30 74 52 37.26 7 63.00 116.7 241.1 0 238 12 adv. hort. sci., 2020 34(2): 213­221 216 compost applied 50 cm away from the trunk of the tree and over the soil surface, after rotating the soil at 10 cm depth for all treatments in march of each year. the physico­chemical properties of the applied compost contained organic carbon (22%), total nitrogen content (1.5%), c/n ratio (15), p2o5 (0.8%), k2o (0.7%), electrical conductivity (6.4 ds. m­1), ph (7.6), moisture content (12%), density (380 kg.m­3), particle diameter (6 mm). the amount of irri­ gation wetted area was adjusted by increasing the number of emitters (6, 9 and 12 per loop with 4 liters per hour discharge) and the irrigation interval was determined by placing tensiometers (irrometer tensiometer model sr manual gauge 12 in) deep down at three irrigations effective root depth (60, 45 and 30 cm, respectively) according to the irrigation experimental factor. each year in february, the com­ posite soil sample were taken from 0­10 cm and the percentage of soil organic matter, porosity and bulk density (g cm­3) were determined. the amount of organic matter was measured by weight loss of oven­ dried (105°c) soil sample after loss on ignition at 400°c. to determine the porosity of the samples, divide the pore space volume by the total volume and multiply the result by 100. soil bulk density was deter­ mined by the weight of dry soil divided by the total soil volume. the amount of consumed irrigation water was measured by contours in each treatment. over the three seasons, the indices of citrus decline were determined on tagged main branches on each tree quadrates. at the late spring, new flush lengths and the percentage of shoot dieback were measured. leaf and fruit drop percentage were determined by counting their number at two separate time (after june drops and pre­harvest time). leaf samples (con­ tain 100 leaves) were collected during july­august from fully expanded new flush sub­terminal leaves from non­fruiting from tagged branches. total chloro­ phyll content was determined by the method worked out by lichtenthaler (1987). leaf fresh weight (wf) were recorded quickly using an analytical balance (mettler toledo al104, switzerland). then, they were dried at 120°c in a circulation oven for 20 minutes, and the temperature dropped to 80°c until the con­ stant weight (dry weight, wd) was reached. then the leaf relative water content (rwc) was calculated as: leaf relative water content (%) = [(wf ­ wd)/wf] x 100 individualleaf areas (la) were determined by li­ cor leaf area meter, and sample were dried and spe­ cific leaf weights, slw (mg cm­2) were measured as: specific leaf weight (mg cm­²) = wd/la root sampling was carried out under emitters from depth of 30, 45 and 60 cm soil depth, based on the effective root depth considered in the irrigation experimental factor, by auger (diameter 9 cm and height 25 cm), in early­august (alves et al., 2012). after washing the root samples, length, weight and number of fibrous root with less than 0.2 cm diame­ ter was determined by digital ruler and weight scale, and length density (cm cm­3), density (mg cm­3) and the percentage of decayed fibrous root were deter­ mined in the soil sample volume harvested by auger (1589.6 cm3) (alves et al., 2012). at the harvesting time total fruit yield per trees was determined by the scale. a random sample of 50 fruits per plot was provided to determine fruit diam­ eter (average of two perpendicular diameters), total soluble solids, ph and titratable acidity of the juice. total soluble solids (brix) were determined by using hand refractometer (wyt portable model) and juice total acid was measured by titration method with 0.1 n s o d i u m h y d r o x i d e u n ti l p h m e t e r r e a d s 8 . 2 (graham, 2017). fruit juice percentage was calculat­ ed by fruit juice weight divided by fruit total weight. water productivity (kg m­3) was calculated based on the yield of trees to the amount of consumed irriga­ tion water. the data set was auto scaled before analysis. all the parameters for three years were sub­ jected to combine analysis of variance (anova) by m s t a t ­ c s o ft w a r e . m e a n s w e r e c o m p a r e d b y duncan’s multiple range test and pearson correlation were determined by spss software. 3. results the results of the combine analysis of variance (anova) over three consecutive seasonal growths from 2016 to 2018, indicated that there was a signifi­ cant effect of experimental factors on improvement of citrus decline deterioration indices in valencia orange (tables 4­8). annual application of compost as organic mulch and adding them to 10 cm top soil in the following years had significant effect on the increment of soil organic matter and soil porosity up to 170.45, 13.18% respectively, and decreasing soil bulk density by 11.98% at 10 cm soil depth in com­ parison with control (without application of compost as mulch) (table 3). annual application of compost as organic mulch significantly increased the fibrous roots length densi­ ty and fibrous roots density of valencia orange trees by 34.63 and 38.27% respectively. also the interac­ tadayon and hosseini ‐ citrus decline amelioration in valencia orange trees 217 tion between expansion the percentage of irrigation wetted area along with and decreasing irrigation effective root depth with annual application of com­ post as organic mulch (w1m1 and w2m1), signifi­ cantly increased the fibrous root density up to 87.09 and 112.9% and decreased the root decay by 43.23 and 46.48% at a lower soil depth respectively in com­ parison with control (w0m0) (table 4). mean separation (± sd) within columns followed by different let­ ters are significantly different at p≤0.05 using duncan’s new mul­ tiple range tests. table 3 ­ effects of experimental treatments on 10 cm top soil organic matter, porosity and bulk density over three consecutive seasonal growths from 2016 to 2018 treatments soil organic matter (%) soil porosity (%) bulk density (g cm­³) w0m0 1.42 ± 0.12 b 42.05 ± 1.21 b 1.49 ± 0.04 a w1m0 1.24 ± 0.14 b 41.86 ± 1.32 b 1.43 ± 0.04 a w2m0 1.30 ± 0.10 b 42.42 ± 1.15 b 1.42 ± 0.05 a w0m1 3.38 ± 0.07 a 47.08 ± 1.01 a 1.31 ± 0.06 b w1m1 3.84 ± 0.08 a 48.51 ± 1.02 a 1.27 ± 0.07 b w2m1 3.50 ± 0.07 a 47.39 ± 0.93 a 1.24 ± 0.06 b mean separation (± sd) within columns followed by different let­ ters are significantly different at p≤0.05 using duncan’s new mul­ tiple range tests. table 4 ­ effects of experimental treatments on root characteri­ stics of declining valencia orange over three consecuti­ ve seasonal growths from 2016 to 2018 treatments fibrous roots length density (cm cm ­³) fibrous root density (mg cm­³) root decay (%) w0m0 0.043 ± 0.003 d 0.31 ± 0.08 d 66.24 ± 6.21 a w1m0 0.054 ± 0.002 c 0.48 ± 0.06 c 53.72 ± 8.60 b w2m0 0.056 ± 0.002 c 0.49 ± 0.06 c 54.25 ± 7.32 b w0m1 0.066 ± 0.003 b 0.53 ± 0.04 bc 48.07 ± 7.56 b w1m1 0.065 ± 0.004 b 0.58 ± 0.04 b 37.60 ± 8.14 c w2m1 0.075 ± 0.003 a 0.66 ± 0.03 a 35.45 ± 8.81 c mean separation (± sd) within columns followed by different letters are significantly different at p≤0.05 using duncan’s new multiple range tests. table 5 ­ effects of experimental treatments on vegetative growth indices of declining valencia orange over three consecutive seasonal growths from 2016 to 2018 treatments total leaf chlorophyll content (mg g­¹ fw) flush length (cm) leaf drop (%) shoot dieback (%) w0m0 0.29 ± 0.03 d 24.82 ± 2.31 d 35.30 ± 1.12 a 35.70 ± 2.11 a w1m0 0.34 ± 0.02 c 35.53 ± 3.78 c 27.63 ± 1.53 b 29.62 ± 3.10 b w2m0 0.36 ± 0.01 c 38.35 ± 3.81 b 21.30 ± 1.71 c 17.60 ± 2.27 d w0m1 0.42 ± 0.03 b 39.47 ± 3.15 b 24.70 ± 1.92 bc 25.25 ± 2.15 c w1m1 0.46 ± 0.02 a 43.63 ± 2.80 a 15.45 ± 1.65 d 15.60 ± 2.84 d w2m1 0.47 ± 0.02 a 44.90 ± 2.66 a 10.20± 1.86 e 8.41 ± 1.90 e in comparison with control (without compost as mulch), application of compost as organic mulchsig­ nificantly increased the total leaf chlorophyll content and new flushes length up to 36.3 and 29.67% respectively and decreased leaf and fruit drop (table 5, 6) and shoot dieback by 40.21, 38.18 and 40.59%, respectively (table 5), in declining valencia orange trees, respectively. increment the irrigation wetted area percentageand decreasing the effective root depth for irrigation, significantly increased leaf chlorophyll contentand flush lengthand reduced leaf and fruit drop and shoot dieback. the interaction between the expansionof irrigation wetted area with annual application of compost as organic mulch (w1m1 and w2m1) had the highest impact on the increment of total leaf chlorophyll contentup to 58.62 and 62% and flush length by 75.78 and 80.9% and decreasing leaf drop 56.17 and 71.1%, fruit drop by 44.62 and 63.3% and shoot dieback by 56.3, 76.44%, respectively, in comparison with control (w0m0). leaf relative water content, specific leaf weight, fruit diameter and tree yield increased with annual application of compost as organic mulchanddevelop­ ing the irrigation wetted area (table 6). the interac­ tion between irrigation wettedarea and annual appli­ cation of compost as organic mulch (w1m1 and w2m1) had the highest impact on the increment of leaf relative water content up to 11.91 and 14.25% and specific leaf weight up to 18.75 and 22.66%, respectively, in comparison with control (w0m0) (table 6). also fruit diameter significantly increased by increment of wetted area and application of mulch by 15.9 and 14.11%, respectively (table 6). the highest yield of valencia orange trees was belonged to the interaction between irrigation wet­ ted area of 70­80% with 30 cm effective root depth for irrigation and application of compost mulch 218 adv. hort. sci., 2020 34(2): 213­221 (table 7). annual application of compost as organic mulch had significant effect on the reduction of con­ sumed irrigation water by 17.39% in comparison with control (without mulch). the interaction between irrigation wetted area and annual application of com­ post as organic mulch (w1m1 and w2m1) had the highest impact on the increment of water productivi­ ty up to 53.06 and 49.79% respectively, in compari­ son with control (w0m0), and there was no signifi­ cant difference between them (table 7). expansion of irrigation wetted area and decrease effective root depth for irrigation under the condition of compost mulch applicationhad the greatest effect on water productivity in valencia orange. annual application of compost as organic mulch sig­ nificantlyincreased the fruit juice content by 11.98%, total soluble solids (brix) by 10.96%, brix/titratable acidity ratio by 24.52% in declining valencia orange trees. fruit acidity was significantly reduced by annual application of mulch. increment of irrigation wetted area percentage significantly, increased fruit juice and brix/titratable acidity ratio. the interaction between increased irrigation wetted area with mulch application (w1m1 and w2m1), resulted in the highest increase in fruit juice percentage by 35.43 and 34%, total soluble solidsby 23 and 26.36% and brix/titratable acidity ratio by 49.6 and 57.6%, respectively in comparison with control (w0m0) (table 8). mean separation (± sd) within columns followed by different letters are significantly different at p≤0.05 using duncan’s new multiple range tests. table 6 ­ effects of experimental treatments on the leaf relative water content, specific leaf weight, fruit drop and fruit diameter of declining valencia orange over three consecutive seasonal growths from 2016 to 2018 treatments leaf relative water content (%) specific leaf weight (mg cm­²) fruit drop (%) fruit diameter cm) w0m0 83.50 ± 5.30 d 3.84 ± 0.12 d 31.15 ± 1.10 a 7.23 ± 0.10d w1m0 89.64 ± 5.24 c 4.12 ± 0.08 c 25.12 ± 1.32 b 7.58 ± 0.08 c w2m0 91.25 ± 4.22 bc 4.36 ± 0.07 bc 21.84 ± 1.25 bc 8.20 ± 0.07 b w0m1 88.36 ± 5.75 c 4.04 ± 0.09 cd 19.61 ± 1.74 c 7.71 ± 0.08 c w1m1 93.45 ± 4.12 ab 4.56 ± 0.06 ab 17.25 ± 2.11 c 8.38 ± 0.06 a w2m1 95.48 ± 4.53 a 4.71 ± 0.06 a 11.43 ± 2.73 d 8.25 ± 0.07 ab mean separation (± sd) within columns followed by different letters are significantly different at p≤0.05 using duncan’s new multiple range tests. table 7 ­ effects of experimental treatments on the leaf characteristics, fruit quantity and water productivity of declining valencia oran­ ge over three consecutive seasonal growths from 2016 to 2018 mean separation (± sd) within columns followed by different letters are significantly different at p≤0.05 using duncan’s new multiple range tests. table 8 ­ effects of experimental treatments on the fruit quality indices of declining valencia orange over three consecutive seasonal growths from 2016 to 2018 treatments total tree yield (kg) consumed irrigation water (m³ ha­¹ year­¹) water productivity (kg m­³) w0m0 62.33 ± 2.14 e 11625.28 ± 136.58 c 2.45 ± 0.11 c w1m0 71.05 ± 2.71 d 13857.14 ± 122.61 b 2.39 ± 0.16 c w2m0 83.10± 1.50 c 14631.67 ± 121.25 a 2.66 ± 0.14 bc w0m1 68.47 ± 2.43 d 10363.64 ±136.84 d 3.01 ± 0.12 b w1m1 89.40± 1.37 b 10904.76 ± 132.70 d 3.75 ± 0.11 a w2m1 94.40± 1.15 a 11869.57 ± 126.39 c 3.67 ± 0.12 a treatments fruit juice (%) titratable acidity (g 100­¹ ml) total soluble solids (brix) brix/titratable acidity ratio w0m0 42.64 ± 2.02 d 1.16 ± 0.01 a 9.56 ± 0.61 d 8.94 ± 0.22 f w1m0 48.80 ± 1.81 c 0.94 ± 0.03 b 11.13 ± 0.35 c 10.26 ± 0.19 e w2m0 53.71 ± 1.60 b 0.95 ± 0.02 b 11.87 ± 0.24 b 11.87 ± 0.17 c w0m1 47.65 ± 2.17 c 0.82 ± 0.02 c 12.29 ± 0.73 a 11.23 ± 0.18 d w1m1 57.75 ± 1.61 a 0.74 ± 0.03 d 11.76 ± 0.40 b 13.37 ± 0.15 b w2m1 57.14 ± 1.55 a 0.84 ± 0.03 c 12.08 ± 0.37 b 14.09 ± 0.14 a tadayon and hosseini ‐ citrus decline amelioration in valencia orange trees 219 results showed that fibrous root density had a significant negative correlation with leaf drops (r= ­ 0.791) and shoot dieback (r= ­0.612) (table 9). also fibrous root density had positive and significant cor­ relation with flush length (r= 0.624), leaf water con­ tent (r= 0.732) and specific leaf weight (r= 0.631). the root decay percentage had significant negative corre­ lation with leaf chlorophyll content (r= ­0.643), flush length (r= ­0.632), leaf water content (r= ­0.603), leaf specific weight (r= ­0.638), yield (r= ­0.691), water productivity (r= ­0.602), and brix/ta (r= ­0.684). also there was a positive and significant correlation between the percentage of root decay with leaf drops (r= 0.784), shoot dieback (r= 0.692), and fruit drops (r= 0.704) (table 9). specific leaf weight had positive and significant correlation with leaf chloro­ phyll content (r= 0.482) (table 9). there was a nega­ tive correlation between fruit drops and relative water content of leaves (r= ­0.774). tree water pro­ ductivity had a positive and significant correlation with the leaf chlorophyll content (r= 0.612), leaf rela­ tive water content (r= 0.766), and specific leaf weight (r= 0.677) (table 9). 4. discussion and conclusions citrus decline was directly related to root decay and decreasing of fibrous root density and root expansion in valencia orange trees. tree perfor­ mance is a function of how the root system is distrib­ uted over a large volume of soil to absorb water and nutrients (lehmann, 2003). in arid and semi­arid regions, the highest density of fibrous roots in the drip irrigation system is located under the emitters and soil wetted area (ciancio and mukerji, 2008; alves et al., 2012). the rate of water adsorption by the tree is reduced when the soil oxygen level is low (levy, 1998; boman et al., 1999) and the most influ­ ential soil condition on citrus decline are soil mois­ ture condition (meena et al., 2018), and soil com­ paction (srivastava and singh, 2009). root develop­ ment is related to the hydrophilicity of roots, soil aer­ ationand distribution of soil moisture (ruiz­sanchez et al., 2005). our results showed that application of compost as organic mulch and rotating them with 10 cm top­ soil improved soil physical properties such as soil organic matter, porosity and bulk density which could provide the best conditions for root development. this treatment could increase the soil aeration and soil moisture retention at topsoil, as two key factors for root health and growth (boman and parsons, 2002; nelson et al., 2008; johnson and graham, 2015). meanwhile, it could reduce surface evapora­ tion, moderate soil surface temperatures and provide the best condition for root development for declining valencia orange trees. application of organic mulches on eureka lemon (citrus limon burm) significantly increased the soil moisture status in various soil table 9 ­ pearson correlation coefficients between the means of citrus decline indices of valencia orange over three consecutive seaso­ nal growths from 2016 to 2018 chlorophyll content flush length leaf drop shoot dieback lrwc 1 slw 2 fruit drop fruit diameter yield water productivity brix/ta. fibrous roots density fibrous roots length density root decay chlorophyll content 1.00 flush length 0.64 ** 1.00 leaf drop ­0.51 * ­0.64 ** 1.00 shoot dieback ­0.55 * ­0.74 ** 0.78 ** 1.00 lrwc 0.54 * 0.64 ** ­0.77 ** ­0.75 ** 1.00 fruit drop ­0.66 ** ­0.25 ns 0.67 ** 0.64 ** ­0.71 ** ­0.69 ** 1.00 fruit diameter 0.43 * 0.49 * ­0.52 * ­0.51 * 0.52 * 0.53 * ­0.53 * 1.00 yield 0.59 ** 0.65 ** ­0.76 ** ­0.67 ** 0.66 ** 0.60 ** 0.26 ns 0.75 ** 1.00 water 0.61 ** 0.50 * ­0.75 ** ­0.65 ** 0.77 ** 0.68 ** ­0.75 ** 0.78 ** 0.78 ** 1.00 productivity brix/t.a. 0.63 ** 0.52 * ­0.70 ** ­0.68 ** 0.15 ns 0.57 ** ­0.66 ** 0.13 ns 0.57 ** 0.46 * 1.00 fibrous roots density 0.71 ** 0.62 ** ­0.79 ** ­0.61 ** 0.73 ** 0.63 ** ­0.71 ** 0.64 ** 0.70 ** 0.71 ** 0.62 ** 1.00 fibrous roots length density 0.58 * 0.48 * ­0.59 ** ­0.54 * 0.68 ** 0.59 ** ­0.54 * 0.61 ** 0.62 ** 0.59 ** 0.52 * 0.78 ** 1.00 root decay ­0.64 ** 0.63 ** 0.78 ** 0.69 ** ­0.60 ** ­0.64 ** 0.70 ** ­0.66 ** ­0.69 ** ­0.60 ** ­0.68 ** ­0.64 ** ­0.71 ** 1.00 1 leaf relative water content. 2 specific leaf weight. *, ** significant at 5 and 1% statistical levels respectively; ns = not significant. adv. hort. sci., 2020 34(2): 213­221 220 depths and farmyard manure were found to be more effective in producing maximum growth extension (kumar et al., 2015). the irrigation wetted area, also affect citrus root system development and distribu­ tion (alves et al., 2012). in our experiment, the expansion of irrigation wetted area and reduction the effective root depth for irrigation with annual appli­ cation of compost as organic mulchand its rotation in the 10 cm soil depth, increased the fibrous root length and root densityat lower soil depths and decreased root decay in valencia orange trees. also, leaf relative water content, specific leaf weight and f r u i t d i a m e t e r i n v a l e n c i a o r a n g e s i g n i fi c a n t l y increased with developing the irrigation wetted area. increment of fibrous root length and root density at lower soil depths with decreasing the irrigation depth and consequent improvement of tree water status were in agreement with the results that showed more root distribution in the drip irrigation method was at the depth of 15 cm, with high water uptake efficiency (kadyampakeni et al., 2014 b). annual application of compost as organic mulch significantly decreased the consumed irrigation water in valencia orange trees. increasing soil organic carbon improve the healthy root system of citrus (sharma et al., 1986). there was a positive and significant correla­ tion between leaf relative water content and leaf chlorophyll content (r= 0.540), and flush lengths (r= 0.643) and a significant negative correlation between leaf water content and leaf drops (r= ­0.771), and shoot dieback (r= ­0.748) (table 8). it has been shown that clementine ‘nules’ vegetative growth and fruit size was higher with increasing numbers of emit­ ters on the double drip­lines treatments (abouatallah et al., 2012). we conclude that annual application of compost as organic mulch under tree canopy area and its rota­ tion at 10 cm soil depth at following years and expanding the irrigation wetted area and decreasing the irrigation effective root depth, significantly improved the indices of citrus decline and increased the water productivity and fruit quality in valencia orange trees. acknowledgements the authors greatly appreciate mr. mojtaba dadgar who provided the commercial orchard for this experiment and fars agricultural research, education and extension center. references a b o u a t a l l a h a . , s a l g h i r . , e l ­ f a d l a . , a f f i n . , ghnizar y., zarrouk a., hammouti b., 2012 ­ impact assessment of drippers distribution around the tree on soil moisture, roots and fruits growth of citrus. ­ der pharma chemica., 4.5: 1969­1981. alves j. jr., bandaranayake w., parsons l.r., evange­ lista a.w., 2012 ­ citrus root distribution under water s t r e s s g r o w n i n s a n d y s o i l o f c e n t r a l f l o r i d a . ­ engenharia 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root distribution of apple tree under various irrigation systems within the hilly region of romania. ­ inter. agrophysics, 18(2): 175­180. usda, 2017 ­ florida citrus statistics. ­ united states department of agriculture, fl, usa. https://www.nass.usda.gov/statistics_by_state/florida/publications/citrus/citrus_statistics/2015-16/fcs1516.pdf impaginato 81 adv. hort. sci., 2020 34(1): 81­91 doi: 10.13128/ahsc­8404 salicylic acid effects on some physio­ chemical properties and secondary metabolite accumulation in mentha piperita l. under water deficit stress g. abdi 1 (*), l. karami 1, 2 1 department of biotechnology, persian gulf research institute, persian gulf university, 7516913817 busheher, iran. 2 department of horticultural sciences, faculty of agriculture and natural resources, university of persian gulf, busheher, iran. key words: mentha piperita l., persian gulf, secondary metabolites, water deficit stress. abstract: salicylic acid (sa) play important roles in different physiological processes of plants such as plant growth, stress response, plant adaptation and secondary metabolite accumulation. this study was conducted to evaluate the effect of exogenous sa applications on the growth measurements such as fresh and dry weight of aerial part and lead dry weight, biochemical properties (mem­ brane permeability, lipid peroxidation, proline content and ros scavenger enzymes) and secondary metabolite accumulation (total phenolic and the flavonoid and essential oil content) in peppermint (mentha piperita l.) plants grown at different levels of water deficit stress (field capacity: fc). for this aim, three different water deficit stress [no stress (100% fc), mild stress (75% fc) and moderate stress (50% fc)] and four different sa concentrations (0, 1, 2 and 2.5 mm) were applied to peppermint plants. results showed that all of the measured parameters were affected by the water deficit stress and sa application. by ele­ vating the level of water deficit stress, fresh and dry weights of aerial parts and dry leaf weight decreased. increasing in the water deficit stress level from mild to moderate stress resulted to reduce the essential oil content while proline, lipid peroxidation, total phenolic contents, flavonoid content, and antioxidant enzyme activities increased depending on water deficit stress. exogenous application of sa obstructed the negative effects of water deficit stress by decreasing the lipid peroxidation and membrane permeability and improving the antioxidant enzymes activities. essential oil content increased significantly in plant treated with sa grown under water deficit stress conditions. application of 2 or 2.5 mm of sa enhanced the plant growth and development without any toxic effects and increased significantly the total phenolic content, dry leaf weight, and the essen­ tial oil content in stressed and even in control (100% fc) peppermint plants. 1. introduction peppermint (mentha piperita l.) is considered as one of the most important medicinal and aromatic herb in the world cultivated in many (*) corresponding author: abdi@pgu.ac.ir citation: abdi g., karami l., 2020 ­ salicylic acid effects on some physiochemical properties and secon‐ dary metabolite accumulation in peppermint (mentha piperita l.) under water deficit stress. ­ adv. hort. sci., 34(1): 81­91. copyright: © 2020 abdi g., karami l. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 27 march 2019 accepted for publication 17 december 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(1): 81­91 82 countries such as india, italy, france, china, hungary and united states among others (lawrence, 2007). during the last two decades peppermint cultivation has experienced a remarkable increase, mainly due to the rediscovery of medicinal properties of pepper­ mint essential oil, which has a lower risk of side effects compared to synthetic drugs (kumar and k u m a r ­ g u p t a , 2 0 0 8 ; h e r r o a n d j a c o b , 2 0 1 0 ) . furthermore, m. piperita is used as flavoring agent for beverages (phenolic and flavonoids compounds group, including eriocitrin, hesperidin and luteolin 7­ o­rutinoside) (mckay and blumberg, 2006; hossain et al., 2010) and food industry (essential oil and differ­ ent type of plant exteract), as a fragrance (higher amounts of high­volatile monoterpenes such as men­ thone and isomenthone (rohloff, 1999), as insecti­ cide (karamaouna et al., 2013) and fungicide in many industrial products. essential oil of peppermint con­ tains very important monoterpenes (essential oil of peppermint contain menthofuran, menthol, men­ t h o n e , i s o m e n t h o n e , p u l e g o n e c a r y o p h y l l e n e , b e t a c a r y o p h y l l e n e , n e o m e n t h o l , 1 , 8 ­ c i n e o l e , sabinene and limonene) with antibacterial, antifungal (edris and farrag, 2003), antioxidant and cytotoxic activities (mimica­dukic et al., 2003; hussain et al., 2010). due to markedly importance of peppermint, maintaining a high and constant essential oil produc­ tion for industries requirements and supplying mar­ ket demands (silva, 2002) under unfavorable condi­ tions such as water deficit stress is very important. water deficit stress is the major yield limiting fac­ tor for many agriculture crop plants affecting many biochemical, morphological and physiological para­ m e t e r s . m a n y r e s e a r c h s t u d i e s i n d i c a t e d t h a t drought stress influenced the growth, yield, sec­ ondary metabolite production and composition in d i ff e r e n t a r o m a ti c a n d m e d i c i n a l p l a n t s . furthermore, between the abiotic stresses, drought stress can exacerbates the effect of the other stress­ es in plants and increase the effects of other stresses such as salinity, cold or hot stress (khalid, 2006; azhar et al., 2011; verma and shukla, 2015). in order to reduce the harmful effects and to increase the resistance of plants to the drought stress, some exogenous plant growth regulator applications such as salicylic acid (sa) can be used (lee et al., 2019). salicylic acid, as a plant messenger molecule, plays a non­enzymatic antioxidant role, regulates many physiological and biochemical mechanisms as cell expansion, vascular differentiation, vegetative and reproductive development, seed germination, flow­ ering, fruit set and secondary metabolite accumula­ tions during stress occurrence (arfan et al., 2007; hayat and ahmad, 2007). this compound belongs to the group of the phytochemicals with beneficial effects on human health (hayat and ahmad, 2007). in addition, sa is a phenolic compound that has a cru­ cial role in plant defense against pathogenic agents (dempsey and klessing, 1994; hayat et al., 2010). apart from growth­promoting and health related effects of sa in different plants for instance in artemisia annua l. (aftab et al., 2010), cuminumcy minum (rahimi et al., 2013) sweet basil (ocimum basilicum l.) and marjoram (majorana hortensis) (gharib, 2007) and salvia macrosiphon (rowshan et al., 2010), sa is also reported to confer resistance to plants against various stresses e.g. in matricaria chamomilla (kovácik et al., 2009), and vigna radiate (khan et al., 2014 a). this experiment was conducted to determine the effect of different concentrations of exogenous sa applications on some physiochemical properties and secondary metabolite accumulation in m. piperita plants grown under different level of water deficit stress. 2. materials and methods plant materials, sa and water deficit stress treat‐ ments plants were initiated from rhizome segments of m. piperita obtained from a commercial nursery located in alborz province, karaj. the plants were grown in a greenhouse at the university of persian gulf in pots with a diameter of 10 cm and irrigated every 2 days during the first 30 days. the daily tem­ perature inside the greenhouse was within optimal ranges for peppermint growth (20­25°c). fertilization was carried out at 15 and 25 days after planting. each pot was fertilized with a solution containing ca(no3)2 (1.12 g/l), mgso4 (0.45 g/l), kno3 (0.35 g/l), kh2po4 (0.30 g/l), ferric edta (0.06 g/l), and mnso4 (0.01 g/l). thirty­day old seedlings were transferred to plastic pots with a diameter of 30 cm filled with soil, cocopeat and perlite (1:1:1; v/v) and kept in an incu­ bation room where the temperature was 25°c ± 1; the relative humidity was 60­80% and photoperiod of 16 h/d. the first sa (synonym: 2­hydroxybenzoic acid; linear formula: c7h6o3 (cas 69­72­7), sigma­ aldrich) treatments (0, 1, 2 and 2.5 mm) was applied to 35 days old plants by spraying aerial parts of the plants. the stock solutions were prepared by dissolv­ ing sa in etoh and final volume was maintained by karami and abdi ‐ salicylic acid effects on peppermint 83 distilled water containing 0.02% of tween 20 as sur­ factant. control plants were sprayed with distilled water including 0.02% of tween 20 and etoh (100%) as in sa solutions. the 2th, 3th and 4th sa application were repeated at days 50 and 65 and 80 days after transplanting on peppermint plants exposed in three different irrigation (100, 75 and 50 fc) treatments as fc (100% field capacity ­ fc), mild stress (75% fc), and moderate stress (50% fc). throughout cultiva­ tion period, moisture levels in the growth media were controlled by daily weighting following the pro­ cedure of yadav et al. (2014). briefly, to calculate the amount of water necessary to bring each soil to determined fc, a 50 g soil sample from randomly chosen pots were collected and the water content was determined by drying at 100°c at 24 h after the pots were watered. the percentage of soil water con­ tent was calculated according to yadav et al. (2014) method. nutrient and water leaching from pots was captured in dish placed under each pot and the leachate was returned to the soil before the addition of any water. at the end of the experiment, all plants within each pot were harvested and then analyzed. determination of biochemical and physical properties the growth response of the plants to elicitor treatments and water deficit stress were determined by measuring the fresh and dry weights of aerial parts per plant. lutts et al. (1996) method was used for determin­ ing the membrane permeability of the excised leaves at the end of the experiment by using a conductivity meter. after harvesting (from three plants per treat­ ment), 5 leaves (randomly chosen full developed leaves from upper part of the plant) were cut into 1 cm segments. leaf segments were washed with three changes of deionized water to remove surface adhered electrolytes. samples were placed in 20 ml vials contain deionized water and incubated at 25°c on a rotary shaker (100 rpm). after 24 h incubated at 25°c, the electrical conductivity of the bathing solu­ tion (l1) was determined by using a conductivity meter. then samples were autoclaved at 120°c for 20 min and the electrical conductivity (l 2) was obtained. the membrane permeability was obtained using l1/l2. all measurements were made in tripli­ cate. lipid peroxidation in leaves was determined by estimating the malondialdehyde (mda). for malondi­ aldehyde (mda) determination, 1 g leaf sample (full developed leaves were collected from the upper part of each plant) was homogenized in 5 ml 1% trichloro­ acetic acid and centrifuged at 10000 g for 10 min. the amount of mda in the supernatant was estimat­ ed by the thiobarbituric reaction as described by madhava et al. (2000). mda concentration was calcu­ lated from the absorbance at 532 nm by using the extinction coefficient of 155 mm cm­1. all measure­ ments were made in triplicate. proline content of treated plant was determined as described by bates et al. (1973). a 1:1:1 solution of proline, ninhydrin acid and glacial acetic acid was incubated at 100°c for 1 hour. for proline colorimet­ ric determinations, the reaction was arrested in an iced bath and the cromophore was extracted with 4 ml toluene and its absorbance at 520 nm was deter­ mined in a biomate spectrophotometer (thermo­ spectronic). after the analyses, following equation: (g proline in extract/115.5) g­1 sample = mol g­1 fw) was used for calculation the proline concentration from a standard curve. enzyme assays at the end of experiment, leaves were collected and frozen in liquid nitrogen and stored at ­80°c before enzyme extraction. ros scavenger enzymes were extracted as described by zhang and kirkham (1996) with some modifications. all operations were carried out at 4°c. intact leaves were ground using mortar and pestle under liquid nitrogen in cold 50 mm sodium phosphate solution (ph 7.5) containing 250 mm sucrose, 10 mm kcl, 1 mm mgcl2, 1.0 mm edta, 0.5 mm 0.1 mm dithiothreitol, 0.1 mm phenyl­ methylsulfonyl fluoride, and 1% (w/v) polyvinyl­ polypyrrolidone in a 6:1 proportion (w/v). the homogenate was then filtered and centrifuged at 25,000 g for 20 min at 4°c. then solid ammonium sulfate (nh4)2so4 added to the supernatant to make up 80% saturated solution and allowed to stir gently for several hours at 4°c. after centrifugation (28,000 g for 45 min at 4°c), pellets, were resuspended in a small volume of 50 mm of sodium phosphate (ph 7.5) and used for enzyme assays. the superoxide dismutase (sod) activity was assayed by observing the inhibition of photochemical reduction of nitro blue tetrazolium according to krishnan et al. (2002) protocol. one ml of assay mix­ ture consisted of 75 μm nitro blue tetrazolium, 2 μm riboflavin, 50 mm na­p buffer (ph 7.8) 0.1 mm edta, 13 mm methionine, and enzyme extract. the sam­ ples were kept 30 cm under a light source (4000 lux) for 15 min and the reaction started during this time. the reaction was stopped by switching off the light. a non­irradiated reaction mixture, served as a control which was run in parallel. the absorbance was read at 560 nm. adv. hort. sci., 2020 34(1): 81­91 84 the ascorbate peroxidase (apx) activity (ε= 2.8 mm­1 cm­1) was determined from the decrease in a290, due to the h2o2­dependent oxidation of ascor­ bate using procedure of zhang and kirkham (1996). one ml reaction mixture contained 50 mm na­p (ph 7.0), 0.5 mm ascorbic acid, 0.1 mm edta, 0.1 mm h2o2, and enzyme. the catalase (cat) activity (ε h2o2 = 39.4 mm ­1 cm­1) were assayed spectrophotometrically according to zhang and kirkham (1996) method by monitoring the change in a240 due to the decreased absorption of h2o2. the reaction mixture contained enzyme extract, 50 mm na­p, ph 7.0, and 15 mm h2o2 (in 1 ml final volume). the reaction was initiated by addi­ tion of h2o2. secondary metabolites determination at the end of the experiment, in all plants within each pot leaves (1 g full developed leaves were col­ lected from the upper part of the each plant) were collected and frozen in liquid n2 immediately and stored at ­80°c before analysis. folin­ciocalteu colourimetric method was used for determining the total phenolic content of the peppermint extract (singleton and rossi, 1965). samples were trans­ ferred into the different test tube and mixed thor­ oughly with folin­ciocalteu reagent (5 ml). after 5 mins, 4 ml of 7.5% sodium carbonate (na2co3) was added and allowed at room temperature to react for 2 hrs. the absorbance was measured using spec­ trophotometer at 765 nm. samples were measured in three replicates. the flavonoid content was deter­ mined according to liu et al. (2002) by aluminium trichloride method using catechin. a volume of 125 μl of extract is added to 75 μl of a 5% nano2 solu­ tion. the mixture was allowed to stand for 6 min, and then 150 μl of aluminium trichloride (10%) was added and incubated for 5 min, followed by the addi­ tion of 750 μl of naoh (1m). the final volume of the solution was adjusted to 2500 μl with distilled water. after 15 min of incubation the mixture turned to pink and the absorbance was measured at 510 nm. total essential oil content h y d r o d i s ti l l a ti o n m e t h o d w a s u s e d . i n t h i s method, at least 10 g of dried m. piperita shoot relat­ ed to the treatment immersed in 150 cc water were submitted to hydro­distillation with a clevenger­type apparatus for 3 h (until no more essential oil was obtained). the essential oil was collected, dried with anhydrous sodium sulfate, and stored at 4°c until used. statistical analysis this experiment was performed in factorial arrangement based a completely randomized design with 3 replicates per treatment and one plant per replicates in which pots were placed on the benches in the incubation room. the factors included differ­ ent sa concentration (0, 1, 2 and 2.5 mm) and water deficit stress in 3 levels. data were analyzed by spss 19 software, and differences among treatments were determined using tukey’s test (p<0.05). 3. results and discussion growth measurement foliar application of salicylic acid (sa) and water deficit stress affected peppermint plants in different ways, including physiological and biochemical prop­ erties. some physiological disorders such as leaf yel­ lowing and abscission were observed in plants at sever water deficit irrigation. also, the high concen­ tration of sa caused burning and drying the edge of some mature leaves in plants (data not shown). the limited water supply is one of the major abiotic fac­ tors that adversely affect agricultural crop production worldwide. water deficit stress significantly reduced the growth measurements (fresh and dry weights of aerial parts of plants and leaf dry weight) in pepper­ mint plants at 50% fc (table 1). however, sa treat­ ments were not caused any growth reduction but in plants under water deficit stress, prevented the growth reduction rate. highest growth values were table 1 ­ effect of foliar spray of sa on some growth parameters * differences between means indicated by the same letters are not statistically significant (p≤0.05). irrigation treatment sa (mm ) fresh weight of aerial parts (g) dry weight of aerial parts (g) dry weight of leaf (g) control 0 30.74 b 5.47 b 2.61 bc 1 31.31 b 7.15 a 3.45 b 2 42.28 a 8.40 a 4.33 a 2.5 41.24 a 8.34 a 4.08 a fc (75%) 0 20.92 cd 4.56 c 1.93 d 1 22.25 cd 4.75 bc 2.13 cd 2 26.66 cde 4.84 bc 2.83 d 2.5 27.71 c 4.65 c 1.91 d fc (50%) 0 19.25 cde 4.44 c 1.58 d 1 19.92 cde 4.26 c 1.62 d 2 18.32 e 3.95 c 1.92 d 2.5 19.21 de 3.91 c 1.64 d karami and abdi ‐ salicylic acid effects on peppermint 85 more than 2­fold compared to the untreated plants in the presence of moderate water deficit stress. responses of plant to environmental stress such as drought stress in terms of volatile emissions are species specific and depend on the duration and intensity of the stress period (jord´anm et al., 2003). eo yield increased under mild water stress in this study. in medicinal and aromatic plants, the effect of obtained from the plants treated only with 2 mm of sa. khorasaninejad et al. (2011) also observed signifi­ cant reduction in growth parameters of peppermint (m. piperita l.) under water stress. sa treatment in non­stress and even in stress conditions showed pos­ itive effect on the growth measurements in compari­ son to the control (table 1). different concentrations of sa (from 10­5 to 10­3 m) improved growth parame­ ters (plant height, number of branches, nodes, and l e a v e s p e r p l a n t , a s w e l l a s l e a f a r e a ) i n b a s i l (ocimum basilicum) and marjoram (origanum majo‐ rana) plants (gharib, 2007) and these authors sug­ gested that such effects could be related to increased photosynthetic activity. the beneficial effects of sa on the growth measurements especially in term of dry weight of mint in this study paralleled with the results of khodary (2004) and hayat and ahmad (2007). this growth enhancement suggests that sa a ff e c t s v a r i o u s p h y s i o l o g i c a l a n d b i o c h e m i c a l processes, including photosynthesis, water process­ es, ion homeostasis, antioxidant capacity generates and a wide array of metabolic responses in plants (hayat and ahmad, 2007). also, sa modulates several physiological responses such as maintain indole‐3‐ acetic acid (iaa, 3‐iaa) and gibberellin levels, inhibit ethylene biosynthesis and prevent auxin oxidation in plants (pierpoint, 1994), which could be one reason for the sa­enhanced vegetative growth of pepper­ mint in our study. essential oils peppermint essential oil (eo) is a rich source of monoterpenes such as menthol and menthone used in pharmaceutical, food, cosmetic and cleaning industries (kamatou et al., 2013) and its commercial value depends on the essential oil contents. in this study, essential oil content was affected with both water stress and sa application (table 2). essential oil content increased from 1.146% (control plants) to 1.192% (mild stress treated plant) and decreased to 0.782% in moderate stress treated plant (table 3). exogenous application of sa alleviated this adverse effect especially in moderate stress condition and also sa application enhanced the eo yield in mild stress condition. in control irrigation conditions, 2 mm sa treatment increased total essential oil con­ tent to 1.256% while application of sa at higher con­ centrations caused a reduction in total essential oil content. under water deficit stress conditions, sa sig­ nificantly increased the total essential oil content when compared to the untreated plants. particularly, 2 and 2.5 mm of sa increased essential oil content table 2 ­ effect of foliar spray of sa on biochemical properties of peppermint plants grown at different level of fc table 3 ­ effect of foliar spray of sa on phenolic and essential oil contents of peppermint plants grown at different level of fc * differences between means indicated by the same letters are not statistically significant (p≤0.05). * differences between means indicated by the same letters are not statistically significant (p≤0.05). irrigation treatment sa (mm) membrane permeability (s) lipid peroxidation (nm g­1) proline (mol g­1) control 0 37.11 def 5.81 d 0.58 ab 1 27.43 efg 6.35 d 0.42 bc 2 28.32 efg 5.38 d 0.28 c 2.5 21.43 fg 6.05 d 0.66 ab fc (75%) 0 41.02 cd 14.48 a 0.67 ab 1 53.74 c 12.01 a 0.59 ab 2 50.26 cd 6.09 d 0.4 bc 2.5 25.49 fg 7.11 cd 0.62 ab fc (50%) 0 87.04 a 11.71 ab 0.83 a 1 41.52 cde 11.29 ab 0.79 a 2 74.28 b 8.66 c 0.59 ab 2.5 49.91 cd 7.03 cd 0.76 a irrigation treatment sa (mm) total phenolic content (mg g­1) flavonoid content (mg gae/g) total essential oil content (%) control 0 8.37 e 17.7g 1.146 d 1 16.51 d 22.2e 1.153 d 2 14.28 de 38.7d 1.256 c 2.5 14.32 de 34.81e 1.092 e fc (75%) 0 28.08 ab 37.2d 1.191 d 1 22.71 bc 42.3c 1.201 d 2 18.08 cd 47.4b 1.418 b 2.5 17. 94 cd 48.2b 1.482 b fc (50%) 0 29.39 ab 40.7c 0.782 f 1 31.99 a 55.3a 1.010 d 2 18.28 cd 53.2a 1.597 a 2.5 33.65 a 55.7a 1.575 a 86 adv. hort. sci., 2020 34(1): 81­91 water deficit stress on essential oil yield is variable and depends on plant species. for example, drought stress had a negative effect on the essential oil yield of m. piperita (khorasaninejad et al., 2011). by con­ trast, water stress had a positive effect on salvia officinalis and showed a higher concentration of monoterpenes such as cineole, camphor and α/ß­ thujone (nowak et al., 2010). increasing in essential oil yield under stress condition may be related to induction changes in morphological or physiological traits such as high trichome density and smaller leaf size that prevent excessive water loss and allow them to survive in arid or semi­arid environmental condi­ tions (nobel, 1999). foliar spray with triazole (a tria­ zole refers to any of the heterocyclic compounds with molecular formula c2h3n3, having a five­mem­ bered ring of two carbon atoms and three nitrogen atoms) decreased the leaf area in m. pulegium (hassanpour et al., 2012) and increased trichome density in bougainvillea spectabilis (mansouri and kurup, 2009). the results suggest that the stimulation of essential oil production under drought stress could be due to low allocation of carbon to the growth and high terpene concentrations under stress conditions. plant growth regulators (pgrs) favorably affect the yield and quantity of essential oil in lemon grass, rose grass, peppermint, spearmint, and sage (shukla and farooqi, 1990; khan et al., 2014 b, 2015). among dif­ ferent concentration of sa tested on m. piperita in none stress condition, 2 mm of sa showed the most effective in improving the essential oil content. the positive effect of sa on essential oil content and essential oil yield may be ascribed to the improve­ ment in overall plant growth and metabolism by sa. it seemed that sa, considered to be a signaling mole­ cule, might be involved in the signal transduction sys­ tem, leading to the balance and improved quantity of the secondary metabolites, i.e., essential oil (hayat and ahmad, 2007). thus, sa­mediated enhanced plant growth, photosynthesis, and the overall plant metabolism might account for oil accumulation in the present study. these results corroborate with the findings on artemisia annua, salvia macrosiphon, cumium cyminum, ocimum basilicum, and majorana hortensis (gharib, 2007; aftab et al., 2010; rowshan et al., 2010; rahimi et al., 2013). moderate stress sig­ nificantly decreased essential oil content in current study (table 3). similarly, khorasaninejad et al. (2011) reported significant decreases in total essen­ tial oil content in water stressed peppermint plants. these results suggest that a high level of water stress can suppresses the essential oil biosynthesis pathway in peppermint. reduction in photosynthesis rate and/or any changes in metabolic pathway could be caused some disorders of oil biosynthesis resulted in reduction in oil content (srivastava et al., 1998). in control irriga­ tion condition, foliar application of sa at 2 mm con­ centration resulted to increase in essential oil con­ tent of plants compared to the control plants while higher concentrations of sa decreased the oil con­ tent. another interesting result is that sa foliar appli­ cations increased the essential oil content in water stressed plants. under water deficit stressed condi­ tions, essential oil contents of peppermint plants treated with sa were higher in comparison with the group exposed to water stress alone. the highest oil content was found in plants treated with sa at 2.5 mm concentration grown under moderate water stress. in this condition, oil content was more than 2 fold greater than that of plants under combination of moderate water stress and 0 mm of sa. according to the commercial importance of peppermint, our results represented that peppermint plants can be grown in mild or in moderate stress in the presence of sa applications. membrane permeability, lipid peroxidation and pro‐ line content both water deficit stresses resulted to a significant increase in the levels of membrane permeability, a sign of injury to the cells (table 2). the maximum increase in membrane permeability was obtained in plants subjected to 50% fc and sa treatments reduced significantly this increase was in under water stress. the maximum reduction in membrane perme­ ability was observed in plants treated with 2.5 mm sa. our results indicated that water stress increased the membrane permeability levels of peppermint plants and they were alleviated significantly by foliar applications of sa. water stress significantly increased the lipid per­ oxidation compared to the control irrigation plants (table 2). the maximum lipid peroxidation content was recorded in plants grown under high level of water deficit stress. however, sa treatments, espe­ cially at 2 mm concentration, significantly overcame the toxicity generated by water stress alone and almost leveled the values with those of control. however, sa did not affect the lipid peroxidation level in control irrigation plants. the proline content increased by increasing the water stress but foliar spraying of sa at 2 mm con­ centration upon water stressed plants reduced the http://https://en.wikipedia.org/wiki/molecular_formula karami and abdi ‐ salicylic acid effects on peppermint 87 proline content. ion leakage is known as a index for evaluating the cell wall damage caused from stress. membrane per­ meability increased in line with the elevating levels of water deficit stress in this study. also, there is a close relationship between producing oxidative damage under water stress condition of cell membrane per­ meability. however sa significantly decreased the membrane permeability in plants under water deficit stress (table 4). this result can approve the capability o f s a s p r a y i n g t r e a t m e n t i n n e g a ti v e e ff e c t o f drought stress. water deficit stress resulted to the displacement of membrane proteins, cellular com­ partmentalization disruption, loss of membrane selectivity, integrity and a loss of activity of enzymes (mahajan and tuteja, 2005). malondialdehyde is one of the main known indi­ cator or reliable biomarker for lipid peroxidation, which is part of the oxidative damage at cell level. malondialdehyde increase the cell permeability by deterioration of membrane integrity. our results s h o w e d t h a t t h e r e i s a s i g n i fi c a n t r e l a ti o n s h i p between lipid peroxidation and water deficit stress (table 4). under water deficit stress conditions the amount of lipid peroxidation increased in peppermint plants, moreover, it was determined that mda level increased as a result of deterioration of the cell struc­ ture. karray­bouraouia et al. (2009) and karlıdag et al. (2011) reported a positive correlation between stress condition and lipid peroxidation. water deficit stress caused considerable membrane injuries lead­ ing to membrane lipid peroxidation. foliar spray of sa did not show any significant changes in lipid per­ oxidation content in control irrigation condition while it significantly decreased lipid peroxidation content in water stress­treated plants. the results showed that sa had an important role in membrane integrity and maintenance of membrane structure via preventing damages caused by water deficit stress. low mem­ brane lipid peroxidation in drought­exposed and sa (0.5 mm)­supplemented t. aestivum reported by kang et al., (2012). amino acids play an important role in plant devel­ opment and metabolism. proline, an amino acid, plays main roles in plants exposed to stressful condi­ tions (like water deficit stress, low temperature, salinity, heavy metal exposure and uv radiations, etc) s u c h a s o s m o p r o t e c ti v e , p r o t e i n c o m p a ti b l e hydrotrope, scavenging free radicals and buffering cellular redox potential, energy supply, stabilize the function and structure of protein and dna, alleviat­ ing cytoplasmic acidosis and maintaining appropriate nadp+/nadph ratios compatible with metabolism, metal chelator, antioxidant properties and a signaling molecule (naidu et al., 1991; bassi and sharma, 1993; schat et al., 1997; hare et al., 1998; rhodes et al., 2002; kavi kishor et al., 2005; munns, 2005; sharma a n d d i e t z 2 0 0 6 ; a s h r a f a n d f o o l a d , 2 0 0 7 ; chookhampaeng, 2011). so, overproduction of pro­ line is a common physiological response of plants against stressful environment. in this study, proline contents were increased with increasing water deficit stress (table 4). so, more proline accumulation has been associated with increasing the stress tolerance of plant. in general, results showed that drought stress affected the proline biosynthesis in the leaves. the foliar spray of sa on stressed plants cause in reduction of proline content but these declines were not statistically significant in some concentration. it could be due to positive effects of sa in maintaining the membrane maintaining cell turgor or osmotic balance as an osmotic regulator (wang and zeng, 1993) or in stabilizing cell membranes, preventing electrolyte leakage and bringing concentrations of ros within normal ranges. phenolic and flavonoids contents total phenolic and total flavonoid contents were increased under water deficit stress significantly as compared to control irrigation (100% fc) (table 3). minimum phenolic content was measured in control plants while its level increased with increasing the table 4 ­ effect of foliar spray of sa on antioxidant enzyme acti­ vities of peppermint plants grown at different level of fc * differences between means indicated by the same letters are not statistically significant (p≤0.05). irrigation treatment sa (mm) sod (unit mg protein­1) cat (mol min­1 mg protein­1) apx (mol min­1 mg protein­1) control 0 25.17 cd 6.44 cde 311.99 c 1 17.47 ef 3.11 e 153.71 d 2 12.31 f 6.63 cde 189.48 d 2.5 21.19 de 4.15 de 279.11 c fc (75%) 0 30.88 bc 15.92b 392.95 b 1 21.17 de 8.52 c 158.21 d 2 23.46 d 7.43 cd 264.01 c 2.5 21.13 de 5.41 cde 314.64 c fc (50%) 0 41.79 a 21.33 a 501.28 a 1 19.94 de 16.21 b 183.23 d 2 31.95 b 9.15 c 331. 53 c 2.5 47.61 a 9.58 c 483.12 a adv. hort. sci., 2020 34(1): 81­91 88 water deficit stress intensity and sa application. foliar application of sa (2 mm) under control irriga­ tion increased total phenolic content less than two times compared to controls. moreover, not only water stress but also sa applications enhanced total phenolic contents compared to controls. the plants under moderate stress were sprayed with a solution of 2.5 mm sa showed the highest phenolic contents. phenolic compounds as stress markers accumulate under stressful environment. it is believed that solu­ ble phenolics compounds act as scavengers of ros and membrane stabilizer during abiotic stress (moyer et al., 2002; taiz and zeiger, 2006). the increase in total phenolic contents in our study is similar to that observed by el­danasouryet al. (2010) in a study with mentha spicata and queslati et al. (2010) with mentha pulegium under salinity stress. also, foliar sa application resulted to increase in total phenolic con­ tents in control plants. foliar sa application at 2 mm was the most effective dose under stress free condi­ tions for peppermint plants. in water stressed plants, foliar spry of sa generally have positive effects on phenolic accumulations. especially at moderate stress and 2 and 2.5 mm concentrations of sa showed the highest amount of phenolics. pepper­mint treated with sa or drought stress showed an increase in total phenolic and flavonoid content in our study. this effect was previously reported in ginger (zingiber officinale) plants, where the total phenolic content increased by approximately 20% after treatment with 10­3 m sa (ghasemzadeh and jaafar, 2012). also, figueroa­pérez et al. (2014) previously reported in peppermint plants, where the flavonoids, increased by approximately 93%, 100% and 56% treated with 0.5, 1.0 and 2.0 mm sa, respectively, when compared with the control. flavonoids were increased in water deficit stress condition and sa treated plants in our study. flavonoids are the main bioactive secondary metabolites in plants and they can serve as scav­ engers of ros by locating and neutralizing radicals before they damage the cells and are thus important for plants under adverse environmental conditions (such as wounding, drought, metal toxicity, and nutri­ ent deprivation) (løvdal et al., 2010; agati et al., 2012). plants often respond to environmental stress­ es with an increase in the endogenous sa level (janda et al., 2014). sa is a promising compound for the reduction of stress sensitivity in the practical agricul­ ture (horváth et al., 2007). ros enzyme activity the activities of sod, cat and apx enzymes were investigated in the leaves of peppermint plants. sod, cat and apx enzymes activities significantly increased in parallel to the water stress in the present study. activity of sod was found at 25.17 unit/mg protein, 30.88 unit mg protein­1 and 41.79 unit mg­1 protein in plants under control, mild and moderate stress, respectively (table 4). in general, foliar application sa on stressed and non­stressed plants alleviated the activity of sod. however, 2.5 mm of sa increased the sod activity in plants under control and moderate stress condition. also, cat activity increased in the plants under water deficit stress (table 4). in control irrigation plants, foliar spray of sa treatments did not show a significant change in cat activity whereas they substantially decreased the cat activity in mild and moderate stress. apx activity almost showed the simi­ lar trend with sod activity and its activity increased with water deficit stress, gradually (table 4). sa appli­ cation at 2.5 mm concentration had no significant effect in terms of the reducing the apx activity in con­ trol and moderate stress treatments. the maximum apx activities were found in under moderate stress without sa application or with 2 mm sa while the highest reductions in apx activity content were recorded in plants treated with 1 mm sa in both mild and moderate water stress treatments. developing enzymatic and non­enzymatic antioxidant defense mechanisms against reactive oxygen species is the main strategy of plants in stressful condition (noctor et al., 2002; gong et al., 2005; cheeseman, 2007). in t h i s s t u d y , r o s e n z y m e a c ti v i ti e s e n h a n c e d b y increasing the intensity of water stress and apx enzyme activity increased with increasing stress con­ ditions and the highest increases occurred in plants under moderate stress condition (table 4). 4. conclusions according to the obtained results, it may be con­ cluded that prolong water stress especially under moderate water deficit stress had negative effect on peppermint. under water deficit stress, peppermint plants showed a significant decline in growth mea­ surement in term of fresh and dry weights of aerial parts and leaf dry weight while increased antioxidant enzyme activities, the membrane permeability and lipid peroxidation. in addition, water stress enhanced total phenolic and flavonoids contents while signifi­ cantly decreased the total essential oil content in moderate stress condition (mild stress increased the total essential oil content but, it was not significant). karami and abdi ‐ salicylic acid effects on peppermint 89 however, the findings of the present study showed that foliar spray of sa increased grown parameters in non­stress plants while there were no significant effects of sa on water stressed plant. severity of water stress has a great impact on the physiological and biochemical process of plants. sa applications in this study reduced the severity of both water stress condition and decreased the level of grown parame­ ters reduction rate. sa significantly reduced the water stress­induced negative effects by improving the antioxidant enzyme activities and decreasing the lipid peroxidation and membrane permeability. in terms of the secondary metabolites, sa treatment increased the total phenolic contents in control plants while, especially in plants exposed to water stress, total essential oil content increased. sa con­ tributed to the plant growth and development with­ out any toxic effects and 2 mm of sa significantly increased the essential oil content in non­stressed plants. also, it was determined that 2 mm of sa for control irrigation and 2 and 2.5 mm of sa for both water stress condition were the optimum concentra­ tions in terms of dry leaf weight, total phenolic con­ tents and essential oil content in peppermint plants. acknowledgements the author wishes to thank dr. m. maffeifor pro­ viding the facilities and technical support. references aftab t., khan m.m,. idrees a.m., naeem m., 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ibadan, nigeria. 4 université polytechnique d’abomey, faculté des sciences et techniques de dassa, laboratoire de biotechnique, ressources génétiques et amélioration des espèces animales et végétales (biorave), bénin. key words: crop diversity, new cocoyam, ssr, taro, togo. abstract: taro and new cocoyam are root and leaf crops commonly grown in tropical to warm temperate regions. in togo, they are neglected and underuti­ lized. here we report the genetic diversity of 26 accessions of taro and 101 accessions of new cocoyam. analysis of simple sequence repeats revealed low polymorphic information content of 0.43 and 0.25 in taro and new cocoyam, respectively. pca scatterplots and neighbour joining dendrograms based on the ssr data clustered accessions into groups that more­or­less correspond to morphological diversity in both species. amova within and between morpho­ logical groups revealed greater variances within groups than between. this indicates weak genetic differentiation between morphological groups, particu­ larly for taro. genetic diversity was greater among taro cultivars. taro has a longer history of introduction and dispersal in africa, and has had more oppor­ tunity for multiple introduction and local cultivar development. different strategies are suggested for future development of these crops in togo and africa. for taro, further studies of existing diversity and recent experimental introductions to africa are likely to be rewarding. new cocoyam, a modern his­ torical introduction, has spread widely in africa with little genetic diversity. for this crop, international collaboration is needed to clarify taxonomy, and to introduce further cultivars for evaluation under local conditions in africa. 1. introduction root and tuber crops are important sources of food and income for household in rural areas of africa. in sub­saharan africa, they provide (*) corresponding author: bdamigou@gmail.com citation: bammite d., matthews p.j., dagnon d.y., agbogan a., agre p., akintayo t.o., odah k., dansi a., abberton m., tozo k., 2021 ­ genetic and morphological diversity in colocasia esculen‐ ta and xanthosoma maffafa in togo, west africa. ­ adv. hort. sci., 35(3): 255­267 copyright: © 2021 bammite d., matthews p.j., dagnon d.y., agbogan a., agre p., akintayo t.o., odah k., dansi a., abberton m., tozo k. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 9 september 2020 accepted for publication 9 july 2021 ahs advances in horticultural science https://doi.org/10.36253/ahsc-9689 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2021 35(3): 255­267 256 about 20% of calories (pinstrup­andersen et al., 1999). taro, colocasia esculenta (l.) schott and new cocoyam, xanthosoma spp., are grown for food and income generation at the household level. both crops are grown in tropical regions of africa, asia, oceania and america, and taro is also common in temperate regions of africa, asia, and oceania (gonçalves, 2011; matthews, 2014; onyeka, 2014; grimaldi, 2016; matthews and ghanem, 2021). taro is considered an ancient crop in africa, with multiple likely routes of introduction from asia (matthews, 2006; fuller et al., 2011; chaïr et al., 2016; grimaldi, 2016). new cocoy­ am is known to have been introduced to ghana in 1843 (karikari, 1971), though earlier introduction fol­ lowing european contact with america has been sug­ gested (bown, 2000). most parts of these plants (corms, side­corms, stolons, petioles, leaf blades, and floral spathes) are edible, the leaves and corms are also commonly used as animal fodder in asia (coursey, 1984; matthews, 2010; mwenye et al., 2010; masuno et al., 2012; matthews, 2014; wada et al., 2017), and medicinal uses are also known (plowman, 1969; ribeiro pereira et al., 2021). the specific parts eaten vary according to cultivar attributes, local food knowledge, and cul­ tural or personal preferences. both crops have great potential for development in africa and globally (okereke, 2020). under cultivation, clonal propagation is universal for both taro and new cocoyam. in a global survey of taro, chaïr et al. (2016) found the greatest genetic diversity and the largest number of private alleles in asian cultivars, especially in india. low genetic diver­ sity was found in western africa, among diploid and triploid cultivars, and also in southern africa, where triploid cultivars were dominant. their observations correspond broadly to what is known about the breeding of taro: flowering, fruiting and seed produc­ tion by wild and cultivated taros are common in trop­ ical regions of asia and oceania (matthews, 2014), but have not been observed in africa despite occa­ sional reports of flowering (e.g. traore, 2013). natural fruiting and seed production by new cocoy­ am has not been reported outside south america, but sterile inflorescences are often produced (obser­ vation by authors), and induced flowering and experi­ mental crosses have been reported in cameroon (onokpise et al., 1992). globally, taro has undergone genetic erosion due to changes in cropping patterns, the spread of improved varieties, and replacement by other crops (lakhanpaul et al., 2003; matthews and ghanem, 2021), including new cocoyam (coursey, 1984). the acridity of taro (and resulting special care needed for cooking) (matthews, 2010), spread of taro leaf blight (tlb) (singh et al., 2012), and shortages of planting materials are contributing factors. in order to identify and preserve cultivars of economic value, maintain living germplasm collections efficiently (without excessive duplication), and provide baseline data for future breeding programs, genetic diversity and mor­ p h o l o g y m u s t b e a s s e s s e d i n c u l ti v a r s o f b o t h species. the taxonomy of cultivated xanthosoma species and relationships with wild species are uncertain, and historically there has been a tendency to use the name x. sagittifolium for all cultivated xanthosoma (giacometti and leon, 1994; castro, 2006; quero­ garcia et al., 2010; doungous et al., 2015). although x. sagittifolium is the name used in many previous studies in africa, the plant is most likely to be x. mafaffa (gonçalves, 2011). the taxonomy of cultivat­ ed xanthosoma spp. in tropical america has been revised by croat and delannay (2017). various culti­ vated species of xanthosoma are also circulating internationally and may have reached africa in the modern historical period. these include x. atrovirens c. koch & bouché (blackish green blades, and “blue” wax on dark green petioles creating a dark purple or black appearance), x. robustum (which can reach 4 m in height, with tall above­ground stems) and x. vio‐ laceum (with violet petiole tissue below a waxy sur­ face) (gonçalves, 2011). in addition to the 1843 intro­ duction of xanthosoma (karikari, 1971), an unsuc­ cessful attempt was made to introduce x. brasiliensis from puerto rico in 1974 (karikari, 1979). in central america, x. mafaffa cultivars vary in corm parenchy­ ma color (red or white), and those with red corms are also tinged with red in the petiole, leaf sheath and spathe (gonçalves, 2011). in togo, a collection of taro and new cocoyam cul­ tivars from throughout the country was assembled, and morphological groups were described in both species (bammite, 2018; bammite et al., 2018 a, b). however, data related to genetic diversity among these crops, based on molecular tools such as ssr, are lacking to enhance effective usage and conserva­ tion of these neglected species and develop a breed­ i n g p r o g r a m t o i m p r o v e t h e q u a l i t y o f t h e i r g e r m p l a s m . t h i s s t u d y a i m e d t o a s s e s s s i m p l e sequence repeat (ssr) diversity in the same collec­ tion of taro and new cocoyam. polymorphic ssr loci have codominant alleles (repeat sequences of vary­ ing length, detected by pcr amplification), and been bammite et al. ‐ genetic diversity of taro and cocoyam 257 used in many studies of taro (devi, 2012; you et al., 2015) and other edible aroids (suppl. table s1), and in genetic linkage mapping of the taro nuclear genome (quero­garcía et al., 2010; soulard et al., 2017). 2. materials and methods plant materials in 2016, cultivars of taro and new cocoyam were collected from 42 localities randomly selected across the five ecological zones of togo (fig. 1). at each vil­ lage, farmers were invited to bring corms of different cultivars grown in their village. for each distinct culti­ var recongnized in group discussions, the local name was recorded and collected corm samples were planted at the centre de recherche agronomique du litoral (cral), an experimental farm of the institut togolais de recherche agronomique (itra) located at davié, at latitude n 6°23’ and longitude e 1°12’ and at 88 m above sea level (table s2). accessions of both species were classified in a binary manner based on morphological characters that are easily observed in the field: taro accessions were identified as either dasheen (with large mother corms, and either stolons or side­corms; pop1), or eddoe (with small mother corms, and few to many side­corms; pop2); new cocoyam accessions were identified as either green (leaves entirely green; pop1) or purple (petioles purple or pink to some extent; pop2). morpological diversity in the same collection was previously analysed with reference to a wide range of agronomic and morphological characters (bammite et al., 2018 b; figs. s1­s3). young leaf tissue from one plant from each of 26 accessions of taro and 101 accessions of new cocoyam was dried on silica gel and taken to the international institute of tropical agriculture (iita) bioscience centre, ibadan, nigeria for dna extraction and genotyping. dna isolation and quantification dna was extracted using an optimized sds proto­ col recommended by iita bioscience centre (2017). about 100 mg of dry leaf tissue was put in a tube with two steel balls and reduced to powder using a spex genogrinder­2000. pre­heated extraction buffer (450 µl of 1m tris­hcl, 0,5m edta, 5m nacl, 20% sds and 1% pvp) was added. tubes were incubated at 65°c for 20 mins and inverted occasionally to homog­ enize each sample. tubes were removed from bath, allowed to cool for two mins, then 200 µl of ice­cold 5m potassium acetate was added and the mixture incubated on ice for 20 mins to precipitate proteins. tubes were then centrifuged at 3500 rpm for 10 mins, and each supernatant was transferred to a new l a b e l e d t u b e . a v o l u m e o f 2 0 0 µ l o f 4 % polyvinylpyrrolidone (pvp) was added to the super­ natant and gently mixed. to precipitate and remove proteins and lipids, 45 µl of chloroform isoamylal­ chohol (24:1) was added, mixed gently and tubes were centrifuged at 3500 rpm for 15 mins. each supernatant was transferred to a new tube, and a 2/3 volume of ice­cold isopropanol was added, mixed, and incubated in ­80°c for 15 mins to precipi­ tate the dna. after centrifuging at 3500 rpm for 15 mins, the dna pellet was washed by adding 400 µl of 70% ethanol, centrifugation at 3500 rpm for 15 mins, and decanting the supernatant until the last drop. the dna pellet was air­dried then resuspended in 100 µl low salt te buffer (10 mm tris­hcl, 1 mm fig. 1 ­ map of togo showing sampling localities in 2016 and ecological zones: zone i, northern lowlands; zone ii, northern togo mountains; zone iii, central lowlands; zone iv, southern togo mountains; zone v, coastal plains of southern togo. figure adopted from bammite et al., 2018 b; zones originally described by ern (1979); base map from ign france, 1990. adv. hort. sci., 2021 35(3): 255­267 258 edta). a volume of 2 µl of rnase a (10 µg/ ml) was added and incubated at 37°c for 40 mins. the quanti­ ty and quality of extracted dna was checked using electrophoresis with 1% agarose gel, and a nanodrop 8000 spectrophotometer, and the extracts were stored at ­20°c until use. pcr amplification initial testing was carried out with 47 primer pairs d e s i g n e d i n p r e v i o u s s s r s t u d i e s : 1 9 f o r f o r amorphophallus paeoniifolius (santosa et al., 2007), 11 for c. esculenta (hu et al., 2009.) and 17 for x. sagittifolium (cathebras et al., 2014) (table s3). the 47 primer pairs were tested first with five samples from each target species to determine which pairs could amplify scorable dna products in each species. pcr amplification was performed in pcr mixture (25 µl) containing 2.5 µl of template dna (20ng/µl), 2.5 µl of 10x nh4 pcr reaction buffer, 1 µl of 50mm mgcl2), 1 µl of 5 µm forward primer, 1 µl of 5 µm reverse primer, 0.2 µl of 5mm each dntp, 0.1 µl of biotaq dna polymerase and 16.1 µl of water. the pcr program consisted of initial denaturation (94°c, 5 mins), 42 cycles each consisting of 20 s denatura­ tion (93°c), 1 min annealing at temperatures ranging from 47 to 59°c (as recommended by the authors above; table s3), and 2 mins elongation (72°c). finally, an extension period of 10 mins was included. after pcr completion, the products were stored at 4°c until gel electrophoresis. ten µl of each pcr product was electrophoresed alongside a 50 bp dna ladder (new england biolab) in polyacrylamide gel (10% instapage gel) at 110 v for one hour, and bands were visualized by silver staining (1l tbe 0,5x buffer + 500 µl of safeview) for 3 mins. amplified fragment sizes were determined by comparison to the 50 bp ladder, and bands were examined and recorded using the endurotm gel documentation system. data analysis recorded gel images with pcr products were analysed the the image studio lite ver 5.2 software, generating binary matrix data for all accessions based on the band patterns observed at each ssr locus: presence of an amplified band was scored as “1”; absence was scored as “0”. summary statistics for each locus were estimated using powermarker 3.25 software. for statistical comparisons within each species, the binary classifications of morphotypes were used: taro ­ dasheen = pop 1, eddoe = pop2; new cocoyam ­ green = pop1, purple = pop2. genalex 6.5 software (peakall and smouse, 2006) was used to calculate the number of polymorphic loci (no. pl), the percentage of poly­ morphic loci (% pl), the observed number of alleles (na), the effective number of alleles (ne), average expected heterozygosity (he) (also known as nei’s gene diversity, nei, 1973), and shannon’s information index (i) (a measure of genetic diversity suitable for codominant data). analysis of molecular variance (amova) was performed to evaluate the genetic varia­ tion within and among morphotype populations by using genalex version 6.503 (peakall and smouse, 2006), and phipt (the proportion of total genetic vari­ ance derived from variance between individuals among populations, i.e. an estimate of population genetic dif­ ferentiation). here, our h0 = no genetic difference among populations, h1 = there is genetic difference among populations, and p= probability of an observed phipt value =/> than that observed by chance, if the null hypothesis (no genetic difference) is true. principal coordinate analysis (pca) was carried out using the genalex version 6.503 (peakall and smouse, 2006). the genetic distance matrix was con­ structed by calculating the shared allele distance for each pair of individuals in powermarker version 3.25. from this matrix, a neighbour joining (nj) tree was constructed using nei’s genetic distances (nei et al., 1983) in the same software. 3. results in the initial test with five samples of each target species, only 27 of 47 ssr primer pairs, amplified and gave polymorphic, scorable bands (fig. 2; table s3): fig. 2 ­ electrophoresis of pcr amplification products to show ssr polymorphism. above: taro tested with primer pair hk29; empty lanes at right are null results for new cocoyam tested with hk29. below: new cocoyam tested with primer pair mxscir10. bammite et al. ‐ genetic diversity of taro and cocoyam 259 two from amorphophallus amplified both target species, 11 from c. esculenta amplified only c. escu‐ lenta and 14 from x. sagittifolium amplified only x. mafaffa. the resulting measures of diversity for each species are shown in tables 1 and 4, and are sum­ marised below. among 26 accessions of taro, 33 alleles were observed at 13 loci, an average of 3.15 alleles per locus (loci are hereafter identified by the primer code names). the frequency of major alleles ranged from 0.38 for locus hk7 to 0.88 for hk5, with an average of 0.62. nei’s gene diversity ranged from 0.21 for hk7 to 0.71 for hk5, with an average of 0.49. polymorphic information content (pic) values ranged from 0.20 to 0.65 with an average of 0.43. the primers hk35, hk26, hk38, ampa9, hk7 with pic values >/= 0.5 were most discriminating (table 1). the percentages of polymorphic loci for each morphotype of taro were 82% (dasheen, pop1) and 58% (eddoe, pop2), with an average of 70% (rounded figures). the num­ bers of different (na) and effective (ne) alleles, shanons information index (i), and nei’s gene diversi­ ty (he) were all higher in dasheen and lower in eddoe (table 2). df = degrees of freedom, ss = sum of squares, ms = mean sum of squares, est. var. = estimated variance, % var = percentage of variation, phipt = proportion of total genetic variance derived from variance between individuals among populations, p = probability value for phipt. table 1 ­ taro: frequency of major alleles, number of alleles, nei's genetic diversity, and polymorphism information content (pic) for 13 primers applied to 26 accessions (togo collection) table 2a ­ taro: statistical measures of genetic diversity in the dasheen and eddoe populations (togo collection) n = no. accessions (test population), na = no. of different alleles, ne = no. of effective alleles, i = shannon's information index, he = nei’s gene diversity, %p = percentage of polymorphic loci (rounded figures) locus freq. major alleles no. alleles genetic diversity pic hk5 0.88 3 0.21 0.2 ampa15 0.85 3 0.27 0.26 hk25 0.77 2 0.36 0.29 hk31 0.65 2 0.45 0.35 hk29 0.65 3 0.48 0.39 ac3 0.5 3 0.54 0.43 hk22 0.65 3 0.51 0.45 hk34 0.65 3 0.51 0.45 hk35 0.65 4 0.52 0.48 hk26 0.54 3 0.59 0.52 hk38 0.42 3 0.64 0.56 ampa9 0.42 5 0.66 0.59 hk7 0.38 4 0.71 0.65 mean 0.62 3.15 0.49 0.43 n na ne i he %p pop1 (dasheen) 15 1.758 1.546 0.451 0.307 82 pop2 (eddoe) 11 1.364 1.234 0.251 0.156 58 mean 1.561 1.39 0.351 0.231 70 se 0.089 0.045 0.033 0.024 12 table 2b ­ taro: summary analysis of molecular variation (amova) in the dasheen and eddoe populations (togo collection) df ss ms est. var. % var. phipt p value between pops 1 15.962 15.962 0.915 17% 0.174 0.014 within pops 24 104.23 4.343 4.343 83% total 25 120.192 ­ 5.258 100% 260 adv. hort. sci., 2021 35(3): 255­267 among the 101 accessions of new cocoyam, 48 alleles were observed at 16 loci, an average of 3.0 alleles per locus. the frequency of major alleles ranged from 0.47 for ampa9 to 0.97 for ampa15, with an average of 0.83. nei’s gene diversity (he) ranged from 0.06 for ampa9 to 0.67 for ampa15, with an average of 0.28. pic values ranged from 0.06 to 0.62, with an average of 0.25. only the ampa9 locus gave a pic value greater than 0.5 (table 3). the percentages of polymorphic loci for each morphotype of new cocoyam were 74% (green, pop1) and 94% (purple, pop2) and with an average of 84% (rounded figures). a lower number of different alleles was recorded in the green population (na = 1.74, n = 23) and a higher number in the purple pop­ ulation (na = 1.87, n = 78), but the number of effec­ tive alleles (ne) and other measures of diversity (i, he) were higher in the green population (table 4). cluster analysis and structuring of genetic diversity analysis of molecular variance (amova) gave per­ centages of molecular variance of 83% within and 17% between the dasheen and eddoe populations of taro, indicating weak differentiation overall (table 2). for new cocoyam, the percentages of molecular vari­ ance were 64% within and 36% between the green and purple population (table 4). the probability value (p) for phipt is higher in taro (0.014) than in new cocoyam (0.001) providing for taro a weaker rejection of the h 0 of no genetic difference between populations. the first and second coordinates of the pca scat­ ter plot (fig. 3) represent, respectively, 48% and 16% (in total 64%; rounded figures) of the detected vari­ ability among taro accessions. for cocoyam acces­ sions, the coordinates represented 41% and 13% (in total 54%) (fig. 4). some dasheen taros formed a dis­ tinct group along the first coordinate, but apart from table 3 ­ new cocoyam: frequency of major alleles, number of alleles, nei's genetic diversity, and polymorphism infor­ mation content (pic) for 16 primers applied to 101 accessions (togo collection) # locus freq. major alleles no. of alleles genetic diversity pic 1 ampa15 0.97 3 0.06 0.06 2 mxscir1 0.95 2 0.09 0.09 3 mxscir1 0.94 2 0.11 0.11 4 mxscir0 0.89 2 0.19 0.18 5 mxscir1 0.87 3 0.23 0.21 6 mxscir1 0.87 3 0.23 0.21 7 mxscir1 0.85 3 0.26 0.23 8 mxscir2 0.86 3 0.25 0.23 9 mxscir2 0.85 3 0.26 0.23 10 mxscir2 0.84 3 0.27 0.24 11 mxscir1 0.83 3 0.29 0.27 12 mxscir2 0.79 3 0.35 0.32 13 mxscir0 0.76 4 0.39 0.35 14 mxscir2 0.77 4 0.38 0.35 15 mxscir1 0.76 3 0.39 0.35 16 ampa9 0.47 4 0.67 0.62 means 0.83 3 0.28 0.25 table 4a ­ new cocoyam: statistical measures of genetic diversity in the green and purple populations (togo collection) n = no. accessions tested, na = average no. of alleles observed per locus, ne = no. of effective alleles, i = shannon's information index, he = nei’s gene diversity, %p = percentage of polymorphic loci (rounded figures). n na ne i he %p pop1 (green) 23 1.742 1.532 0.418 0.287 74 pop2 (purple) 78 1.871 1.176 0.23 0.128 94 mean 1.806 1.354 0.324 0.208 84 se 0.06 0.05 0.033 0.024 10 table 4b ­ new cocoyam: summary analysis of molecular variation (amova) in the green and purple populations (togo collection) source df ss ms est. var. % var. phipt p value between pops 1 54.566 54.566 1.463 36% 0.36 0.001 within pops 99 257.255 2.599 2.599 64% total 100 311.822 ­ 4.061 100% df = degrees of freedom, ss = sum of squares, ms = mean sum of squares, est. var. = estimated variance, % var = percentage of variation, phipt = proportion of total genetic variance derived from variance between individuals among populations, p = probability value for phipt. bammite et al. ‐ genetic diversity of taro and cocoyam 261 this, there is no clear separation of dasheen and eddoe accessions overall in the ssr data. in contrast, the green and purple new cocoyam formed very dis­ tinct groups along the first pca coordinate. these relationships between ssr diversity in pca scatter­ plot and simple morphotype classification are mir­ rored in the nj dendrograms. the nj dendrogram of ssr diversity in taro (fig. 5) revealed one larger cluster (c1) that includes a mix of dasheen (7 accessions) and eddoe types (10 acces­ sions), and a two clusters (c2, c3) that include dasheen types only (9 accessions). the accessions in c3 were all dasheen types, from wet, flooded envi­ ronments, with purple petiole and petiole junction, a n d g e n e r a l l y p r o d u c i n g m a n y s t o l o n s ( s e e discussion and conclusions) (fig. s1a, fig. s2). the nj dendrogram of ssr diversity in new cocoy­ am revealed two large clusters (c1 and c2) (fig. 6) that largely correspond to the green and purple mor­ photypes of this species (13 and 88 accessions in each category; and many identical haplotypes). with regard to the more specific morphological groups previously reported, c1 includes mostly g1, and c2 includes a mix of mostly g2 and g3 (purple morphotypes; fig. s3). 4. discussion and conclusions the present ssr results indicate few duplicate accessions in the smaller collection of taro (fig. 5) and many apparent duplicates in the larger collection of x. mafaffa (fig. 6). qualitative and quantitative traits for morphological and agronomic characters of taro and new cocoyam were previously recorded and analysed by bammite et al. (2018 b). thirty­eight characters were selected from the descriptor list of i p g r i ( 1 9 9 9 ) f o r c . e s c u l e n t a a n d 2 8 f r o m t h e descriptor list of ibpgr (1989) for x. saggitifolium. based on these detailed observations, morphological groups within each species were identified using upgma analysis (fig. s1). although these groups (g1­ fig. 3 ­ principal coordinate analysis (pca) of taro accessions classified as dasheen or eddoe. fig. 4 ­ principal coordinate analysis (pca) of new cocoyam accessions classified as green or purple. fig. 5 ­ taro: neighbour joining tree based on ssr data from 13 loci in 26 accessions. cluster 1 includes both dasheen (closed circles) and eddoe (open circles) morphotypes. clusters 2 and 3 include only dasheen morphotypes. morphological groups identified by bammite et al. (2018 b) (figs. s1a and s2) are mixed in cluster 1 (mostly g3, g4 and a few g2) and not mixed in clusters 2 and 3. tgul (togo, university of lomé) accession numbers are shown. adv. hort. sci., 2021 35(3): 255­267 262 g4 in taro, g1­g3 in new cocoyam) do not always correspond as expected to the single­character cate­ gories used in the present study (corm size and shape in taro, plant colour in new cocoyam) (figs. s2 and s3), congruences are apparent between ssr genetic diversity and morphological diversity, however the latter is defined. complete correspondence between single­character and multi­character classifications is not expected, but future studies of morphological diversity can be improved by ensuring greater unifor­ mity in the planting materials used. ahmed et al. (2020) found that the dasheen/ eddoe classification corresponds to two distinct evo­ lutionary lineages within c. esculenta, and suggested that the existence of many intermediate or mixed morphotypes may reflect hybridisation between these lineages. in togo, ssr cluster c1 includes both dasheen and eddoe forms, consistent with the sug­ gestion of mixing (hybridisation) between eddoe and dasheen lineages (see also lakhanpaul et al., 2003). clusters c2 and c3 corresponded entirely to dasheen forms with large mother corms and either stolons or side­corms. the three accessions in c2 had morpho­ logical above­ground traits similar to those typical of eddoe in the togo collection (large pendant or drooping leaves) and underground morphological traits of dasheen type (g2, with large central corm with small side­corms). these accessions were col­ lected in home gardens of farmers from kabyè ethnic g r o u p i n t h e e c o l o g i c z o n e i v ( s o u t h e r n t o g o mountains) (fig. 1). they reported having introduced t h e m f r o m e c o l o g i c a l z o n e i i ( n o r t h e r n t o g o mountains), which is consistent with the report by ern (1979) of an expansion of banana, cassava and taro production on steep slopes in zone iv by kabyè settlers from the north. the observation of two major ssr clusters in new cocoyam (c1­c2) suggests that more than one species of xanthosoma is present in togo, not just x. mafaffa (bammite et al., 2018 a, b). a survey of amplified fragment length polymorphism (aflp) in new cocoyam in ethiopia also revealed two major clusters (wada et al., 2018). the existence of such distinct lineages and the general uncertainty of iden­ tification of xanthosoma species suggest an urgent need for direct and detailed comparisons between cultivated xanthosoma spp. in africa, and the wild and cultivated species of xanthosoma in tropical america. for both species, the neighbour joining analysis of ssr allelic diversity at a small number of loci pro­ vides tree diagrams in which terminal branching (near tree tips) is not a reliable indicator of phyloge­ ny. much of the “within population” variation in both species may reflect somatic mutation within clonal cultivar lineages. the small numbers of loci analysed here (13 in taro, 14 in new cocoyam) make it inher­ ently difficult to distinguish clones with certainty, as there are 14 chromosomes in the haploid comple­ ment of c. esculenta (coates et al., 1988; cusimano et al., 2012), and 13 in xanthosoma spp. (cusimano et al., 2012; wada et al., 2018) giving rounded aver­ ages of just 0.9 loci (taro) and 1.1 loci (new cocoyam) fig. 6 ­ new cocoyam: neighbour joining tree based on ssr data from 16 loci in 101 accessions collected in togo. cluster 1 includes green morphotypes (open circles) and cluster 2 i n c l u d e s p u r p l e m o r p h o t y p e s ( c l o s e d c i r c l e s ) . morphological groups identified by bammite et al. (2018b) (figs. s1b and s3) are slightly mixed in both clu­ sters (a few g2 and g3 in cluster 1, and a few g1 in cluster 2). tgul (togo, university of lomé) accession numbers are shown. bammite et al. ‐ genetic diversity of taro and cocoyam 263 sampled per chromosome. for taro, actual coverage is less than 0.9 loci/chromosome, as two hk loci have been mapped to one linkage group and chromosome (table s4). although chaïr et al. (2016) introduced a method to estimate clonality based on just 11 loci, the same research group also employed a much m o r e r o b u s t m e t h o d u s i n g d i v e r s i t y a r r a y technology (darttm) to screen polymorphic loci (pos­ sibly thousands) across the entire taro genome (vandenbrouke et al., 2016). by combining the latter method with a detailed survey of morphological diversity in an assemblage of vanuatu cultivars, vandenbrouke et al. (2016) could unequivocally iden­ tify clonal lineages within which somatic mutation has produced distinct phenotypes that are recog­ nised, selected, and maintained by farmers. most recently, soulard et al. (2017) have mapped polymor­ phic snp and ssr loci across the entire taro nuclear genome, while yin et al. (2021) have published nearly complete sequences for all 14 chromosomes in taro. somatic mutation may explain some of the mor­ phological and genetic diversity found in taro in togo and africa generally, but spontaneous breeding a m o n g d i p l o i d c u l ti v a r s i n a f r i c a m a y a l s o b e involved. the chromosome numbers of togo cultivars h a v e n o t b e e n s t u d i e d , b u t t r i p l o i d t a r o s a r e widespread and common in africa (chaïr et al., 2016) a n d t h e n e i g h b o u r i n g c o u n t r y o f b u r k i n a f a s o (traore, 2013), and can be assumed to be inherently sterile because they are triploids. the eddoe­type taros (c. esculenta var. antiquorum) in togo are likely to be triploids, as this morphotype is generally triploid in neighbouring burkina faso (traore, 2013) and eastern asia (plucknett, 1983; matthews, 2014; wang et al., 2020), but this cannot be assumed ­ if the diploid (fertile) progenitors of triploid eddoe cul­ tivars still exist, some might share the eddoe mor­ photype. it also cannot be assumed that the dasheen types are diploid. there are multiple triploid lineages in taro, and some dasheen and intermediate mor­ photypes are also triploid (kreike et al., 2004). the results of our initial primer screening corrobo­ rate those of traore (2013), who found that primers designed for c. esculenta are not transferable to xanthosoma spp. accessions. we also found that, conversely, the primers designed for xanthosoma do not amplify c. esculenta accessions. in their original report of the hk primers, hu et al. (2009) surveyed 30 plants from several provinces of china. chaïr et al. (2016) screened 64 primer pairs developed from c. esculenta and a. paeoniifolius, and selected 11 from c. esculenta, of which three were from the hk primer series. this study (the largest survey of ssr diversity in taro) included 321 cultivars from 19 countries in asia, africa, america and the pacific. several hk p r i m e r s w e r e a l s o u s e d b y h u n t e t a l . ( 2 0 1 3 ) . including the present togo survey, results for hk7, hk22 and hk26 can now be compared across four studies (table 5). the largest number of alleles was found in the largest sample set representing many countries (chaïr et al., 2016), which is not surprising. the surprise here is that togo, a relatively small country far from asia, displayed only slightly fewer alleles than a similar number of plants from across china (hu et al., 2009), a much larger country that is also a candidate region for the origin of triploid taros (matthews, 2014; wang et al., 2020; zhu et al., 2000). the number of alleles in two wild breeding populations in papua new guinea and an adjacent region of northern australia was larger than in the china and togo cultivars, but also much less than in the large survey by chaïr et al. (2016). the relatively low number of alleles detected in togo presumably reflects the small number of plants table 5 ­ sample size (n) and number of alleles at ssr loci in taro, in four different studies using the hk primer series designed by hu et al. (2009) (­) = loci not studied locus number of alleles hu et al. (2009), n=30, china hunt et al. (2013), n=42–49, australia & png (two wild populations) chaïr et al. (2016), n= 321 (19 countries excl. china) present study n= 26, togo hk5 6 10 ­ 3 hk7 4 2 12 4 hk22 3 ­ 18 3 hk26 5 8 28 3 hk31 3 4 ­ 2 hk34 3 9 ­ 3 hk35 3 11 ­ 4 adv. hort. sci., 2021 35(3): 255­267 264 tested, and the relatively low genetic diversity of taro in africa generally (chaïr et al. 2016). nevertheless, the overall diversity of taro in togo, and in the neigh­ bouring countries of ghana and burkina faso (traore, 2013) does suggest a complex history of the crop in the region, and in africa. among published studies of ssr diversity in taro (table s1), no two studies have used the same meth­ ods to collect, maintain and test plants, and no stan­ dard set of primer pairs and target loci has emerged. crucially, different sample sets differ in whether they represent initial collections created to assess diversi­ ty in possibly­identical cultivars from different loca­ tions (as in the present study), or later­stage collec­ ti o n s i n w h i c h a p p a r e n t d u p l i c a t e s h a v e b e e n removed. observed diversity depends on how plants are collected, how many are collected, and how each collection is maintained over time. taro and new cocoyam collections are constructed “populations” of clones, not random samples from freely breeding populations. for all of these reasons, we do not com­ pare our statistical estimates (calculated data) with those of other small­scale studies. in the near future, new techniques for large­scale and low­cost dna sequencing may allow more accurate, comprehen­ sive and direct comparison of genotypes in different c u l ti v a r a s s e m b l a g e s . a l r e a d y f o r t a r o , p u b l i c databases contain records of thousands of ssr and snp (single nucleotide polymorphism) loci revealed by whole­genome and transcriptome studies (liu et al., 2015; you et al., 2015; helmkampf et al., 2017; soulard et al., 2017; wang et al., 2020), and a draft sequence for all 14 chromosomes has been published (yin et al., 2021). in the togo collection, flowering occurred among accessions of both species, but fruiting and seed pro­ duction were not observed. togo has a tropical savannah climate, with distinct wet and dry seasons, and annual rainfall ranging from around 800 mm to 1,600 mm (djaman et al., 2017). in burkina faso, in t h e s a m e g e n e r a l c l i m a t e z o n e , b u t f u r t h e r northwest, most taro is mostly grown in provinces with annual rainfall ranging from around 700 mm to 1,100 mm, near togo (traore, 2013). although condi­ ti o n s d u r i n g t h e w e t s e a s o n i n t o g o ( a p r i l t o october) might be suitable for breeding by taro, dry and windy conditions during the winter harmattan (ern, 1979) would be fatal for unprotected seedlings. these are very different circumstances from those in the natural range of taro, in the tropical rainforest zone of asia and the western pacific, where wild breeding populations are found (matthews, 1991; hunt et al., 2013; matthews, 2014). togo itself lies in a dry savannah corridor (the dahomey gap) flanked by tropical rainforest (the upper and lower guinean forests) where spontaneous breeding by taro may be possible. breeding and selection of taro cultivars in these nearby forest regions might have contributed to some of the diversity found in togo. different strategies are suggested here for future development of taro and new cocoyam in togo. for taro, it will be rewarding to study the existing range of eddoe and dasheen cultivars further, and to make experimental introductions of new cultivars from outside africa, following the example of ouedraogo et al. (2018). efforts will be needed to produce dis­ ease­free stocks of existing cultivars so that fair com­ parisons can be made with newly­introduced plants that are disease free. new cocoyam, a relatively modern historical introduction, has spread widely in africa, and a lack of diversity is clear among the accessions collected in togo. international collabora­ tion is needed to identify and introduce new cultivars for evaluation under local conditions. this will be dif­ ficult, as there are no international breeding pro­ grammes for the crop, and little is known about the origins and diversity of cultivated xanthosoma species in central and south america. xanthosoma spp. are even more neglected as orphan crops than taro (matthews and ghanem, 2021). together, taro and new cocoyam offer a range of cultivars suitable for cultivation in wetland to dryland environments. agriculture in togo is predominantly rainfed and often experiences both flooding and drought (djaman et al., 2017). in seasons and loca­ tions when water is abundant, the flooding tolerance of dasheen taro (onwueme, 1999) is a positive attribute that can enhance the food security of farm­ ers working in riverine flood plains. when irrigation is provided, very good yields of taro can be expected in otherwise dry environments: this is shown by the success of taro as an irrigated summer crop in the eastern mediterranean (matthews, 2006), a region with long dry summers and relatively little annual rainfall (approx. 400 mm per year in the main agricul­ tural districts of cyprus). over the last 50 years, cli­ mate change has been very obvious throughout togo, with the wet season becoming 1­2 months shorter (djaman et al., 2017; gadédjisso­tossou, 2018). during the period 1961­2001, for example, annual precipitation decreased at 80% of weather stations across the country (djaman et al., 2017). bammite et al. ‐ genetic diversity of taro and cocoyam 265 under these circumstances, maintaining or expand­ ing the 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1 1 department of horticultural sciences, faculty of agriculture, university of zanjan, zanjan, iran. 2 department of water engineering, faculty of agriculture, university of zanjan, zanjan, iran. key words: anthocyanin, antioxidant enzymes, leaf water status, nutrient uptake. abstract: the field experiment was conducted to evaluate the effect of foliar spray of calcium lactate (ca) on fresh yield and biochemical attribute of lettuce (lactuca sativa l.) under water deficit stress, in a split plot form based on a ran­ domized complete block design with three irrigation regimes (70, 85 and 100% etc) and three calcium lactate treatment levels (0, 0.75 and 1.5 g l­1) in three replicates. results revealed that water deficit stress significantly reduced the growth and yield of plant, leaf relative water contents, excised leaf water retention and n, p and mg absorption while led to increase anthocyanin, phe­ nol and flavonoids contents, antioxidant activity, peroxidase and catalase activ­ ity and water use efficiency. the results of our research indicated that the application of cal 1.5 g l­1 is capable of increasing lettuce yield, under field con­ ditions with 30% less than optimal irrigation. cal treatment showed a clearly protective effect in stressed plants, enhancing their leaf water status, antioxi­ dant capacity and n and ca contents in comparison to untreated plants. therefore, feeding leaves by cal with increasing antioxidant activity and nutri­ ents content especially n led to increase growth and fresh yield of lettuce under normal irrigation and water deficit conditions. 1. introduction abiotic stresses such as high temperature, drought, salinity and chemi­ cal toxicity, are the most important limiting factors to crop productivity. drought is undoubtedly one of the most important stresses that have huge impact on growth and productivity of the crops (fahad et al., 2017; hussain et al., 2018). water stress is the most prominent abiotic stress limiting agri­ cultural crop growth and productivity (gholipoor et al., 2013; ihsan et al., 2016). deficit irrigation stress as a consequence of the progressive decrease in water availability has been a hot topic regarding food security during the last two decades (unesco, 2012). growth and development of plants is influenced by reduction in turgor that result in decreased nutrient acquisi­ (*) corresponding author: tbarzegar@znu.ac.ir citation: khani a., barzegar t., nikbakht j., ghahre­ mani z., 2020 ­ effect of foliar spray of calcium lactate on the growth, yield and biochemical attribute of lettuce (lactuca sativa l.) under water deficit stress ­ adv. hort. sci., 34(1): 11­24. copyright: © 2020 khani a., barzegar t., nikbakht j., ghahremani z. this is an open access, peer reviewed article published by firenze university press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and its supporting information files. competing interests: the authors declare no competing interests. received for publication 31 october 2019 accepted for publication 14 may 2019 ahs advances in horticultural science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ adv. hort. sci., 2020 34(1): 11­24 12 tion from dry soil (luo et al., 2011). due to the threat of climate change, there is a need to limit the use of water resources in arid and semi­arid climates. it is therefore important to find new approaches to avoid crop productivity losses in ‘limited fresh­water’ areas. lettuce (lactuca sativa l.), an annual plant of asteraceae family, is considered as one of the most important salad vegetables as a cool season crop. lettuce leaves contain vitamins c and e, carotenoids, phenolic acids with anti­free radical activity, and min­ erals with a lot of fiber, which are an important part of the human diet. moreover, lettuce contains lac­ tocin and lactucopicrin which improve the quality of sleep (chakraborti et al., 2002; faostat, 2016). since most of vegetable species are shallow­root­ ed, they are sensitive to mild water stress. in lettuce production, it is particularly important to preserve optimal growth through a well­scheduled irrigation program, where the harvested part of the plant is the photosynthetic leaf area, (ahmed et al., 2000; casanova et al., 2009). its leaves have high water content and it is sensitive to mild water deficit stress due to its shallow root system (kizil et al., 2012). therefore, in lettuce, new strategies will become crit­ ical to enhance productivity under deficit irrigation (malcom et al., 2012). foliar application of agro­chemicals has widely been used in agriculture as a rapid, low­cost and effective way for enhancing growth and productivity of many vegetable crops under water deficit stress especially green leafy vegetables like lettuce. calcium lactate is considered as one of important agro­chemi­ cal which can be spray and play important roles in physiological and biochemical processes. calcium (ca) is the mineral nutrient most commonly decrease absorption under water deficit condition, so increas­ ing the calcium content in the leafy vegetables could further improve ca concentrations in plant tissues (grusak, 2002). ca is an essential macronutrient for plant growth and development, and is considered as an important intracellular messenger, mediating responses to hor­ mones, stress signals and a variety of developmental processes. furthermore, ca is an important compo­ nent in the structure of cell walls and cell membranes (hepler and winship, 2010). ca plays a role in the regulation of various mechanisms of plants under environmental conditions such as water stress, heat, cold and salinity. in addition, calcium signaling is required for acquisition of tolerance or resistance to the stress (cousson, 2009). positive effect of calcium in improving stress tolerance can be attributed to regulate of water status, antioxidant systems activity, osmolytes accumulation, improving photosynthetic pigment content, and nutritional balances (kurtyka et al., 2008). ca plays an important role in oxidative stress signaling, linking h2o2 perception and induc­ tion of antioxidant genes in plants (rentel and knight, 2004). ca participates in most cellular signaling processes (sanders et al., 2002) and interacts strong­ ly with reactive oxygen species (evans et al., 2005). since the combined effects of ca and water deficit stress have hardly been reported, the current study was, therefore, designed to evaluate the influence of foliar application of calcium lactate on the growth, yield and biochemical attribute of lettuce cv. new red fire under water deficit stress. 2. materials and methods experimental design the field experiment was carried out at the research farm of the agriculture faculty, university of zanjan, iran, during 2017. the experiment was performed using a split plot based on a randomized complete block design with three irrigation regimes (70, 85 and 100% etc) as the main plot and three cal­ cium lactate (cal) treatment levels (0, 0.75 and 1.5 g l­1) as the sub­plot in three replicates. the soil prop­ erties of experimental filed as well as average daily climatic data during the growing seasons was shown in tables 1 and 2, respectively. plant material seeds of lettuce (lactuca sativa l.) cv. new red fire was obtained from a “takii seed” company. lettuce seeds were sown in the nursery on the 2nd of august. seedlings were transplanted at the 3­4 leaf stage when the seedlings were four weeks old with 2 5 c m s p a c i n g w i t h i n r o w a n d 3 5 c m s p a c i n g between rows that there were about 11.5 plants per square meters (plants m­2). table 1 ­ soil physical and chemical properties on the site of experimental field soil texture organic matter (%) ph ec (ds m­1) n (%) ca (g kg­1) na (g kg­1) k (g kg­1) loam clay 0.94 7.4 1.49 0.07 0.12 0.13 0.20 khani et al. ‐ protective effect of calcium lactate on lettuce 13 irrigation treatments and calcium lactate applications after plant establishment, lettuce plants were sprayed with different concentration calcium lactate at 6­7th leaf stage, 10 and 20 days after first spraying for 3 times, during the plant growth. irrigation treat­ ments were applied one week after the first spraying. all foliar sprayings time were the same and distilled water was used for control treatment. the three irri­ gation levels were calculated based on actual evapo­ transpiration (etc): (1) control, irrigated 100% crop water requirement (i100), (2) deficit irrigation 85% etc (i85), and (3) deficit irrigation 50% etc (i50). the water requirement of the plant for control treatment was estimated using long­term average daily data of meteorological parameters recorded at zanjan meteorological station and following relation. etc = et0 × kc etc: water requirement of lettuce (mm/day), et0: e v a p o t r a n s p i r a ti o n o f g r a s s r e f e r e n c e p l a n t (mm/day) and kc: vegetable coefficient of lettuce (no unit). it is necessary to explain that et0 values were estimated based on the standard fao­penman­ monteith method. table 2 shows the long­term average of meteorological parameters of zanjan synoptic station during the period of plant growth which was used to calculate et0 and etc values. after calculating the etc values, the net and gross i r r i g a ti o n w a t e r r e q u i r e m e n t s o f l e tt u c e w e r e estimated based on cropping intervals, type of irrigation system and irrigation interval and then give the plant at each irrigation time. based on the calculations, the amount of irrigation water given to the control plants was estimated to be 895.7 m3.ha­1. water requirement of other treatments was estimat­ ed and distributed based on the water requirement of control treatment and water stress (allen et al., 1998). all necessary management practices such as weeds control were done according to recommend­ ed practices during the crop growth. measurements anthocyanin content. anthocyanin content in leaf tissue was determined according to the method of mita et al. (2000). fresh weight of leaves (0.1 g) was homogenized in methanol containing 1% (v/v) hcl and then was filtrated. the filtration was stored at 4°c for 24 hours in dark conditions. the absorbance of filtration was recorded at 550 nm using uv­vis spectrophotometer (specorp 250 jena­history) and the anthocyanin was expressed as µmol g­1 fw. total phenols and flavonoids contents the fresh leaf tissue (2.0 g) was washed with deionized water, and homogenized in 80% cold methanol (20:80, v/v). the homogenate was cen­ trifuged at 10,000 rpm for 10 min, and the super­ natant was collected for the measurement of, total phenolic and flavonoid content. total phenolics assay was carried out according to the procedure described in the literature (meda et al., 2005). the results were expressed as mg of gallic acid equivalents (gae) per 100 g of fresh weight based on a standard curve using gallic acid as standard. total flavonoids were determined by the colorimetric method (kim et al., 2002). quercetin was used as a reference standard, and the results were expressed as mg quercetin equivalents per 100 g fresh weight of leaf. antioxidant activity as mentioned in the previous paragraph, 2.0 g of leaves were homogenated in methanol and then was centrifuged. the filtration was used to determine free radical scavenging activity using the 2, 2,­ diphenyl­2­picryl­hydrazyl (dpph) method at optical density 517 nm (sun et al., 2007). antioxidant activity (%) was calculated using the following equation: antioxidant activity = a dpph ­ a sample (517 nm)/a dpph × 100 catalase (cat) and peroxidase (pox) enzymes activity samples were taken from the fully expanded leaf and transferred to the laboratory in the ice. leaf sample (0.5 g) was frozen in liquid nitrogen and ground using a porcelain mortar and pestle. catalase (cat) activity was measured by following the decomposition of h2o2 at 240 nm with a uv spec­ trophotometer (cakmak and horst, 1991). samples table 2 ­ average daily climatic parameters of zanjan synoptic station during the growth seasons (2017) of lettuce meteorological parameter may june july august september rainfall (mm) 0.01 1.11 5.00 0.00 0.02 average temperature (˚c) 22.94 25.71 27.68 24.79 15.73 minimum temperature (˚c) 11.29 16.8 17.61 14.68 7.89 maximum temperature (˚c) 32.47 33.96 36.82 35.12 25.05 adv. hort. sci., 2020 34(1): 11­24 14 without h2o2 were used as blank. the activity of cat was calculated by the differences obtained at od240 values at 30 second interval for 2 min after the initial biochemical reaction. peroxidase (pox) activity was measured using modified method of the tuna et al. (2008) with guaiacol at 470 nm. a change of 0.01 units per minute in absorbance was considered to be equal to one unit pox activity, which was expressed as unit g­1 fw min­1. leaf water status (rwc, elwr) t h e f r e s h w e i g h t o f y o u n g l e a v e s ( f w ) w a s recorded and then was kept in petri dishes for 24 hours immersed in distilled water. the turgid weight (tw) was measured after saturation of leaves with water. the leaves were dried at 70˚c to constant weight and then weighted (dw). leaf relative water contents (rwc) were calculated according to the fol­ lowing formula reported by hanson and hitz (1982). (%) rwc= (fw­dw) / (tw­dw) × 100 for the determination of excised leaf water reten­ tion (elwr), the youngest leaves collected for each treatment were weighed to record fresh weight (fw), kept at room temperature (25˚c) for 6 hours and reweighed (wl). elwr was calculated using the following formula suggested by lonbani and arzani (2011). elwr= [1­(fw­wl)/fw] ×100 nutrient contents the lettuce leaf samples from each treatment were collected at the end of the experiment. for mineral analysis, leaf samples were taken and oven­ dried at 70°c until constant weight. then 0.3 g of the dry samples was taken and digested using a mixture of sulphuric acid (h2so4) and hydrogen peroxide (h2o2) as described by allen et al. (1974). all the stud­ ied elements were assayed in the digest of the con­ cerned plant samples. total nitrogen was determined using kjeldahl method as described by piper (1950). phosphorus determination was done by complexing it with ammonium molybdate, which on reduction with ascorbic acid gives stable blue colour, the con­ tent of p was measured by spectrophotometer at 882 n m a c c o r d i n g t o w a t a n a b e a n d o l s e n ( 1 9 6 5 ) . potassium, calcium and magnesium content were analyzed by flame photometer (chapman and pratt, 1961). yield and water use efficiency (wue) lettuce plants were weighed after harvest with a digital gravimetric scale. the average weight of single plant was calculated in grams and total yield was estimated in kg/m2. also water use efficiency (wue) was obtained from the ratio of the amount of yield of each treatment to the amount of water consumed by the same treatment in kg m­3. statistical analyses of the data the analysis of variance (two­way anova) and least significant difference (lsd) test (p≤0.05 and p≤0.01) used to compare means within each sam­ pling date. the statistical analysis and standard error calculation were carried out using sas software (v. 9.1). 3. results anthocyanin content the data in table 3 and figure 1, displays the anthocyanin contents of lettuce leaf applied with dif­ ferent concentrations of cal under water deficit. mean comparisons of data showed that deficit irriga­ tion led to a significant increase in antioxidant activi­ ty compared to control. however, the effect of cal on the anthocyanin contents was depended to the ** and * represent significance at the 1 and 5% probability levels, respectively, and ns represents non­significance at p<0.05. table 3 ­ variance analysis (anova) of effect of calcium lactate on physiological characteristics in lettuce under deficit irrigation s.o.v df mean of squares anthocyanin total phenols flavonoids cat activity pox activity antioxidant activity rwc elwr replication 2 1.003 0.002 0.308 0.057 0.003 16.725 8.614 23.677 irrigation 2 2.162 ** 6.187 ** 11.932 ** 1.953 ** 0.851 ** 132.47 ** 42.799 * 238.260 ** error (a) 4 5.648 0.135 0.330 0.009 0.001 23.524 9.036 14.106 calcium lactate 2 4.292 * 1.057 ** 24.265 ** 0.702 ** 0.052 * 318.416 ** 186.691 ** 420.124 ** calcium lactate × irrigation 4 2.770 * 0.475 * 1.149 * 0.075 * 0.030 * 43.004 * 1.302 ns 29.661 * error (b) 12 6.388 0.121 0.338 0.023 0.008 9.686 9.323 6.176 coefficient of variation (%) ­ 11. 18 2. 31 4.19 8.87 16.93 3.73 3.9 3.4 khani et al. ‐ protective effect of calcium lactate on lettuce 15 total phenols and flavonoids contents the exposure to water deficit stress significantly (p<0.05) increased total phenols and flavonoids con­ tents (table 3, figs. 2a, b). besides, the results of the present study also showed that foliar application of cal increased total phenols and flavonoids contents under normal and deficit irrigation, however, the effects of cal was dependent to the irrigation levels. the maximum value of phenols and flavonoids con­ tents was recorded in plant treated with 0.75 g l­1 cal under irrigation 70% etc. in all levels of irrigation, application of 0.75 g l­1 cal had the greatest effect on total flavonoid content, although did not show significant difference with cal 1.5 g l­1 under irriga­ tion 70% etc. phenolic compounds include many secondary metabolites in plants that display antioxidant proper­ ties (barbagallo et al., 2012). some of the phenolic compounds, such as phenolic acids or flavonoids, are widely recognized in most of the plant species (jwa et a l . , 2 0 0 6 ) . p h e n o l i c c o m p o u n d s a r e i m p o r t a n t because of their contribution to the nutritional quali­ ty attributes of fruits and vegetables such as color, astringency, bitterness and flavor (vinson et al., 2001). the role of phenols as antioxidant is support­ ed by several researches and the recovery methods h a v e a g r e a t i m p o r t a n c e f o r i n d u s t r i a l u s e (barbagallo et al., 2012). environmental stress can cause an increase in the content of phenolic com­ pounds of cell (weidner et al., 2009). aghdam et al. (2013) reported that the total phe­ nols and flavonoids contents increased in the cor­ nelian cherry fruit with cacl2 treated. their results suggested that cacl2 treatment may stimulate the accumulation of phenols and flavonoids fruits by acti­ vating their biosynthetic pathways. biosynthesis of phenols such as flavonoids in plants carried out via the shikimate­phenylpropanoid pathways. ca2+ plays irrigation regime treatment. the highest value of anthocyanin content was obtained from plant treat­ ed with 1.5 g l ­1 cal under irrigation 85% etc. according to the results, there was no significant dif­ ference between different levels of cal under irriga­ tion 100% etc. however, anthocyanin content at 85% etc was significantly increased by using cal while under irrigation of 70% etc with the increase of anthocyanin content did not show any significant dif­ ference between different levels of cal. secondary metabolic products, which are inten­ sively biosynthesized under drought, are antioxidants (do nascimento and fett­neto, 2010). anthocyanin pigments as one of secondary metabolites and antioxidative systems play many important eco­phys­ iological roles in plants, including roles in stress pro­ tection (winkel­shirley, 2002). increased anthocyanin contents are thought to mask chlorophyll and/or act as a filter for preventing high light absorption by leaves and thus minimize photoinhibition (farrant, 2000). therefore anthocyanin accumulation in drought­stressed leaves confirms a possible protec­ tive role of anthocyanins as sun­screens and reactive oxygen species (ros) scavengers in stressed plants (merzlyak and chivkunova, 2000), that similar results have also been reported by jazizadeh and mortezaei nejad (2016) in chicory. the obtained results indicated that cal was effec­ tive in preserving and increasing anthocyanin con­ tent. these findings were in agreement with abd­ elhady (2014) findings who also observed that cal pretreatments proved to be effective for increasing the retention of anthocyanin in frozen strawberry. fig. 2 ­ effects of cal treatments on total phenol and flavonoids contents of lettuce under deficit irrigation. values are means with standard errors (n= 3). data were subjected to two­way anova and different letters mean that values are statistically different p<0.05. fig. 1 ­ effects of cal treatments on anthocyanin content of let­ tuce under deficit irrigation. values are means with stan­ dard errors (n= 3). data were subjected to two­way anova and different letters mean that values are stati­ stically different p<0.05. http://https://www.sciencedirect.com/science/article/pii/s0570178314000116" /l "! http://https://www.sciencedirect.com/science/article/pii/s0570178314000116" /l "! 16 adv. hort. sci., 2020 34(1): 11­24 a d i r e c t r o l e i n t h e b i o s y n t h e s i s o f p h e n o l s (castañeda and perez, 1996). cal might be a poten­ ti a l m o l e c u l e f o r a c ti v a ti n g p h e n y l p r o p a n o i d ­ flavonoids pathways of fruits by increasing the pal activity (jacobo­velazquez et al., 2011; aghdam et al., 2013). catalase (cat) and peroxidase (pox) enzymes activity significant differences among irrigation treat­ ments were observed for cat and pox enzyme activi­ ty (table 3, figs. 3a, 3b). the antioxidant enzyme activates increased with the decrease of irrigation water applied. as the results showed cat and pox enzymes activity increased with increasing cal con­ centration under deficit irrigation, although no signif­ icantly differences was observed in normal irrigation. the highest cat and pox enzymes activity were recorded in plant treated with 1.5 g l­1 cal under irri­ gation 70% etc, which had no significant difference with 0.75 g l­1. the production of ros (vurukonda et al., 2016) is another major factor that impairs plant growth under water deficit (liting et al., 2015). plants employ a number of mechanisms, at molecular, cellular and p h y s i o l o g i c a l l e v e l s t o p e r s i s t s t r e s s c o n d i ti o n (shinozaki and yamaguchi­shinozaki, 2000). the acti­ vation of antioxidant enzymes is one of the major types of these mechanisms which enable plants to control ros (shahid et al., 2014). cat, ascorbate per­ oxidase (apx), superoxide dismutase (sod) and pox are the key antioxidant enzymes involved in detoxifi­ c a ti o n o f s u p e r o x i d e a n d h y d r o g e n p e r o x i d e (kadkhodaie et al., 2014). a relationship between antioxidant enzymes activity and water stress or salinity tolerance was confirmed by comparison of a tolerant cultivar with a sensitive cultivar in several plant species, such as tomato (mittova et al., 2002). calcium is known to regulate different metabo­ lisms in plants mediating signaling pathways, which modulate gene expression in response to stress and its adaptation (upadhyaya et al., 2011). upadhyaya et al. (2011), observed pox activity was increased in the stressed plant as compared to controls, but recovering plants showed pox activity increasing after rehydration, which was enhanced by cacl2 and reported that cacl2 treatment resulted in increased non enzymatic antioxidant and enhanced activities of enzymatic antioxidant, including sod, pox and cat, and thus reduced ros accumulation and lipid peroxi­ dation ultimately leading to improved post­drought recovery potential in camellia sinensis. calcium applied alleviation of drought­induced damage has been clarified in numerous plants e.g. zoysia japonica (xu et al., 2013), and phaseolus vulgaris (abou el­ yazied, 2011). antioxidant activity (aa) aa was affected significantly by the irrigation treatments, and water deficit stress increased aa, w h i c h n o s i g n i fi c a n t d i ff e r e n c e w a s o b s e r v e d between irrigation 100 and 85%etc (table 3, fig. 4). in present study, the exogenous application of cal significantly (p<0.05) increased aa of lettuce under different irrigation regimes compared to control plant. the highest aa (93.03%) was observed in cal 0.75 g l−1 under irrigation 85% etc, however had no significant difference with cal 1.5 g l­1 treatment under irrigation 70% etc (fig. 4). the antioxidant activity in lettuce arises from phe­ nolic compounds, secondary plant products, such as fig. 3 ­ effects of cal treatments on catalase (cat) and peroxida­ se (pox) enzymes activity of lettuce under deficit irriga­ tion. values are means with standard errors (n= 3). data were subjected to two­way anova and different letters mean that values are statistically different p<0.05. fig. 4 ­ effect of cal treatments on antioxidant activity of lettuce under deficit irrigation. values are means with standard errors (n = 3). data were subjected to two­way anova and different letters mean that values are statistically dif­ ferent p<0.05. khani et al. ‐ protective effect of calcium lactate on lettuce 17 flavonoids and phenols, and also anthocyanin. also, the antioxidant activity strongly correlated to the presence of efficient oxygen radical scavengers, such as vitamin c and phenolic compounds (tulipani et al., 2008). in current study a significant correlation was found between antioxidant activity and antho­ cyanins, phenols and flavonoids contents; which the anthocyanin, phenolic and flavonoids content and cat and pox enzymes activity, as well as the total antioxidant activity also increased with increasing water deficit stress and cal concentration. this find­ ing described that phenolic compounds and antho­ cyanin, and antioxidant enzyme activity makes an important contribution to the antioxidant capacity in lettuce leaf. velioglu et al. (1998) reported a strong relationship between total phenolic content and antioxidant activity in fresh fruits and vegetables. leaf water status (rwc, elwr) based on the findings (table 3, figs. 5a, 6), deficit irrigation caused a significant reduction in rwc and elwr contents. the application of cal significantly ameliorated relative water content (rwc) and excised leaf water retention (elwr) contents (figs. 5b, 6). mean comparisons of data, displayed that pre­ treatment with cal markedly reduced the effects of water deficit stress and also improved elwr under control irrigation and water deficit stress. the highest value of elwr content was obtained in plant treated with cal 1.5 g l­1 under irrigation of 85 and 100% etc. rwc and elwr are among the main physiological criteria that influence plant water relations and have been used for assessing drought tolerance (xing et al., 2004). under drought stress, leaf rwc plays an important role in the tolerance of plants to stress by inducing osmotic adjustments due to the accumula­ tion of osmoprotectants (barnabás et al., 2008; zhang et al., 2012). the maintenance of a high plant water status during stress is a significant defensive mechanism to maintain enough water by minimizing water loss (e.g. caused by stomatal closure, tri­ chomes, reduced leaf area, senescence of older leaves, etc.) and maximizing water uptake (e.g. by increased root growth) (barnabás et al., 2008). because of the decrease in leaf area, the accumula­ tion of chlorophyll has increased, but due to high transpiration, the plant loses more water and as a result, the rwc of leaf and consequently photosyn­ thesis decreases (farooq et al., 2012). farooqi et al. (2000) indicated that rwc of lemongrass leaves decreased in all the cultivars due to drought but after rehydration, rwc gradually increased to pre­stress level, which has also been reported in several crop species such as melon (mani, 2014). as well as drought stress significantly decreased rwc and elwr in spring safflower (balian et al., 2015). ruiz­lozano and azcon (1997) reported that calci­ um application significantly increased rwc in lettuce. the results of this research showed that the rwc of leaves increased with calcium application. increasing relative water content means increasing water hold­ ing capacity, which can prevent water loss in leaves in a dry environment (ma et al., 2005). nutrient contents according to the results (table 4, fig. 7), n con­ tent in lettuce leaves increased with increasing cal concentration, indeed the highest value of n was obtained at cal 1.5 g l­1 under irrigation 100 %etc that had significant difference with deficit irrigation treatments (85 and 70% etc), whereas in other treat­ ments there were not any significant differences. mean comparisons of data, showed that deficit irri­ fig. 5 ­ effects of irrigation (a) and cal (b) treatments on leaf relative water content (rwc) of lettuce. data were subjected to two­way anova and different letters mean that values are statistically different p<0.05. fig. 6 ­ effects of cal treatment on excised leaf water retention (elwr) content of lettuce under deficit irrigation. values are means with standard errors (n= 3). data were subjec­ ted to two­way anova and different letters mean that adv. hort. sci., 2019 33(3): 11­24 18 gation significantly increased p content in lettuce leaves and decreased k content compared to control irrigation (table 4, figs. 8a, 8b). the ca content increased with the deficit irriga­ tion treatments, in particular with moderate deficit irrigation (85% etc). ca content in lettuce leaves increased in response to higher cal concentration ( f i g . 9 a ) . i n f a c t , t h e h i g h e s t v a l u e o f c a w a s observed in treatments with application of 1.5 g l­1 cal under irrigation 70 and 85% etc. the overall effect of deficit irrigation on mg content was nega­ tive, with a decrease of 0.79% (fig. 9b) under deficit i r r i g a ti o n 7 0 % e t c . m g c o n t e n t i n t h e l e a v e s decreased when the concentration of cal applied was increased. the lowest value of mg content was obtained in plant applied with 1.5 g l­1 cal under deficit irrigation 70% etc. drought stress and associated reduction in soil moisture can decrease plant nutrient uptake by reducing nutrient supply through mineralization (sanaullah et al., 2012), and nutrient diffusion in the soil (chapin iii, 1991; lambers et al., 2008). drought can depress plant growth by reducing n and p uptake, transport and redistribution (rouphael et al., 2012). a majority of studies have indicated that plants decrease n and p uptake with a decline in soil moisture (sardans and peñuelas, 2012). n uptake was reduced in maize under stress conditions, which indicates that the absorption of nutrients is limited in conditions of water deficit stress, which may be reduced due to reduced transpiration rate, active transfer and membrane permeability (naeem et al., 2017). owing to a reduction in stomatal conduc­ tance, photosynthesis and transpiration rates also table 4 ­ variance analysis (anova) of effects of calcium lactate on nutrient contents and fresh yield in lettuce under deficit irrigation ** and * represent significance at the 1 and 5% probability levels, respectively, and ns represents non­significance at p<0.05. source of variations df mean of squares n content p content k content ca content mg content fresh yield wue replication 2 0.030 0.008 0.008 0.002 0.004 206838.82 0.380 irrigation 2 0.208 ** 0.111 ** 0.153 ** 0.621 ** 2.157 ** 21349043.15 ** 3.293 ** error (a) 4 0.008 0.008 0.003 0.005 0.004 83934.71 0.183 calcium lactate 2 1.158 ** 0.024 ns 0.025 ns 0.291 ** 0.095 ** 6105376.18 ** 10.144 ** calcium lactate × irrigation 4 0.116 ** 0.006 ns 0.010 ns 0.047 ** 0.018 * 413961.66 * 0.317 ns error (b) 12 0.012 0.006 0.008 0.004 0.005 101407.46 0.170 coefficient of variation (%) ‐ 1.47 3.48 1.43 2.09 3.4 2.82 2.78 fig. 7 ­ effects of cal treatments on nitrogen content of lettuce leaves under deficit irrigation. values are means with standard errors (n= 3). data were subjected to two­way anova and different letters mean that values are stati­ stically different p<0.05. fig. 8 ­ effect of irrigation treatments on phosphorus (a) and potassium (b) contents of lettuce leaves. values are means with standard errors (n= 3). data were subjected to two­way anova and different letters mean that values are statistically different p<0.05. fig. 9 ­ effect of cal treatments on calcium (a) and magnesium (b) contents of lettuce leaves under deficit irrigation. values are means with standard errors (n= 3). data were subjected to two­way anova and different letters mean that values are statistically different p<0.05. khani et al. ‐ protective effect of calcium lactate on lettuce 19 decrease, and co2 assimilation rates progressively decline in response to drought (farooq et al., 2012). therefore, drought effects on plant may depend on the reduction in n and p uptake relative to the decrease in co2 assimilation (he and dijkstra, 2014). based on the current findings, increasing k and ca contents and decreasing mg content of lettuce leaves under water deficit stress as compared to well­ watered conditions that also reported by tadayyon et al. (2018) in castor plants. potassium has a positive correlation with the physiological effects of plants, such as water use efficiency, stomatal control, air and underground body biomass, and is likely to play an important role in photosynthesis (sardans et al., 2012). increasing k content of leaves with decreasing irrigation rate maybe due to role of this cation in the regulation of osmotic pressure and stomatal control, (zhao et al., 2000). nahar and gretzmacher (2002) reported that with increasing deficit irrigation, mg concentration in tomato tissues decreased, which is similar to results of the present study. the same results were reported from other authors, that high concentrations of ca often result in increased leaf ca along with a marked reduction in leaf mg (nassery et al., 1979; borghesi et al., 2011). as well as, naeem et al. (2018) revealed that concen­ tration of macronutrients (n, k, ca) in maize grains was markedly improved by foliar supply of calcium which indicates its synergistic effect on uptake and translocation of these nutrients. tuna et al. (2007) also observed leaf n, k and ca content increased in tomato plants supplemented with calcium under stress conditions. in safflower, by decreasing soil moisture k and mg content decreased. following this reduction, there was a significant increase in calcium concentration, which is justified by the antagonistic relationship between ca and mg (vafaie et al., 2013). morard et al. (1996) reported an intense antagonistic relation­ ship between ca and mg, that mg transfer to leaves was affected by calcium. fresh yield lettuce plants grown under control and deficit irrigation conditions exhibited significant differences between cal treatments in fresh yield (table 4, fig. 10). water deficit stress caused significant reductions in yield. in fact, deficit irrigated plants showed a 6.8 and 15.8% decrease in fresh yield, respectively. as the results showed, with increasing cal concentra­ ti o n s , l e tt u c e y i e l d s i g n i fi c a n t l y i n c r e a s e d a n d reached to highest value (1.37 kg.m­2) at cal 1.5 g l­1 under irrigation 100% etc (fig. 10). lettuce is one of the leaf­edible vegetables that it is extremely sensitive to water deficit stress due to shallow root system (sabedze and wahome, 2010). our results are in agreement with many open­field studies on lettuce (jiménez­arias, 2019) and lettuce (sayyari et al., 2013). deficit irrigation defined as a practice that applies water below full crop­water requirements, deliberately exposes plants to a cer­ tain level of moisture stress. it is well known that drought stress results in dehydration of the cell and osmotic imbalance that impairs numerous metabolic and physiological processes in plants (mahajan and tuteja, 2005). reduction in fresh yield of lettuce with deficit irrigation might be attributed to the suppres­ sion of cell division and expansion, and growth due to the low turgor pressure and also closure of stomata leaf and more leaf senescence under drought stress (sayyari et al., 2013). foliar application of cal enhanced fresh yield of lettuce. naeem et al. (2013) reported that crop pro­ ductivity and photosynthetic efficiency in senna occi‐ dentalis was improved under ca application. with low calcium availability, a reduction in bean plant height, leaf area and shoot and root growth has been reported (leal and prado, 2008). foliar applied of chelated calcium enhanced the seed yield and relat­ ed attributes in common bean under water­deficit conditions (abou el­yazied, 2011). water use efficiency (wue) according to the results (table 4, figs. 11a, 11b), irrigation and cal treatments significantly affected wue, but their interaction showed no significant dif­ ferences. water deficit stress significantly increased wue and the highest wue was recorded in 70% etc deficit­irrigated plants that had no significant differ­ fig. 10 ­ effect of cal treatments on fresh yield of lettuce under deficit irrigation. values are means with standard errors (n= 3). data were subjected to two­way anova and dif­ ferent letters mean that values are statistically different p<0.05. adv. hort. sci., 2019 33(3): 11­24 20 ence with deficit irrigation 85% etc (fig 8a). wue increased with increasing cal concentration and the highest wue (15.8 kg m­3) was obtained at 1.5 g l­1 cal (fig. 8b). w u e , t h e p h y s i o l o g i c a l p a r a m e t e r o f c r o p , describes the relationship between plant water use a n d d r y m a tt e r p r o d u c ti o n ( c a i e t a l . , 2 0 1 2 ) . regarding water resource constraints, it is essential to find ways to preserve water and increase water use efficiency in plants (topcu et al., 2007; alenazi et al., 2015). the highest wue value was determined in 70% etc irrigation. it was calculated that wue values increased with the decrease in the amount of water. these results are similar to the previous finding of şimşek et al. (2004), who reported that the maximum wue for watermelon was obtained with low irriga­ tion. with increased wue, there is a greater biomass production per amount of water transpired, and less w a t e r i s n e e d e d f o r g r o w t h a n d d e v e l o p m e n t (nemali and van iersel, 2008). wue is strongly related to photosynthetic activity and transpiration efficiency, and can be affected by irrigation (monneveux et al., 2006). ca is directly involved in photosynthesis processes, and its deficit reduces the plant’s biomass by reducing the efficien­ cy of carboxylation and photosynthesis (alarcon et al., 1999). the results of the current experiment showed that n content was increased with ca appli­ cation, which increasing n content leads to increase dry matter production as well as the wue. therefore, ca maybe increased the wue by increasing the amount of n and ca in lettuce plants. 4. conclusions the results obtained in this investigation pro­ posed that lettuce is sensitive to 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